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Am J Physiol Heart Circ Physiol 289: H432-H438, 2005. First published February 11, 2005; doi:10.1152/ajpheart.01195.2004
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Permissive contributions of NO and prostacyclin in CO-induced cerebrovascular dilation in piglets

Charles W. Leffler, Alexander L. Fedinec, Helena Parfenova, and Jonathan H. Jaggar

Laboratory for Research in Neonatal Physiology, Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee

Submitted 30 November 2004 ; accepted in final form 7 February 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Endogenously produced CO is an important dilator in newborn cerebrovascular circulation. CO dilates cerebral arterioles by activating Ca2+-activated K+ channels, but modulatory actions of other effectors and second messenger inputs are unclear. Specifically, the mechanisms behind the obligatory permissive roles of prostacyclin and NO are uncertain. Therefore, the present study was performed using acutely implanted, closed cranial windows in newborn pigs to address the hypothesis that the permissive roles of NO and prostacyclin in cerebrovascular dilation in response to CO involve a common mechanism. The NO donor sodium nitroprusside restored dilation in response to CO after inhibition of that dilation with the prostaglandin cyclooxygenase inhibitor indomethacin. The stable prostacyclin analog iloprost restored CO-induced dilation blocked by the NO synthase inhibitor N{omega}-nitro-L-arginine. Restoration of dilation in response to CO by the cGMP-dependent phosphodiesterase inhibitor zaprinast and blockade of CO dilation by the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazole-[4,3-a]quinoxalin-1-one (ODQ) suggests involvement of the cGMP/PKG pathway. Iloprost or the cAMP-dependent dilator isoproterenol restored dilation in response to CO after ODQ administration. However, CO-induced dilation blocked by the cGMP-dependent PKG inhibitor Rp-8-[(4-chlorophenyl)thio]-cGMPS triethylamine could not be reversed by administration of sodium nitroprusside, iloprost, or isoproterenol. Conversely, PKA inhibition did not block dilation in response to CO. Overall, data indicate that activation of PKG is the predominant mechanism of the permissive actions of NO and prostacyclin for CO-induced pial arteriolar dilation.

carbon monoxide; nitric oxide; protein kinase G; newborn; cyclic AMP; GMP

CARBON MONOXIDE (CO), produced by cellular heme metabolism catalyzed by heme oxygenase, is a gaseous signaling molecule analogous to nitric oxide (NO; Ref. 20). CO is a major component in the regulation of cerebrovascular circulation in newborn infants, mediating or contributing to dilation in response to excitatory amino acids and hypoxia and providing a modulatory vasodepressor influence that attenuates vasoconstrictor responses (17, 27, 36).

In the intact newborn pig cerebral circulation, dilation in response to CO involves both NO and prostacyclin (16). Specifically, either indomethacin (Indo), which blocks prostaglandin synthesis, or N{omega}-nitro-L-arginine (L-NNA), which blocks production of NO, prevents cerebrovascular dilation in response to CO. Prostacyclin and NO play permissive roles in allowing dilation in response to CO, because constant background levels of the prostacyclin receptor agonist iloprost and the NO-releasing molecule sodium nitroprusside (SNP), which do not themselves induce substantial dilation, restore dose-dependent dilation in response to CO after Indo and L-NNA administration, respectively (16). That NO has a permissive contribution to CO-induced dilation may explain the apparent discrepancy that inhibition of NO synthase (NOS) blocks dilation in response to glutamate in newborn pig cerebral circulation (21) similar to inhibition of heme oxygenase (27). An action (or actions) of NO is necessary for CO to mediate dilation in response to glutamate (14). The action of CO is to increase the Ca2+ sensitivity of large-conductance, Ca2+-activated K+ (KCa) channels, which increases effective coupling to Ca2+ sparks and leads to smooth muscle hyperpolarization (9).

Whether the mechanisms of permissive roles of NO and prostacyclin are the same or distinct is unknown. It appears that part of the permissive action of NO in CO-induced dilation results from the maintenance of a minimum cGMP concentration (14). Mediators of the permissive action of prostacyclin for CO-induced dilation are unknown. The permissive role of prostacyclin in cerebral vasodilation in response to hypercapnia in piglets involves protein kinase C (PKC) rather than guanylyl cyclase (25), so the permissive mechanisms of action for NO and prostacyclin could be different. On the other hand, prostacyclin increases not only cAMP in piglet cerebral circulation but also cGMP (22), so that prostacyclin could contribute to maintenance of cGMP. In addition, cAMP can activate protein kinase G (PKG) without involvement of protein kinase A (PKA; Refs. 11, 19, 34).

The present experiments address the hypothesis that the permissive roles of NO and prostacyclin in cerebrovascular dilation in response to CO involve a common mechanism.


   METHODS AND MATERIALS
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 ABSTRACT
 METHODS AND MATERIALS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
All procedures that involve animals were reviewed and approved by the Animal Care and Use Committee of The University of Tennessee Health Science Center. Newborn pigs (1–3 days of age; 1–2.5 kg body wt) were anesthetized with ketamine hydrochloride (33 mg/kg im) and acepromazine (3.3 mg/kg im) and maintained on {alpha}-chloralose (50 mg/kg iv). The animals were intubated and ventilated with air. Catheters were inserted into the femoral vein for maintenance of anesthesia and drug injections and into the femoral artery to record blood pressure and draw samples for blood gas measurements and pH analysis. Blood gas levels, pH, and body temperature were maintained within normal ranges. The scalp was retracted, and a hole (2-cm diameter) was made in the skull over the parietal cortex. The dura was cut without touching the brain, and all cut edges were retracted over the bone so that the periarachnoid space was not exposed to bone or damaged membranes. A stainless steel and glass cranial window was placed in the hole and cemented into place with dental acrylic. The space under the window was filled with artificial cerebrospinal fluid (aCSF) that was equilibrated with 6% CO2 and 6% O2, which produced blood gas values and pH measurements within the normal range for CSF (pH, 7.33–7.40; PCO2, 42–46 mmHg; and PO2, 43–50 mmHg). Fluid under the window could be exchanged via needle ports on the sides of the window. Pial vessels were observed with a dissecting microscope. Diameters were measured with a video micrometer coupled to a television camera mounted on the microscope and a video monitor. Data from one arteriole of ~60 µm diameter are reported from each piglet.

Materials. CO was purchased as compressed gas (99.5%). Water was saturated with CO to produce a 10–3 M stock solution. The stock was diluted in aCSF for injection under the cranial window at concentrations from 10–11 to 10–7 M. Water-soluble Indo (indomethacin trihydrate) was a gift from Merck (Rahway, NJ). Iloprost was a gift from Schering (Berlin, Germany) or was purchased from Cayman Chemical (Ann Arbor, MI). Other reagents were purchased from Sigma Chemical (St. Louis, MO).

Experiments. CO was applied directly to pial arterioles, and the maximal diameter attained over a 5-min period was recorded as the response to each dose. Repeat ascending dose-response curves to CO were produced before and after no treatment or treatment with Indo (5 mg/kg iv), L-NNA (10–3 M, topically), or both Indo and L-NNA.

Responses to isoproterenol (10–6 M; topically, 5 min) were measured before any other treatment had been given and again at the very end of each experiment.

Experiments to investigate potential permissive enabling capacities of prostacyclin, NO, or isoproterenol were conducted by applying iloprost (10–11 to 10–8 M), SNP (1–7 x 10–7 M), both iloprost and SNP, or isoproterenol (10–8 to 10–7 M) topically during treatment with CO.

Guanylyl cyclase was inhibited with topical administration of 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxaline-1-one (ODQ; 2.5 x 10–4 M; Ref. 14). cGMP phosphodiesterase was inhibited with zaprinast (10–5 M; Ref. 22), PKG was inhibited with Rp-8-[(4-chlorophenyl)thio]-cGMPS triethylamine (Rp-8-pCPT-cGMPS; 2 x 10–7 M), and PKA was inhibited with Rp-8-CPT-cAMP (10–6 M; Ref. 34). Blockade of PKG and PKA at these doses was confirmed by selective inhibition of dilations in response to 8-Br-cGMP and 8-Br-cAMP, respectively.

Statistical analysis. Values for each variable are presented as means ± SE. Comparisons among populations within each experimental group were made using ANOVA with repeated measures. Fisher’s protected least-significant difference test was used to determine differences between populations within each group. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Throughout the duration of the experiments, there were no significant changes in piglet blood gas levels, pH, body temperature, or arterial pressure when the values were compared at the beginning and end of the experiments. Neither pial arteriolar diameters nor responses to topical isoproterenol (10–6 M) were different when measured at the beginning (control, 55 ± 3; isoproterenol, 71 ± 3 µm) and at the end (control, 58 ± 3; isoproterenol, 68 ± 3 µm) of the experiments.

Figure 1 shows the effect of NOS inhibition with L-NNA on vasodilation in response to CO in the absence and presence of iloprost and/or SNP. Dilation in response to CO was abolished by L-NNA. Iloprost (10–10 M) partially restored dilation in response to CO in arterioles treated with L-NNA. As expected and as shown previously (16), SNP (5 x 10–7 M) also restored dilation in response to CO after inhibition of NOS. Iloprost and SNP in combination had an effect on dilation in response to CO that was similar to SNP alone. These data suggest that prostacyclin appears in part to substitute for the permissive role of NO in CO-induced dilations.



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  		Fig. 1. Dilation of pial arterioles in response to carbon monoxide (CO) before and in the presence of N{omega}-nitro-L-arginine (L-NNA), L-NNA with iloprost (ILO; 10–10 M), and L-NNA with sodium nitroprusside (SNP; 2 x 10–7 M) and iloprost. Values are means ± SE; n = 5. *P < 0.05 compared with no CO.

 
As reported before (16), inhibition of prostaglandin cyclooxygenase (COX) with Indo, similar to inhibition of NOS, blocked dilation in response to CO and after inhibition of COX, and iloprost (10–11 M) restored dilation in response to CO. In addition, SNP (2 x 10–7 M) produced similar restoration to CO dilation as did iloprost in arterioles treated with Indo [54 ± 6 to 59 ± 6, 49 ± 6 to 55 ± 6, and 52 ± 5 to 56 ± 5 µm for CO (0–10–9 M) in control, iloprost (10–11 M) after Indo, and SNP (2 x 10–7 M) after Indo, respectively]. Iloprost and SNP in combination had the same effect after Indo administration on dilation in response to CO as did either alone. Thus similar to prostacyclin substituting for NO, NO appears to substitute for prostacyclin in permitting CO-induced dilation.

As was the case with either L-NNA or Indo alone, when both are given together, dilation in response to CO is blocked (Fig. 2). After combined NOS and COX inhibition, SNP alone restored the dilatory response, and iloprost alone partially returned the dilatory response in response to CO (Fig. 2). Adding iloprost to SNP was no more effective at restoring the dilation in response to CO than was SNP alone. The combination of iloprost (10–11 M) and SNP (3 x 10–7 M) restored dilation in response to CO as expected after combined NOS and COX inhibition. These data show that providing a necessary constant background concentration of NO alone restores dose-dependent dilation in response to CO even when both NOS and COX are blocked. Substitution of the prostacyclin agonist at 10–11 M for SNP was less effective but did reintroduce CO dilation that had not been there before.



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  		Fig. 2. Dilation of pial arterioles in response to CO before and in the presence of L-NNA with indomethacin (Indo), L-NNA with Indo and SNP (3 x 10–7 M), L-NNA with Indo and iloprost (10–11 M), and L-NNA with Indo, iloprost, and SNP. *P < 0.05 compared with no CO; n = 6.

 
Both prostacyclin and NO can increase cGMP (22). We previously showed that inhibition of guanylyl cyclase blocks, and cGMP clamp with 8-Br-cGMP restores, dilation to CO (14). Therefore, we examined the possibility that an increase in cellular cGMP by inhibition of cGMP-specific phosphodiesterase (PDE) might restore dilation in response to CO after Indo and L-NNA administration. The PDE inhibitor zaprinast at the highest concentration that did not markedly dilate pial arterioles (10–5 M) restored vasodilation in response to CO in piglets treated with Indo (Fig. 3) or L-NNA (Fig. 4). These data suggest that the permissive roles of both NO and prostacyclin can be at least partially replicated by elevating cellular cGMP concentration.



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  		Fig. 3. Dilation of pial arterioles in response to CO before and in the presence of Indo, Indo with zaprinast, and Indo with zaprinast (10–5 M) and SNP (3 x 10–7 M). *P < 0.05 compared with no CO; n = 5.

 


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  		Fig. 4. Dilation of pial arterioles in response to CO before and in the presence of L-NNA, L-NNA with zaprinast (10–5 M), and L-NNA with zaprinast and Indo. *P < 0.05 compared with no CO; n = 5.

 
Similar to COX and NOS inhibition, guanylyl cyclase inhibition with ODQ blocked dilation in response to CO (Fig. 5). As expected, ODQ also blocked SNP-induced pial arteriolar dilation thereby demonstrating effective blockade of guanylyl cyclase (Fig. 6). Surprisingly, SNP could dose dependently return dilation in response to CO in the presence of ODQ (see Fig. 5), which suggests that NO may have dilatory actions independent of cGMP. Conversely, constant background levels of CO did not restore dose-dependent dilation in response to SNP after ODQ administration (Fig. 6). Only when a sufficient concentration of SNP was present with a dilator concentration of CO did the combination produce dilation (Fig. 6). These data show that blockade of guanylyl cyclase, which would reduce cGMP, prevents dilation in response to CO and attenuates the permissive efficacy of NO in dilation in response to CO. NO may also have a permissive effect independent of guanylyl cyclase.



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  		Fig. 5. Dilation of pial arterioles in response to CO before and in the presence of 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxaline-1-one (ODQ), ODQ with 3 x 10–7 M SNP (SNP 1), and ODQ with 7 x 10–7 M SNP (SNP 2). *P < 0.05 compared with no CO; n = 7.

 


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  		Fig. 6. Dilation of pial arterioles in response to SNP before and in the presence of ODQ, ODQ with 10–9 M CO, and ODQ with 10–8 M CO. *P < 0.05 compared with no CO; n = 5.

 
We next examined whether the permissive action of iloprost toward CO-induced dilation may, similar to that of NO, involve activation of guanylyl cyclase. Activation of guanylyl cyclase by the prostacyclin receptor agonist is conceivable because iloprost increases cGMP as well as cAMP (22). After inhibition of vasodilator responses to CO by ODQ, iloprost was added to the CSF, and CO-induced dilation was reexamined (Fig. 7). In the presence of ODQ, iloprost restored dilation in response to CO. Furthermore, a maximal permissive effect of iloprost was reached at 10–9 M, since further increasing the iloprost concentration did not enhance the dilation in response to CO (Fig. 7). These data suggest that the permissive mechanism of iloprost for CO-induced dilation is not via activation of soluble guanylyl cyclase.



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  		Fig. 7. Dilation of pial arterioles in response to CO before and after administration of ODQ alone and ODQ with different concentrations of iloprost (10–10, 10–9, and 10–8 M). *P < 0.05 compared with no CO; {dagger}P < 0.05 compared with same CO concentration before ODQ administration; n = 6.

 
On the piglet brain surface, iloprost increases cAMP much more than cGMP (22). Therefore, iloprost may enable CO to cause dilation due to an increase in cAMP. To examine this hypothesis, we used isoproterenol, which increases cAMP, to examine restoration of dilation in response to CO after inhibition of COX and NOS (Fig. 8). Isoproterenol restored concentration-dependent dilation in response to CO after COX and NOS inhibition. Mechanisms of permissive actions of isoproterenol and NO may be the same, because addition of SNP in the presence of isoproterenol did not further increase the dilator response to CO (Fig. 8).



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  		Fig. 8. Dilation of pial arterioles in response to CO before and after administration of Indo with L-NNA, Indo with L-NNA and isoproterenol (10–8 and 10–7 M), and Indo with L-NNA, isoproterenol (10–7 M), and SNP (3 x 10–7 M). *P < 0.05 compared with no CO; n = 6.

 
Actions of cGMP typically result from PKG-mediated phosphorylations. In addition, cAMP can activate PKG directly (11, 19, 34). Therefore, Rp-8-pCPT-cGMPS was used to inhibit PKG (34) to investigate whether CO-induced dilation is blocked when PKG is inhibited. In vivo, a lower concentration of Rp-8-pCPT-cGMPs (2 x 10–7 M) was used than in isolated cells (10–5 M; Ref. 34), because higher concentrations of Rp-8-pCPT-cGMPS caused very large and sustained dilations (data not shown). KT-5823, another selective PKG inhibitor, was not used as an alternative, because it had the same vasodilatory effect. Although the concentration was low, 2 x 10–7 M Rp-8-pCPT-cGMPS blocked dilation to 10–4 M 8-Br-cGMP, which is a direct activator of PKG (before Rp-8-pCPT-cGMPS administration, 58 ± 4 to 68 ± 4; in the presence of Rp-8-pCPT-cGMPS, 59 ± 4 to 59 ± 3 µm; n = 6). The PKG inhibitor also blocked dilation in response to CO (Fig. 9) and largely prevented isoproterenol from returning CO-induced dilation. However, surprisingly, the Rp-8-CPT-cGMPS concentration that blocked CO-induced dilation did not prevent SNP from restoring that dilation (Fig. 10). These data in toto suggest that PKG provides a permissive signal to allow CO to cause dose-dependent dilation via a mechanism that does not require progressive increases in PKG-catalyzed phosphorylations.



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  		Fig. 9. Dilation of pial arterioles in response to CO before administration and in the presence of the protein kinase G (PKG) inhibitor Rp-8-[(4-chlorophenyl)thio]-cGMPS triethylamine (Rp-8-pCPT-cGMPs; 2 x 10–7 M) and Rp-8-pCPT-cGMPs with either SNP (3 x 10–7 M) or isoproterenol (10–7 M). *P < 0.05 compared with no CO; {dagger}P < 0.05 compared with SNP; n = 6.

 


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  		Fig. 10. Schematic model of potential permissive mechanisms for NO and prostacyclin (PGI2) in CO-induced vascular smooth muscle hyperpolarization leading to dilation. AC, adenylyl cyclase; GC, guanylyl cyclase; IP, prostacyclin receptor; KCa, Ca2+-activated K+ channels. See text for explanation and details.

 
In contrast with the abolition of CO-induced dilation by inhibition of PKG, inhibition of PKA with Rp-8-pCPT-cAMPS had no effect on dilation in response to CO. Pial arteriolar diameters before PKA inhibition were 60 ± 2 and 73 ± 1 µm at 10–9 M CO and 81 ± 1 µm at 10–7 M CO; after PKA inhibition, diameters were 63 ± 1 and 71 ± 1 µm at 10–9 M CO and 78 ± 1 µm at 10–7 M CO (n = 4). The lack of effect was not due to failure to block PKA, because dilation in response to 8-Br-cAMP but not 8-Br-cGMP was blocked by Rp-8-pCPT-cAMPS (data not shown). Furthermore, in the presence of Rp-8-pCPT-cAMPS, which abolished dilation to 8-Br-cAMP, ODQ totally blocked and isoproterenol (10–7 M) in the continued presence of ODQ restored dilation in response to CO. These data suggest that the mechanism by which isoproterenol is permissive for CO dilation is via cAMP increasing PKG-catalyzed phosphorylation instead of phosphorylation catalyzed by PKA.


   DISCUSSION
TOP
 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We previously showed that either inhibition of COX with Indo or inhibition of NOS with L-NNA blocks the dilatory responses of piglet cerebral arterioles in response to CO (16). Furthermore, we reported that constant, low levels of iloprost and SNP restored the dilatory responses to arterioles whose responses to CO had been blocked by Indo and L-NNA, respectively. The present study suggests that prostacyclin and NO may share common mechanisms of permissive action, because SNP can restore dilation in response to CO when COX is blocked, and iloprost can restore dilation when NOS is blocked. At least partial commonality of mechanism is further suggested by the finding that the effects of iloprost and SNP are not additive, and they would not be expected to be if a common on/off mechanism were employed. The present results also suggest that the mechanism by which prostacyclin and NO permit the dilatory response to CO has a PKG component, since the cGMP PDE inhibitor zaprinast restores dilation in response to CO after either Indo or L-NNA administration. Furthermore, the guanylyl cyclase inhibitor ODQ and the PKG inhibitor Rp-8-pCPT-cGMPS block CO-induced dilation. However, the ability of NO to partially restore the dilatory response to CO even when guanylyl cyclase is blocked suggests, as did a previous study (14), that NO may have actions independent of cGMP (see below).

Our interpretation of the present and previously reported data is summarized pictorially in Fig. 10 and discussed below. The ultimate mechanism leading to CO-induced dilation is an increase in KCa channel activity. Indeed, a direct effect of CO on KCa channels is elevation of Ca2+ sensitivity (37). However, sufficiently elevated localized Ca2+ must be provided to open KCa channels even with increased Ca2+ sensitivity. PKG may increase Ca2+ sparks by actions on the sarcoplasmic reticulum Ca2+ load and/or ryanodine receptor channel activity. PKG can also phosphorylate the KCa channel itself. That could increase channel activity directly or increase CO activation of the channel. The predominant permissive action of both NO and prostacyclin is to stimulate PKG via cGMP and cAMP, respectively. In addition, NO may have actions independent of PKG that involve nitrosylation of KCa channels and/or ryanodine receptor channels.

Superficially, inhibition of dilation in response to CO by the guanylyl cyclase inhibitor ODQ might be interpreted to suggest that CO activates guanylyl cyclase, thereby increasing cGMP, which leads to dilation. However, two sets of experimental findings show that this interpretation is incorrect. First, at concentrations that cause vasodilation, SNP causes dose-dependent increases in cortical periarachnoid CSF concentrations of cGMP (3). Conversely, doses of CO that cause equivalent dilations in response to SNP do not increase cGMP (17). This is not surprising, because guanylyl cyclase is much more sensitive to NO than CO (20). Second, if, with ODQ treatment, 8-Br cGMP is added to clamp the cGMP level at a constant, dilation in response to CO is restored although cGMP concentration cannot change (14). Such a permissive requirement of NO via cGMP also has been shown in hypercapnia-induced dilation in adult rats (8, 30). These results show that cGMP can cause changes in cerebral arterioles that allow CO to cause dilation, but dose-dependent dilation in response to CO is caused by another mechanism after those changes have been made.

Data from the present study suggest that a key permissive mechanism for both prostacyclin and NO is a PKG-mediated phosphorylation event. These data include the findings that 1) PKG inhibition but not PKA inhibition blocked CO-induced dilation; and 2) the cAMP-dependent vasodilator isoproterenol restored CO-induced dilation in the presence of guanylyl cyclase inhibition, and this effect was prevented by Rp-8-pCPT-cGMPS. The capacity for direct PKG activation by cAMP to mediate reduction of vascular smooth muscle Ca2+ concentration, coronary and cerebral vasodilation, and inositol 1,4,5-trisphosphate receptor phosphorylation in intact vessels is well known (11, 13, 19, 23). Somewhat surprising at first is the ability of the PKG blocker to inhibit the permissive action of the cAMP-dependent dilator isoproterenol but less so that of the cGMP-dependent dilator SNP. However, such a result is less perplexing when one considers the numerous isoforms of PKG, certainly with differing sensitivities to selective PKG inhibitors, and the intricately constructed cellular compartmentalization and tethering to effectors of these kinases (23). Indeed, the {alpha}-isoform of type I PKG associates with KCa channels and enhances KCa channel activity (Ref. 29; see below for additional significance). Differential inhibition of specific cGMP-dependent protein kinases in the present experiments is likely, because a less-than-optimal inhibitor concentration had to be used to prevent loss of tone. The dilation in response to the PKG inhibitors presumably results from elimination of a phosphorylation event necessary for maintenance of vascular tone. Both PKG inhibitors Rp-8-pCPT-cGMPS and KT-5823 but not the PKA inhibitor (data not shown) had the same vasodilatory effect, which suggests that a nonspecific action of the inhibitors is unlikely. It is not only possible but likely that the PKGs most sensitive to cAMP and those most sensitive to cGMP are not the same, are compartmentalized differently in the cell, and/or are tethered to different targets. Thus the low dose of inhibitor used may inhibit PKGs that are sensitive to cAMP and responsible for the isoproterenol action, whereas other PKGs preferentially sensitive to cGMP are not blocked. cAMP, which inhibits cGMP efflux, could also increase intracellular cGMP by this mechanism, and independent of guanylyl cyclase activation, lead to PKG activation (38, 39).

CO activates KCa channels (32), which causes hyperpolarization (15), reduction in voltage-dependent Ca2+ channel activity, and reduction in intracellular Ca2+ concentration, thereby leading to dilation (10). CO-induced dilations in rat gracilis muscle and renal arterioles and piglet cerebral arterioles are inhibited by blockers of large-conductance KCa channels (12, 17, 41). When cerebral arteriolar dilation in response to CO is blocked by KCa channel inhibitors, such blockade is not reversible by treatment with iloprost or SNP (16).

In piglet cerebral arteriolar myocytes, CO increases outward K+ currents (STOCs), which are the result of KCa channel openings caused by Ca2+ sparks (9, 37). Because the direct action of CO that results in dilation is via the KCa channel but the permissive roles of prostacyclin and NO appear to involve PKG, it seems reasonable to suggest that PKG activity is necessary to allow CO to increase STOCs and promote cellular hyperpolarization.

The phosphorylation event catalyzed by PKG would likely involve a component of STOC production, namely, KCa channels and/or Ca2+ sparks, because CO-induced smooth muscle hyperpolarization and thus dilation are produced by increasing STOC frequency and amplitude. Considerable literature supports the concept of PKG-mediated stimulation of KCa channels (2, 26, 33). Furthermore, it is becoming increasingly apparent that cAMP-dependent vasodilators can produce vasodilation via a PKG-mediated mechanism that activates KCa channels (5, 7, 34). Thus a highly plausible mechanism by which PKG activity permits CO to induce dilation may involve phosphorylation of the KCa channel itself, thereby altering the response of the channels to CO. In contrast with KCa channel effects, less has been reported on the effects of PKG-catalyzed phosphorylation on Ca2+ sparks, but ryanodine receptors that cause sparks could be a target. It has been shown in cerebral arteries that cGMP-dependent dilators increase Ca2+ spark frequency (24). Such increases would be another explanation for a permissive action of cGMP for CO-induced dilation. A higher spark frequency and amplitude combined with KCa channels that are more Ca2+ sensitive would cause greater K+ efflux and dilation in response to CO. It is likely that this action can be attributed to PKG, because cGMP is a poor activator of PKA (6, 18, 19). Furthermore, inhibition of PKA did not block dilation to CO.

Our previous results showed that SNP is more effective than 8-Br-cGMP in returning dilation in response to CO after inhibition of NOS (14). The present data suggest that NO has actions in addition to providing the stimulus for basal cGMP maintenance. SNP was more effective in restoring dilation in response to CO after NOS inhibition than was iloprost, but iloprost and SNP had similar restorative actions after COX inhibition. Furthermore, SNP had permissive actions toward CO-induced dilation even when guanylyl cyclase was blocked. The most likely mechanisms include nitrosylation of KCa channels and/or ryanodine receptors (1, 28, 40).

The cellular source(s) of the permissive prostaglandin(s) and NO cannot be determined from the experimental design of the present experiments. The brain surface exposed to inhibitors and agonists placed under the cranial window is composed of the intact system of vascular endothelium and smooth muscle, perivascular nerves, astrocytes, oligodendricytes, pericytes, microglia, adventitia, and meninges. COX and cell-specific downstream prostaglandin synthases are ubiquitously expressed and NOS isoforms are constitutively expressed in endothelium, vascular smooth muscle, neurons, and glia. Previous investigations related specifically to hypercapnia-induced cerebrovascular dilation suggest neuronally provided permissive NO signals in adult rats (31), endothelially provided permissive NO signals in adult pigs (35), and endothelially provided permissive prostacyclin signals in newborn pigs (35). Available data do suggest that a critical source of both prostacyclin and NO for CO-induced dilation may be endothelium. Specifically, isolated, pressurized pial arterioles from newborn pigs dilate in response to CO. Endothelial damage blocks this dilation (4). After endothelial damage, dilation in response to CO is restored by SNP (4), 8-Br-cGMP (4), or iloprost (data not shown). Similar to the present experiment, in isolated arterioles, ODQ blocked CO-induced dilation, and the dilation was restored by addition of 8-Br-cGMP to the superfusate (4). Nevertheless, contributions of neurons and glia, specifically, to the functioning of the intact cerebrovascular circulatory system require much additional study. Furthermore, the functional significance of paracrine, permissive communication to regulation of cerebral blood flow remains largely unknown, but the rapidly growing collection of discoveries of such intercellular signaling suggests the importance is not trivial.


   GRANTS
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 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
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REFERENCES
 
This research was supported by the National Heart, Lung, and Blood Institute.


   ACKNOWLEDGMENTS
 
The authors thank G. Short for figures and M. Lester for clerical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. W. Leffler, Dept. of Physiology, 894 Union Ave., Memphis, TN 38163 (E-mail: cleffler{at}physio1.utmem.edu)

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.


   REFERENCES
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 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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