| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Departments of 1 Obstetrics and Gynecology, 2 Pharmacology, and 3 Medicine, University of Vermont, College of Medicine, Burlington, Vermont 05405
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
The effects of activating protein kinase C
(PKC) with indolactam V (Indo-V) and
1,2-dioctanoyl-sn-glycerol (DOG) on
smooth muscle intracellular Ca2+
concentrations
([Ca2+]i)
and arterial diameter were determined using ratiometric
Ca2+ imaging and video edge
detection of pressurized rat posterior cerebral arteries. Elevation of
intraluminal pressure from 10 to 60 mmHg resulted in an increase in
[Ca2+]i
from 74 ± 5 to 219 ± 8 nM and myogenic constriction.
Application of Indo-V (0.01-3 µM) or DOG (0.1-30 µM)
induced constriction and decreased
[Ca2+]i
to 140 ± 11 and 127 ± 12 nM, respectively, at the highest
concentrations used. In the presence of Indo-V, the dihydropyridine
Ca2+-channel-blocker nisoldipine
produced nearly maximum dilation and decreased
[Ca2+]i
to 97 ± 7 nM. In 
-toxin-permeabilized arteries, the constrictor effects of Indo-V and DOG were not observed in the absence of Ca2+. Both PKC activators
significantly increased the degree of constriction of permeabilized
arteries at different
[Ca2+]i.
We conclude that 1) Indo-V- or
DOG-induced constriction of pressurized arteries requires
Ca2+ influx through
voltage-dependent Ca2+ channels,
and 2) PKC-induced constriction of
pressurized rat cerebral arteries is associated with a decrease in
[Ca2+]i,
suggesting an increase in the Ca2+
sensitivity of the contractile process.
protein kinase C activators; calcium ion imaging; -toxin-
permeabilized arteries; dihydropyridine; protein kinase C
| |
INTRODUCTION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
PROTEIN KINASE C (PKC) has been implicated in the regulation of a variety of intracellular processes, including smooth muscle contraction (18, 32, 33, 37). Its role in the control of vascular tone was considerably strengthened by the identification of PKC as a physiological target for diacylglycerol (DAG), a second messenger in a number of signal transduction pathways (18, 22, 32, 33, 37).
Indirect evidence indicates that PKC may play an important role in regulating cerebrovascular tone under both physiological and pathological conditions (5, 8, 23, 26, 27, 29, 34, 35). An increase in PKC activity of cerebral arteries in response to vasoconstrictor agonists has been recently reported (8, 26), and upregulation of PKC is thought to contribute to cerebral vasospasm after subarachnoid hemorrhage (23, 27). PKC activation has also been implicated in pressure-induced myogenic vasoconstriction in some (7, 19, 29) but not all (21, 24) studies.
The activation of PKC by phorbol esters, by synthetic analogs of DAG, or by indolactam V (Indo-V) invariably results in vasoconstriction both in vivo and in vitro; however, the underlying mechanisms remain largely unknown (5, 8, 17, 23, 26, 27, 29, 34). In some vascular smooth muscle preparations, phorbol ester-induced contraction was associated with an increased influx of Ca2+ into cells that might result from enhanced activity of voltage-dependent Ca2+ channels or secondary to membrane depolarization due to inhibition of potassium channels (1, 3, 6, 9, 25, 31, 32). Indo-V-induced constriction in pressurized mesenteric arteries, however, occurred in the absence of any measurable changes in cytosolic Ca2+ (17), suggesting Ca2+ sensitization of the contractile process to be the major mechanism.
We previously reported that activators of PKC, such as Indo-V, produced a significantly augmented constriction of pressurized cerebral arteries (29). Therefore, the main objective of this study was to determine the role of intracellular Ca2+ concentration ([Ca2+]i) in vasoconstriction of pressurized cerebral arteries induced by Indo-V and 1,2-dioctanoyl-sn-glycerol (DOG). Experiments were conducted using isolated pressurized cerebral arteries with myogenic tone, a physiologically essential property that is thought to play an important role in the regulation of cerebral blood flow. Our results provide direct support for the idea that activators of PKC constrict cerebral arteries through an increase in the Ca2+ sensitivity of the contractile process.
| |
METHODS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Animals. Adult (16- to 24-wk old) male and female Wistar-Kyoto rats (n = 29) were anesthetized by an intraperitoneal injection of methohexital sodium (Brevital, 50 mg/kg) or pentobarbitone (150 mg/kg) and killed by decapitation. The brain was removed and immersed in a dissection dish filled with physiological salt solution (PSS, composition presented in Solutions and drugs). The entire posterior cerebral artery, including its branches, was removed and carefully dissected free from surrounding connective tissues under a stereo dissection microscope. All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication 85-23, 1985). The experimental protocols were approved by the Institutional Animal Use and Care Committee of the University of Vermont.
Experimental protocol: Intact arteries. Detailed descriptions of simultaneous measurements of arterial diameter and [Ca2+]i in smooth muscle cells of the arterial wall (arterial wall [Ca2+]i) have been published previously (21). Briefly, arteries were loaded with fura 2, a Ca2+-sensitive fluorescent dye. Fura 2-AM (1-2 µl of 1 mM stock solution) was premixed with an equal volume of a 25% solution of pluronic acid in DMSO and then was diluted in PSS to yield a final concentration of 2-4 µM. The excised posterior cerebral artery, including its branches, was incubated in the fura 2-AM/PSS loading solution at room temperature in the dark for 45 min. Fura 2-loaded arteries were then washed with PSS and kept on ice in PSS. An arterial segment was cannulated, mounted in a specially designed arteriograph (21), and continuously superfused at 3-6 ml/min with oxygenated PSS (95% O2-5% CO2) at 37°C.
Ratio images were obtained at a rate of 0.2 Hz from background-corrected, four-frame averaged images of the 510 ± 40-nm emission from the arteries when alternatively excited at 340 and 380 nm using the Image-1/FL quantitative ratio imaging software (Universal Imaging, West Chester, PA). Arterial diameter was measured in micrometers by means of a length-calibrated edge detector.
After a 30-min equilibration period at 10 mmHg, intravascular pressure was increased to 60 mmHg. Typically, elevation of intraluminal pressure resulted in an immediate distension of the artery followed by myogenic constriction. After a 15- to 20-min stabilization period, Indo-V or DOG was applied in increasing concentrations.
In a separate set of experiments, the endothelium was removed by placing an air bubble in the lumen of the artery for 1-2 min, followed by perfusion with regular PSS. The effectiveness of this denudation procedure was confirmed by the absence of dilatory response to an application of ACh as described previously (21 and see RESULTS).
Experimental protocol: Permeabilized
arteries. Small arterial segments (0.5-1.0 mm in
length) were prepared from tertiary branches of the posterior cerebral
artery and were transferred to an arteriograph filled with
Ca2+-free relaxing solution. A
dual-chamber arteriograph containing two cannulated arterial segments
was placed on the stage of an inverted microscope equipped with a video
camera. Detailed descriptions of the pressurizing and lumen diameter
analyzing systems have been published previously (28). Arteries were
pressurized to 50 mmHg in
Ca2+-free relaxing solution
containing ryanodine (10 µM) after pretreatment with caffeine (10 mM)
to deplete intracellular Ca2+
stores. Permeabilization of arteries was performed in relaxing solution
by the addition of Staphylococcus
aureus -toxin (800 U/ml) for 20 min as previously
described (14). Arterial segments were then rinsed two to three times
with relaxing solution to remove remaining toxin from the bath
solution. Vessels were allowed to equilibrate for 30 min. During this
period, arterial segments were warmed to 37°C and, where
appropriate, pretreated with activators of PKC for 20 min. Constrictor
responses to different concentrations of
Ca2+ were obtained in a
cumulative fashion by applying each concentration of Ca2+
until arterial diameter reached a new steady-state level (usually 5-10 min). Only one Ca2+ concentration-response curve
was constructed for each artery segment. Permeabilization with
-toxin was performed at room temperature, whereas Ca2+
concentration-response curves were obtained after warming to 37°C.
Solutions and drugs. The PSS contained (in mM) 119 NaCl, 4.7 KCl, 24.0 NaHCO3, 1.2 KH2PO4, 1.6 CaCl2, 1.2 MgSO4, 0.023 EDTA, and 11.0 glucose equilibrated with a mixture of 95% O2- 5% CO2, pH = 7.4. High-potassium solution (60 mM) was prepared by substituting equimolar amounts of NaCl with KCl. The composition of HEPES-buffered PSS was (in mM) 141.8 NaCl, 4.7 KCl, 1.7 MgSO4, 0.05 EDTA, 2.8 CaCl2, 1.2 KH2PO4, 10.0 HEPES, and 5.0 glucose (pH = 7.4). Relaxing (Ca2+-free) solution contained the following (in mM): 63.6 potassium methanesulfonate (KMS), 2.0 MgCl2, 4.5 MgATP, 2.0 EGTA, 10.0 phosphocreatine, and 30.0 PIPES; pH was adjusted to 7.1 with 10.0 N KOH.
The composition of the activating solution was similar to that of the relaxing solution, except that it contained 10.0 mM EGTA and CaCl2, which was added to produce a specific free Ca2+ concentration. The amount of CaCl2 needed to yield the desired free ionic concentrations was calculated by a computer program (2). Both relaxing and activating solutions contained 1.0 µM FCCP, a mitochondrial blocker, 1.0 µM leupeptin, a protease inhibitor, and 10.0 µM ryanodine. Ionic strength was kept constant at 200 mM by adjusting the concentration of KMS.
All chemicals were purchased from Sigma Chemical (St. Louis, MO) with
the exception of S. aureus -toxin,
Indo-V, and ryanodine, which were obtained from Calbiochem (La Jolla,
CA). Fura 2-AM and pluronic acid were purchased from Molecular Probes.
Nisoldipine was a gift from Dr. A. Scriabine of Miles Laboratories
(West Haven, CT). Fura 2-AM was dissolved in dry DMSO as a 1 mM stock
solution and was frozen in 50-µl aliquots until used. Lyophilized
-toxin was reconstituted in deionized water to yield a stock
solution of 12,500 hemolytic U/ml and either used on the same day or
frozen (
20°C) and used on the following day. Indo-V,
ryanodine, nisoldipine, and FCCP were all prepared as 10 mM stock
solutions in alcohol. Caffeine was dissolved directly in relaxing
solution just before use.
Data analysis and statistics. Arterial wall [Ca2+]i was calculated using the following equation (from Ref. 15)
![[Ca<SUP>2+</SUP>]<SUB>i</SUB> = <IT>K</IT><SUB>d</SUB>&bgr;(R − R<SUB>min</SUB>)/(R<SUB>max</SUB> − R)](/content/vol277/issue3/fulltext/H1178/img001.gif)
|
is the ratio of
fluorescence at 380 nm recorded in the absence of
Ca2+ to the 380-nm fluorescence
recorded in a saturating concentration of
Ca2+.
Rmin and
Rmax were measured from
ionomycin-treated arteries, and was determined as previously
described (21). These values were then pooled and used to convert the
ratio values into a
[Ca2+]i.
The dissociation constant
(Kd) was
determined separately by using in situ titration of
Ca2+ in fura 2-loaded arteries.
The pooled mean values for Rmax,
Rmin, and were 2.31 ± 0.14, 0.43 ± 0.03, and 2.08 ± 0.15 (mean ± SD), respectively, and
Kd was 282 nM
(21). Diameter, pressure, and ratio values were simultaneously recorded
using Axotape 2.0 (Axon Instruments). All data then were imported as
ASCII files into Sigma Plot or Sigma Stat for graphical representation,
calculations, and statistical analysis. Data are expressed as means ± SE, where n is the number of
arterial segments studied. Usually, two arterial segments were used
from the same animal. Diameter values of intact arteries were expressed
in micrometers as means ± SE.
Ca2+-induced constriction of
permeabilized arteries was expressed as a percentage of maximum
arterial diameter in the relaxing solution at the same intravascular
pressure. A Student's t-test was used to determine the significance of differences between sets of data with
P < 0.05 considered significant.
| |
RESULTS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
PKC activators decrease arterial wall
[Ca2+]i
and constrict intact pressurized cerebral arteries.
After equilibration at 10 mmHg, arterial wall
[Ca2+]i
was 74 ± 5 nM (n = 28). When
intraluminal pressure was elevated from 10 to 60 mmHg, the initial
arterial distension was followed by a maintained increase in the level
of arterial wall
[Ca2+]i
to 219 ± 8 nM (n = 28) and a
decrease in arterial diameter (Fig. 1,
A and
B, and Fig.
2A).
Application of the synthetic PKC activator Indo-V (1 nM-3 µM)
resulted in concentration-dependent constriction and reduction of
arterial wall
[Ca2+]i.
Indo-V (3 µM) constricted the arteries from 175 ± 8 to 99 ± 8 µm and decreased
[Ca2+]i
from 226 ± 11 to 140 ± 11 nM
(n = 9; Figs.
1A and 2,
B-D). DOG, a stable
membrane-permeable analog of the endogenous activator of PKC, DAG,
evoked an effect similar to that of Indo-V (Figs. 1B and
3A). For example, 30 µM DOG caused
a vasoconstriction from 171 ± 7 to 96 ± 8 µm and a decrease
in
[Ca2+]i
from 202 ± 11 to 127 ± 12 nM
(n = 4; Fig.
3, B and
C). These results suggest that
elevation of arterial wall
[Ca2+]i
is not responsible for the constriction of pressurized small cerebral
arteries induced by PKC activators.
|
|
|
21 mV (21), and changes in membrane potential are unlikely.
Figure 4A
illustrates the effect of high-potassium solution in arteries with
myogenic tone at an intraluminal pressure of 60 mmHg. Application of
high-potassium solution (60 mM) resulted in significant elevation of
[Ca2+]i
from 220 ± 18 to 393 ± 45 nM
(P < 0.05), which was accompanied by
vasoconstriction from 177 ± 8 to 123 ± 11 µm
(n = 11). After stabilization of
arterial diameter and arterial wall
[Ca2+]i,
the application of Indo-V (1 µM) resulted in an additional constriction of 20 ± 4% in high-potassium solution and a
significant decrease in
[Ca2+]i
from 395 ± 66 to 319 ± 50 nM
(n = 6; Fig. 4, A,
B, and
D). Similarly, in the presence of
high-potassium solution, DOG (10 µM) caused a comparable decrease in
arterial diameter of 13 ± 2% and a reduction in
[Ca2+]i
from 391 ± 67 to 254 ± 26 nM
(n = 5; Fig. 4,
C and
D). These findings suggest that
Indo-V- or DOG-induced decreases in arterial wall
[Ca2+]i
can occur without changes in membrane potential. Furthermore, these
experiments suggest that PKC activators may inhibit
Ca2+ entry and/or stimulate
Ca2+ extrusion from the cytosol
into the sarcoplasmic reticulum or extracellular space.
|
Ca2+
dependence of PKC-induced responses in intact pressurized arteries.
Inhibitors of voltage-dependent
Ca2+ channels such as
dihydropyridines dilate pressurized cerebral arteries to an extent
similar to that caused by external
Ca2+ removal (21, 24), suggesting
that voltage-dependent Ca2+
channels are the major Ca2+ entry
pathway in cerebral arteries. Consistent with this observation are
numerous studies that demonstrate that pressure-induced myogenic constriction of cerebral arteries is largely, if not exclusively, dependent on the influx of Ca2+
through voltage-dependent Ca2+
channels (4, 21, 24, 30). In this study, Indo-V- or DOG-induced
constriction was associated with a decrease in arterial wall
[Ca2+]i.
However, the measured levels of
[Ca2+]i
at 140-150 nM were still substantially higher than those observed before elevation of intraluminal pressure (74 ± 5 nM). We therefore sought to evaluate the Ca2+
dependence of the PKC-mediated vasoconstriction. We reexamined the
effects of Indo-V and DOG in the presence of nisoldipine, a
dihydropyridine inhibitor of voltage-dependent
Ca2+ channels. The concentration
of nisoldipine applied (0.5 µM) has been shown to be maximally
effective in preventing voltage-dependent Ca2+ influx and
myogenic tone of cerebral arteries (21). As mentioned previously,
elevation of intraluminal pressure from 10 to 60 mmHg resulted in an
increase in
[Ca2+]i
from 76 ± 9 to 223 ± 7 nM that was accompanied by
vasoconstriction (Fig.
5A).
Indo-V (1 µM) induced an additional constriction from 174 ± 18 to
117 ± 4 µm and lowered arterial wall
[Ca2+]i
from 223 ± 7 to 154 ± 10 nM
(n = 7; Fig. 5,
B and
C). In the presence of 1 µM
Indo-V, application of nisoldipine reduced
[Ca2+]i
to 97 ± 7 nM (n = 7), a level not
significantly different from that measured before pressure elevation,
and caused nearly maximal vasodilation. In nisoldipine-treated arteries
and in the continued presence of Indo-V, application of papaverine at a
concentration of 0.1 mM induced a little additional dilation and
reduced arterial wall
[Ca2+]i
to 86 ± 8 nM (n = 3). These
results, summarized in Fig. 5, B and
C, suggest that
Ca2+ influx through
voltage-dependent Ca2+ channels is
necessary for PKC-induced vasoconstriction of pressurized cerebral
arteries.
|
-toxin-permeabilized and pressurized (50 mmHg) arteries. In
Ca2+-free EGTA-containing (2 mM)
relaxing solution, neither Indo-V (0.1, 1, and 5 µM) nor DOG (10 µM) produced any significant constriction (maximal constriction of
4.6 ± 0.7% of baseline diameter at the highest concentration of
Indo-V; Fig.
6A).
Increasing the bath concentration of
Ca2+ to 100 nM constricted
arteries by 11 ± 2%. Under these conditions, application of Indo-V
or DOG resulted in a strong additional constriction in all vessels
(Fig. 6B). The decreases in arterial
diameter induced by Indo-V at concentrations of 0.1, 1, and 5 µM were
25 ± 6% (n = 6), 47 ± 11%
(n = 4), and 41 ± 5%
(n = 6) of baseline diameter in
Ca2+-free relaxing solution,
respectively. Similarly, a maximal concentration of DOG (10 µM)
produced a constriction of 49 ± 10%
(n = 6) of baseline diameter. Original
traces illustrating effects of Indo-V and DOG in permeabilized arteries
preconstricted with 100 nM of Ca2+
are shown of Fig. 6, C and
D.
|
|
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
PKC-induced vasoconstriction is associated with reduction in arterial wall [Ca2+]i. The main finding of this study is that PKC activation in small cerebral arteries results in vasoconstriction that is accompanied by a significant reduction in arterial wall [Ca2+]i (Figs. 1-3). This effect is concentration dependent, graded, and sustained. The similarity in effects observed in response to Indo-V and DOG, two structurally different isozyme-nonselective activators of PKC (10, 13), strengthens the specificity of the underlying mechanism with respect to PKC activation. Endothelial removal was without effect (Fig. 8); hence, vascular smooth muscle appears to be the principal target for the actions of both PKC activators.
Normally, vasoconstriction is associated with elevations in intracellular Ca2+. The observed effects of PKC activation therefore seem both paradoxical and intriguing. Ca2+ is clearly requisite for cerebral artery pressure-induced (myogenic) constriction, as demonstrated by earlier studies that detail the inhibitory effects of Ca2+ entry blockers (dihydropyridines and diltiazem) on myogenic tone (4, 21, 24, 30). In this study, the vasoconstrictor effects of Indo-V and DOG could be inhibited by the application of nisoldipine (Fig. 5), demonstrating that a certain level of [Ca2+]i is required and that the estimated Ca2+ threshold for PKC activation lies between 80 and 130 nM. Thus Ca2+ is clearly required not only for cerebral artery pressure-induced (myogenic) constriction but also for the contractile response resulting from PKC activation.Mechanism of PKC-induced decrease in arterial wall [Ca2+]i. The mechanism by which PKC activation lowers intracellular Ca2+ is not known. The ability of Indo-V and DOG to produce similar effects (decreased [Ca2+]i and vasoconstriction) in vessels bathed in 60 mM KCl solution (Fig. 4) minimizes the possibility that the decrease is due to membrane hyperpolarization. Two other potential mechanisms, a decrease in Ca2+ influx or an acceleration of Ca2+ extrusion from the cytosol of smooth muscle cells, should therefore be considered to explain these findings. Direct effects of PKC activation on Ca2+ channels have been described, with both activation and inhibition reported, depending less on the type of preparation and experimental conditions but more notably on the type and concentration of the PKC activator used (9, 12, 17, 31, 32). For example, drastic and sustained inhibition of L-type Ca2+ channels by phorbol esters and synthetic analogs of DAG has been reported in single aortic smooth muscle cells (12). Some vasoconstrictors (e.g., bombesin, vasopressin) that increase the breakdown of polyphosphoinositides and generate a DAG also reduce activity of Ca2+ channels of aortic smooth muscle cells through a PKC-dependent mechanism (12). On the other hand, Indo-V increased inward Ca2+ currents in single smooth muscle cells from mesenteric arteries (17). In smooth muscle cells from human umbilical arteries, a dual modulator effect (transient activation and subsequent inhibition) has been described (31). The coexistence of differentially regulated, multiple PKC isoenzymes can perhaps account for the reported discrepant effects of PKC activators on L-type Ca2+ channels (18, 22, 37).
In the present study, effects of Indo-V and DOG were examined in arteries subjected to intraluminal pressure (60 mmHg). The resulting myogenic constriction is dependent on Ca2+ influx through voltage-dependent Ca2+ channels in response to pressure-induced membrane depolarization (4, 16, 21). Therefore, inhibition of Ca2+ channel activity would result in reduction of cytosolic Ca2+ and might be one of the possible explanations for the PKC-induced [Ca2+]i decrease observed in our experiments. Phorbol esters and synthetic DAG have also been shown to stimulate Na+-dependent Ca2+ efflux and activate the low-capacity sarcolemmal Ca2+-ATPase in some vascular smooth muscle preparations (11, 22, 36). An acceleration of Ca2+ extrusion from the cytoplasm or enhanced Ca2+ sequestration into the sarcoplasmic reticulum may be an alternative or additional mechanism for PKC-induced lowering of [Ca2+]i in pressurized cerebral arteries.Ca2+
dependence of PKC-induced vasoconstriction.
To dissociate
[Ca2+]i-lowering
and membrane potential effects of Indo-V and DOG from their influence
on arterial diameter (constriction), a separate group of vessels was
permeabilized with -toxin. The advantage of this approach is that
cytoplasmic Ca2+ can be
"clamped," thereby allowing direct observation of the effects of
PKC activation without the complications of changing membrane potential
or ionic fluxes across plasma membrane. When vessels were treated with
Indo-V or DOG after Ca2+ was added
to the bath, a significant potentiating effect was observed (Fig. 6).
As in intact vessels, a certain level of
Ca2+ was required, since the
sizable constriction was only observed in solutions containing 100 nM
or higher concentrations of Ca2+.
The effects of PKC activation on permeabilized arteries were minimal or
entirely absent in Ca2+-free
solution (Fig. 6A).
PKC-induced Ca2+ sensitization of the contractile process. We interpret our findings showing that PKC activation potentiates Ca2+-dependent vasoconstriction in intact and permeabilized cerebral arteries as evidence for a PKC-mediated increase in the Ca2+ sensitivity of the contractile process. This augmentation was particularly clear in intact vessels, since they constricted without any increase but, rather, a decrease in arterial wall [Ca2+]i. Both Indo-V and DOG are reportedly equally potent in activating different PKC isoenzymes (20). The Ca2+ dependence of PKC-induced constriction in small cerebral arteries might therefore reflect a preferential expression of Ca2+-dependent isoenzymes. This finding is in contrast to that reported for large cerebral arteries and the ferret aorta, where PKC activation resulted in substantial Ca2+-independent contraction (8, 37).
Several mechanisms have been implicated in PKC-induced sensitization of the contractile process in smooth muscle (17, 18, 22, 33, 37). Inhibition of myosin light chain phosphatase is one potential way for enhancing force production at a given level of cytosolic Ca2+ (33). This mechanism is probably involved in PKC-mediated constriction of small mesenteric arteries, since Hill and co-workers (17) demonstrated that exposure to indolactam significantly increased myosin light chain phosphorylation and could be abolished by ML-7, an inhibitor of myosin light chain kinase. Conversely, in a different report, phorbol ester-induced constriction of rat basilar artery was insensitive to ML-7, suggesting the involvement of some other mechanism(s) (26). PKC-induced phosphorylation of caldesmon or calponin, actin-binding proteins that inhibit actin-activated ATPase of smooth muscle myosin, may be another important mechanism for Ca2+ sensitization of the contractile process and has been implicated in Ca2+-independent force production by vascular smooth muscle (18, 22, 37). The specific mechanisms underlying PKC-induced sensitization of the contractile process in pressurized rat cerebral arteries, as shown in present study, remain to be elucidated. Finally, it is worth noting that smooth muscle [Ca2+]i in unstimulated cerebral arteries maintained at a pressure well below the myogenic range (10 mmHg) was 74 ± 5 nM. As evident from Fig. 4, 60 mM potassium solution induced nearly maximal constriction, and the calculated concentration of [Ca2+]i was ~400 nM. In our previous paper (14), and also in the present study, Ca2+ in concentrations below 100 nM did not induce any measurable constriction in permeabilized cerebral arteries, and near-maximal constriction was observed at Ca2+ concentrations of 300-400 nM (Fig. 7). Therefore, the levels of Ca2+ in intact pressurized cerebral arteries at different levels of constriction are in good agreement with the data obtained in vessels that were permeabilized with-toxin. The Ca2+-diameter
curve was quite steep in both preparations, such that the entire range
of constriction occurred over a relatively narrow range of
Ca2+ concentrations (100-300
nM). The shift in the
Ca2+-diameter response of
permeabilized vessels as a function of Indo-V and DOG concentration is
shown in Fig. 7 as well, for comparison with data from intact arteries.
Although arterial sensitivity to
Ca2+, in terms of constriction,
was similar in intact vs. permeabilized arteries, the
Ca2+ sensitivity of PKC activation
may differ. The Ca2+ requirement
for PKC-induced constriction in intact arteries appeared to be somewhat
higher than that of permeabilized arteries. Nevertheless, the
constriction induced by PKC activators required intracellular Ca2+ in both cases [the
present study and also our previous study (14)]. The minimal
level of
[Ca2+]i
necessary to observe an indolactam effect in permeabilized arteries was
between 60 and 100 nM (14), whereas it was between 100 and 140 nM in
intact arteries. Because the relationship between arterial diameter and
[Ca2+]i
was quite steep (see Fig. 7), small changes in a number of factors
could contribute to this apparent difference. For example, during
permeabilization, the artery was bathed in high-potassium, Ca2+-free EGTA- and
-toxin-containing solution for 30 min, and the [Ca2+]i
was manipulated by changing bath
Ca2+. If this procedure slightly
altered the degree of PKC activation, the
Ca2+ dependency of PKC activation,
any of the targets of PKC activation, or the dephosphorylation of the
target, this could change the apparent
Ca2+ requirement of PKC activation.
In summary, PKC activation augments cerebral artery constriction by
significantly increasing the Ca2+
sensitivity of the contractile apparatus in cerebral vascular smooth
muscle. In intact vessels, this effect is accompanied by a marked
decrease in smooth muscle
[Ca2+]i
through as-yet-unknown mechanisms. These appear to be independent of
membrane potential and most likely involve a combination of inhibition
of voltage-dependent Ca2+ channels
and stimulation of Ca2+ extrusion
from the cytosol.
| |
ACKNOWLEDGEMENTS |
|---|
This study was supported by Grant-in-Aid 93014090 (G. Osol) and 9860037T (H. J. Knot) from the American Heart Association and National Heart, Lung, and Blood Institute Grants HL-44455 (M. T. Nelson) and HL-51728 (G. Osol).
| |
FOOTNOTES |
|---|
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: N. I. Gokina, Dept. of Obstetrics and Gynecology, The Univ. of Vermont, College of Medicine, Burlington, VT 05405 (E-mail: gokina{at}salus.med.uvm.edu).
Received 16 December 1998; accepted in final form 19 May 1999.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Aiello, E. A.,
O. Clement-Chomienne,
D. P. Sontag,
M. P. Walsh,
and
W. C. Cole.
Protein kinase C inhibits delayed rectifier K+ current in rabbit vascular smooth muscle cells.
Am. J. Physiol.
271 (Heart Circ. Physiol. 40):
H109-H119,
1996
2.
Andrews, M. A.,
D. W. Maughan,
T. M. Nosek,
and
R. E. Godt.
Ion-specific and general ionic effects on contraction of skinned fast-twitch skeletal muscle from the rabbit.
J. Gen. Physiol.
98:
1105-1125,
1991
3.
Bonev, A. D.,
and
M. T. Nelson.
Vasoconstrictors inhibit ATP-sensitive K+ channels in arterial smooth muscle through protein kinase C.
J. Gen. Physiol.
108:
315-323,
1996
4.
Brayden, J. E.,
and
M. T. Nelson.
Regulation of arterial tone by activation of calcium-dependent potassium channels.
Science
256:
532-535,
1992
5.
Clark, A. H.,
and
C. J. Garland.
5-Hydroytryptamine-stimulated accumulation of 1,2-diacylglycerol in the rabbit basilar artery: a role for protein kinase C in smooth muscle contraction.
Br. J. Pharmacol.
102:
415-421,
1991[Medline].
6.
Clement-Chomienne, O.,
M. P. Walsh,
and
W. C. Cole.
Angiotensin II activation of protein kinase C decreases delayed rectifier K+ current in rabbit vascular myocytes.
J. Physiol. (Lond.)
495:
689-700,
1996[Medline].
7.
D'Angelo, G.,
and
G. A. Meininger.
Transduction mechanisms involved in the regulation of myogenic activity.
Hypertension
23:
1096-1105,
1994
8.
Ferrer, M.,
A. Encabo,
J. Marin,
C. Peiro,
J. Redondo,
M. R. de Sagarra,
and
G. Balfagon.
Comparison of the vasoconstrictor responses induced by endothelin and phorbol 12,13-dibutyrate in bovine cerebral arteries.
Brain Res.
599:
186-196,
1992[Medline].
9.
Fish, R. D.,
G. Sperti,
W. S. Colucci,
and
D. E. Clapham.
Phorbol ester increases the dihydropyridine-sensitive calcium conductance in a vascular smooth muscle cell line.
Circ. Res.
62:
1049-1054,
1988
10.
Fujiki, H.,
M. Suganuma,
M. Nakayasu,
T. Tahira,
Y. Endo,
K. Shudo,
and
T. Sugimura.
Structure-activity studies on synthetic analogues (indolactams) of the tumor promoter teleocidin.
GANN
75:
866-870,
1984
11.
Furukawa, K.-I.,
Y. Tawada,
and
M. Shigekawa.
Protein kinase C activation stimulates plasma membrane Ca2+ pump in cultured vascular smooth muscle cells.
J. Biol. Chem.
264:
4844-4849,
1989
12.
Galizzi, J.-P.,
J. Qar,
M. Fosset,
C. Van Renterghem,
and
M. Lazdunski.
Regulation of calcium channels in aortic muscle cells by protein kinase C activators (diacylglycerol and phorbol esters) and by peptides (vasopressin and bombesin) that stimulate phosphoinositide breakdown.
J. Biol. Chem.
262:
6947-6950,
1987
13.
Geiges, D.,
T. Meyer,
B. Marte,
M. Vanek,
G. Weissgerber,
S. Stabel,
J. Pfeilschifter,
D. Fabbro,
and
A. Huwiler.
Activation of protein kinase C subtypes alpha, gamma, delta, epsilon, zeta, and eta by tumor-promoting and nontumor-promoting agents.
Biochem. Pharmacol.
53:
865-875,
1997[Medline].
14.
Gokina, N. I.,
and
G. Osol.
Temperature and protein kinase C modulate myofilament Ca2+ sensitivity in pressurized rat cerebral arteries.
Am. J. Physiol.
274 (Heart Circ. Physiol. 43):
H1920-H1927,
1998
15.
Grynkiewicz, G.,
M. Poenie,
and
R. Y. Tsien.
A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J. Biol. Chem.
260:
3440-3450,
1985
16.
Harder, D. R.
Pressure-dependent membrane depolarization in cat middle cerebral artery.
Circ. Res.
55:
197-202,
1984
17.
Hill, M. A.,
M. J. Davis,
J. Song,
and
H. Zou.
Calcium dependence of indolactam-mediated contractions in resistance vessels.
J. Pharmacol. Exp. Ther.
276:
867-874,
1996
18.
Horowitz, A.,
C. B. Menice,
R. Laporte,
and
K. G. Morgan.
Mechanisms of smooth muscle contraction.
Pharmacol. Rev.
76:
967-1003,
1996.
19.
Karibe, A.,
J. Watanabe,
S. Horiguchi,
M. Takeuchi,
S. Suzuki,
M. Funakoshi,
H. Katoh,
M. Keitoku,
S. Satoh,
and
K. Shirato.
Role of cytosolic Ca2+and protein kinase C in developing myogenic contraction in isolated rat small arteries.
Am. J. Physiol.
272 (Heart Circ. Physiol. 41):
H1165-H1172,
1997
20.
Kazanietz, M. G.,
L. B. Areces,
A. Bahador,
H. Mischak,
J. Goodnight,
J. F. Mushinski,
and
P. M. Blumberg.
Characterization of ligand and substrate specificity for the calcium-dependent and calcium-independent protein kinase C isozymes.
Mol. Pharmacol.
44:
298-307,
1993[Abstract].
21.
Knot, H. J.,
and
M. T. Nelson.
Regulation of arterial diameter and wall [Ca2+] in cerebral arteries of rat by membrane potential and intravascular pressure.
J. Physiol. (Lond.)
508:
199-209,
1998
22.
Lee, M. W.,
and
D. L. Severson.
Signal transduction in vascular smooth muscle: diacylglycerol second messengers and PKC action.
Am. J. Physiol.
267 (Cell Physiol. 36):
C659-C678,
1994
23.
Matsui, T.,
M. Sugawa,
H. Johshita,
Y. Takuwa,
and
T. Asano.
Activation of the protein kinase C-mediated contractile system in canine basilar artery undergoing chronic vasospasm.
Stroke
22:
1183-1187,
1991
24.
McCarron, J. G.,
C. A. Crichton,
P. D. Langton,
A. MacKenzie,
and
G. L. Smith.
Myogenic contraction by modulation of voltage-dependent calcium currents in isolated rat cerebral arteries.
J. Physiol. (Lond.)
498:
371-379,
1997[Medline].
25.
Minami, K.,
K. Fukuzawa,
and
Y. Nakaya.
Protein kinase C inhibits the Ca2+-activated K+ channel of cultured porcine coronary artery smooth muscle cells.
Biochem. Biophys. Res. Commun.
190:
263-269,
1993[Medline].
26.
Murray, M. A.,
F. M. Faraci,
and
D. D. Heistad.
Signal transduction pathways in constriction of the basilar artery in vivo.
Hypertension
19:
739-742,
1992
27.
Nishizawa, S.,
N. Nezu,
and
K. Uemura.
Direct evidence for a key role of protein kinase C in the development of vasospasm after subarachnoid hemorrhage.
J. Neurosurg.
76:
635-639,
1992[Medline].
28.
Osol, G.,
and
W. Halpern.
Myogenic properties of cerebral blood vessels from normotensive and hypertensive rats.
Am. J. Physiol.
249 (Heart Circ. Physiol. 18):
H914-H921,
1985
29.
Osol, G.,
I. Laher,
and
M. Cipolla.
Protein kinase C modulates basal myogenic tone in resistance arteries from the cerebral circulation.
Circ. Res.
68:
359-367,
1991
30.
Osol, G.,
R. Osol,
and
W. Halpern.
Effects of diltiazem on myogenic tone in pressurized brain arteries from spontaneously hypertensive rats.
In: Essential Hypertension, Calcium Mechanism and Treatment, edited by K. Aoki. Tokyo: Springer, 1986, p. 107-114.
31.
Schuhmann, K.,
and
K. Groschner.
Protein kinase-C mediates dual modulation of L-type Ca2+ channels in human vascular smooth muscle.
FEBS Lett.
341:
208-212,
1994[Medline].
32.
Shearman, M. S.,
K. Sekiguchi,
and
Y. Nishizuka.
Modulation of ion channel activity: a key function of the protein kinase C enzyme family.
Pharmacol. Rev.
41:
211-237,
1989[Abstract].
33.
Somlyo, A. P.,
and
A. V. Somlyo.
Signal transduction and regulation in smooth muscle.
Nature
372:
231-236,
1994[Medline].
34.
Sugawa, M.,
T. Koide,
S. Naitoh,
M. Takato,
T. Matsui,
and
T. Asano.
Phorbol 12,13-diacetate-induced contraction of the canine basilar artery: role of protein kinase C.
J. Cereb. Blood Flow Metab.
11:
135-142,
1991[Medline].
35.
Tanaka, Y.,
H. Ishiro,
T. Nakazawa,
M. Saito,
K. Ishii,
and
K. Nakayama.
Potentiation by endothelin-1 of Ca2+sensitivity of contractile elements depends on Ca2+ influx through L-type Ca2+ channels in the canine cerebral artery.
Gen. Pharmacol.
26:
855-864,
1995[Medline].
36.
Vigne, P.,
J.-P. Breittmayer,
D. Duval,
C. Frelin,
and
M. Lazdunski.
The Na+/Ca2+ antiporter in aortic smooth muscle cells: characterization and demonstration of an activation by phorbol esters.
J. Biol. Chem.
263:
8078-8083,
1988
37.
Walsh, M. P.,
A. Horowitz,
O. Clement-Chomienne,
J. E. Andrea,
B. G. Allen,
and
K. G. Morgan.
Protein kinase C mediation of Ca2+-independent contractions of vascular smooth muscle.
Biochem. Cell Biol.
74:
485-502,
1996[Medline].
This article has been cited by other articles:
|
|
 |
|
  C. A. Cobine, B. P. Callaghan, and K. D. Keef Role of L-type calcium channels and PKC in active tone development in rabbit coronary artery Am J Physiol Heart Circ Physiol, June 1, 2007; 292(6): H3079 - H3088. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Adebiyi, G. Zhao, S. Y. Cheranov, A. Ahmed, and J. H. Jaggar Caveolin-1 abolishment attenuates the myogenic response in murine cerebral arteries Am J Physiol Heart Circ Physiol, March 1, 2007; 292(3): H1584 - H1592. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Ito, Y. P. R. Jarajapu, M. B Grant, and H. J Knot Characteristics of myogenic tone in the rat ophthalmic artery Am J Physiol Heart Circ Physiol, January 1, 2007; 292(1): H360 - H368. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Xiao, J. N. Buchholz, and L. Zhang Pregnancy attenuates uterine artery pressure-dependent vascular tone: role of PKC/ERK pathway Am J Physiol Heart Circ Physiol, June 1, 2006; 290(6): H2337 - H2343. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Ding and P. A. Murray Cellular mechanisms of thromboxane A2-mediated contraction in pulmonary veins Am J Physiol Lung Cell Mol Physiol, November 1, 2005; 289(5): L825 - L833. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. P. R. Jarajapu and H. J. Knot Relative contribution of Rho kinase and protein kinase C to myogenic tone in rat cerebral arteries in hypertension Am J Physiol Heart Circ Physiol, November 1, 2005; 289(5): H1917 - H1922. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. I. Gokina, K. M. Park, K. McElroy-Yaggy, and G. Osol Effects of Rho kinase inhibition on cerebral artery myogenic tone and reactivity J Appl Physiol, May 1, 2005; 98(5): 1940 - 1948. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Zhang, K. Rhinehart, W. Kwon, E. Weinman, and T. L. Pallone ANG II signaling in vasa recta pericytes by PKC and reactive oxygen species Am J Physiol Heart Circ Physiol, August 1, 2004; 287(2): H773 - H781. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. P. V. Dantas, J. Igarashi, and T. Michel Sphingosine 1-phosphate and control of vascular tone Am J Physiol Heart Circ Physiol, June 1, 2003; 284(6): H2045 - H2052. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Y. Su, K. M. Reber, C. A. Nankervis, and P. T. Nowicki Development of the myogenic response in postnatal intestine: role of PKC Am J Physiol Gastrointest Liver Physiol, March 1, 2003; 284(3): G445 - G452. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. P. Massett, Z. Ungvari, A. Csiszar, G. Kaley, and A. Koller Different roles of PKC and MAP kinases in arteriolar constrictions to pressure and agonists Am J Physiol Heart Circ Physiol, December 1, 2002; 283(6): H2282 - H2287. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Lagaud, N. Gaudreault, E. D. W. Moore, C. van Breemen, and I. Laher Pressure-dependent myogenic constriction of cerebral arteries occurs independently of voltage-dependent activation Am J Physiol Heart Circ Physiol, December 1, 2002; 283(6): H2187 - H2195. [Abstract] [Full Text] [PDF]
|
|
|
|
, regular physiological salt solution at 60 mmHg)
resulting from the exposure of intact arteries to either DOG (
, 10 µM) or Indo-V (
, 3 µM), illustrating the similarity between
intact vs. permeabilized vessels. (Note that outer diameter values from
intact arteries were corrected by subtracting average vessel wall
thickness to enable comparisons with luminal diameter means obtained in
experiments with 