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Am J Physiol Heart Circ Physiol 279: H2704-H2712, 2000;
0363-6135/00 $5.00
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Vol. 279, Issue 6, H2704-H2712, December 2000

Role of inwardly rectifying K+ channels in K+-induced cerebral vasodilatation in vivo

Sophocles Chrissobolis1, James Ziogas1, Yi Chu2, Frank M. Faraci2, and Christopher G. Sobey1

1 Department of Pharmacology, The University of Melbourne, Parkville, Victoria 3010, Australia; and 2 Departments of Internal Medicine and Pharmacology, Cardiovascular Center, University of Iowa College of Medicine, Iowa City, Iowa 52242


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We tested whether activation of inwardly rectifying K+ (Kir) channels, Na+-K+-ATPase, or nitric oxide synthase (NOS) play a role in K+-induced dilatation of the rat basilar artery in vivo. When cerebrospinal fluid [K+] was elevated from 3 to 5, 10, 15, 20, and 30 mM, a reproducible concentration-dependent vasodilator response was elicited (change in diameter = 9 ± 1, 27 ± 4, 35 ± 4, 43 ± 12, and 47 ± 16%, respectively). Responses to K+ were inhibited by ~50% by the Kir channel inhibitor BaCl2 (30 and 100 µM). In contrast, neither ouabain (1-100 µM, a Na+-K+-ATPase inhibitor) nor NG-nitro-L-arginine (30 µM, a NOS inhibitor) had any effect on K+-induced vasodilatation. These concentrations of K+ also hyperpolarized smooth muscle in isolated segments of basilar artery, and these hyperpolarizations were virtually abolished by 30 µM BaCl2. RT-PCR experiments confirmed the presence of mRNA for Kir2.1 in the basilar artery. Thus K+-induced dilatation of the basilar artery in vivo appears to partly involve hyperpolarization mediated by Kir channel activity and possibly another mechanism that does not involve hyperpolarization, activation of Na+-K+-ATPase, or NOS.

barium; basilar artery; nitric oxide synthase; ouabain; sodium-potassium adenosine triphosphatase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

VASODILATATION INDUCED BY K+ is thought to be important in the cerebral circulation, where release of K+ from activated neurons is proposed to be a major factor linking increases in cerebral metabolism and blood flow (3, 18, 25). Extracellular K+ concentration may increase in the brain during neuronal activity, from ~3 to >10 mM (33), and in this concentration range K+ dilates cerebral arteries (10, 15, 17, 21) and arterioles (2, 14, 19, 22). It is thought that small increases in extracellular K+ stimulate relaxation of vascular muscle primarily through hyperpolarization of the cell membrane via activation of inwardly rectifying potassium (Kir) channels (5, 17, 23), but the importance of this mechanism in the cerebral circulation in vivo is not known. Ba2+ (at <100 µM) is currently the most selective and effective inhibitor of Kir channels (28) and has been reported to inhibit K+-induced vasodilatation in isolated cerebral vessels (15, 17, 20, 21). In vitro data suggest that activity of Kir channels (15, 17) may modulate basal tone of cerebral arteries. Although very high concentrations of Ba2+ (4-20 mM) were reported to constrict cerebral arterioles in anesthetized mice (29), we are not aware of any in vivo study that has examined cerebral vascular effects of Ba2+ in concentrations selective for inhibition of the Kir channel. Although multiple types of Kir channels are known to exist, the Kir2.1 channel is reportedly present in vascular muscle (1), and new evidence using Kir2.1-deficient mice suggests that this K+ channel may be the mediator of dilator responses to K+ in isolated cerebral arteries (37).

Hyperpolarization produced by increased activity of the Na+-K+-ATPase pump has also been suggested to contribute to K+-induced vasodilatation (21). Increased Na+-K+-ATPase activity results in membrane hyperpolarization due to the net movement of three Na+ out of the cell for two K+ entering the cell. The function of the Na+-K+-ATPase can be investigated pharmacologically using ouabain, and this inhibitor has been reported to constrict the basilar artery in vivo (10) and inhibit relaxation of isolated cerebral vessels in response to increases in K+ of <5 mM (21). In addition, nitric oxide (NO) may play a role in increases in cerebral blood flow in response to topical application of K+ (4, 13). Thus the relative importance of Kir channels, Na+-K+-ATPase, and NO synthase (NOS) in cerebral vasodilatation during increases in cerebrospinal fluid (CSF) K+ concentration in vivo is presently unknown.

There were four main goals of the present study. First, we confirmed that K+ dilates the basilar artery in vivo. Second, we investigated the importance of Kir channels, Na+-K+-ATPase, and NOS activity on basilar artery diameter under baseline conditions and on K+-induced vasodilator responses. Third, we tested whether K+ causes hyperpolarization of the basilar artery and, if so, by which mechanism. Fourth, we used RT-PCR to test for the presence of mRNA for the Kir2.1 channel in the basilar artery. The rationale for these latter experiments was on the basis of the observations that mRNA for Kir2.1 (not Kir2.2 or Kir2.3) channels is present in vascular muscle, and that the properties of cloned Kir2.1 are similar to native Kir current (1). In addition, a recent study (37) of Kir2.1-deficient mice suggests that K+-induced hyperpolarization and relaxation is mediated by the Kir2.1 channel. Thus we sought to confirm in our experiments that this channel subtype is expressed in the basilar artery of the rat.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Eighty-seven male Sprague-Dawley rats (250-520 g) were used in the study. The study was approved by The University of Melbourne, Departments of Pharmacology and Physiology Animal Experimentation Ethics Committee, in accordance with the guidelines of the National Health and Medical Research Council of Australia and the University of Iowa Animal Care and Use Committee.

In Vivo Measurement of Basilar Artery Diameter

The rats (n = 70) were anesthetized with pentobarbital sodium (50 mg/kg ip) supplemented with 10-20 mg · kg-1 · h-1 iv. A tracheostomy was performed on the rats, and the animals were mechanically ventilated with room air and supplemental O2. Depth of anesthesia was regularly monitored by applying pressure to a paw. If changes in heart rate or blood pressure were observed, additional anesthetic was administered.

A catheter was placed in a femoral artery to measure systemic arterial pressure and to obtain arterial blood samples. Blood gases were monitored and maintained within normal levels during the experiment (pH = 7.35 ± 0.01; PCO2 = 38 ± 1 and PO2 = 158 ± 3 mmHg). A femoral vein was cannulated for infusion of supplemental anesthetic. Body temperature was maintained at 37-38°C with the use of a heating pad.

A craniotomy was performed over the ventral brain stem, as described in detail previously (6). The cranial window was superfused with artificial CSF at a rate of 3 ml/min using inlet and outlet ports. The artificial CSF, which was bubbled with 5% CO2 in N2 and maintained at 37-38°C, contained the following (in mM): 2.95 KCl, 132 NaCl, 3.69 dextrose, 1.7 CaCl2, 0.64 MgCl2, and 23.2 NaHCO3. The CSF sampled from the cranial window was composed of pH = 7.35 ± 0.01 and PCO2 = 37 ± 1 mmHg, and PO2 = 116 ± 1 mmHg. The basilar artery was visualized with the use of a microscope equipped with a television camera coupled to a video monitor. The diameter was continuously measured using a computer-based tracking program (Diamtrak; Montech, Australia) and recorded on a chart recorder.

In Vivo Experimental Protocol

After preparation of the cranial window, an equilibration period of at least 20 min was allowed before commencing the experimental protocol to ensure that all variables were stable. Vasoactive drugs were superfused over the basilar artery within the CSF in increasing concentrations. No more than three vasodilator drugs were tested in each experiment, and they were applied in random order. The diameter of the basilar artery was recorded under baseline conditions and during application of each drug concentration (2-3 concentrations of each agent), and the steady-state change in diameter, which was usually achieved within 3-5 min, was recorded. A washout period of at least 15 min was allowed between applications of each drug, when the diameter returned to baseline level. When pharmacological inhibitor treatments were used, control responses to vasodilators were first established. The inhibitor was then applied to the vessel for at least 20 min beforehand and then continued during application of vasodilator drugs. The effect of the inhibitor on each vasodilator was determined by comparing the second response with the initial (control) response. No more than one inhibitor treatment was studied per rat. In a group of "time control" rats (n = 7), we verified that responses to each vasodilator are normally reproducible when repeated in the absence of a pharmacological inhibitor. In ~75% of arteries, increasing the concentration of K+ caused rhythmic changes in vessel diameter. When rhythmic activity was elicited in response to K+, the oscillations were regular in nature, and vascular responses were measured at the midpoint between the maximum and the minimum. Data were qualitatively identical whether the midpoint or maximum diameter was considered; however, we deemed that the midpoint was more representative of the magnitude of the response to K+.

Measurement of Membrane Potential in Basilar Artery Smooth Muscle

Isolated basilar artery preparation. The rats (n = 14) were rendered unconscious after inhalation of 80% CO2-20% O2 and were then killed by decapitation. The brain was quickly removed and placed into cold artificial CSF solution. The basilar artery was carefully dissected from the brain and pinned down to the Sylgard base of a 5-ml petri dish and superfused constantly (4 ml/min) with CSF at 37°C. The artery was allowed to equilibrate in the chamber for 30 min before commencement of the experiment.

Electrophysiological measurements. Capillary glass microelectrodes (borosilicate glass capillaries, GC 120-F-10) were made using a Flaming-Brown micropipette puller (model P-87, Sutter Instruments) and backfilled with KCl (0.5 M). Microelectrodes with resistances between 80 and 180 mOmega were used. An Ag/AgCl electrode connected to a headstage (model HS-2, Axon Instruments) was placed in the microelectrode to transmit changes in membrane potential, relative to a reference Ag/Ag-Cl electrode present in the organ bath, to an amplifier (Axoprobe, Axon Instruments). The potentials were amplified (model NL106, Neurolog), filtered (DC-3KHz, model NL125, Neurolog), and observed on an oscilloscope (model BWD845, BWD Instruments). The signal was digitized by an analog-to-digital converter (model TL-1 DMA, Axon Instruments) for recording and computer analysis.

Smooth muscle cells of the basilar artery were impaled with the microelectrodes using a Leitz micromanipulator. A successful electrode impalement was indicated by a rapid fall in membrane potential to approximately -45 mV or lower. Membrane potential was then allowed to stabilize over the next 5-7 min, and only cells that had a stable resting potential lower than -50 mV were used.

In Vitro Experimental Protocol

A 2-min recording of resting membrane potential was made, and K+ (5, 10, or 15 mM) or aprikalim (10 µM) was then perfused over the basilar artery for 5-10 min. A washout period of at least 30 min followed before another concentration was tested. When the effect of Ba2+ on the response to 10 mM K+ or 10 µM aprikalim was being tested, Ba2+ was superfused for at least 10 min and was present during application of the vasodilator.

RT-PCR

Total RNA was extracted from basilar arteries of three rats after the method described in detail previously (11, 12). RNA (0.5-1 µg) was reverse transcribed to produce cDNA using random hexamers as primers. The RT product of 0.1-0.2 µg RNA equivalent was used for the PCR reaction.

The primers for Kir2.1 were the following: sense, forward (nucleotides no. 862-881), 5'-GACAATGCAGACTTTGAAAT-3'; antisense, reverse (nucleotides no. 1171-1188); and 5'-CTCTGGAACTCCGTTCTC-3' (Gene Accession no. L48490 in Genbank). The expected length of the amplification product was 327 base pair. Brain samples were used as a positive control for mRNA of Kir2.1.

Drugs

The vasoactive drugs used in the study were KCl, acetylcholine (ACh), sodium nitroprusside (SNP), barium chloride (BaCl2), ouabain, NG-nitro-L-arginine (L-NNA), cromakalim, and aprikalim. Aprikalim was obtained from Rhône-Poulenc Rorer (Paris, France). All other drugs were obtained from Sigma Chemical (St. Louis, MO). Stock solutions (1 mM) of cromakalim or aprikalim were prepared by dissolving the drug in 50% dimethyl sulfoxide and 50% 0.9% saline. Subsequent dilutions were made in saline. All other drugs were dissolved and diluted in saline.

Data Presentation and Statistics

Changes in basilar artery diameter are expressed as percent change over baseline diameter (means ± SE) and were found to be normally distributed with the use of Prism software (GraphPad Software). Membrane potential changes are expressed as absolute change from the resting value (in mV). Comparisons between single concentrations in control and treatment groups were made with the use of Student's paired or unpaired t-test as appropriate. P < 0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Basilar Artery Diameter in Vivo

Mean arterial pressure was 98 ± 2 mmHg, and baseline diameter of the basilar artery was 180 ± 3 µm (n = 70).

Control vasodilator responses. Figure 1 shows the group data for the concentration-dependent effects of K+ on basilar artery diameter. Increases in K+ from the control level of 3 mM caused marked dilator responses of the basilar artery in vivo. Alternatively, eliminating K+ from the artificial CSF by replacement with Na+ caused a small constriction. Time-control experiments confirmed that vasodilator responses to increased K+ were reproducible within the same animal (Fig. 2A). These effects were specific for K+ and not due to changes in osmolarity because increasing Na+ concentration in the CSF by 12 mM produced only a 3 ± 1% (n = 7) increase in basilar artery diameter. In contrast, increasing K+ by 12 mM caused a 35 ± 4% increase in vessel diameter (n = 53). SNP, ACh, and cromakalim each also elicited concentration-dependent dilatation of the basilar artery (Fig. 3, A-C, respectively).


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  		Fig. 1.   Change in basilar artery diameter in response to K+ (n = 44 for 5 mM; n = 53 for 10 and 15 mM; n = 14 for 20 mM; n = 7 for 30 mM). Decreasing K+ concentration to 0 mM resulted in a mild constriction (n = 4). All values are means ± SE.



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  		Fig. 2.   Data from experiments showing time-control responses to K+ (n = 7) (A), and the effect of 30 µM Ba2+ (n = 10-11) (B) and 100 µM Ba2+ (n = 8) (C) on dilator responses to K+. Baseline diameters: (A) 1st = 167 ± 9 µm, 2nd = 160 ± 16 µm; (B) control = 186 ± 7 µm, Ba2+ (30 µM) treated = 171 ± 9 µm*; and (C) control = 173 ± 10 µm, Ba2+ (100 µM) treated = 154 ± 13 µm*. All values are means ± SE. *P < 0.05 vs. control.



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  		Fig. 3.   Effect of Ba2+ (30 µM) on vasodilator responses to sodium nitroprusside (SNP) (n = 9) (A), ACh (n = 6) (B), and cromakalim (n = 7) (C). Baseline diameters: control = 184 ± 9 µm, Ba2+ treated = 167 ± 10 µm* (A); control = 187 ± 5 µm, Ba2+ treated = 175 ± 12 µm (B); control = 182 ± 10 µm, Ba2+ treated = 157 ± 11 µm* (C). All values are means ± SE. *P < 0.05 vs. control.

Effect of Ba2+ on vasodilator responses. BaCl2 (30 and 100 µM) caused constriction of the basilar artery, which tended to be concentration dependent (30 µM: baseline = 177 ± 5 µm, % change = -6 ± 2%, n = 19, 100 µM: baseline = 167 ± 5 µm, Delta  = -8 ± 1%, n = 13). At 30 µM, a concentration considered selective for inhibition of the Kir channel (23, 27), Ba2+ inhibited vasodilator responses to K+ by 40-50% (Fig. 2B). A higher Ba2+ concentration (100 µM) had no additional inhibitory effect on vasodilator responses to K+ (Fig. 2C). Vasodilator responses to SNP (Fig. 3A), ACh (Fig. 3B), and cromakalim (Fig. 3C) were unaffected by 30 µM Ba2+, indicating that its inhibitory effect was selective for K+-induced vasodilator responses.

Effect of ouabain on vasodilator responses. Ouabain (1-100 µM) caused constriction of the basilar artery, which tended to be concentration dependent (1 µM: baseline = 163 ± 6 µm, Delta  = -5 ± 2%, n = 7, 10 µM: baseline = 169 ± 5 µm, Delta  = -8 ± 2%, n = 4, 100 µM: baseline = 169 ± 5 µm, Delta  = -9 ± 2%, n = 4). Ouabain (1 µM - n = 3, data not shown; and 100 µM) had no inhibitory effect on vasodilator responses to either K+ (Fig. 4) or SNP (n = 4, data not shown).


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  		Fig. 4.   Effect of ouabain (Ouab, 100 µM) on vasodilator responses to K+ (n = 4). Baseline diameters: control = 179 ± 4 µm, ouabain treated = 159 ± 5 µm. All values are means ± SE.

Effect of combined treatment with Ba2+ and ouabain. Application of BaCl2 (30 µM) together with ouabain (1 µM) decreased diameter of the basilar artery in an additive manner (baseline = 177 ± 1 µm, Delta  = -18 ± 2%, n = 7). This treatment also inhibited vasodilator responses to K+ (Fig. 5A) to a similar degree as did BaCl2 alone (Fig. 2B) and had no effect on responses to SNP (Fig. 5B).


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  		Fig. 5.   Combined effect of Ba2+ (30 µM) and ouabain (1 µM) on vasodilator responses to K+ (n = 7) (A) and SNP (n = 7) (B). Baseline diameters: control = 175 ± 8 µm, Ba2+ + ouabain treated = 143 ±7 µm* (A); control = 173 ± 9 µm, Ba2+ + ouabain treated = 144 ± 6 µm* (B). All values are means ± SE. *P < 0.05 vs. control.

Effect of L-NNA on vasodilator responses. Treatment with L-NNA (30 µM) decreased diameter of the basilar artery (baseline = 185 ± 8 µm, Delta  = -15 ± 1%, n = 6). Vasodilator responses to K+ were not affected by L-NNA (Fig. 6A), whereas responses to ACh were abolished (Fig. 6B), indicating that L-NNA was effective in preventing NO production in endothelium. With the use of Prism software (GraphPad), statistical power calculations indicated that it is very unlikely that the effect of L-NNA on responses to K+ would have reached statistical significance after additional experiments.


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  		Fig. 6.   Effect of NG-nitro-L-arginine (L-NNA, 30 µM) on vasodilator responses to K+ (n = 6) (A) and ACh (n = 6) (B). Baseline diameters: control = 188 ± 11 µm, L-NNA treated = 157 ± 8 µm* (A); control = 182 ± 5 µm, L-NNA treated = 129 ± 11 µm* (B). All values are means ± SE. *P < 0.05 vs. control.

Basilar Artery Membrane Potential

Effect of K+ on membrane potential. Resting membrane potential was -57 ± 2 mV under control conditions (n = 21 cells, 14 arteries). K+ (5-15 mM) caused marked hyperpolarization of the basilar artery (range = 6-24 mV) (Figs. 7A and 8A). The K+-induced decrease in membrane potential consisted of an initial maximum response, which decayed to a lower steady-state response after 3-5 min (Figs. 7A and 8A). The maximum response was concentration dependent, whereas the steady-state response was similar at all concentrations tested (Fig. 8A).


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  		Fig. 7.   Recordings of changes in membrane potential (Em) of the basilar artery in response to 10 mM K+ in the absence (A) and presence (B) of 30 µM Ba2+.



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  		Fig. 8.   Changes in Em of the basilar artery in response to K+ in the absence (A) and presence (B) of 30 µM Ba2+. A: n = 4-7 for maximum values and n = 3-7 for steady-state values. Em at baseline was -57 ± 2 mV (n = 17 cells, 9 arteries). B: baseline membrane potentials were control = -52 ± 3 mV, n = 7 for both maximum and steady state (the same data as in A), Ba2+-treated = -56 ± 3 mV, n = 4, for both maximum and steady state. All values are means ± SE. *P < 0.05 vs. control response.

Effect of Ba2+ on K+-induced hyperpolarization. Treatment with Ba2+ (30 µM) caused depolarization (6 ± 2 mV, n = 4) of the basilar artery (Fig. 7B). Ba2+ virtually abolished both maximum and steady-state phases of the hyperpolarization response caused by K+. Data for 10 mM K+ are shown in Figs. 7B and 8B.

Effect of Ba2+ on aprikalim-induced hyperpolarization. The activator of ATP-sensitive K+ channels, aprikalim (10 µM), caused a 20 ± 3 mV hyperpolarization of the basilar artery under control conditions (n = 3). Aprikalim caused a similar 20 ± 1 mV hyperpolarization in the presence of 30 µM Ba2+ (n = 4), indicating that Ba2+ did not inhibit vascular hyperpolarization in a nonselective manner.

RT-PCR

PCR products corresponding to mRNA for Kir2.1 were present in the basilar artery, aorta, and brain (Fig. 9). Thus data confirming expression of Kir2.1 in the basilar artery and the inhibitory effects of Ba2+ on K+-induced vasodilator and hyperpolarization responses are consistent with a role for Kir channels in the functional effects of K+ in the cerebral circulation.


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  		Fig. 9.   Agarose gel with RT-PCR products corresponding to in vivo expression of the Kir2.1 channel in cerebral arteries. Lane 1, size markers; lane 2, negative control. Primers for rat Kir2.1 produce positive amplification products of 327 base pairs in rat basilar artery (lane 3), rat aorta (lane 4), and rat brain (lane 5, positive control). No bands were detected if reverse transcription was omitted (data not shown). This result was reproduced in another 2 experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

There are several findings of the present study. First, small increases in the CSF concentration of K+ (of <30 mM) caused up to ~50% dilatation of the basilar artery, confirming that K+ powerfully dilates this artery in vivo (10). Second, this response to K+ was selectively inhibited by 30 µM Ba2+, which, at this concentration, is considered a selective inhibitor of the Kir channel (23, 27). By contrast, neither ouabain nor L-NNA had a significant effect on K+-induced vasodilatation, suggesting that neither the Na+-K+-ATPase nor NOS, respectively, are involved in K+-induced cerebral vasodilatation. Third, BaCl2, ouabain, and L-NNA each caused concentration-dependent constriction of the basilar artery, suggesting that activity of Kir channels, Na+-K+-ATPase, and NOS normally contribute to regulation of basilar artery diameter. Fourth, K+ caused marked hyperpolarization of the isolated basilar artery. Ba2+ abolished this hyperpolarization, suggesting that Kir channels exclusively mediate K+-induced cerebral vascular hyperpolarization. Because only about one-half of the vasodilator response to K+ was inhibited by the same concentration of Ba2+ (30 µM) that abolished K+-induced hyperpolarization, an additional mechanism seems likely to contribute to the vasodilatation in vivo. Fifth, RT-PCR experiments confirmed the presence of mRNA for Kir2.1 in the basilar artery, consistent with this subunit being functional in cerebral arteries (1, 37).

Effect of Ba2+ on K+-induced Dilatation of Cerebral Arteries

Topical application of low concentrations of K+ to the basilar artery elicited substantial increases in diameter, consistent with previous reports of K+-induced relaxation of isolated large cerebral arteries (15, 17, 21, 26, 35) and dilatation of cerebral arteries (10) and arterioles (2, 4, 19, 22) in vivo. Constriction and depolarization of the basilar artery occurred during treatment with Ba2+, suggesting that Kir channel activity normally modulates vessel tone. An influence of Kir channel activity on resting cerebral artery diameter is further evidence for the importance of K+ channels in regulation of cerebrovascular tone, because inhibitors of the voltage-dependent (31) and calcium-activated K+ channels (16, 24, 30) also cause basilar artery constriction in vivo. Whereas constriction of the middle cerebral artery in response to 40 µM Ba2+ has been reported in vitro (15), the present data are the first evidence that Kir channel activity influences resting cerebral vascular tone in vivo.

Activation of the Kir channel and conduction of an outward K+ current in response to small increases in extracellular K+ is thought to occur because of unique gating properties of Kir channels (23, 28). Larger increases in K+ (by >30 mM) cause smooth muscle depolarization and subsequent constriction of both cerebral and noncerebral arteries due to marked membrane depolarization and Ca2+ entry via voltage-operated Ca2+ channels (28). Recent findings in isolated cerebral arteries suggested that the Kir channel is indeed involved in mediating cerebral smooth muscle hyperpolarization and vasorelaxation in response to K+ (15, 17, 20, 21, 26).

This is the first study to provide evidence that K+-induced cerebral vasodilatation in vivo is Ba2+ sensitive, and this response is thus likely to be at least partly mediated by activation of Kir channels. Previous studies in vitro have shown that 30-50 µM Ba2+ can selectively abolish smooth muscle relaxation in response to K+, whereas selective inhibitors of other K+ channel types do not inhibit K+-induced relaxation (15, 17, 21), suggesting that these concentrations of Ba2+ are sufficient to fully inhibit Kir channels and that K+ is likely to activate only this type of K+ channel.

Interestingly, only partial (~50%) inhibition of K+-induced vasodilatation was produced by Ba2+ in this study, in contrast to findings from some in vitro studies in which Ba2+ was reported to abolish K+-induced cerebral vasorelaxation (15, 17, 21, 26, 37). The discrepancy in the sensitivity to Ba2+ of the K+-induced vasodilatation versus hyperpolarization could be related to the in vivo versus in vitro approaches. Additional factors, including the presence of blood flow and the stimulation of paracrine signals from nearby parenchymal tissues, could potentially have an influence. Thus it is possible that additional mechanisms are involved in K+-induced vascular responses in vivo. Although several previous studies have been performed using a basal concentration of 5 to 6 mM K+ (15, 17, 26), rather than ~3 mM K+, which is more representative of normal levels in CSF (33), this would not seem to account for the apparent differences in in vivo and in vitro mechanisms observed in this study, because we used 2.95 mM K+ in both preparations. Given that 100 µM Ba2+ caused no greater inhibition of vasodilatation than did 30 µM Ba2+, and that 30 µM Ba2+ completely inhibited the hyperpolarization, we speculate that Kir channel activation may be only one of two or more mechanisms contributing to the in vivo vasodilator response to K+. When administered in higher concentrations (typically >100 µM), Ba2+ can also inhibit ATP-sensitive K+ channels (23). However, 30 µM Ba2+ had no effect on either cromakalim-induced vasodilatation or on aprikalim-induced hyperpolarization in this study, indicating that Ba2+ did not nonspecifically inhibit ATP-sensitive K+ channels.

Expression of mRNA for Kir2.1 Channel in Basilar Artery

Very little molecular data has so far been reported on expression of K+ channels, including Kir channels, in cerebral vessels. A recent study (32) reported immunohistochemical evidence that three Kir channel subtypes (Kir2.1, Kir2.2, and Kir2.3) are expressed in the rat middle cerebral artery. In contrast, Bradley et al. (1) have reported that mRNA for the Kir2.1, not Kir2.2 or Kir2.3, channel is expressed in several vessels, including cerebral arteries. A recent study by Zaritsky et al. (37) suggested that K+-induced hyperpolarization and relaxation in response to K+ is absent in cerebral arteries isolated from Kir2.1-deficient mice but normal in Kir2.2-deficient mice. With the use of RT-PCR, we also confirmed the presence of mRNA for the Kir2.1 channel in the basilar artery. Thus, these molecular data together with our in vivo and in vitro data, which shows Ba2+-sensitive dilatation and hyperpolarization in response to K+, suggests that Kir channels are functionally important in the basilar artery. The findings are consistent with previous data implicating the Kir2.1 channel in mediating cerebral hyperpolarization and vasorelaxant responses to K+ (1, 37). Nevertheless, our findings do not prove that this channel subtype is responsible for the K+-induced responses observed, and further work will be necessary to confirm the functional importance of Kir channel subtypes in the basilar artery in vivo.

Effect of Ouabain on K+-induced Dilatation of Cerebral Arteries

In addition to the Kir channel, Na+-K+-ATPase has been implicated in K+-induced cerebral vasodilatation, especially in response to the lowest concentrations of K+ (21). Increased activation of this pump by K+ is thought to result initially in a large net extrusion of positive charge, as Na+, and consequently transient vascular smooth muscle hyperpolarization. However, we found that up to 100 µM ouabain had no effect on K+-induced vasodilatation, suggesting no involvement of Na+-K+-ATPase in these responses. The concentrations of ouabain used here (1-100 µM) are likely to be sufficient to markedly inhibit the Na+-K+-ATPase in the rat basilar artery because ouabain inhibits Na+-K+-ATPase activity in the rat aorta with an IC50 of 50 nM (8). Application of ouabain produced vasoconstriction, as expected (10, 34), also suggesting effective inhibition of Na+-K+-ATPase. There may be regional differences in the role of Na+-K+-ATPase in the cerebral circulation because 100 µM ouabain has been reported to inhibit relaxation of the rat isolated posterior cerebral artery in response to increases of <5 mM K+ (21). However, up to 1 mM ouabain had no effect on K+-induced relaxation in the middle cerebral artery of the rat (15). The complete block of K+-induced hyperpolarization by Ba2+ observed in the present study is further evidence against any such role for Na+-K+-ATPase activity in the basilar artery. Overall, our data suggest no important role for increased Na+-K+-ATPase activity in K+-induced cerebral vasodilatation in vivo.

Evidence Against a Role for NO in K+-induced Dilatation of Basilar Artery

NO release from endothelial cells and/or neurons was considered to be a candidate mechanism of K+-induced vasodilatation, on the basis of findings from a previous study (4) in which increases in cortical perfusion in response to topical application of K+ were attenuated by a NOS inhibitor. In contrast, we found that inhibition of NOS by L-NNA, which was sufficient to abolish ACh-induced vasodilatation, had no effect on K+-induced increases in basilar artery diameter. Thus it is unlikely that NO played an important role in mediating responses to K+. We considered the possibility that Kir channels are on endothelial cells of the basilar artery. If this then is the case, our data suggest that endothelial Kir channel activation by extracellular K+ does not produce significant release of endothelial-derived NO, as might be predicted after endothelial cell hyperpolarization (36). Our data are therefore consistent with findings from previous studies (15, 17, 21) that reported K+-induced vasodilatation to be endothelium independent. A major aim of future studies will be to attempt to identify the Ba2+-insensitive component of the vasodilator response to K+.

Effect of K+ on Membrane Potential of Basilar Artery

Small changes in membrane potential (<5 mV) under basal conditions are known to cause significant changes (20-40%) in cerebral artery diameter (7). We observed marked dilatation of the basilar artery in response to 5-15 mM K+ in vivo and large hyperpolarizations (6-24 mV) in vitro. We cannot be certain of the magnitude of the hyperpolarization in vivo, because we studied membrane hyperpolarization in quiescent arteries in vitro in which resting potential was probably more negative than under in vivo conditions where there is normal myogenic tone (7). We are not aware of any laboratory that measures membrane potential of cerebral arteries in vivo. Nevertheless, the marked changes in vascular membrane potential recorded in response to K+ in vitro suggest that the effect of K+ on membrane potential in vivo was also likely to be substantial.

K+-induced vascular relaxation is thought to be mediated by hyperpolarization of the smooth muscle membrane (23). In the present study, hyperpolarization in response to K+ in vitro was virtually abolished by Ba2+ (30 µM), as has been reported previously (17). This finding strongly suggests that Kir channels exclusively mediate K+-induced smooth muscle hyperpolarization of the basilar artery, but because Ba2+ (30-100 µM) did not completely abolish K+-induced vasodilatation in vivo, we speculate that another mechanism, independent of smooth muscle hyperpolarization, also contributes to the vasodilator response to K+.

Importantly, both the electrophysiological approach (in vitro) and the vessel diameter approach (in vivo) yielded the following consistent findings: 1) both K+ and ATP-sensitive K+ channel openers (aprikalim and cromakalim) caused profound vasodilatation and hyperpolarization, 2) Ba2+ caused vasoconstriction and depolarization, and 3) Ba2+ selectively inhibited responses to K+ but not to ATP-sensitive K+ channel openers. Thus measurements of membrane potential in quiescent vessels in vitro appear to be informative for interpreting complementary pharmacological data obtained in vivo. We suggest that when in vivo studies are not included, the use of pressurized vessels may become more important for predicting the physiological relevance of data from electrophysiological experiments.

We have used pentobarbital anesthesia to perform the present study. In studies of cerebral circulation, pentobarbital (or other related barbiturates) is used very commonly. In a study that compared responses of cerebral vessels to K+ with different anesthetics, it was found that dilatation to low concentrations of K+ was similar with pentobarbital and alpha -chloralose (9). These findings suggest that pentobarbital is not having a selective inhibitory effect on responses of cerebral vessels to K+ in vivo.

In conclusion, the results of this study demonstrate that K+ elicits marked dilatation of the basilar artery in vivo. This effect is, in part, Ba2+ sensitive and therefore likely to be mediated by a Kir channel. Kir2.1 was found to be expressed in this artery. K+-induced vascular hyperpolarization measured in vitro was abolished by Ba2+ and may contribute to the vasodilator response measured in vivo. However, the data may also suggest evidence for an unidentified Ba2+-insensistive mechanism(s), probably independent of hyperpolarization, which also contributes to K+-induced cerebral vasodilatation in vivo.


    ACKNOWLEDGEMENTS

We thank Lilly Quan for technical assistance.


    FOOTNOTES

This work was supported by a Project Grant from the National Health and Medical Research Council of Australia and National Institutes of Health Grants HL-38901 and NS-24621.

Address for reprint requests and other correspondence: C. G. Sobey, Dept. Pharmacology, Univ. of Melbourne, Parkville, Victoria 3010, Australia (E-mail: c.sobey{at}pharmacology.unimelb.edu.au).

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.

Received 13 April 2000; accepted in final form 11 July 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 279(6):H2704-H2712
0363-6135/00 $5.00 Copyright © 2000 the American Physiological Society


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