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Departments of *Anesthesiology and Forensic Medicine, Wakayama Medical University, Wakayama, Japan
Address correspondence and reprint requests to Hiroyuki Kinoshita, MD, PhD, Department of Anesthesiology, Wakayama Medical University, 811-1 Kimiidera, Wakayama 641-0012, Japan. Address e-mail to hkinoshi{at}pd5.so-net.ne.jp.
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Abstract |
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(5 x 10–7 M), the arterioles within brain slices were evaluated using computer-assisted microscopy. Potassium chloride (KCl) induced vasodilation in cerebral arterioles from normotensive (5–10 mM) and hypertensive (5–15 mM) rats, whereas an inward rectifier K+ channel antagonist barium chloride (BaCl2; 10–5 M) completely abolished the vasodilation in both strains. In arterioles of hypertensive rats, vasodilator responses to KCl were augmented compared with those in normotensive rats. In contrast, the vasodilator responses induced by sodium nitroprusside (3 x 10–8 to 3 x 10–6 M) in these two strains were similar. These results suggest that in cerebral cortex parenchymal microvessels, inward rectifier K+ channels play a crucial role in vasodilation produced by extracellular K+ and that the dilation of cerebral arterioles via these channels is augmented in chronic hypertension. |
Introduction |
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Chronic hypertension causes both smooth muscle and endothelial malfunctions in the vasculature (7) and, therefore, is a major risk factor for all stroke subtypes, including cerebral infarction and cerebral hemorrhage (8). Importantly, cerebral microvessels are targeted in chronic hypertension, resulting in hypertensive angiopathy (6). Therefore, it is crucial to note possible modification of cerebral microvascular function in this pathophysiological condition. However, in chronic hypertension, vasodilation mediated by inward rectifier K+ channels has not been assessed in cerebral microvessels. In addition, it is unclear, even in larger cerebral arteries, whether inward rectifier K+ channels play a major role in vasodilation produced by K+ in this condition (9).
The present study was designed to examine whether increased levels of extracellular K+ induce vasodilator responses in cerebral parenchymal arterioles in hypertensive (spontaneously hypertensive [SHR]) and normotensive (Wistar-Kyoto [WKY]) rats and whether the roles of inward rectifier K+ channels in the vasodilation of these two strains are different.
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Methods |
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Brains were cut freehand into blocks containing the neocortex, followed by immediate sectioning into slices (150-µm thick) with a mechanical tissue slicer (Vibratomes 1000; Ted Pella. Inc., Redding, California). Throughout the slicing procedure, brain blocks were continuously bathed in the control solution bubbled with 93% O2 + 7% CO2 at 4°C. Individual slices were then transferred to a recording chamber filled with control solution, which was mounted on an inverted microscope (Olympus IX70; Olympus, Tokyo, Japan). The recording system consisted of a recording chamber (3 mL) and a tubing compartment (7 mL), including the perfusion chamber. The slices were continuously superfused with control solution at the flow rate of 1.5 mL/min, bubbled with 93% O2 + 7% CO2 (Pco 2 = 40 mm Hg; pH = 7.4; 37°C). An intraparenchymal arteriole (5.0- to10.0-µm internal diameter and 11.5- to 21.0-µm external diameter) was located within the neuronal tissue, and its internal diameter was continuously monitored by live computerized videomicroscopy (10). The videomicroscopy equipment consisted of an inverted microscope, a 40x objective (Olympus), and a 2.25x video projection lens (Olympus). The image of a parenchymal arteriole was transmitted to video camera (C6790-81; Olympus) and displayed on a computer via a media converter (Physio-Tech, Tokyo, Japan).
The differentiation between the arteriole and the venule in the brain is based on previous studies documenting that, in the brain, one or several layers of smooth muscle cells should be identified in the arteriole, and the venule resembles a large capillary with no more in its wall than endothelial cells resting on a basal lamina (11,12). In some studies, this was confirmed by hematoxylin-eosin stain of the slice after each experiment (10). Moreover, immunohistochemical analysis was performed, as previously described (13). Briefly, brain samples were fixed in 4% formaldehyde buffered with phosphate-buffered saline (pH value 7.2), followed by making paraffin-embedded sections (4-µm thick). After deparaffinization, sections were incubated with mouse anti--smooth muscle actin monoclonal antibody (Sigma Aldrich Inc., St Louis, Missouri) at a concentration of 10 µg/mL. After incubation with horse-radish peroxidase-conjugated goat anti-mouse immunoglobulin G antibody diluted 1:200 (ZYMED Inc., San Francisco, California), the immunocomplex was visualized by the HistoMark®TrueBlue TM peroxidase system (KPL Inc., Gaitherberg, Maryland), according to the manufacturer’s recommendations.
We calculated the ratio of internal to external vessel diameters. A vessel with a ratio <0.5 was used as an arteriole for the following experiments. We defined the internal diameter as the length between the internal margins of arteriolar walls. Changes of internal diameter in cerebral microvessels were recorded on computer image files and then analyzed using the image analysis software with the sensitivity to 0.01 µm (Physio-Tech) (10). Microvessel diameters were derived as an average of four measurements taken along approximately 20 µm of vessel length.
Each slice was equilibrated for at least 30 min before the start of the experimental protocols. All experiments were performed during constriction in response to prostaglandin F2 (5 x 10–7 M). We have found that this concentration of prostaglandin F2 produces approximately 70% vasoconstriction compared with maximal contraction induced by prostaglandin F2 (10–5 M) in cerebral parenchymal arterioles (10). Barium chloride (BaCl2; 10–5 M) was applied 15 min before the addition of prostaglandin F2 (5 x 10–7 M). Concentration-responses to KCl (5–15 mM), levcromakalim (3 x 10–8 to 3 x 10–7 M), or sodium nitroprusside (3 x 10–8 to 3 x 10–6 M) were obtained. Concentration responses were obtained cumulatively by adding a vasoconstrictor or a vasodilator substance into the bubbling chamber connected to, but separated from, the recording chamber. Only one concentration response was made for each slice. The duration of experiment for each slice was within 3 h because we have shown that 3 h after the preparation of brain slice, the vasodilator function mediated by endothelial or neuronal nitric oxide synthase seems to be intact in our experimental condition (10). The amount of dilation of the cerebral arteriole induced by vasodilators was normalized using the constriction produced by prostaglandin F2 (5 x 10–7 M) in each arteriole. Therefore, the percent dilation was calculated by the following equation: % dilation = 100 x (Ddilator – DPGF)/(Dcontrol – DPGF), % constriction to prostaglandin F2 = 100 x (DPGF – Dcontrol)/Dcontrol. Dcontrol, DPGF, and Ddilator were the arteriolar diameters of control condition after administration of prostaglandin F2 (5 x 10–7 M) or the vasodilator, respectively.
The following pharmacological drugs were used in our study. BaCl2, prostaglandin F2, and sodium nitroprusside were all obtained from (Sigma Aldrich Inc.). Levcromakalim was a generous gift from GlaxoSmithKline plc (Greenford, United Kingdom). Drugs were dissolved in distilled water such that volumes of < 60 µL were added to the perfusion system. The stock solution of levcromakalim (10–5 M) was prepared in dimethyl sulfoxide (3 x 10–4 M). The concentrations of drugs are expressed as final molar concentration.
The data were expressed as mean ± sd; n refers to the number of rats from which the brain slice was taken. Statistical analysis was performed using repeated-measures of analysis of variance, followed by Student-Newman-Keuls test as a post hoc analysis. Differences were considered to be statistically significant when P < 0.05.
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Results |
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Figure 2 demonstrates immunohistochemical analysis of an arteriolar smooth muscle layer in the brain slice. The arteriole demonstrates a smooth muscle layer (stained with black) as well as an endothelial cell layer (without stain). Importantly, the venule, which has similar external diameter to this arteriole, is without a smooth muscle layer but with an endothelial layer. These results confirmed anatomical differences between arterioles and venules in the brain, which were previously reported (11,12).
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During submaximal constriction in response to prostaglandin F2 (5 x 10–7 M), the addition of KCl (5–15 mM for SHR and 5–10 mM for WKY rats) produced vasodilation in the cerebral parenchymal arterioles, whereas the inward rectifier K+ channel antagonist BaCl2 (10–5 M) completely abolished the vasodilation in both strains (Fig. 3A). In the arterioles of SHR, vasodilator response to KCl (10 and 15 mM) was significantly augmented compared with that in normotensive rat arterioles. In the normotensive rat arterioles, KCl (15 mM) instead produced vasoconstrictor effects, resulting in only minimal vasodilation compared with the control condition (Fig. 3A).
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In arterioles contracted with prostaglandin F2 (5 x 10–7 M), a nitric oxide donor, sodium nitroprusside (3 x 10–8 to 3 x 10–6 M), induced vasodilation in a concentration-dependent fashion (Fig. 3B). No significant difference was noted between SHR and WKY rats in vasodilation produced by sodium nitroprusside.
BaCl2 (10–5 M) did not alter vasodilation induced by a selective adenosine triphosphate (ATP)-sensitive K+ channel opener levcromakalim (3 x 10–8 to 3 x 10–7 M) in arterioles contracted with prostaglandin F2 (5 x 10–7 M) (Fig. 3C).
Control internal and external diameters of cerebral parenchymal arterioles did not differ between WKY rats and SHR (internal diameters, 6.4 ± 0.8 µm for WKY rats; n = 10; or 6.9 ± 1.1 µm for SHR; n = 10; external diameters, 15.3 ± 2.6 µm for WKY rats; n = 10; or 14.8 ± 2.4 µm for SHR; n = 10; respectively).
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Discussion |
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Previous studies documented variable results of vasodilator responses to KCl in rat cerebral arterioles of the middle cerebral arterial branch (approximately 35–45 µm in diameters). KCl from 5 to 30 mM is capable of inducing vasodilation (15), and extracellular K+ (5 mM) induces marked vasodilation, whereas that of 10 mM produces minimal vasodilation (16). Our recent study of the cerebral parenchymal arterioles (5- to 10-µm internal diameter) from WKY rats demonstrated vasodilator responses produced by the addition of KCl (5–10 mM) (17). Methodological (pressurized or nonpressurized setting and existence of perfusion) and regional or size differences among the above studies may contribute to the diverse vasodilator responses of cerebral arterioles to extracellular K+ in normotensive rats.
In the current study, the addition of KCl (10 and 15 mM) produced a larger vasodilation of cerebral parenchymal arterioles in SHR. In SHR arterioles, KCl (15 mM) was capable of producing approximately 60% vasodilation, whereas it did not produce any vasodilation in normotensive rats. The augmented cerebral vasodilation seen in SHR was completely abolished by BaCl2, indicating that inward rectifier K+ channels solely contribute to this increased component of vasodilation (14). An in vivo study has documented that K+ (5 mM, but not 10 mM) produces limited augmentation of vasodilation in the basilar artery of SHR, and this increased dilation is independent of inward rectifier K+ channels (9). In the posterior cerebral artery (approximately 184-µm diameter) from the stroke-prone SHR, vasodilation induced by increased concentrations of K+ from 7 to 15 mM was virtually abolished (18). These findings suggest that the vessel size itself may be one of the determinants of differential cerebral vasodilator responses via inward rectifier K+ channels in SHR. Changes in the resting membrane potential of the vascular smooth muscle cell have an impact on the rectification through inward rectifier K+ channels (14). In SHR, the augmented activity of voltage-dependent Ca2+ channels results in the increase of the resting membrane potential of the vascular smooth muscle cell (19). Although in some specific conditions of chronic hypertension, roles of voltage-dependent Ca2+ channel and K+ channel conductance in the determination of the resting membrane potential of cerebral vascular smooth muscle cells seem to be rather limited (7), the increased membrane potential of smooth muscle cells may also cause the augmented vasodilation in response to increased levels of extracellular K+ in SHR. Several studies have demonstrated that Kir2.1 gene expression in arterial smooth muscle is required for inward rectifier K+ channel currents and K+-induced dilations in cerebral arteries (20). In basilar arteries from SHR and normotensive rats, the subtype of inward rectifier K+ channel (Kir2.1) is similarly expressed, indicating that the augmentation of cerebral vasodilation in SHR is not due to the increased expression of inward rectifier K+ channels in chronic hypertension (9). Indeed, we cannot exclude the possibility that the sensitivity of inward rectifier K+ channels, which apparently contribute to levels of the vascular smooth muscle membrane potential, may produce the differential vascular responses between normotensive rats and SHR (14).
Several previous studies in the brain demonstrated that increased extracellular K+ is capable of inducing ouabain-sensitive and BaCl2-insensitive vasodilation, indicating involvement of Na+/K+ ATPase in this vasodilation (15,16). In contrast to studies demonstrating that BaCl2-sensitive and ouabain-insensitive vasodilation produced by extracellular K+ (2,3,9), the above two studies were performed using propane sulfonic acid-buffered saline as artificial cerebrospinal fluid (15,16). Therefore, such methodological difference may at least partly play a role in the differential results regarding the effects of ouabain on vasodilation induced by extracellular K+ in normotensive rats. More importantly, in the basilar artery, ouabain does not alter vasodilation produced by KCl, suggesting a negative role of Na+/K+ ATPase in K+-induced cerebral vasodilation in chronic hypertension (9).
The nitric oxide donor sodium nitroprusside produced similar vasodilation of cerebral parenchymal arterioles from SHR and normotensive rats. Vasodilator responses of larger cerebral arteries to sodium nitroprusside are not modulated by chronic hypertension, suggesting that this pathophysiological condition may not alter the sensitivity of cerebral vascular smooth muscle cells in response to nitric oxide (9). In view of our results regarding vasodilation induced by KCl, it is unlikely that augmented cerebral vasodilation produced by K+ via inward rectifier K+ channels in chronic hypertension is caused by nonselective changes in the dilator properties of vascular smooth muscle cells.
We have recently introduced a system that is capable of evaluating precapillary arterioles with an internal diameter of <10 µm in brain parenchyma using computer-assisted microscopy (10,17). Importantly, the responses of parenchymal arterioles in our preparation to vasoactive substances were similar to those seen in pressurized cerebral arterioles (10,17). The parenchymal arterioles of the brain slice exist in the intact tissues composed of neurons and astrocytes attached to the arteriole, indicating that the circumstance of arterioles in the current study seems to be closer to that seen in the in vivo condition, as opposed to the condition of the isolated cerebral artery (21). However, parenchymal arterioles in our experimental condition were not perfused and, thus, their reactivity to the vasodilator and vasoconstrictor substances may have been altered as compared with the in vivo vasomotor reactivity. Therefore, it may be difficult to directly extrapolate our results to the in vivo condition. The current study indicates that in cerebral microvessels, inward rectifier K+ channels of cerebral vascular smooth muscle cells play an important role in vasodilation produced by extracellular K+ and that dilation of cerebral microvasculature via these channels is augmented during chronic hypertension. However, one cannot completely exclude the possibility that extracellular K+ may act via neuronal cells on eliciting cerebral arteriolar vasodilation because previous studies demonstrated that inward rectifier K+ channels are expressed in neurons and glial cells (22,23).
In the cerebral circulation, where extracellular K+ increases as a consequence of neuronal activity, K+-induced vasodilation is regarded as one of the important mechanisms linking increased metabolic demand with increased cerebral blood flow (24). Indeed, in the brain, extracellular K+, which is normally 3–5 mM, increases to 10–12 mM when neurons are activated (24). Also, it is crucial to note that extracellular K+ in the cerebral vasculature markedly increases during stresses such as cerebral hypoxia, ischemia, and hypoglycemia (25–27). Therefore, K+ concentrations in the present study seem to be appropriate to evaluate cerebral vasodilation via inward rectifier K+ channels. Our results suggest that increased vasodilation produced by extracellular K+ during chronic hypertension should be beneficial to preserve cerebral blood flow in this diseased state. We speculate that in brain parenchyma, the augmented vasodilation via inward rectifier K+ channels may counteract the tendency towards increase in cerebral vascular tone during chronic hypertension, although future in vivo studies are warranted to verify this hypothesis. The current study of cerebral microvessels may contribute to understanding the adaptive mechanisms required to maintain cerebral perfusion in patients with chronic hypertension.
The authors thank Nobuyuki Kakimoto, Department of Forensic Medicine, Wakayama Medical University for helping with the immunohistochemical analysis in this study.
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Footnotes |
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Supported, in part, by Grant-in-Aid, 16390458 (H.K.), 16659426 (H.K.), 14770786 (Y.K.), 15790829 (M.D.), 16591558 (K.M.), 16591557 (K.O.), and 13470327 (Y.H.) for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan, Tokyo Japan, and 11-7 for Medical Research from Wakayama prefecture, Wakayama, Japan (H.K.).
Accepted for publication September 20, 2005.
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References |
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–smooth muscle actin: a new probe for smooth muscle differentiation. J Cell Biol 1986;103:2787–96.This article has been cited by other articles:
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