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Division of Neurobiology, Department of Neurology and Neuroscience, Weill Medical College of Cornell University, New York, New York 10021
Submitted 21 May 2003 ; accepted in final form 27 June 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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cerebral circulation; hypertension; somatosensory activation; field potentials; laser-Doppler flowmetry
The octapeptide angiotensin (ANG) II plays a central role in the mechanisms of HTN (for a review, see Ref. 28). The precursor protein angiotensinogen, secreted into the circulation by the liver, is cleaved by the kidney-derived aspartyl protease renin to form the decapeptide ANG I. ANG I is then converted to ANG II by the metalloprotease angiotensin-converting enzyme, which is released by the lungs (28). The "classic" renin-ANG system (RAS) produces circulating ANG II, but a tissue RAS producing ANG II locally is present in all major organs, including the blood vessels and the brain (28). ANG II has powerful short- and long-term effects on many organs. In systemic vessels, ANG II is a potent vasoconstrictor and produces endothelial dysfunction and smooth muscle proliferation (27). In the cerebral circulation, ANG II is a vasoconstrictor in many animal species and has powerful effects on cerebrovascular autoregulation (for a review, see Ref. 30). Evidence from transgenic mice overexpressing angiotensinogen and renin suggests that ANG II alters endothelial function in cerebral blood vessels (4). However, it is not known whether ANG II also impairs the increases in CBF produced by neural activity. Such alteration would provide further insight into the deleterious effects of ANG II and may suggest new therapeutic opportunities to counter the consequences of HTN.
In the present study, we used a well-established model of functional activation of the somatosensory cortex to determine whether ANG II alters the increases in CBF produced by neural activity. We found that systemic administration of ANG II markedly attenuates the increase in somatosensory cortex blood flow produced by stimulation of the facial whiskers but not those produced by hypercapnia or by a nitric oxide (NO) donor. The alteration in functional hyperemia could not be attributed to mechanical effects of the ANG II-induced elevation in AP or to neuronal effects of the peptide. The findings suggest a heretofore unrecognized effect of ANG II on the coupling between synaptic activity and CBF that has important implications for the deleterious actions of ANG II on cerebral function.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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General Surgical Procedures
All procedures were approved by the Institutional Animal Care and Use Committee. Studies were conducted in 90 C57BL/6J male mice (age: 2–3 mo, 20–30 g body wt) obtained from Jackson Laboratories (Bar Harbor, ME). Mice were anesthetized with isoflurane in 100% O2 (induction: 5%; maintenance: 2%). The trachea was intubated, and mice were artificially ventilated with an oxygen-nitrogen mixture. The O2 concentration in the mixture was adjusted to provide an arterial PO2 (PaO2) of 120–140 mmHg (Table 1). One of the femoral arteries was cannulated for recording of mean AP (MAP) and collection of blood samples. Rectal temperature was maintained at 37°C using a thermostatically controlled rectal probe connected to a heating lamp. End-tidal CO2, monitored by a CO2 analyzer (Capstar-100, CWE), was maintained at 2.6–2.7% (21). After surgery, isoflurane was discontinued, and anesthesia was maintained with urethane (750 mg/kg ip) and chloralose (50 mg/kg ip). Throughout the experiment, the level of anesthesia was monitored by testing corneal reflexes and motor responses to tail pinch. To minimize confounding effects of anesthesia on vascular reactivity, the time interval between the administration of urethane-chloralose and the testing of CBF responses was kept consistent among the different groups of mice studied.
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A small craniotomy (2 x 2 mm) was performed to expose the parietal cortex, the dura was removed, and the site was superfused with a modified Ringer solution (37°C, pH: 7.3–7.4; see Ref. 10 for composition). CBF was continuously monitored at the site of superfusion with a laser-Doppler probe (Vasamedic; St. Paul, MN) positioned stereotaxically on the cortical surface. The outputs of the flowmeter and blood pressure transducer were connected to a data-acquisition system (MacLab) and saved on a computer for off-line analysis. CBF values were expressed as percent increases relative to the resting level. Zero values for CBF were obtained after the heart was stopped by an overdose of isoflurane at the end of the experiment. Although laser-Doppler flowmetry is not quantitative, it monitors relative changes in CBF quite accurately (for a review, see Ref. 11).
Recording of Field Potentials
Field potentials were recorded using an electrode placed on the somatosensory cortex contralateral to the activated whiskers. To avoid disruption of the blood-brain barrier and penetration of ANG II into the brain at the site of recording, a superficial electrode was used. The recording electrode was fed to an amplifier (Axon Instruments), the output of which was connected to a computerized data-acquisition system (Mac Lab). The somatosensory cortex was activated by electrical stimulation of the whisker pad (2 V, 0.5 Hz, pulse duration: 1 ms). Ten stimulation trials were averaged using a data-acquisition system and stored on a hard drive for off-line analysis (35). Only recording sites from which the greatest field potential was obtained were used.
Implantation of Osmotic Minipumps for Sustained Delivery of ANG II or Phenylephrine
Osmotic minipumps containing saline, ANG II, or phenylephrine (PE) were implanted subcutaneously in mice (n = 5 mice/group) under isoflurane anesthesia. Systolic AP and heart rate were monitored daily in awake mice using tail-cuff plethysmography (16). To decrease the stress associated with the measurements and to accustom the mice to the procedure, systolic AP was measured daily for 4 days before pump implantation. Although the tail-cuff method may underestimate absolute AP by 
5 mmHg compared with carotid measurements, it detects accurately relative AP changes (29). Concentrations and delivery rates of ANG II (2.74 mg · kg–1 · day–1) and PE (27.4 mg · kg–1 · day–1) were adjusted to produce comparable levels of AP elevation. Seven days after implantation, mice were anesthetized and instrumented for assessment of cerebrovascular reactivity by laser-Doppler flowmetry as described in Experimental Protocols.
Experimental Protocols
Effect of systemic ANG II on CBF responses to whisker stimulation, hypercapnia, S-nitroso-N-acetyl penicillamine, or adenosine. After stabilization of MAP and blood gases (Table 1), the whisker barrel region of the somatosensory cortex was activated for 60 s by stroking of the contralateral facial whiskers (21, 22), and the evoked changes in CBF were recorded. After a stable response was achieved, ANG II (ANG II acetate, Sigma) was administered intravenously. The ANG II infusion was adjusted to elevate MAP by 20–25 mmHg gradually over 10–15 min until a stable increase was obtained. At this time, the infusion rate was 0.25 ± 0.02 µg · kg–1 · min–1, which produced elevations in plasma ANG II within the range of that produced by endogenous activation of the RAS in rodents (19). The response to whisker activation was tested again after 30 min of ANG II infusion. In a separate group of mice (n = 5), the CBF response to systemic hypercapnia [arterial PCO2 (PaCO2) = 50–60 mmHg] and to neocortical superfusion with the NO donor S-nitroso-N-acetyl penicillamine (SNAP; 50 µM) or with adenosine (400 µM) was tested. Whisker stimulation and hypercapnia were carried out in separate mice because prior hypercapnia enhances the CBF response to whisker stimulation (31). Less than 0.3 ml of fluid were infused over the entire duration of the experiment.
Effect of systemic PE on CBF responses to whisker stimulation. The methods for these experiments were identical to those described in Effect of systemic ANG II on CBF responses to whisker stimulation, hypercapnia, S-nitroso-N-acetyl penicillamine, or adenosine with the exception that either vehicle (saline) or PE was infused intravenously. Because sudden increases in MAP influence cerebrovascular responses irrespective of the mechanism by which HTN is induced (14), MAP increases similar in rate and magnitude to those obtained with ANG II were produced. Responses to whisker stimulation were tested before and at least 30 min after PE infusion. At the time when MAP was steadily elevated, the PE infusion rate was 0.34 ± 0.05 µg · kg–1 · min–1.
Effect of MAP elevation on cerebrovascular responses evoked by ANG II. First, the increases in CBF produced by whisker stimulation, hypercapnia, or SNAP were assessed during intravenous infusion of vehicle. The infusion was then switched to ANG II or PE, and the elevation of MAP was prevented by slow removal of arterial blood. After at least 30 min of ANG II or PE infusion, responses to whisker stimulation, hypercapnia, or SNAP were tested again.
Effect of ANG II on somatosensory field potentials evoked by whisker stimulation. First, evoked potentials were recorded during intravenous infusion of vehicle. ANG II was then infused intravenously as described in Effect of MAP elevation on cerebrovascular responses evoked by ANG II, and field potentials were recorded at least 30 min later. After the completion of the recordings with ANG II, the infusion was discontinued, the Na+ channel blocker tetrodotoxin (TTX; 1 µM) was applied topically to the somatosensory cortex, and the field potentials evoked by the stimulation were recorded again.
Effect of topical neocortical application of ANG II on CBF responses to whisker stimulation or hypercapnia. After stabilization of MAP and blood gases, the window was superfused with Ringer solution. First, we sought to determine whether ANG II would affect resting CBF. ANG II was topically applied at increasing concentrations (from 50 nM to 100 µM), and CBF responses were recorded after a stable change had occurred. Responses to whisker stimulation or hypercapnia were first tested with Ringer solution superfusion. Next, the superfusion solution was changed to ANG II, and cerebrovascular responses were tested at least 30 min later. Finally, the solution was switched back to Ringer solution, and responses were tested again 30 min later.
Effect of sustained ANG II or PE infusion on CBF responses to whisker stimulation, hypercapnia, or SNAP. Osmotic minipumps for the infusion of saline, ANG II, or PE were implanted in separate groups of mice (n = 5 mice/group), and MAP was measured daily as described in Implementation of Osmotic Minipumps for Sustained Delivery of ANG II or Phenylephrine. Seven days after minipump implantation, mice were instrumented for the assessment of cerebrovascular reactivity to whisker stimulation, hypercapnia, or SNAP.
Data Analysis
Data are expressed as means ± SE. Two-group comparisons were analyzed by two-tailed Student's t-test. Multiple comparisons were evaluated by ANOVA and Tukey's test. Probability values of <0.05 were considered statistically significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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In mice that received an infusion of vehicle (n = 5), whisker stimulation produced increases in CBF that were comparable in magnitude to those reported from this and other laboratories in anesthetized mice (Fig. 1C) (2, 22). Systemic administration of ANG II (n = 5) elevated MAP by 20–25 mmHg (P < 0.05) but did not affect resting CBF (P > 0.05; Fig. 1, A and B). However, ANG II attenuated the increase in CBF produced by whisker stimulation (P < 0.05; Fig. 1C). In contrast, ANG II did not affect the increases in CBF produced by systemic hypercapnia (PCO2:50–60 mmHg; Fig. 1D), by the NO donor SNAP (50 µM; Ringer solution: +24 ± 2%; ANG II: +24 ± 1%, P > 0.05, n = 5), or by adenosine (400 µM; Ringer solution: +31 ± 1%; ANG II: +32 ± 1%, P > 0.05, n = 5). Administration of PE produced increases in MAP that were similar in magnitude and time course to those produced by ANG II (P > 0.05 from ANG II; Fig. 1E). PE did not affect resting CBF (before PE: 17.3 ± 2 perfusion units; after PE: 17.8 ± 1.3 perfusion units, P > 0.05) and did not attenuate the increase in CBF produced by whisker stimulation (P > 0.05; Fig. 1F).
Systemic Administration of ANG II Attenuates the CBF Response to Whisker Stimulation in the Absence of MAP Elevation
With ANG II infusion, the removal of blood did not alter resting CBF (before blood removal: 16.0 ± 0.3 perfusion units; after blood removal: 16.3 ± 0.8 perfusion units, P > 0.05). However, the CBF response to whisker stimulation was attenuated (P < 0.05) despite no change in MAP (P > 0.05; Fig. 2, A and B). In contrast, with PE infusion, the functional hyperemic response was not affected (P > 0.05; Fig. 2, A and B). ANG II or PE infusion did not alter the CBF response to hypercapnia or SNAP (P > 0.05; Fig. 2, C and D). The amount of blood removed (ANG II: 146 ± 9 µl; PE: 143 ± 11 µl) and the resulting reduction in hematocrit (ANG II: from 41.7 ± 1.0 to 39.9 ± 0.8%; PE: from 42.1 ± 0.2 to 40.7 ± 1.1%) did not differ between the animals that received ANG II and PE (P > 0.05).
Topical Application of ANG II to the Somatosensory Cortex Attenuates the CBF Response to Whisker Stimulation
First, we sought to establish a concentration of ANG II that does not affect resting CBF. Topical application of ANG II produced concentration-related reductions in CBF (Fig. 3). At 50 nM, however, resting CBF was not reduced (P > 0.05 from Ringer solution; Fig. 3). This ANG II concentration was then used in subsequent experiments. Superfusion with ANG II did not affect MAP or resting CBF (P > 0.05; Fig. 4, A and B). However, ANG II attenuated the increase in CBF produced by whisker stimulation (P < 0.05; Fig. 4C). When the solution was changed back to Ringer solution, the response was fully reestablished (P > 0.05 from Ringer solution before ANG II). The increase in CBF produced by hypercapnia was not affected by ANG II (P > 0.05; Fig. 4D).
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Systemic Administration of ANG II Does Not Affect the Field Potentials Evoked by Whisker Activation
Systemic administration of ANG II elevated MAP from 80 ± 4 to 100 ± 2 mmHg (P < 0.05, n = 5) but did not affect the amplitude (P1 wave: vehicle 4.0 ± 0.7 mV; ANG II 4.2 ± 0.7 mV, P > 0.05, n = 5) or the shape of the field potentials (Fig. 5). Topical neocortical application of TTX (1 µM) abolished the field potentials (Fig. 5).
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Sustained Infusion of ANG II But Not PE Attenuates the CBF Response to Whisker Stimulation
Implantation of ANG II and PE pumps increased AP (Fig. 6). The elevations were similar in time course and magnitude to those previously reported (e.g., Refs. 8 and 33). At 7 days, the increase was 39 mmHg for ANG II and 36 mmHg for PE (P < 0.05; Fig. 6). ANG II but not PE attenuated the increase in CBF produced by whisker stimulation (Fig. 7B). In these experiments, MAP was lower than in awake mice as a result of anesthesia (cf. Figs. 6 and 7A). CBF responses to SNAP and hypercapnia were not affected by ANG II or PE (Fig. 7, C and D).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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The reduction of the hemodynamic response to functional activation produced by ANG II cannot result from differences in MAP or blood gases because these variables were carefully controlled and did not differ among the groups of mice studied. Furthermore, the effect cannot be a consequence of a nonspecific alteration in cerebrovascular reactivity because the increase in CBF produced by other stimuli, such as systemic hypercapnia and topical application of SNAP or adenosine, was not affected. Therefore, the reduction in functional hyperemia cannot be attributed to changes in systemic variables or nonspecific alterations in vascular reactivity.
After we established that ANG II attenuates functional hyperemia, we began to investigate the potential mechanisms of the effect. There are several mechanisms by which systemic administration of ANG II could attenuate the increase in CBF produced by somatosensory activation. One possibility is that the mechanical effects of the elevations in MAP on cerebral blood vessels alter vascular reactivity. It is well established that acute increases in MAP alter the reactivity of the cerebral circulation (14, 15). For example, in the cat and rat, large and rapid elevations in MAP (50–60 mmHg) attenuate the vasodilation produced by endothelium-dependent vasodilators or hypercapnia (14, 15). These effects are independent of the pressor agent used to induce hypertension and are associated with increased permeability of the blood-brain barrier and morphological alterations in cerebral blood vessels (14, 15, 20). However, it is unlikely that the effect of ANG II is due to a mechanical effect of HTN on cerebral blood vessels because the attenuation in functional hyperemia 1) persists if MAP is not allowed to rise; 2) occurs also with topical application of the peptide to the somatosensory cortex, a treatment that does not alter MAP; 3) is not observed when MAP is elevated by the administration of the pressor agent PE; and 4) is not associated with alterations in the hypercapnic vasodilation, a response attenuated by the vascular injury induced by acute HTN (14). Furthermore, the elevations in MAP used in the present study were slow in time course and smaller in magnitude than those producing the cerebrovascular alterations mentioned above. Therefore, mechanical effects of the MAP elevation are unlikely to contribute to the actions of ANG II on functional hyperemia.
A second mechanism by which ANG II could alter the increase in CBF produced by whisker stimulation is by acting directly on neurons to attenuate the synaptic activity produced by the stimulation. ANG receptors have been described on neurons, and, through these receptors, ANG II can have profound effects on neuronal function (for reviews, see Refs. 7 and 32). In several neuronal types, ANG II attenuates K+ conductance and activates calcium currents leading to increased neuronal excitation, although direct or indirect inhibitory actions have also been reported (7, 32). However, direct neuronal effects of ANG II are unlikely because 1) ANG II does not cross the blood-brain barrier in amounts sufficient to influence neuronal activity (5), and 2) we found that circulating ANG II, while attenuating functional hyperemia, does not alter the field potential produced by whisker stimulation.
On the other hand, circulating ANG II could act through the circumventricular organs (CVOs), which are permeable to ANG II and are largely responsible for mediating the central effects of the peptide (5). CVOs are enriched with ANG receptors and have extensive neural projections involving multiple brain regions (26). However, it is unlikely that ANG II attenuates functional hyperemia by acting on CVOs because the application of ANG II directly to the cerebral cortex, a treatment that bypasses CVOs, reproduces the effects of systemic administration of the peptide. Furthermore, the observation that ANG II does not alter somatosensory field potentials also provides evidence that indirect neural effects through CVOs are unlikely.
Therefore, the most likely scenario is that the effects of ANG II on functional hyperemia are mediated through direct vascular actions of the peptide. The fact that ANG II is effective when administered perivascularly (superfusion) or intravascularly (intravenously) suggests that the cellular targets of ANG II are accessible to the peptide both from the luminal and abluminal sides of the vessel. Investigation of the cellular and subcellular distribution of ANG receptors in the vessel wall would help with the interpretation of this finding. Multiple factors are involved in mediating the increases in CBF produced by synaptic activity (1, 17). While vasoactive substances released by active neurons and glia produce vasodilation in local microvessels, retrograde propagation of the vasodilation through intravascular mechanisms is thought to amplify the local vascular response leading to sustained increases in CBF (12). Several vasodilators released by neuronal activity have been implicated in the process, including NO, cyclooxygenase products, cytochrome P-450 metabolites, adenosine, and K+ and H+, among others (1, 17, 24). Our findings suggest that ANG II does not affect the neural processes that generate the vasodilators, but it inhibits their vascular effects, resulting in attenuation of the vascular response associated with neural activation. In the systemic circulation, ANG II is well known to attenuate endothelium-dependent responses, an effect mediated by scavenging of NO by ANG II-generated superoxide (27). Considering that ANG receptors have been reported on cerebral arteries (for a review, see Ref. 30), it is conceivable that similar mechanisms are responsible for the attenuation of functional hyperemia. In apparent contrast with this hypothesis is our finding that ANG II does not attenuate the increase in CBF produced by the NO donor SNAP. However, SNAP releases large amounts of NO with spatial and temporal profiles that do not reflect the pattern of NO release during functional activation. Therefore, it is difficult to draw conclusions from the experiments with SNAP beyond the fact the vascular reactivity to large amounts of NO is preserved. Future studies will have to further investigate this complex issue.
The neuronal microenvironment is carefully regulated by the controlled delivery of energy substrates and timely removal of byproducts of cellular metabolism (1). The alterations in the increase in CBF evoked by synaptic activity produced by ANG II could perturb this delicate homeostatic balance and, if the perturbation is protracted in time, could lead to neuronal dysfunction. Therefore, the findings of the present study raise the possibility that the AP-independent alterations in cerebrovascular homeostasis produced by ANG II contribute to the cerebral dysfunction observed in hypertensive patients. For example, essential HTN leads to a cognitive decline in otherwise healthy elderly individuals (for a review, see Ref. 25). Recent clinical studies have demonstrated that antihypertensive agents that act by blocking the RAS, e.g., angiotensin-converting enzyme inhibitors, exert beneficial cerebrovascular effects that cannot be explained by the associated reduction in AP (for a review, see Ref. 9). These clinical findings support the contention that ANG II produces deleterious cerebral effects independently of the associated HTN. However, additional clinical and experimental studies are needed to provide further evidence in support of this hypothesis.
In conclusion, we demonstrated that systemic administration of ANG II elevates MAP and attenuates the increases in CBF produced by whisker stimulation but not those produced by systemic hypercapnia and by topical application of SNAP or adenosine. The mechanisms of this effect of ANG II are not related to the elevation of MAP, to direct actions on the neural activity evoked by the stimulation, or to effects mediated through CVOs. Rather, the evidence suggests that ANG II interferes with the vascular action of the mediators responsible for the increases in CBF evoked by neural activity. The ANG II-induced attenuation of functional hyperemia could alter the balance between substrate delivery and energy consumption in the working brain and contribute to the deleterious effect of HTN on the central nervous system.
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DISCLOSURES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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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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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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