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Center for Perinatal Biology, Departments of Physiology/Pharmacology and Obstetrics and Gynecology, School of Medicine, Loma Linda University, Loma Linda, California 92350
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The present study was designed to test the
hypothesis that in cerebral arteries of the fetus, ATP-sensitive
(KATP) and Ca2+-activated K+
channels (KCa) play an important role in the regulation of
intracellular Ca2+ concentration
([Ca2+]i) and that this differs significantly
from that of the adult. In main branch middle cerebral arteries (MCA)
from near-term fetal (~140 days) and nonpregnant adult sheep,
simultaneously we measured norepinephrine (NE)-induced responses of
vascular tension and [Ca2+]i in the absence
and presence of selective K+-channel openers/blockers. In
fetal MCA, in a dose-dependent manner, both the
KATP-channel opener pinacidil and the
KCa-channel opener NS 1619 significantly inhibited
NE-induced tension [negative logarithm of the half-maximal inhibitory
concentration (pIC50) = 5.0 ± 0.1 and 8.2 ± 0.1, respectively], with a modest decrease of
[Ca2+]i. In the adult MCA, in contrast, both
pinacidil and NS 1619 produced a significant tension decrease
(pIC50 = 5.1 ± 0.1 and 7.6 ± 0.1, respectively) with no change in [Ca2+]i.
In addition, the KCa-channel blocker iberiotoxin
(10
7 to 106 M) resulted in increased
tension and [Ca2+]i in both adult and fetal
MCA, although the KATP-channel blocker glibenclamide
(107 to 3 × 105 M) failed to do so.
Of interest, administration of 107 M iberiotoxin totally
eliminated vascular contraction and increase in
[Ca2+]i seen in response to 105
M ryanodine. In precontracted fetal cerebral arteries, activation of
the KATP and KCa channels significantly
decreased both tension and [Ca2+]i,
suggesting that both K+ channels play an important role in
regulating L-type channel Ca2+ flux and therefore vascular
tone in these vessels. In the adult, KATP and the
KCa channels also appear to play an important role in this
regard; however, in the adult vessel, activation of these channels with
resultant vasorelaxation can occur with no significant change in
[Ca2+]i. These channels show differing
responses to inhibition, e.g., KCa-channel inhibition,
resulting in increased tension and [Ca2+]i,
whereas KATP-channel inhibition showed no such effect. In addition, the KCa channel appears to be coupled to the
sarcoplasmic reticulum ryanodine receptor. Thus differences in plasma
membrane K+-channel activity may account, in part, for the
differences in the regulation of contractility of fetal and adult
cerebral arteries.
cerebrovascular circulation; vascular smooth muscle; sympathetic nervous system; norepinephrine; intracellular calcium; potassium channels; fetus; adult
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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PREVIOUSLY, WE AND OTHERS have reported significant differences in cerebral artery contractility with development from fetus to adult. Fetal arteries develop less tone but have greater aminergic activity than those of adult (33); they also show greater calcium sensitivity (26), rely less on Ins(1,4,5)P3-mediated contractile mechanisms (27, 38), and have relatively small intracellular Ca2+ stores compared with the adult (25). In cerebral (and other) arteries, K+ channels play a major role in modulating membrane potential, voltage-gated Ca2+-channel activity, intracellular Ca2+ concentration ([Ca2+]i), and vascular tone. The activation of K+ channels leads to K+ efflux, resulting in hyperpolarization and decreased open probability of L-type Ca2+ channels, thereby reducing Ca2+ influx, [Ca2+]i, and vascular tone. Conversely, norepinephrine (NE)-induced depolarization with inhibition of K+ efflux leads to increased open probability of L-type Ca2+ channels, increased influx of extracellular Ca2+, increased [Ca2+]i, and contraction. Because of the ionic properties of the cell membrane at rest, activity change of only a few K+ channels is sufficient to alter membrane potential and thereby tone (30, 34). In general, the relationship of vessel diameter to membrane potential is linear, i.e., a decrease in membrane potential (less negative) is associated with increased tone and contraction (21).
A growing literature has considered the interactions of K+ and Ca2+ channels in several cell types, including the smooth muscle cells from main branch cerebral arteries (18, 24, 30). Four types of K+ channels have been identified, and each is present in cerebral arteries: ATP sensitive (KATP), Ca2+ activated (KCa), voltage dependent (Kv), and inward rectifier (KIR) (10, 11, 24, 30, 31). A wide variety of vasoactive stimuli have been shown to alter cerebral vessel K+-channel activity, including NE, which acts to inhibit K+ efflux (depolarization) and thereby open Ca2+ channels (10, 11). Several studies have demonstrated the sensitivity of the main branch middle cerebral artery (MCA) to K+-channel activation in the adult (30, 34, 35). By comparison, few studies have examined responses to K+-channel openers and blockers in cerebral arteries of the fetus or changes with development.
In previous studies, we have shown that, in contrast to the adult, fetal cerebral arteries are almost completely dependent on extracellular Ca2+ via L-type Ca2+ channels for contraction (25, 26). As noted above, in cerebral and other arteries, K+ channels play a key role in regulating L-type Ca2+-channel activity. Because of this dependence on L-type Ca2+ channels for Ca2+ flux and the fact that essentially nothing is known about K+-channel function in developing cerebral vasculature, the present study was designed to test the hypothesis that in cerebral arteries of the fetus, the several K+ channels, particularly the KATP and KCa channels, play an important role in the regulation of [Ca2+]i and vascular tone and that this role differs significantly from that of the adult.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Experimental animals and tissues.
For these studies, we used main branch MCAs from near-term fetus
(~140 days) and nonpregnant adult sheep (
2 yr) obtained from
Nebeker Ranch (Lancaster, CA), as we have previously described (27, 28). The ewes were anesthetized and killed with 100 mg/kg intravenous pentobarbital sodium, after which we obtained
isolated cerebral artery segments. We have shown that this method of
death has no significant effect on vessel reactivity, compared with use
of other anesthetic agents (33). To avoid the complication of endothelial-mediated effects, we removed the endothelium by carefully inserting a small wire three times (28).
Cerebral arteries (fetus 300-350 µM, adult 400-450 µM)
were then used immediately for simultaneous measurements of the
[Ca2+]i and tensions (26). To
confirm endothelium removal, we contracted the vessel with
105 M 5-hydroxytryptamine and, at the plateau, added
106 M ADP. Vessels that relaxed >20% after this
treatment were rejected for further study. Unless otherwise noted, all
chemical compounds were purchased from Sigma Chemical (St. Louis, MO).
Contractility and intracellular calcium measurements. We cut the MCAs into rings 2 mm in length and mounted them on two tungsten wires (0.13-mm diameter; A-M Systems, Carlsborg, WA). We attached one wire to an isometric force transducer (Kent Scientific, Litchfield, CT) and the other to a post attached to a micrometer used to vary resting tension in a 5-ml tissue bath mounted on Jasco CAF-110 intracellular Ca2+ analyzer (Jasco, Easton, MD) and measured vascular tension, as previously described (25, 26). This, with measurements of vessel inside diameter, wall thickness, length, and potassium-induced force, enabled calculation of force-per-unit cross-sectional area, as previously described (33). MCA rings were equilibrated under 0.3 g tension at 25°C for 40 min before loading with the acetoxymethyl ester of fura 2 (fura 2-AM; Molecular Probes, Eugene, OR), a fluorescent Ca2+ indicator that is a measure of mean cytoplasmic [Ca2+]i (14). Fura 2 fluorescence and force were measured simultaneously at 38°C, as previously described (26). As we have noted, although some investigators may prefer the transformation of fluorescence to [Ca2+]i, in tissues such as cerebral arteries, the presentation of the ratio is less ambiguous (26). During all contractility experiments, we continuously digitized, normalized, and recorded contractile tensions and the fluorescence ratio (R340/380) using an online computer. For all vessels, we evaluated the contractile response for tension and fluorescence ratio by measuring the maximum peak height and expressing it as percent Kmax (a measure of "efficacy") and calculated pD2 [the negative logarithm of the mean effective concentration at half-maximal response (EC50) for NE and an index of tissue "sensitivity" or "potency"] (26). In the presence of fura 2, neither K+- nor NE-induced tensions were significantly different from those contractions in the absence of the dye (25).
Relative roles of the several K+ channels and their change with development. Because little is known of the role of the several types of K+ channels in modulating NE-induced changes in [Ca2+]i and vascular tension in the cerebral arteries and, particularly, in the fetus, we quantified these variables in the presence of selective K+-channel activators or blockers. For all studies, after initial K+ (120 mM) depolarization to determine Kmax, we performed NE dose-response curves to establish the maximum contraction, %Kmax, and pD2.
To examine the role of activation of KATP on [Ca2+]i and tension, we first stimulated the vessel with 105 M NE. Then, on the plateau of the
response curve, we added increasing concentrations of pinacidil
(107 to 104 M) to stimulate K+
efflux, hyperpolarize the vessel, and cause relaxation. From these
data, we plotted the percent inhibition (e.g., %decrease of tension
and [Ca2+]i) as a function of agonist dose.
Then, in a related study, we gave 105 M pinacidil and,
after 15 min, performed an NE dose-response study (109 to
104 M). From these latter data, we plotted the shift in
the NE dose-response curve. To examine the effect of
KATP-channel inhibition, we quantified NE-induced change in
[Ca2+]i and tension after the
KATP-channel blocker glibenclamide (3 × 105 M). In a related study, we first gave 3 × 107 M NE to achieve a 30% maximum response, then
administered glibenclamide in increasing doses (107 to
3 × 105 M) to increase tension and
[Ca2+]i. In addition, to examine the effect
of glibenclamide alone, we administered 107 to 3 × 105 M of that compound.
To examine the role of activation of KCa on NE-induced
[Ca2+]i and vascular tension, we quantified
these after administration of 105 M NE. Then, on the
plateau of the response, we administered increasing doses of the
KCa-channel opener NS 1619 (109 to
106 M) to stimulate K+ efflux. In a related
study, we first gave NS 1619 (107 M) and, after 15 min,
determined the NE dose response (109 to 104
M). To examine the role of inhibition of the KCa channel on
[Ca2+]i and tension, we performed NE
dose-response curves in the presence of the channel blocker (15 min)
iberiotoxin (107 M). In another study, we gave 3 × 107 M NE to achieve ~30% maximum response and then
gave increasing doses of iberiotoxin (109 to
106 M). In addition, to examine the effect of iberiotoxin
alone, we administered 109 to 106 M of that compound.
To examine the potential role of inhibition of Kv on
Ca2+-channel activity in fetal and adult cerebral arteries,
we measured NE-induced [Ca2+]i and tension
after administration of 4-aminopyridine (4-AP; 104 M).
Alternatively, we administered 3 × 107 M NE and
then gave increasing doses of 4-AP (105 to
103 M). Finally, to determine the possible role of
KIR in modulating Ca2+-channel activity, we
measured NE-induced [Ca2+]i and tension after
addition of KCl (1.5 × 102 M) to activate the
channel or barium chloride (105 M) to block it. In
addition, we administered 3 × 107 M NE to achieve
~30% maximum response, then gave increasing doses of
BaCl2.
Statistical analysis.
All values were calculated as means ± SE. In all cases,
n refers to the number of vessel segments (that corresponds
to the number of animals) studied. The n values for the
different experiments are given in Tables
1 and 2.
Because of the nature of these studies, several statistical tests were
used to test for significant differences. For testing differences
between two groups, we used a simple unpaired Student's
t-test. For multiple comparisons, one- and two-way ANOVA
(vessel, age) coupled with Duncan's multiple-range test were used.
Where appropriate, we used ANOVA with repeated measures. A P
value of <0.05 was considered significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Role of KATP in vascular
[Ca2+]i and tension.
To examine the role of ATP-sensitive K+-channel activation
on tension and fluorescence ratio (R340/380, an index of
[Ca2+]i) in adult MCA, we first contracted
the vessel with 105 M NE, then, on the plateau of the
response, administered pinacidil in increasing half-log increments
(107 to 104 M). As seen in Fig.
1A, at concentrations
>106 M, pinacidil inhibited tension with a half-maximal
inhibitory concentration (pIC50) value of 5.1 ± 0.1. In contrast, fluorescence ratio showed essentially no change (Fig.
1B; n = 4). As also shown in Fig. 1, in
contrast to the lack of pinacidil-induced decrease in
[Ca2+]i in the adult, the fetal MCA responded
to increasing pinacidil concentrations with a decrease of both tension
(Fig. 1A) and [Ca2+]i (Fig.
1B), with pIC50 values of 5.0 ± 0.1 and
4.6 ± 0.1, respectively (Table 1).
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9 to 104 M). As
shown in Fig. 2, in response to
increasing NE dose, adult MCA showed typical increases in vascular
tension (Fig. 2A) and [Ca2+]i
(Fig. 2B). The mean maximal NE-induced tension and
[Ca2+]i were 1.7 ± 0.1 g and
0.15 ± 0.01 units, respectively, values that were close to 100%
Kmax (n = 12). In other experiments, we first administered 105 M pinacidil, then, after 15 min,
repeated the NE concentration response. As shown in Fig. 2A,
after KATP-channel activation by pinacidil, the NE-induced
contractile response was attenuated to 1.0 ± 0.2 g
(P < 0.05), whereas the fluorescence ratio was unchanged (Fig. 2B). There was no significant change in
pD2 (n = 3).
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5 M pinacidil was given followed, after 15 min, by NE
in increasing half-log doses, neither the maximal responses of tension
and [Ca2+]i nor their pD2 values
were significantly different from control values (n = 3; see Table 2 for values).
To examine to what extent KATP-channel inhibition resulted
in further contraction of adult and fetal cerebral arteries, we first
contracted the vessel to ~30% maximum with 3 × 107 M NE and then administered glibenclamide in half-log
doses (107 to 3 × 105 M). In neither
adult nor fetal MCA did such glibenclamide treatment show a significant
effect on either NE-induced tension or
[Ca2+]i (n = 3 each;
data not shown).
To examine the effect of KATP-channel inhibition on
NE-induced responses, we administered 3 × 107 M
glibenclamide and then, after 15 min, determined the NE
concentration-response curves. As seen in Fig.
3, for adult MCA, glibenclamide had a modest effect in lowering maximum tension (Fig. 3A) and
fluorescence ratio (Fig. 3B) 20-30%. Nonetheless,
pD2 values were not significantly different from control.
In a similar manner, in fetal MCA, 3 × 107 M
glibenclamide slightly attenuated the NE-induced response with slight
decreases in tension and [Ca2+]i (Fig. 3,
C and D). Again, pD2 values were not
significantly different from NE controls. When given alone in half-log
doses (109 to 106 M) to either adult or
fetal MCA, glibenclamide showed no effect on tension or
[Ca2+]i (n = 3 each; data not
shown).
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Role of KCa in vascular
[Ca2+]i and tension.
Figure 4 shows the effect of the
KCa-channel opener NS 1619 (109 to
106 M) on adult and fetal MCA precontracted with
105 M NE. In the adult, at NS 1619 concentrations
>3 × 108 M, vascular tension was modestly
decreased with ~40% inhibition at 106 M
(pIC50 = 7.6 ± 0.1; Fig. 4A). In
contrast, NS 1619 did not affect the fluorescence ratio (Fig.
4B). Compared with the adult, Fig. 4 shows the profound
effect of KCa-channel activation in decreasing fetal MCA
tension (essentially 100% inhibition at 107 M NS
1619; pIC50 = 8.2 ± 0.1; Fig. 4A)
and, to a lesser extent, fluorescence ratio (pIC50 = 7.3 ± 0.1; Fig. 4B; Table 1). In addition, because NS
1619 may also block Ca2+ channels, in both adult and fetal
cerebral artery, we administered 107 M NS 1619, and,
after 15 min, depolarized the cells with 120 mM K+. Under
these conditions, the K+-induced increases in tension and
fluorescence ratio were the same as control. Finally, we showed that in
the presence of zero extracellular Ca2+ or after
administration of 105 M nifedipine (L-type
Ca2+-channel blocker), K+ failed to increase
either tension or fluorescence ratio (data not shown).
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7 M NS 1619 and then, after 15 min,
examined the NE dose response. As shown in Fig.
5, under these conditions, NE-induced
tension (Fig. 5A) and fluorescence ratio (Fig.
5B) were minimally affected. Figure 5 also shows the fetal
NE dose response after administration of 107 M NS 1619, with a 10% decrease in vascular tension (Fig. 5C) and a
minimal decrease in fluorescence ratio (Fig. 5D). Again, however, there was no significant change in pD2.
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9 to 106 M). As seen in Fig.
6, in adult MCA at 107 M
iberiotoxin, both tension (Fig. 6A) and
[Ca2+]i increased significantly (Fig.
6B; n = 3). In a similar manner, in the
fetal artery, at 107 M iberiotoxin and above, tension
(Fig. 6C) and [Ca2+]i (Fig.
6D) increased significantly. In another study, to determine to what extent this increase in tension and
[Ca2+]i were in fact due to increased
Ca2+ flux through the L-type Ca2+ channels, we
first administered nifedipine (105 M) and then,
after 15 min, determined the iberiotoxin dose-response relationship
(n = 3). Under these circumstances, neither adult nor
fetal MCA showed a significant increase in either tension or
[Ca2+]i.
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7 M iberiotoxin, neither tension (Fig. 7A)
nor fluorescence ratio (Fig. 7B) were significantly altered.
pD2 values were the same as control (n = 3 each). Similarly, in fetal MCA, as shown in Fig. 7, C and
D, for tension and fluorescence ratio, respectively, KCa-channel inhibition by 107 M iberiotoxin
also had no significant effect (n = 3 each).
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Role of Kv in
[Ca2+]i and tension.
We also examined the role of inhibition of Kv on the
tension and fluorescence ratio of adult MCA, measuring these variables after the administration of 104 M 4-AP followed by NE
dose response. In the adult MCA, 4-AP was associated with a modest 10%
decrease in tension with no significant change in fluorescence ratio.
For fetal MCA, administration of 104 M 4-AP also had a
negligible effect on the NE dose response of tension and fluorescence
ratio, with no change in pD2 values (n = 3 each; data not shown).
Role of KIR in
[Ca2+]i and tension.
To explore the possible role of KIR in modulating
Ca2+-channel activity, we administered 105 M
barium chloride to inhibit these channels, after which we performed an
NE dose response. As seen in Fig. 8, in
adult MCA, vascular tension (Fig. 8A) was not significantly
altered. Fluorescence ratio (Fig. 8B) modestly increased,
the pD2 values being 5.2 ± 0.2 and 4.9 ± 0.2, respectively (P < 0.05). In contrast, for fetal MCA, Fig. 8, C and D, shows the profound effect
of KIR inhibition by 105 M BaCl2
both on tension and fluorescence ratio. The pD2 values were
not significantly altered.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The present studies offer several important observations. First,
activation of plasma membrane KATP channels by pinacidil, with presumed closure of L-type Ca2+ channels, resulted in
significant inhibition (e.g., decrease) of NE-induced tension both in
fetal and adult MCA (Fig. 1). In the fetal vessel, this occurred
concurrently with a significant decrease in
[Ca2+]i; however,
[Ca2+]i did not decrease significantly in the
adult. This illustrates the greater dependence of fetal cerebral
arteries on extracellular Ca2+ for maintenance of tone. In
addition, it is an example of the much greater Ca2+
sensitivity (e.g., tension as a function of
[Ca2+]i) of the fetal cerebral artery
compared with the adult. Second, in a somewhat similar manner, NS 1619 activation of KCa channels (again with presumed closure of
L-type Ca2+ channels) resulted in a significant
inhibition of tension in the fetal vessel with only a moderate
inhibition in that of the adult (Fig. 4). This occurred with only
slight decrease in [Ca2+]i in the fetal
vessel but no significant decrease in the adult. Again, this
illustrates the extracellular Ca2+ requirement of the fetal
cerebral artery and its great Ca2+ sensitivity. Third, in
contrast to these findings, KATP-channel inhibition by
glibenclamide (which inhibits the KATP channel and thereby
activates or opens L-type Ca2+ channels) had
essentially no effect on either vascular tension or
[Ca2+]i. Nonetheless, KCa-channel
inhibition by iberiotoxin resulted in a significant increase in both
tension and [Ca2+]i (Fig. 6), a response that
was eliminated by the L-type Ca2+-channel blocker
nifedipine (105 M). In addition, whereas glibenclamide
modestly attenuated NE dose response (Fig. 3), iberiotoxin failed to do
so (Fig. 7). Fourth, inhibition of KV channels also had
little effect on NE-induced contraction. Fifth, BaCl2
inhibition of KIR channels demonstrated only a small effect
on [Ca2+]i in adult MCA. In contrast, in the
fetal artery, BaCl2 significantly inhibited both tension
and [Ca2+]i, presumably as a result of
blockade of L-type Ca2+ channels (Fig. 8).
Role of K+ channels in cerebral artery smooth muscle. Smaller arteries, including those of the cerebral vasculature, exist in a partially contracted state and can constrict further or dilate depending on the tissue requirements for blood and/or oxygen. This basal tone is an important determinant of vascular resistance and blood pressure and, to a great extent, is regulated by the smooth muscle cell membrane potential, which, in turn, is regulated by the plasma membrane K+ channels (30). Increasing the open probability of K+ channels increases K+ efflux, resulting in membrane hyperpolarization as K+ approaches its equilibrium potential. This results in decreased open probability of L-type Ca2+ channels, decreasing Ca2+ influx, and vasodilatation. Conversely, inhibition of K+ channels results in membrane depolarization and vasoconstriction. As noted earlier, four distinct types of K+ channels have been identified, each of which serves a unique function in the regulation of smooth muscle membrane potential. Although these channel types are fairly well defined structurally and relatively selective pharmacological blockers exist for each, selective activators have been described for only the KATP and KCa channels.
Role of KATP channels. These channels respond to changes in cellular metabolic state, opening in response to a decrease in intracellular ATP concentration. Activation by pinacidil or lemakalim results in hyperpolarization and vasorelaxation, whereas inhibition by sulfonylurea drugs such as glibenclamide may result in vasoconstriction (5) and may disrupt cerebral autoregulation (17). Several endogenous substances produce hyperpolarization and relaxation of cerebral arteries that may be mediated by activation of KATP channels (10). For instance, the vasodilators adenosine and calcitonin gene-related peptide increased glibenclamide-sensitive currents, whereas vasoconstrictors serotonin and histamine inhibited pinacidil-induced potassium currents (20). Nonetheless, the overall regulation of these channels by physiological mediators is poorly understood (30).
As noted in Fig. 1, a key observation of the present studies is the inhibition (e.g., decrease) of vascular tension both in adult and fetal cerebral arteries after pinacidil-induced activation of KATP channels (Fig. 1A). In fetal MCA, the fluorescence ratio decreased pari passu with the decrease in tension, suggesting relatively great Ca2+ sensitivity (e.g., dependence of tension on [Ca2+]i) in the fetal artery. In contrast, in the adult vessel, this decrease in tension was accompanied by no significant change in [Ca2+]i (Fig. 1B). In view of the significant effect of pinacidil in decreasing tension and in fetal MCA fluorescence ratio (Fig. 1), it is somewhat surprising that opening the KATP channel, with relative closure of the L-type Ca2+ channel, had less effect on inhibiting the NE dose response (Fig. 2). This was particularly the case in the fetal vessel that showed such a great response to pinacidil. In 5- to 7-day-old lambs, Pearce and Elliott (32) reported that the KATP-channel agonist lemakalim resulted in relaxation of 106 M serotonin-induced contraction in
second and fourth branch MCAs with pIC50 values of 5.5 ± 0.3 and 5.2 ± 0.2, respectively. In the adult sheep, the
values were 7.2 ± 0.4 and 6.6 ± 0.4 in these vessels,
respectively. In neither newborn nor adult were the differences between
second and fourth branch vessels significant. These somewhat different
results from those of the present study might be anticipated as a
consequence of different KATP-channel agonists used
(pinacidil vs. lemakalim), different contractile agent (NE vs.
serotonin), different developmental age (fetus vs. newborn), or the
different manner in which the studies were conducted (cumulative
half-log dose on plateau of contraction vs. repeated contraction of
vessel with administration of lemakalim at each contraction).
Also, inhibition of the KATP channel by glibenclamide
itself (109 to 106 M; with presumed opening
of L-type Ca2+ channels) failed to increase tension or
[Ca2+]i in either adult or fetal MCA. In
regard to the responses seen in Fig. 3, one might argue that inhibition
of the KATP channel with opening of the L-type
Ca2+ channel should result in no further augmentation of
NE-induced opening of Ca2+ channels (with increases in
[Ca2+]i and tension). Nonetheless, the
decrease in [Ca2+]i and tension seen at
3 × 106 M NE and above may have been a nonspecific effect.
Role of KCa channels. Large-conductance K+ channels activated by membrane depolarization and an increase in [Ca2+]i are located in essentially all vascular and other smooth muscle cells (29). Activated by NS 1619 and inhibited by iberiotoxin, charybdotoxin, and related peptides (10), the role of physiological regulators in mediating KCa-channel activity is largely unknown (30). In ovine pulmonary arteries, Reeve et al. (36) demonstrated a significant maturational change with development from fetus to adult, with a change from KCa to KV channels regulating resting membrane potential. Although several studies have examined KCa-channel activity in cerebral (4, 6, 8, 12, 16, 23) and pial (2, 3, 13) arteries, none of these examined the interrelations of tension and [Ca2+]i or changes with development.
As shown in Fig. 4A, another critical observation of the present study is that NS 1619 activation of the KCa channel profoundly inhibited vascular tension in fetal MCA but decreased tension to a lesser extent in the adult vessel. In the fetal vessel, this KCa-channel activation was associated with a modest inhibition of fluorescence ratio (in contrast to the case for KATP-channel activation). By comparison, in adult MCA, this was accompanied by no significant decrease in [Ca2+]i (as was also the case with KATP-channel activation). Nonetheless, these results again suggest a greater dependence of tension on [Ca2+]i (e.g., sensitivity) of fetal cerebral arteries compared with those of the adult. Again, it may be surprising that opening of KCa channels with resultant blockade of the Ca2+ channel did not show more effect on NE-induced contraction and increase in [Ca2+]i (Fig. 5). A related issue is to what extent NS 1619 blocks plasma membrane Ca2+ channels in addition to activating the KCa channel. Such an effect has been reported in rat cerebral arteries (16). As noted above, both in adult and fetal MCA, tension and fluorescence ratio depolarization responses to 120 mM K+, after administration of 107 M NS 1619, were not significantly different from
control. This suggests that, indeed, the decreased tension and
fluorescence ratio in response to NS 1619 (Fig. 4) was the result of
activation of the KCa channel rather than to block the
L-type Ca2+ channel.
In addition, as shown in Fig. 6, iberiotoxin inhibition of the
KCa channel (with opening of the L-type Ca2+
channel) resulted in significant increases in tension and
[Ca2+]i in cerebral arteries of both age
groups. This is in marked contrast to the lack of such an effect by
KATP-channel blockade, suggesting that they exert their
effect on Ca2+ channels by different mechanisms.
Importantly, iberiotoxin-induced contraction was totally blocked by
administration of the L-type Ca2+-channel blocker
nifedipine (105 M). Also, in view of iberiotoxin's
profound effect in increasing tension and fluorescence ratio, it is
somewhat surprising that it had such a minimal effect on the NE
dose-response relationship (Fig. 7). A related phenomenon of interest
is the coupling of the KCa channel to the sarcoplasmic
reticulum ryanodine receptor in cerebral arteries that we have recently
reported (25) and that may be more common than is
generally appreciated (18). In both fetal and adult MCA,
administration of 107 M iberiotoxin followed after 15 min
by 105 M ryanodine totally blocked the increase in
tension and fluorescence ratio seen after ryanodine alone
(25).
Role of KV channels. When the plasma membrane is depolarized, these channels (also called delayed rectifier channels) open to allow K+ efflux and membrane repolarization (15); thus they play a key role in the regulation of membrane potential and vascular tone (21, 22). Although there exist several KV-channel subtypes, in vascular smooth muscle, these all are inhibited by 4-AP. Nonetheless, in the present study, 4-AP failed to demonstrate significant effects on either tension or [Ca2+]i when given alone or in concert with an NE dose-response curve.
Role of KIR channels. In contrast to KCa and KV channels, which are activated by membrane depolarization, the KIR channels (named for greater inward than outward K+ movement when voltage clamped) are activated by membrane hyperpolarization. As such, they may play a role in maintaining resting membrane potential and K+-induced vasodilatation, although this regulation is poorly understood (10, 30). Several groups have examined the role of these channels in determining resting tone in cerebral arteries (9, 19), membrane potential (34), and in the metabolic regulation of cerebral blood flow in response to changes in K+ concentration (24). We know of no studies on the possible changing role of these channels with development. As noted above, the BaCl2 inhibition of tension and Ca2+ in the fetal MCA was probably secondary to Ba2+ blockade of L-type Ca2+ channels rather than to inhibition of KIR channels per se.
Perspectives
The role of the several plasma membrane K+ channels and their sensitivity to various agents may differ greatly as a function of vessel type, species, and developmental age. The marked dependence of fetal cerebral arteries on external [Ca2+] following activation of KATP or KCa channels fits with previous studies from our group (25, 26) and others on developmental differences in cerebral artery contractility. Fetal cerebral arteries develop less tone but have greater aminergic activity than those of adult (33); the newborn MCA requires more transmembrane calcium uptake than the adult (39); fetal arteries show greater calcium sensitivity (1, 26); and fetal arteries rely less on Ins(1,4,5)P3-mediated contractile mechanisms (27, 38). In addition, the presence of relatively small intracellular stores in immature vessels (7, 25, 37) further emphasizes the dependence of fetal cerebral arteries on extracellular Ca2+ compared with the adult.By quantifying simultaneously [Ca2+]i and tension, the present studies are the first to demonstrate the role of the several K+ channels in the NE-induced contractile response of fetal and adult cerebral arteries. As we have shown previously, for their contraction, fetal cerebral arteries demonstrate considerable dependence on extracellular Ca2+ and Ca2+ flux via L-type Ca2+ channels (26). This dependence is associated with the fetal vessels being exquisitely sensitive to KATP- and KCa-channel activation (with presumed blockade of L-type Ca2+ channels), compared with the adult. Overall, contractility of fetal cerebral arteries is highly dependent on Ca2+ flux through L-type Ca2+ channels, with a less important role for SR Ca2+ store-dependent mechanisms (25, 26). With maturation to the adult, SR Ca2+ store-dependent mechanisms increase in importance (25). On the basis of data of the present study, KATP and KCa channels each play an important role in the regulation of vascular tone in fetal and adult cerebral arteries. These roles appear to differ, however, as evidenced by their somewhat different responses to inhibition, e.g., KCa-channel blockade resulting in increased tension and [Ca2+]i, whereas KATP-channel blockade shows no such effect. Importantly, the KCa channel appears to be coupled to the SR ryanodine receptor (17, 25).
The obvious question arises as to when during the course of development from fetus to newborn to adult the role of plasma membrane K+-channel activity and interactions with L-type Ca2+ channels alter and cerebral vessels become less dependent on extracellular [Ca2+]. Additional questions relate to the role of developmental changes in the density, affinity, and isoforms of the several K+ channels and their relative roles in maintaining membrane potential. These areas are the subject of current studies in the elucidation of the mechanisms of cerebral vascular reactivity and their change with development.
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ACKNOWLEDGEMENTS |
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We thank Brenda Kreutzer for preparing the manuscript.
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FOOTNOTES |
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This work was supported by United States Public Health Service Grants HD/HL-03807 and PO1-HD-31226 to L. D. Longo.
Address for reprint requests and other correspondence: L. D. Longo, Center for Perinatal Biology, Loma Linda Univ., School of Medicine, Loma Linda, CA 92350 (E-mail: llongo{at}som.llu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 24 March 2000; accepted in final form 3 August 2000.
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ABSTRACT
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METHODS
RESULTS
DISCUSSION
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) and fetal (
) main
branch middle cerebral artery (MCA) in response to the ATP-sensitive
K+ (KATP)-channel activator pinacidil
(n = 4 each). Vessels were first contracted with
10
