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Neuroanesthesia Research, University of Illinois at Chicago, Chicago, Illinois
Submitted 20 July 2004 ; accepted in final form 14 September 2004
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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-aminoadipic acid (L-AAA), 24 h before the study. In both intact and Ovx females, L-AAA had no effect on responses to the NO donor, S-nitroso-N-acetyl penicillamine, but ADP-induced pial arteriolar dilations were significantly reduced (by 33–90%), compared with vehicle-treated controls. When NG-nitro-L-arginine (L-NNA) was administered to L-AAA-treated rats, the ADP response was virtually lost in intact females, but no further reductions were observed in the Ovx rats. On the other hand, in L-AAA-treated Ovx females, when the gap junction blocker, Gap 27, was subsequently added to the suffusate, ADP reactivity fell to very low levels. In vehicle-treated control rats, L-NNA and Gap 27 reduced ADP reactivity by 
50% in intact and Ovx females, respectively. An earlier study indicated that the endothelium was a key site of influence for L-NNA (intact) and Gap 27 (Ovx). Thus present and previous results imply that the ADP response in pial arterioles represents the additive actions of an endothelial and a GL component. That supposition was confirmed in the present study by the finding that combining endothelial and GL injury produced an essentially complete loss of ADP reactivity in both intact and Ovx females. Finally, topical application of the selective P2Y1 antagonist, MRS-2179, was associated with a nearly complete suppression of the ADP response in both intact and Ovx females. These results suggest that 1) ADP-induced pial arteriolar dilation involves additive contributions from P2Y1 receptors present in both vascular endothelium and the GL; 2) the influence of the GL component is not altered by ovariectomy; and 3) the gap junction-dependent component of the ADP response in Ovx females is unlikely to include the GL and probably resides in the vessels themselves.
endothelium-derived hyperpolarizing factor; gap junction; nitric oxide; purinergic receptor
50% of the ADP response in both intact and Ovx rats was insensitive to endothelial injury and, in endothelium-intact vessels, NOS inhibition or gap junctional blockade (20). Indeed, in that study, a major gap junctional protein, connexin 43 (Cx43), was found to be well expressed not only in pial arterioles, but also in the underlying layer of glial processes, termed the glia limitans (GL). In another recent study, we obtained evidence suggesting that the GL and gap junctions play significant roles in hypercapnia-induced relaxation of pial arterioles (19).
The central hypothesis of this investigation is that the GL plays a key role in ADP-induced pial arteriolar dilation. To address that hypothesis, we examined whether the GL is important to the endothelium-independent portion of ADP-induced pial arteriolar relaxation; whether GL contributions are altered by chronic depletion of reproductive hormones; whether the gap junctional contributions to the ADP response in Ovx females involve the GL; and whether the ADP response is mediated by P2Y1 receptors present in both vascular tissue (principally endothelium) and the GL. To that end, we compared the effects of topical applications of the gliotoxin L--aminoadipic acid (L-AAA) on the ADP response in intact vs. Ovx females. The GL-injured rats were studied in the absence and presence of NG-nitro-L-argninine (L-NNA) and the gap junctional blocker Gap 27 (see Ref. 6). Because recent findings from our laboratory indicated that L-NNA and Gap 27 influenced the endothelial component of the ADP response in intact and Ovx females, respectively (20), we hypothesized that combining L-AAA with L-NNA or Gap 27 would result in the disappearance of ADP reactivity. As a further test of that hypothesis, pial arteriolar ADP reactivity was examined in rats subjected to combined endothelial and GL injury. Finally, ADP responses were monitored in the absence and presence of the selective P2Y1 antagonist MRS-2179. The findings of these experiments suggested that pial arteriolar ADP-induced dilations in female rats occur as the result of additive contributions from P2Y1 receptors present in endothelium and GL, irrespective of hormone status.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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4 wk after the date of arrival. Pial arteriolar reactivities were evaluated through closed cranial windows placed on the day preceding study (19). For window placement, the rats were anesthetized with halothane, intubated, paralyzed with a short-acting muscle relaxant (vecuronium), and mechanically ventilated with a 0.8% halothane-70% N2O-30% O2 gas mixture. After window preparation, 300 µl of an artificial cerebrospinal fluid (aCSF) solution with or without L-AAA (2 mM) were injected into the space under the cranial window (19). The cranial window access ports were then plugged, and the skin overlying the skull was sutured together. The animal was permitted to recover from anesthesia, extubated, and returned to its cage. On the day of study, after anesthesia induction with halothane and paralysis (curare), the rats were tracheotomized and mechanically ventilated. Bilateral femoral arterial and venous catheters were inserted under anesthesia with 0.8% halothane-70% N2O-30% O2. After catheterization, the rat was placed in a head holder, and the cranial window inflow, outflow and intracranial pressure monitoring cannulae were connected. Halothane was discontinued, and a 10 µg/kg fentanyl bolus was given intravenously. During the study, the rats were maintained with fentanyl (25 µg·kg–1·h–1 iv) and 70% N2O-30% O2. The space under the window was filled with aCSF (pH 
7.35–7.40) that was equilibrated with a gas mixture consisting of 20% O2-5% CO2-balance of N2. The 37°C aCSF solution was suffused at 0.5 ml/min. Body temperature was controlled at 37°C, and mean arterial pressure and intracranial pressure were monitored continuously during the experiment. Measurements of the diameters of pial arterioles (25–50 µm) were made using video microscopy (see Ref. 18). In all experiments, the initial diameter measurements were made 1 h posthalothane and after 40-min drug-free aCSF suffusion.
The rats were divided into four principal experimental subgroups: intact vehicle; intact L-AAA; Ovx vehicle; and Ovx L-AAA. For the intact females, after measurement of baseline diameter values, ADP, at concentrations of 10 and 100 µM, was suffused into the space under the cranial window (5 min each concentration). After 10 min of drug-free aCSF suffusion, a baseline measurement was made, and the NO donor S-nitroso-N-acetyl penicillamine (SNAP) was sequentially suffused at 0.1 and 1.0 µM (5 min at each dose). After a return to baseline, L-NNA (1 mM) was added to the aCSF and suffused for 60 min, and the responses to ADP and SNAP were again evaluated. Time control rats, from vehicle and L-AAA-treated groups, where L-NNA was omitted from the aCSF, were also studied. No changes in ADP or SNAP reactivity were noted in these animals (data not shown). In the Ovx rats, a similar protocol was used, but with one additional step. Thus the responses to ADP (10 and 100 µM) and SNAP (0.1 and 1.0 µM) were evaluated as in the intact females: first in the absence and then in the presence of L-NNA. Subsequently, a suffusion of the Cx43 and Cx37 gap junction inhibitory peptide, Gap 27 (300 µM), was initiated and maintained for 1 h before reevaluation of ADP responses. No changes in ADP or SNAP reactivities were seen in time controls, where L-NNA and L-NNA + Gap 27 were omitted from the aCSF solution (not shown).
Two additional experimental protocols were performed. In the first, we sought to obtain further evidence in support of the hypothesis that the pial arteriolar vasodilating response to ADP, in intact and Ovx females, is a reflection of the additive contributions from vascular endothelium and the GL. To that end, L-AAA-treated rats were subjected to a light plus dye (L/D) endothelial injury strategy, utilized in several previous studies in our laboratory (e.g., Ref. 20). For these experiments, at 24 h after L-AAA treatment, ADP (10 and 100 µM) and SNAP (0.1 and 1.0 µM) reactivities were measured. Subsequently, the animals were subjected to L/D injury, where mercury light was passed through a filter that allowed transmission only at 450–490 nm. The light passed coaxially through the microscope objective and was focused on an area including the vessels being studied. Two percent fluorescein dye (0.8 ml/100 g body wt) was given intravenously, and the mercury light was turned off 55 s after the fluorescein dye infusion was initiated. The responses to ADP or SNAP were then reevaluated. For the second protocol, the P2Y1 receptor dependency of ADP-induced pial arteriolar dilations was examined in vehicle-injected intact and Ovx females. In these rats, dose-related responses to suffusions of ADP (10, 30, and 100 µM) and then adenosine (10 and 100 µM) were measured. After reestablishment of baseline diameters, via suffusion of drug-free aCSF, a suffusion of MRS-2179 (10 µM) was initiated. After 60 min, ADP and adenosine reactivities were again evaluated. The EC50 for MRS-2179 in vitro has been reported to be in the range of 0.1–0.3 µM, but is ineffective toward other P2Y receptors at concentrations up to 30 µM (1, 8).
Arterial blood samples were taken at 30-min intervals for PaO2, PaCO2, and pH analysis using a blood gas/pH analyzer (model ABL 520, Radiometer; Copenhagen, Denmark). PaO2 (
100 mmHg), PaCO2 (30–40 mmHg), and pH (7.40) were maintained during the study.
All reagents were obtained from Sigma (St. Louis, MO) and dissolved in aCSF unless otherwise stated. Gap 27 peptide (SRPTEKTIFII) was synthesized by the Protein Research Laboratory at the University of Illinois at Chicago. The purity of the peptide was >95%. Values are presented as means ± SE. Comparisons of arteriolar diameter values within groups were made using one-way repeated-measures ANOVA, combined with a post hoc Tukey analysis. Analyses of diameter changes between groups were made using Student's t-test. A P value < 0.05 was considered as significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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AAA-treated intact and Ovx female rats (Fig. 1). The absence of any change in vascular reactivity to SNAP suggests that neither ovariectomy nor L-AAA alters vascular smooth muscle function. The absence of any effect of L-NNA (1 mM, topical application) confirms that SNAP acts independently of endogenous generation of NO.
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AAA-treated intact females, in the absence and presence of L-NNA, are shown in Fig. 2. As previously shown by us, in the intact females, NOS inhibition reduced the ADP response by 50%. In rats exposed to topically applied L-AAA for 24 h, ADP reactivity was reduced, relative to vehicle controls, by 33% (100 µM ADP) to 70% (10 µM ADP) in the absence of NOS inhibition. Moreover, on addition of L-NNA, ADP reactivity was reduced substantially further, by 82–100% from the initial response observed in the vehicle group. These seemingly additive effects of L-NNA and L-AAA suggest that NO and the GL support separate components of the ADP response in intact females.
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AAA-treated Ovx females, in the absence and presence of L-NNA and L-NNA + Gap 27, are summarized in Fig. 3. As previously shown by us (20, 21), although pial arteriolar reactivity to ADP is not significantly different in Ovx vs. intact females (see Fig. 2), NOS inhibition has no effect on the ADP response in Ovx rats (consistent with the loss of eNOS dependency). In Ovx females treated with L-AAA, the pial arteriolar response to ADP was reduced by 35–90% from the response seen in vehicle-treated Ovx females, with no further changes on addition of L-NNA. However, when the gap junctional inhibitor Gap27 (which preferentially affects Cx43- and Cx37-containing gap junctions) was applied, ADP reactivity at the lower concentration completely disappeared and was markedly repressed (by >80% from the initial response in the vehicle group) at the higher ADP concentration. It should be noted that Gap 27 has no effect on pial arteriolar reactivity to ADP in intact females (20). The effects of GL injury and gap junctional blockade seem to be generally additive, although the combination of GL injury and gap junction blockade appears to exceed what one might expect if the effects of L-AAA and Gap 27 were completely independent of one another. Nevertheless, the mostly additive nature of these results suggests that the gap junctional dependency and astrocytic (GL) influence, to a large degree, involve separate components of the pial arteriolar response to ADP in the Ovx female. The fact that the gap junctional component does not seem to be substantially affected by injury to the GL suggests that the gap junctions involved in the ADP response in Ovx females are mostly nonastrocytic. A likely location for these gap junctions would be within the vessels themselves.
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AAA-induced GL injury and, subsequently, after acute L/D-induced endothelial injury. These data are provided in Fig. 4. Similar to the results presented earlier, L-AAA treatment was accompanied by ADP responses that were 40–70% lower than the control responses (control values given in Figs. 2 and 3). On L/D treatment, the response at the lower ADP concentration (10 µM) completely disappeared, and the response at 100 µM ADP was reduced to 10% of the control value. The absence of any changes in the pial arteriolar responses to SNAP (Fig. 5) confirms that the L/D procedure did not injure vascular smooth muscle.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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AAA-induced GL injury, elicits a nearly complete loss of ADP reactivity.
In a recently published report (19), we established that L-AAA selectively damages the GL while (functionally) sparing vascular cells and neurons. In that study, we also obtained evidence that the GL plays an important role in supporting hypercapnia-induced pial arteriolar dilation. Additional findings indicated that Cx43, and presumably gap junctions, participated in that response. However, in that instance, results suggested that gap junctions residing in astrocytes were involved.
Although both involve multicellular influences, there are major differences in stimulus/vascular response coupling when comparing in vivo pial arteriolar responses to hypercapnia vs. ADP. One obvious difference is that ADP acts via a purinergic receptor (mostly P2Y1; see Ref. 16), whereas hypercapnia does not. As such, one might look to the sites of P2Y1 expression to derive clues as to the mechanisms at play in an ADP response that includes a vascular, as well as an extravascular, component. Of particular relevance to the present investigation, P2Y1 receptors have been shown to be well expressed in rodent astrocytes (e.g., Ref. 22). Endothelial cells are reported to be another site of P2Y1 expression, which is consistent with the purported role of this receptor subtype in cerebral endothelium-derived NO generation (7). In the brain, evidence points to the endothelial presence of P2Y1 message in arteries (11), although immunohistochemical analysis has not confirmed endothelial expression of P2Y1 protein (14). Nevertheless, in the present study, P2Y1 blockade with the selective antagonist MRS-2179 was associated with a virtually complete loss of ADP reactivity in pial arterioles. Because, in female rats, endothelium (see Fig. 4 and Ref. 20) and astrocytes appear to contribute roughly equally (i.e., 50/50) to the pial arteriolar ADP response, the results obtained with P2Y1 antagonist applications would seem to support the presence of endothelial P2Y1 receptors, at least in pial vessels. This apparent incongruity between results obtained using pharmacological and immunohistochemical approaches may simply be a function of an insufficient sensitivity to P2Y1 antibodies, although we cannot completely discount the possibility, however unlikely (1), that MRS-2179 may be interacting with another P2Y variant or that P2Y1 expression may be relatively greater in pial arterioles compared with the rest of the cerebral vasculature. The similar efficacies of MRS-2179 and combined endothelial and GL injury in effecting a nearly total blockade of pial arteriolar ADP reactivity also imply that the P2Y1 receptors involved reside almost exclusively in endothelium and the GL. This leaves little, if any, room for contributions from the P2Y1 receptors that appear to be present in neurons (12) and cerebral vascular smooth muscle (11). Furthermore, the finding that L-AAA- and L/D-induced injuries do not damage cortical neurons and pial arteriolar smooth muscle (Fig. 5 and Ref. 19), yet ADP reactivity is lost nonetheless, makes it even less likely that P2Y1 receptors in those cells contribute in any meaningful way.
The fact that application of the P2Y1 antagonist was associated with a loss of ADP reactivity in both intact and Ovx females suggests that activation of endothelial P2Y1 receptors not only can stimulate eNOS-derived NO generation but also, under the "right" circumstances, is capable of promoting a gap junction-related vasodilation that has characteristics of an EDHF (20) (see also Ref. 6). We can only speculate as to what common factor links the endothelium-dependent portion of the P2Y1-mediated ADP response in intact vs. Ovx females. The metabotropic, heterotrimeric G protein-coupled P2Y1 receptor is known to promote intracellular Ca2+ mobilization. This appears to occur via activation of phospholipase C and inositol 1,4,5-trisphosphate-mediated release of Ca2+ from intracellular stores, with the increase in intracellular Ca2+ concentration ([Ca2+]i) being sustained by capacitative Ca2+ entry (9). Thus it is likely that ADP engagement of the endothelial P2Y1 receptor promotes eNOS activation, or, in the presence of repressed eNOS function [e.g., estrogen-depleted states (13)], increased activity of a gap junction-dependent, EDHF-like mechanism (20). Both "targets" would require acute upstream elevations in endothelial [Ca2+]i (see Refs. 10 and 17).
The mechanistic features of astrocytic P2Y1 receptor contributions to ADP-induced pial arteriolar dilation are even less clear. However, it is probably safe to assume that this also involves mobilization of [Ca2+]i because P2Y1 receptor activation has been shown to elicit Ca2+ wave propagation between adjacent astrocytes (4). While this implies a Ca2+-dependent astrocyte-derived factor, the nature of this apparent factor is unknown. Certainly, paracrine factors generated by Ca2+-activated enzymes must be given serious consideration. Candidate enzymes include nNOS and the Ca2+-dependent PLA2 isoforms. NO generated via nNOS action seems unlikely, in light of recent findings from our laboratory showing that topical application of the neuronal NOS-selective inhibitor ARR-17477 (at least in male rats) had no effect on pial arteriolar responses to ADP suffusions (17). With regard to PLA2, one might consider the downstream metabolism of a principal product of PLA2 action, i.e., arachidonic acid (AA). Indeed, evidence exists linking P2Y1 receptor activation to AA release in astrocytes (3). There are well-established vasodilating substances generated via AA metabolic pathways. These include cyclooxygenase-1 (COX-1) products, like prostacylin, or products of the cytochrome P-450-2C11 epoxygenase. However, in earlier studies, we failed to observe any effects of COX-1 or epoxygenase inhibitors on the ADP response of pial arterioles (21). Nevertheless, whereas the absence of any effect of epoxygenase inhibition would be consistent with a lack of any influence from de novo epoxide generation, it does not negate the participation of epoxides released from preformed pools. There are additional PLA2-related candidates one might consider. The possibility exists that AA itself, in the absence of further metabolism, may promote vasodilation, for example, via interaction with AA-sensitive membrane ion channels (2). AA has also been shown to be a potent vasodilator when applied directly to cerebral arteries and arterioles in vivo (e.g., Refs. 5 and 15). However, that AA action appears to be attributable to its metabolism via the cyclooxygenase or lipoxygenase pathway. Although a COX-1-generated AA metabolite has been essentially eliminated as a potential mediator of ADP-induced pial arteriolar relaxation in our animals, it may be of some value, in future studies, to examine whether a lipoxygenase pathway product or AA itself is involved.
In conclusion, the GL appears to have a significant influence on ADP-induced pial arteriolar dilation in female rats, irrespective of hormone status. That contribution involves the NO-insensitive component of the ADP response. In fact, in the intact female, the eNOS-dependent and the astrocyte-derived elements, in an additive manner, seem to account for nearly all of the pial arteriolar response to ADP (Fig. 8A). In the Ovx female, the NO-dependent component is replaced by a mechanism related to gap junctions and EDHF. Thus a substantial portion of the ADP response in the Ovx female can be accounted for (additively) by a gap-junctional (EDHF) element and an astrocytic element (Fig. 8B). In contrast to the pial arteriolar response to hypercapnia (19), the gap junctions involved would appear to be nonastrocytic (probably vascular). In either case, it appears that astrocytes comprising the GL contribute to ADP-induced pial arteriolar relaxation through a P2Y1 receptor-initiated mechanism. Whether that mechanism involves a diffusible or a mechanical factor (or something else) remains to be established.
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ACKNOWLEDGMENTS |
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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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P < 0.05 vs. vehicle.
