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Laboratory for Research in Neonatal Physiology, Departments of Physiology and Pediatrics/Obstetrics and Gynecology, University of Tennessee Health Science Center, Memphis, Tennessee 38163
Submitted 22 August 2003 ; accepted in final form 8 September 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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carbon monoxide-releasing molecule; cranial window; cerebrovascular circulation; glutamate; nitric oxide
CO, at relatively high concentrations, can stimulate soluble guanylyl cyclase (4, 10, 24) and increase cGMP in the vascular tissue (4, 10, 29). The vasodilator effect of CO has been attributed to cGMP, because inhibitors of soluble guanylyl cyclase attenuate CO-induced vasodilation (10, 13). However, other mechanisms can produce CO-induced vasodilation. CO can hyperpolarize the vascular smooth muscle by directly activating large-conductance Ca2+-activated K+ (KCa) channels (32). CO increases the Ca2+ sensitivity of KCa channels (14) and may also decrease the production of vasoconstrictors such as endothelin-derived (26) or cytochrome P-450-derived (7) products.
Previously we showed that inhibition of nitric oxide (NO)-synthase (NOS) with N
-nitro-L-arginine (L-NNA) blocked cerebral vasodilation to CO in piglets (21). This blockade was reversed by sodium nitroprusside (SNP). CO-induced vasodilation also was blocked by tetraethylammonium chloride and iberiotoxin, KCa channel blockers (22). Dilation blocked by tetraethylammonium chloride or iberiotoxin was not restored by SNP. Periarachnoid cerebrospinal fluid (CSF) cGMP levels do not increase in response to the topical application of dilator concentrations of CO in piglets, in contrast to SNP, where dilation is accompanied by cGMP increases (2). The excitatory neurotransmitter glutamate causes dilation of newborn pig cerebral arterioles via a mechanism that involves the HO/CO pathway (9, 30). Glutamate increases CO production by cerebral microvessels from endogenous and exogenous heme (20).
Therefore, we hypothesized that CO-induced vasodilation may require a permissive enabling action of cGMP that can be produced by basal NO, but not a CO-induced elevation of cGMP. The objective of this study was to investigate the role of cGMP and NO in CO- and glutamate-induced cerebral vasodilations.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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-chloralose during the experiment (30–40 mg/kg iv initially, supplemented with 7 mg·kg–1·h–1 iv). A femoral artery was catheterized to monitor blood pressure and heart rate and to obtain blood samples for the measurement of pH, PCO2, PO2. A second catheter was placed in a femoral vein for anesthetic and fluid administration. A tracheotomy was done, and an endotracheal tube was inserted. The pig was mechanically ventilated and supplemented with O2 if needed to maintain arterial pH, PCO2, and PO2 in the normal range. Core temperature was monitored with a rectal probe and maintained at 37.5–38.5°C. Cranial window placement and pial arteriolar monitoring. The scalp was surgically removed, and a 2-cm-diameter hole was cut in the skull over the parietal cortex. The dura was incised and reflected over to the bone to prevent contact between the brain surface and the cut edge of the bone. A stainless steel-and-glass window was implanted into the hole and cemented sequentially with bone wax and dental acrylic. A 45- to 150-µm-diameter pial arteriole was selected and monitored through the window with a dissecting microscope and an attached videocamera. Vessel diameter was measured with a video micrometer.
The space under the window was filled with artificial CSF (aCSF) composed of (in meq/l) 3 K+, 1.2 Mg+, 2.5 Ca+, 150 Na+, 132 Cl–, 6 urea, 3.7 dextrose, and 25 
. The aCSF was warmed in a water bath to 37°C and bubbled with a mixture of N2, O2, and CO2 to maintain pH at 
7.33 and PCO2 and PO2 at 45 mmHg.
Experimental design. The baseline pial arteriolar diameter, along with heart rate, mean arterial blood pressure, and core temperature, was recorded. At the end of each tested response, an arterial blood gas sample was drawn, and the area under the window was gently flushed with fresh aCSF to remove the previous stimulus. The pial arteriole was allowed to return to the baseline diameter before the next stimulus or a treatment was given.
Pial arteriolar responses to isoproterenol, SNP, and CO. Isoproterenol (Iso) and SNP were dissolved in fresh aCSF and applied topically (10–6 M). The pial arteriolar diameter was recorded at 1, 3, and 5 min. Then the Iso or SNP was replaced with fresh aCSF. The responses to Iso and SNP were measured in the absence and presence of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). CO was purchased in a compressed gas cylinder of 100% CO. Water was initially saturated with CO at room temperature (10–3 M). The CO solution was then diluted with aCSF to a concentration of 10–5 M. The space under the window was flushed with 10–5 M CO dissolved in aCSF, and pial arteriolar responses were measured at 1, 3, and 5 min.
CO-releasing molecule, Mn2(CO)10 (dimanganese decacarbonyl). Dimanganese decacarbonyl (DMDC, 10–5 M) was used as a CO-releasing compound. DMDC was dissolved in DMSO and aCSF and topically applied to the pial arterioles. The pial arteriolar response to DMDC was measured at 1, 3, and 5 min. DMDC is a light-sensitive compound. It releases CO in the presence of light. Therefore, DMDC was protected from light at all times, except when under the cranial window, where it was activated with light pipes. Pial arteriolar responses to inactivated DMDC (10–5 M) were determined by placing under the window DMDC that had been dissolved in DMSO and aCSF and exposed to light for 1 wk. The pial arteriolar diameter was not affected by the inactivated DMDC (n = 5 pigs).
Guanylyl cyclase inhibitor. Once all the control responses were established, the guanylyl cyclase inhibitor ODQ was applied. ODQ was initially dissolved in DMSO to a concentration of 4 x 10–2 M. The ODQ solution was then diluted with aCSF to a concentration of 2.5 x 10–4 M. The space under the window was filled with 2.5 x 10–4 M ODQ. A baseline pial arteriolar diameter with ODQ was measured after 45 min of exposure to ODQ. Then the space under the window was flushed with 10–5 M CO in aCSF containing 2.5 x 10–4 M ODQ, and the pial arteriolar response was measured at 1, 3, and 5 min. The above-described procedure was repeated with 10–6 M SNP and Iso instead of CO. In other piglets in which DMDC was used instead of gaseous CO, at the end of each pial arteriolar response to DMDC, ODQ, or DMDC + ODQ, CSF from under the cranial windows was collected for the measurement of cGMP. The DMSO concentration was maintained constant throughout the experiments.
The pial arteriolar response to 5 x 10–3 M DMSO was determined by placing DMSO under the window without ODQ or DMDC. The pial arteriolar diameter was not affected by DMSO. DMSO alone had no effect on responses to Iso and SNP.
8-Bromo-cGMP. 8-Bromo-cGMP (8-BrcGMP, 10–4 M) was dissolved in aCSF and placed under the window. The pial arteriolar diameters were measured at 1, 3, and 5 min. Responses to DMDC were also determined in the presence of ODQ and ODQ + 8-BrcGMP (10–4 M), thus maintaining a constant level of cGMP.
Glutamate. Responses to topical application of 10–6 M SNP and 10–4 M glutamate for 5 min were measured before and after treatment with 10–3 M L-NNA. Then 10–4 M 8-BrcGMP + L-NNA was placed under the window, and the pial arteriolar diameter was measured at 1, 3, and 5 min. Responses to glutamate were determined in the presence of L-NNA and L-NNA + 8-BrcGMP (10–4 M), thus maintaining a level of cGMP but preventing NO from increasing and activating guanylyl cyclase.
In other piglets, SNP replaced 8-BrcGMP. SNP at 5 x 10–7 and 7 x 10–7 M was placed under the window with L-NNA, and the pial arteriolar diameter was measured at 1, 3, and 5 min. Responses to glutamate were determined in the presence of L-NNA and then L-NNA + 5 x 10–7 and 7 x 10–7 M SNP sequentially, thus maintaining constant levels of NO. cGMP assay by ELISA. cGMP levels in CSF were measured with a commercial ELISA kit (Stratagene, La Jolla, CA). CSF samples were collected from the side ports of the cranial window as described above. The space under the cranial window has a volume of 500 µl, and 300 µl of CSF were collected for the detection of cGMP. The aCSF samples were acetylated with 2:5 acetic anhydride-triethylamine before they were applied to a 96-well plate. After the samples were placed into the wells, they were incubated with cGMP-horseradish peroxidase conjugate for 1 h at room temperature. The plates were then washed, and tetramethylbenzidine-H2O2 substrate was added and incubated for 30 min. Concentrations of cGMP were calculated by measuring the absorption at 650 nm.
Statistical analysis. Values are means ± SE. Comparisons among groups were made by ANOVA with repeated measures followed by Fisher's protected least significant difference test. P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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In subsequent experiments, CO was replaced with a light-activated CO donor, DMDC. DMDC dilated the pial arterioles in a dose-dependent fashion (Fig. 3). Repeated dose-response curves were superimposable (data not shown).
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Even at 10–5 M, DMDC did not increase the cortical CSF concentration of cGMP: 443 ± 151 and 265 ± 188 fmol/ml before and after DMDC administration, respectively. However, similar to CO-induced dilation, dilation to DMDC was abolished by the guanylyl cyclase inhibitor ODQ (Fig. 4).
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8-BrcGMP at 10–4 M, which mildly dilates the pial arterioles (7 ± 2%), restored a vasodilator response to DMDC to pial arterioles treated with ODQ (Fig. 5).
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Glutamate (10–4 M) and SNP (10–6 M) dilated pial arterioles similarly (Fig. 6). The NOS inhibitor L-NNA tended to decrease the baseline pial arteriolar diameter (–13% ± 3) and blocked the dilation caused by glutamate, but not the dilation caused by SNP (Fig. 6). 8-BrcGMP at 10–4 M, which mildly dilates the pial arterioles (6 ± 2%), partially restored a vasodilator response to glutamate to pial arterioles treated with L-NNA (Fig. 7). When the concentration of 8-BrcGMP (2 x 10–4 M) was doubled, there was no greater restoration of the response to glutamate. SNP at a concentration that minimally dilates the pial arterioles (7 ± 3%), i.e., 5 x 10–7 M, completely restored the vasodilator response to glutamate to pial arterioles treated with L-NNA (Fig. 8). Increasing the concentration of SNP (7 x 10–7 M) did not increase the vasodilator response to the same level of glutamate (Fig. 8).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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CO dilates pial arterioles of the newborn pigs similarly to the dose-dependent dilation of the renal and gracilis muscle arterioles (15, 22, 33). CO also contributes to maintenance of ductus arteriosus patency (6). CO is an autocrine and paracrine vasodilator in cerebral and systemic circulations (23), and endogenously produced CO can play a role in the regulation of basal tone in resistance vessels throughout the body (18).
DMDC, a CO donor, produces dose-dependent dilation of the pial arterioles similar to that produced by CO. Transitional metal carbonyls can release CO in a concentration-dependent manner (28). DMDC is a transitional metal carbonyl complex that releases CO by photodissociation in cold light when dissolved in DMSO and water. DMDC produces dose-dependent dilation of the isolated pressurized pial arterioles in vitro (9), and DMDC markedly attenuates L-NNA-mediated increases in the coronary perfusion pressure, similar to hemin (28). Another transitional metal carbonyl complex Ru(CO)3Cl2, which releases CO spontaneously, caused sustained vasodilation in precontracted rat aortic rings and significantly reduced acute hypertension in vivo, similar to CO (28). No major cytotoxic effects were observed after 24 h of exposure of smooth muscle cells to DMDC or Ru(CO)3Cl2 (28). DMDC was selected as a CO donor for our study, because it releases CO continuously when illuminated through the cranial window, but not in the dark, before injection under the window.
CO is a paracrine mediator with multiple similarities to NO. The vascular wall produces both gases by inducible and constitutive forms of HO and NOS, respectively. Depending on the vessel or the tissue involved, the mechanism by which CO mediates its action may involve activation of soluble guanylyl cyclase (27), stimulation of KCa channels (14, 32), or inhibition of endothelin release from endothelial cells (7). Similar to NO, CO can inhibit platelet aggregation and relax blood vessels by activating soluble guanylyl cyclase and elevating intracellular levels of cGMP (5). In olfactory receptor neurons, which have a high level of CO production and no NOS activity, endogenous CO stimulates cGMP production (31). CO and NO could cause dilation by activating guanylyl cyclase, but guanylyl cyclase is eight times more sensitive to NO than to CO. We could not detect any increase in cerebral cGMP production coincident with DMDC-induced vasodilation. Similarly, in an earlier study, we did not detect an increase in cGMP coincident with pial arteriolar dilation to CO- or heme-L-lysinate-induced vasodilation (22). Nor did exogenous CO stimulate cGMP production in rat cerebral cortex (19). In contrast, other studies have shown an increase in cGMP level in pulmonary vessels, isolated smooth muscle cells, and platelets (27) on exposure to CO. Clearly, CO can activate guanylyl cyclase and increase cGMP, but at levels of CO that strongly dilate piglet pial arterioles, neither CO nor DMDC increases cGMP.
Nevertheless, it has been frequently assumed that vasodilator effects of CO are caused by stimulation of guanylyl cyclase. However, as noted above, dose-dependent increases in cGMP in response to exogenously administered CO typically have not been demonstrated. Most studies have assumed that because ODQ decreased the dilation to CO or to its precursors, activation of guanylyl cyclase by CO produced the vasodilation but did not measure cGMP levels.
NO or SNP, which releases NO, induces vasodilation that is mediated by cGMP. In the piglet cerebral circulation, pial arteriolar dilations to SNP and Iso are accompanied by pronounced elevations in CSF cGMP and cAMP, respectively (2, 16). In contrast, dilation to CO is not coincident with marked elevation of CSF cyclic nucleotides (22).
ODQ is a soluble guanylyl cyclase inhibitor and was used to block cGMP production. ODQ significantly decreased the CSF cGMP levels, indicating efficacy and blocked dilations to SNP. ODQ did not affect the vasodilation to Iso that is mediated by cAMP.
An important aspect of this study is that DMDC caused dilation of pial arterioles when cGMP was clamped constant with 8-BrcGMP + ODQ. 8-BrcGMP is a stable cGMP analog that mimics the actions of cGMP. The treatment of piglets with ODQ completely abolished the vasodilation to exogenously administered CO (DMDC). 8-BrcGMP, at a constant level that caused little dilation, permitted dilation to CO to occur in the piglets treated with ODQ. Under these conditions, cGMP was present but could not increase. Therefore, we conclude that cGMP has a permissive role in CO-induced vasodilation.
Inhibition of NOS with L-NNA blocked the vasodilation to glutamate, as reported previously by Meng et al. (25). One of the new findings of this study was that clamping cGMP constant with 8-BrcGMP + L-NNA partially restored the dilation to glutamate. Under these conditions, increasing NO could not activate guanylyl cyclase that was blocked by ODQ, but cGMP was present in the form of 8-BrcGMP. Glutamate also dilated pial arterioles when NO was clamped constant with exogenous SNP after NOS inhibition. NO, at a constant level that caused little dilation, completely restored the dilation to glutamate in the piglets treated with L-NNA. Under these conditions, NO was present but could not increase because NOS was inhibited. Furthermore, increases in SNP concentration (from 5 x 10–7 to 7 x 10–7 M) did not increase the response to glutamate. These data suggest that the role of NO in glutamate-induced vasodilation is a permissive one. The mechanism of action appears to be, at least in part, by maintaining a necessary cGMP "tone." The finding that SNP is more effective than 8-BrcGMP at restoring dilation to glutamate may suggest that NO can have actions in addition to activating guanylyl cyclase. For example, NO can have direct, cGMP-independent actions on channel proteins by S-nitrosylation (1).
CO has been reported to interact with a histidine residue on KCa channels in cultured smooth muscle cells (32). CO-induced dilations in rat gracilis muscle, renal arterioles, and piglet cerebral arterioles are inhibited by blockers of large-conductance KCa channels (15, 21, 33). When activated, KCa channels cause hyperpolarization that inhibits voltage-dependent Ca2+ channels (12). Inhibition of voltage-dependent Ca2+ channels causes a reduction in intracellular Ca2+ leading to dilation (12).
Ca2+ sparks are localized intracellular Ca2+ transients. In arterial smooth muscle, Ca2+ sparks activate KCa channels, thereby inducing hyperpolarizing outward currents called STOCs (3). It has been shown that CO dilates cerebral arterioles in vascular smooth muscle cells by elevating the coupling of Ca2+ sparks to KCa channels, which increases STOCs, hyperpolarizing the smooth muscle cell (14).
In conclusion, cerebrovascular dilation to CO is completely blocked by ODQ, but CO does not increase cGMP. Because a minimal level of 8-BrcGMP restores the dilation to CO after guanylyl cyclase inhibition with ODQ, it appears that a certain threshold level of cGMP is necessary to allow CO to increase KCa channel activity to produce dilation. We previously showed that CO dilates pial arterioles via activation of KCa channels and that NO can provide a necessary permissive signal. Because NOS inhibition, like HO inhibition, blocks dilation to glutamate and a constant level of SNP restores the dilation to glutamate after NOS inhibition, it appears that the contribution of NO to glutamate dilation is permissive, with CO increasing KCa channel activity as the terminal effector. The contribution of NO is via activation of guanylyl cyclase and possibly also a cGMP-independent action. Therefore, we conclude that cGMP and NO play permissive roles in CO-induced vasodilation.
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ACKNOWLEDGMENTS |
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GRANTS
This research was supported by the National Heart, Lung, and Blood Institute and the Vascular Biology Center of Excellence.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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]quinoxalin-1-one (ODQ) inhibits relaxation of rabbit aortic rings induced by carbon monoxide, nitric oxide, and glyceryl trinitrate. Can J Physiol Pharmacol 75: 1034–1037, 1997.[CrossRef][ISI][Medline]
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P < 0.05 vs. no change.