Glutamate is the principal cerebral excitatory neurotransmitter and dilates cerebral arterioles to match blood flow to neural activity. Arterial contractility is regulated by local and global Ca2+ signals that occur in smooth muscle cells, but modulation of these signals by glutamate is poorly understood. Here, using high-speed confocal imaging, we measured the Ca2+ signals that occur in arteriole smooth muscle cells of newborn piglet tangential brain slices, studied signal regulation by glutamate, and investigated the physiological function of heme oxygenase (HO) and carbon monoxide (CO) in these responses. Glutamate elevated Ca2+ spark frequency by ∼188% and reduced global intracellular Ca2+ concentration ([Ca2+]i) to ∼76% of control but did not alter Ca2+ wave frequency in brain arteriole smooth muscle cells. Isolation of cerebral arterioles from brain slices abolished glutamate-induced Ca2+ signal modulation. In slices treated with l-2-α-aminoadipic acid, a glial toxin, glutamate did not alter Ca2+ sparks or global [Ca2+]i but did activate Ca2+ waves. This shift in Ca2+ signal modulation by glutamate did not occur in slices treated with d-2-α-aminoadipic acid, an inactive isomer of l-2-α-aminoadipic acid. In the presence of chromium mesoporphyrin, a HO blocker, glutamate inhibited Ca2+ sparks and Ca2+ waves and did not alter global [Ca2+]i. In isolated arterioles, CORM-3 [tricarbonylchloro(glycinato)ruthenium(II)], a CO donor, activated Ca2+ sparks and reduced global [Ca2+]i. These effects were blocked by 1H-(1,2,4)-oxadiazolo-(4,3-a)-quinoxalin-1-one, a soluble guanylyl cyclase inhibitor. Collectively, these data indicate that glutamate can modulate Ca2+ sparks, Ca2+ waves, and global [Ca2+]i in arteriole smooth muscle cells via mechanisms that require astrocytes and HO. These data also indicate that soluble guanylyl cyclase is involved in CO activation of Ca2+ sparks in arteriole smooth muscle cells.
- calcium spark
- calcium wave
- global calcium
regional neuronal activity modulates local brain blood flow through a response termed functional hyperemia (12, 16). Such local blood flow regulation functions to match O2 and nutrient demands to cellular requirements by altering the contractility of vascular smooth muscle cells (SMCs) within the arteries and arterioles that supply the vicinity of the active neurons. In the brain, vascular SMCs can receive regulatory input from multiple cell types, including the endothelium, neurons, astrocytes, and pericytes (16). Each of these cell types can modulate vascular SMC contractility, thereby contributing to functional hyperemia.
Glutamate is the principal excitatory cerebral neurotransmitter. One important physiological function of glutamate is to dilate cerebral arterioles to match blood flow to neural activity. Astrocytes are one candidate cell type for the mediation of the glutamate-induced neurovascular coupling to elicit vasodilation (13, 24, 32, 39, 40). Parenchymal arterioles are surrounded by astrocytic endfeet, and pial arterioles lay on the glia limitans and are covered with astrocyte processes and endfeet (13, 24, 27, 42). Astrocytes express both metabotropic glutamate receptors (mGluRs) and ionotropic glutamate receptors (iGluRs) and are uniquely positioned for sensing neuronal activity and regulating cerebral blood flow. In brain slices, glutamate elevates astrocyte intracellular Ca2+ concentration ([Ca2+]i) but reduces adjacent vascular SMC [Ca2+]i, a response that is associated with vasodilation (2, 7, 8, 43). Astrocyte-derived vasoactive mediators that have been described to elicit this response include epoxyeicosatrienoic acids (3, 30), K+ (8, 36), ATP (34), and heme oxygenase (HO)-2-derived carbon monoxide (CO) (29).
CO appears to be a major astrocyte-derived cerebral vasodilator, particularly in newborns (27, 29). Exogenous CO applied to SMCs or HO-derived CO generated within SMCs elevates Ca2+ spark frequency and increases the Ca2+ sensitivity of SMC large-conductance Ca2+-activated K+ (KCa) channels (18, 19, 41). These changes elevate transient KCa current frequency and enhance KCa channel coupling to localized intracellular Ca2+ transients termed Ca2+ sparks, which elevates transient KCa current amplitude. The resulting increase in KCa current causes membrane hyperpolarization, leading to a reduction in voltage-dependent Ca2+ channel activity, and vasodilation (9, 18, 29). Astrocyte-generated, HO-2-derived CO activates transient KCa currents in closely opposed arterial SMCs, leading to a reduction in global [Ca2+]i and vasodilation (29).
Ca2+ sparks are only one type of Ca2+ signal that occurs in SMCs. Indeed, SMCs generate multiple local and global Ca2+ signals that differ with respect to spatial, temporal, and amplitude properties and physiological functions (17, 21). The regulation of these different local and global Ca2+ signals in arteriole SMCs by glutamate and the physiological function of astrocytes in mediating the responses are unclear.
Here, we tested the hypothesis that glutamate regulates local and global Ca2+ signals in arteriole SMCs through astrocyte- and HO-dependent mechanisms. Using high-speed confocal imaging of pial arteriole SMCs in newborn pig brain slices, we show that glutamate-induced local and global Ca2+ signal modulation is mediated by functional astrocytes and HO activation. In the presence of a HO inhibitor, a different pattern of Ca2+ signal modulation by glutamate was observed. The data also indicate that soluble guanylyl cyclase (sGC) is involved in CO activation of Ca2+ sparks in arteriole SMCs. These data identify glutamate modulation of Ca2+ signaling in newborn arteriole SMCs.
The animal procedures used were approved by the Animal Care and Use Committee of the University of Tennessee. Newborn pigs (1–3 days old, 1–2.5 kg) were anesthetized with ketamine hydrochloride (33 mg/kg im) and acepromazine (3.3 mg/kg im). The brain was removed and placed in M199 with 10 mM HEPES and 10 mM glucose at 4°C (pH 7.37). Tangential slices (∼200 μm thick) were cut from the cerebral cortical surface and maintained in M199. Where required, cerebral arteries (50–200 μm in diameter) were manually harvested, cleaned, and maintained in ice-cold (4°C) physiological saline solution (PSS) containing (in mM) 112 NaCl, 4.8 KCl, 26 NaHCO3, 1.8 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, and 10 glucose and gassed with 74% N2-21% O2-5% CO2 (pH 7.4). To selectively injure astrocytes in brain slices, slices were treated with l-2-α-aminoadipic acid (l-AAA; 2 mM, 2 h). Control slices were incubated with d-2-α-aminoadipic acid (d-AAA), an inactive isomer.
Confocal Ca2+ imaging.
Brain slices and isolated arterioles were incubated in HEPES-buffered PSS containing fluo-4-AM (10 μM) for 25 min at room temperature followed by a 30-min wash. Brain slices were transferred to a recording chamber with constant perfusion (3–5 ml/min) of PSS containing U-46619 (10 nM), a thromboxane A2 receptor agonist, at 35°C to promote arterial tone. Isolated arterioles were imaged in an extracellular bath solution of the following composition (in mM): 110 NaCl, 30 KCl, 10 HEPES, 2 CaCl2, 1 MgCl2, and 10 glucose (pH 7.4 with NaOH). This methodology has been previously performed to depolarize arteries to approximately −40 mV, a membrane potential similar to that of cerebral arteries pressurized to 60 mmHg (5, 18, 20, 22, 23). SMCs within the wall of in situ and isolated arterioles were imaged using a Noran Oz laser scanning confocal microscope (Noran Instruments, Middleton, WI) and a ×60 water-immersion objective (numerical aperture = 1.2) attached to a Nikon TE300 microscope. Cells were illuminated with a krypton-argon laser at 488 nm, and emitted light >500 nm was collected. Planar images (56.3 × 52.8 μm) were recorded every 16.7 ms, i.e., 60 images/s. Laser intensity was kept low to prevent cell damage during arteriole SMC Ca2+ imaging. Individual arteriole SMCs were identified based on their perpendicular orientation to the arteriole wall. At least two different representative areas of each arteriole were scanned for at least 10 s under each condition to determine spark and wave frequency and global [Ca2+]i. The same arteriole area was scanned only once to avoid any laser-induced changes in Ca2+ signaling. Imaging was performed at least 15 min (unless otherwise stated) after exposure to drugs so that measurements reflect the steady-state responses of SMCs. Control data and the effects of drugs were measured using the same arteriole segment (i.e., paired experiments). Therefore, each n value refers to data obtained from a single brain slice. Ca2+ sparks, waves, and global [Ca2+]i were analyzed in SMCs using custom analysis software written by Drs. M. T. Nelson and A. D. Bonev (University of Vermont) using IDL 5.2 (Research Systems, Boulder, CO). Automated and manual detection of Ca2+ sparks were performed by dividing an area of 1.54 μm (7 pixels) × 1.54 μm (7 pixels) (i.e., 2.37 μm2) in each image (F) by a baseline (F0) that was determined by averaging 10 images without Ca2+ spark activity. A Ca2+ spark was defined as a localized increase in F/F0 that was >1.2 (5). Ca2+ waves were defined as a F/F0 elevation >1.2 that propagated for at least 10 μm. Global Ca2+ fluorescence was calculated from the same images used for Ca2+ spark analysis and was the mean arteriole pixel value of 600 different images acquired over 10 s. To calculate global Ca2+ changes, the mean global pixel value in glutamate was divided by the corresponding control value.
Values are expressed as means ± SE. Statistical significance was calculated using one-way ANOVA followed by Student-Newman-Keuls test for multiple comparisons and Student's t-test for comparing paired and unpaired data. P < 0.05 was considered significant.
Glutamate modulates local and global Ca2+ signals in arteriole SMCs of brain slices.
Localized Ca2+ sparks and propagating Ca2+ waves occurred in SMCs of newborn pig brain slice arterioles (Fig. 1, A–C). Glutamate (30 μM), an excitatory amino acid, increased Ca2+ spark frequency from ∼0.12 to 0.22 events·cell−1·s−1, or 188%, but did not alter Ca2+ spark amplitude (F/F0: control 1.56 ± 0.01, n = 154; glutamate 1.54 ± 0.01, n = 330; Fig. 2A). In contrast to effects on Ca2+ spark frequency, glutamate did not alter Ca2+ wave frequency (Fig. 2B). Glutamate reduced global [Ca2+]i to ∼76% of control (Fig. 2C). These data indicate that glutamate regulates both local and global Ca2+ signals in arteriole SMCs of brain slices.
Isolation of arterioles abolishes glutamate modulation of local and global Ca2+ signals in SMCs.
To investigate the mechanisms mediating glutamate-induced Ca2+ signal modulation in cerebral arteriole SMCs, arterioles were dissected from within brain slices and carefully cleaned of extravascular brain tissue. In contrast to effects in brain slices, glutamate did not alter Ca2+ spark or wave frequency or global [Ca2+]i in isolated arterioles (Fig. 3, A–C). These data indicate that extravascular brain cells are required for glutamate modulation of local and global Ca2+ signals in cerebral arterioles.
l-AAA prevents glutamate-induced Ca2+ spark and global Ca2+ modulation in arteriole SMCs of brain slices.
To determine whether astrocytes mediate glutamate-induced Ca2+ signal modulation in arteriole SMCs, slices were exposed to l-AAA, a selective astrocyte toxin, or d-AAA, an inactive isomer, for 2 h before being imaged. This treatment protocol produces histological evidence of injury to the superficial glia limitans and loss of astrocyte-dependent cerebrovascular responses without altering responses in general (27, 29, 42). l-AAA prevented both glutamate-induced Ca2+ spark activation and the reduction in global [Ca2+]i (Fig. 4, A and C). In contrast, in l-AAA-treated slices, glutamate increased Ca2+ wave frequency by ∼129% (Fig. 4B). In d-AAA-treated slices, glutamate activated Ca2+ sparks, did not alter Ca2+ wave frequency, and reduced global [Ca2+]i, similarly to in control slices (Figs. 2, A–C, and 4, A–C). These data indicate that astrocytes are required for glutamate modulation of Ca2+ sparks and global [Ca2+]i and that in the absence of functional astrocytes, glutamate activates Ca2+ waves.
Chromium mesoporphyrin blocks glutamate-induced Ca2+ spark and global Ca2+ modulation in arteriole SMCs in brain slices.
We tested the hypothesis that HO activation mediates glutamate-induced Ca2+ signal modulation in cerebral arterioles. Slices were treated with chromium mesoporphyrin (CrMP), a HO blocker, before exposure to glutamate. In CrMP-treated slices, glutamate reduced Ca2+ spark and wave frequency to ∼81 and 77% of control, respectively (Fig. 5). In contrast, in CrMP-treated brain slices, glutamate did not alter global [Ca2+]i. These data suggest that functional HO is required for glutamate-induced Ca2+ spark activation and the reduction in global [Ca2+]i. In the absence of HO activation, glutamate reduces Ca2+ sparks and waves and does not alter global [Ca2+]i.
CO regulates local and global Ca2+ signals in arteriole SMCs.
To investigate the mechanism by which HO modifies Ca2+ signals, we tested the hypotheses that CO is a signaling factor. The regulation of SMC Ca2+ signals by CO-releasing molecule (CORM)-3 [tricarbonylchloro(glycinato)ruthenium(II)], a CO donor, was measured in isolated arterioles to avoid CO-mediated effects that could occur via extravascular brain cells. CORM-3 elevated mean Ca2+ spark frequency to ∼220% of control, did not alter Ca2+ wave frequency, and reduced global [Ca2+]i to ∼83% of control (Fig. 6). These data indicate that CO regulates local and global Ca2+ signals and support evidence that glutamate activates Ca2+ sparks and reduces global [Ca2+]i through HO-derived CO generation.
We next studied the signaling pathways by which CO regulates local and global Ca2+ signals in arteriole SMCs. 1H-(1,2,4)-oxadiazolo-(4,3-a)-quinoxalin-1-one (ODQ), a sGC blocker, did not alter Ca2+ spark frequency or global [Ca2+]i but elevated Ca2+ wave frequency to ∼190% of control (Fig. 6). When applied in the presence of ODQ, CORM-3 did not alter Ca2+ spark frequency, Ca2+ wave frequency, or global [Ca2+]i (Fig. 6).
These data indicate that CO stimulation of Ca2+ sparks and the reduction of global [Ca2+]i in arteriole SMCs requires active sGC. These data also suggest that although CO does not modulate Ca2+ waves, basal sGC activated by a CO-independent mechanism attenuates Ca2+ waves under control conditions.
Here, we measured glutamate and CO modulation of local and global intracellular Ca2+ signals in SMCs of intact arterioles in brain slices and arterioles isolated from brain tissue. The major findings of this study are that in newborn pigs 1) glutamate elevates Ca2+ spark frequency and reduces global [Ca2+]i but does not alter Ca2+ waves in SMCs; 2) isolation of arterioles abolishes Ca2+ signal modulation by glutamate; 3) l-AAA abolishes glutamate modulation of Ca2+ sparks and global Ca2+ and leads to glutamate-induced Ca2+ wave activation; 4) when HO is inhibited, glutamate reduces Ca2+ sparks and Ca2+ waves and does not alter global Ca2+; and 5) CO activation of Ca2+ sparks and the reduction of global [Ca2+]i involves sGC. These data indicate that glutamate modulates different local and global Ca2+ signals in arteriole smooth muscle through astrocytic and nonastrocytic mechanisms that are both HO dependent and independent. The data also indicate that HO-derived CO activates Ca2+ sparks and lowers global [Ca2+]i.
Previous studies have characterized local and global Ca2+ signals that occur in either isolated vascular SMCs or SMCs located within intact arterial segments (e.g., Refs. 5, 15, 17, 21, 26, 33, and 38). Studies measuring Ca2+ signals in SMCs of arterioles within brain slices did not describe the occurrence of Ca2+ sparks (7, 8). Rather, global intracellular Ca2+ oscillations were reported in neonatal (postnatal days 1–7) and juvenile (>20 days old) rat parenchymal arterioles and adult mouse arterioles in cortical brain slices (7, 8). Here, localized Ca2+ sparks and propagating Ca2+ waves were observed in newborn piglet (postnatal days 1–3) tangential brain slice arterioles. In contrast, we did not observe global intracellular Ca2+ oscillations, which by definition are a homogeneous intracellular Ca2+ transient. Ontogeny alters both the generation and physiological functions of rat arterial SMC Ca2+ sparks (10). Differences in Ca2+ signals observed in these reports could also be explained by the following: 1) the cerebral arteriole preparations studied; 2) conditions (e.g., U-46619 concentration: 100–150 nM in Refs. 7 and 8 vs. 10 nM in the present study); 3) Po2, which modifies astrocyte-mediated responses to glutamate (Ref. 11; solutions in the present study were gassed with 21% O2 vs. 95% in Refs. 7 and 8); 4) animal species (rat and mouse vs. piglet in the present study); and 5) imaging system temporal and spatial resolution, which can limit the ability to observe rapid and localized Ca2+ signals, such as Ca2+ sparks. Given the small number of studies that measured local and global Ca2+ signals in SMCs of brain slice arterioles, future investigations will be required to better understand these different observations.
Global brain intracellular glutamate concentrations are ∼1 mM, and extracellular concentrations are within the range of 1–10 μM (6, 31). Pathological conditions, including hypoxia and brain injury, can elevate brain glutamate concentrations 50-fold (6). Under the conditions used here, glutamate concentrations that reach cells within brain slices are likely to be far lower than those applied. Furthermore, local glutamate concentrations within the vicinity of cells nearby those that release glutamate are likely to be higher than global estimates. Therefore, we studied Ca2+ signal regulation by 30 μM glutamate, which is likely to produce a local physiological glutamate concentration within the slice. Glutamate elevated Ca2+ spark frequency and reduced global [Ca2+]i in arteriole SMCs in brain slices. These data are consistent with glutamate acting as a vasodilator (27, 29). Glutamate is the physiological glutamate receptor agonist and activates all glutamate receptor subtypes. It was not a goal of this study to identify the glutamate receptor subtypes mediating Ca2+ signal regulation, but conceivably mGluRs and iGluRs may be involved. A future investigation would be appropriate to study the glutamate receptor subtypes that mediate astrocyte regulation of SMC Ca2+ signals.
Ca2+ sparks activate KCa currents, which reduce voltage-dependent Ca2+ channel activity and lower global [Ca2+]i, leading to vasodilation (21). Ca2+ sparks occur due to ryanodine-sensitive Ca2+ release [ryanodine receptor (RyR)] channel activation. Global Ca2+ can be modulated by both extracellular Ca2+ influx and intracellular Ca2+ release (21). These data suggest that glutamate-induced vasodilation in newborn cerebral arterioles occurs due to KCa channel activation and the subsequent decrease in global [Ca2+]i, as recently proposed (29). In contrast, glutamate did not alter Ca2+ wave frequency. In SMCs, Ca2+ waves can occur due to the activation of sarcoplasmic reticulum membrane RyR channels and/or inositol (1,4,5)-trisphosphate-gated Ca2+ release channels (14, 20). The physiological function of intracellular Ca2+ waves in SMCs is unclear, but these signals have been proposed to either contribute to contraction or to have no effect on contractility (14, 15, 38).
Glutamate did not alter intracellular Ca2+ signals in SMCs of isolated cerebral arterioles, indicating that an extravascular cell type(s) was the glutamate receptor and signal mediator to SMCs. U-46619 was applied to slices, whereas isolated arterioles were studied in a solution containing 30 mM K+. Each of these stimuli causes SMC depolarization and arterial constriction. U-46619 has been used as a vasoconstrictor in studies using brain slices, and 30 mM K+ has been used in many studies to depolarize arterial SMCs to a voltage (−40 mV) similar to that in arteries at physiological pressure (5, 7, 8, 11, 18, 20, 22, 23). U-46619 likely activates thromboxane receptors on multiple cell types within the slice, including on SMCs. U-46619 was not used in isolated arterioles because vasoconstrictors block Ca2+ sparks, activate Ca2+ waves, and elevate global [Ca2+]i, which is a different Ca2+ signaling phenotype than that observed in SMCs within brain slices exposed to U-46619 (20). Therefore, we used U-46619 to depolarize brain slice arterioles and 30 mM K+ to depolarize isolated arterioles. These protocols allow a comparison of these new data with those in previous studies. We anticipate that depolarization with U-46619 or 30 mM K+ is unlikely to alter responses to glutamate.
To investigate a role for astrocytes, we used l-AAA, a glial cell-specific toxin that at the concentration and time period of exposure used, kills astrocytes, disrupts cortical glia limitans, and blocks arteriole dilation to glutamate but not to sodium nitroprusside (a nitric oxide donor), isoproterenol (a β-adrenergic receptor agonist), or CO (27, 29). l-AAA also strongly inhibits cerebrovascular dilation to glutamate in newborn cortical slices and in vivo (27, 29). l-AAA prevented glutamate-induced Ca2+ spark activation and the reduction in global [Ca2+]i. These data indicate that astrocytes mediate glutamate-induced Ca2+ spark activation and the reduction in global [Ca2+]i. In contrast to the effects in control and d-AAA-treated brain slices, glutamate activated Ca2+ waves in SMCs of l-AAA-treated brain slices. Conceivably, l-AAA may have damaged, but not fully removed, astrocytic regulation of SMC Ca2+ signals. However, sufficient residual astrocyte activity does not appear to be present because l-AAA blocked Ca2+ spark and global [Ca2+]i regulation by glutamate and changed the regulation of waves from no response to activation. These data suggest that glutamate-induced Ca2+ sparks inhibit waves or that glutamate may act through a nonastrocytic brain cell type, including endothelial cells, to activate Ca2+ waves (1, 35). Since cerebral arterioles receive input from both neurons and astrocytes (4, 16, 37), another explanation for these data is that in the absence of functional astrocytes, glutamate activates arteriole SMC Ca2+ waves through a neuron-dependent mechanism. This conclusion is consistent with our finding that glutamate did not activate Ca2+ waves in SMCs of isolated arterioles where astrocytes are absent. These data also indicate that, in the intact brain, glutamate typically reduces Ca2+ wave frequency through effects on astrocytes, which is also consistent with this excitatory neurotransmitter acting as a vasodilator. Conceivably, in glutamate, functional astrocytes may inhibit Ca2+ waves and another brain cell type may activate Ca2+ waves, leading to no net change in Ca2+ wave frequency, as observed. Although not the focus of this study, this hypothesis deserves future attention. When the control data in Fig. 4, A and B, were compared, there was a small difference in absolute mean Ca2+ spark and wave frequency between d-AAA and l-AAA, but this was not statistically significant. In support of a lack of effect, d-AAA (control: 53 ± 2 μm, 5 h; and d-AAA, 52 ± 3 μm, n = 9, P > 0.05) and l-AAA did not alter pial arteriole diameters in anesthetized piglets (27). In summary, findings indicate that astrocyte-dependent and -independent pathways fine tune glutamate modulation of different local and global Ca2+ signals in arteriole SMCs.
We have previously shown that in newborn pigs, glutamate-induced cerebral arteriole dilation requires astrocyte-derived HO-2-generated CO (29). CO causes vasodilation by enhancing the apparent Ca2+ sensitivity of SMC KCa channels, leading to enhanced coupling to Ca2+ sparks (18, 19, 41). Consistent with these observations, astrocyte-derived CO and exogenous CO cause vasodilation that is inhibited by KCa channel blockers (28, 29). In addition, heme-l-lysinate, a HO substrate, elevated Ca2+ spark frequency in cerebral artery SMCs (18). However, it was unclear whether CO was the mediator of Ca2+ spark activation and whether astrocyte-derived CO would activate Ca2+ sparks. Here, we show that CrMP blocks glutamate-induced Ca2+ spark activation and the reduction in global [Ca2+]i. CrMP was the only HO inhibitor used in this study. All HO inhibitors are porphyrin rings containing different metal ions. Substitution of the metal ion from chromium to tin, another HO inhibitor, does not alter the effectiveness of HO inhibition (25). CrMP was chosen to complement previous work, which used this blocker to study cerebral arteriole diameter regulation by HO-2. We also demonstrated that exogenous CO activates Ca2+ sparks in SMCs of isolated arterioles. Thus, data suggest that astrocyte-derived CO activates Ca2+ sparks in arteriole SMCs. In response to an elevation in astrocyte-derived CO, the Ca2+ spark frequency elevation and increased Ca2+ spark to KCa channel coupling would combine to significantly elevate SMC KCa current.
ODQ, a highly selective, membrane-permeant sGC inhibitor, was used to inhibit sGC. We (25) have previously shown that ODQ blocks vasodilation to sodium nitroprusside in anesthetized piglets and that this effect can be reversed with 8-bromo-cGMP. ODQ elevated Ca2+ wave frequency but did not change Ca2+ sparks in SMCs. These data suggest that a sGC activator, perhaps basal nitric oxide from endothelial cells, may suppress Ca2+ waves without altering Ca2+ sparks. Data also indicate that CO activation of Ca2+ sparks requires sGC. In contrast to the requirement of sGC for CO activation of Ca2+ sparks, CO directly activates KCa channels (19). Our data provide one explanation to an important question regarding the necessity of sGC for CO-induced vasodilation and the “permissive-enabling” function of the cGMP/PKG pathway. Although sGC inhibition does not prevent CO-induced KCa channel activation in SMCs, sGC inhibition blocks CO-induced vasodilation (19, 25, 41). However, clamping cGMP constant returns the dilation to CO (25). Our data indicate that for CO to cause vasodilation, it must both activate KCa channels directly and stimulate Ca2+ sparks through sGC. CO-induced KCa channel activation alone in the absence of the Ca2+ spark frequency elevation appears to be insufficient to significantly alter membrane potential and reduce voltage-dependent Ca2+ channel activity. Similarly, KCa channel inhibition blocks CO-induced vasodilation (18). Thus, CO-induced sGC-mediated Ca2+ spark activation and direct KCa channel activation by CO are both essential for the functional vasodilation caused by this gasotransmitter.
In the presence of CrMP, glutamate reduced both Ca2+ sparks and Ca2+ waves and did not change global [Ca2+]i. This was unexpected given that in the control, glutamate activated Ca2+ sparks, did not alter Ca2+ waves, and reduced global [Ca2+]i. In this experiment, HO would be inhibited in all cell types within the brain slice. An explanation for this finding is that glutamate also suppresses arteriole Ca2+ sparks and Ca2+ waves, but this effect can only be resolved in the absence of HO activation. Our data raise the concept that glutamate activates HO-independent Ca2+ spark and Ca2+ wave inhibitory mechanisms that may be mediated by neurons and/or astrocytes.
In summary, we examined glutamate regulation of local and global intracellular Ca2+ signals in brain slice arteriole SMCs. The data indicate that glutamate modulates local and global Ca2+ signals in arteriole smooth muscle through both astrocyte- and HO-dependent mechanisms. In the absence of astrocytes or functional HO, glutamate does not change global [Ca2+]i and the effects on arterial Ca2+ signals are absent or the opposite of those in the intact system, suggesting other brain cell types can modulate these events.
This work was supported by National Heart, Lung, and Blood Institute Grants HL-67061, HL-77678, and HL-094378 (to J. H. Jaggar) and HL-042851 and HL-034059 (to C. W. Leffler). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health.
No conflicts of interest are declared by the author(s).
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