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Center for Perinatal Biology, Departments of Physiology, Pharmacology and Obstetrics and Gynecology, Loma Linda University, School of Medicine, Loma Linda, California, 92350
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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This study tested the hypothesis that
protein kinase C (PKC) has dual regulation on norepinephrine
(NE)-mediated inositol 1,4,5-trisphosphate [Ins
(1,4,5)P3] pathway and vasoconstriction in
cerebral arteries from near-term fetal (~140 gestational
days) and adult sheep. Basal PKC activity values (%membrane
bound) in fetal and adult cerebral arteries were 38 ± 4% and
32 ± 4%, respectively. In vessels of both age groups, the PKC
isoforms 
, 
I, II, and 
were
relatively abundant. In contrast, compared with the adult, cerebral
arteries of the fetus had low levels of PKC-
. In response to
10
4 M phorbol 12,13-dibutyrate (PDBu; PKC agonist), PKC
activity in both fetal and adult cerebral arteries increased
40-50%. After NE stimulation, PKC activation with PDBu exerted
negative feedback on Ins(1,4,5)P3 and
intracellular Ca2+ concentration
([Ca2+]i) in arteries of both age groups. In
turn, PKC inhibition with staurosporine resulted in augmented
NE-induced Ins(1,4,5)P3 and [Ca2+]i responses in adult, but not fetal,
cerebral arteries. In adult tissues, PKC stimulation by PDBu increased
vascular tone, but not [Ca2+]i. In contrast,
in the fetal artery, PKC stimulation was associated with an increase in
both tone and [Ca2+]i. In the presence of
zero extracellular [Ca2+], these PDBu-induced
responses were absent in the fetal vessel, whereas they remained
unchanged in the adult. We conclude that, although basal PKC activity
was similar in fetal and adult cerebral arteries, PKC's role in
NE-mediated pharmacomechanical coupling differed significantly in the
two age groups. In both fetal and adult cerebral arteries, PKC
modulation of NE-induced signal transduction responses would appear to
play a significant role in the regulation of vascular tone. The
mechanisms differ in the two age groups, however, and this probably
relates, in part, to the relative lack of PKC- in fetal vessels.
cerebrovasculature; smooth muscle; signal transduction; adrenergic; norepinephrine; protein kinase C; protein kinase C isoenzymes; inositol 1,4,5-trisphosphate; intracellular Ca2+, Ca2+ channels; vascular tone; fetus; development
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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REGULATION OF
THE CEREBRAL CIRCULATION and the mechanistic basis for
differences in cerebrovascular contractility in different vessels,
although of critical importance in health and disease, involves
multiple factors of the signal-transduction cascade, the interrelations
of which are poorly understood (15). In addition to
mobilization of inositol 1,4,5-trisphosphate
[Ins(1,4,5)P3] via G proteins and
phospholipase C- (PLC), noradrenergic stimulation of
1-adrenergic receptors (AR) is associated with synthesis
of diacylglycerol (DAG), which results in activation of protein kinase C (PKC) (29). Members of the PKC family of isoenzymes play
a key regulatory role in a variety of cell functions including cell growth and differentiation, gene expression, membrane function, and so
forth (29). In cerebral and other blood vessels, PKC has
been shown to play a dual role in the regulation of tone. By negative
feedback, PKC may inhibit PLC, thereby attenuating the agonist-induced
increases in Ins(1,4,5)P3, intracellular
Ca2+ concentration ([Ca2+]i), and
contraction (2, 27). In addition, PKC activation per se
can increase vascular tone (6, 31, 34). PKC may also
modulate vascular smooth muscle Ca2+ sensitivity to
-adrenergic and other agonists (28). Nonetheless, the
role of PKC-mediated mechanisms in cerebral vessels and how these
change with development from fetus to adult is unclear.
To test the hypothesis that in fetal cerebral arteries, the dual role of PKC activation in modulating norepinephrine (NE)-induced stimulation of Ins(1,4,5)P3, [Ca2+]i, and [Ca2+]i-independent increases in vascular tone differs significantly from that of the adult, we performed the following studies. In fetal and adult cerebral arteries, we quantified PKC in both absolute amounts and as percent membrane bound (active form) under basal conditions and after PKC stimulation or inhibition. We also measured relative levels of the several PKC isoforms in adult and fetal cerebral arteries. We also quantified Ins(1,4,5)P3 levels in response to PKC stimulation or inhibition. In addition, in middle cerebral arteries, we simultaneously measured vascular tension and [Ca2+]i responses to NE after PKC activation or inhibition.
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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 cerebral arteries from near-term fetal
(~140 days) and nonpregnant adult sheep (
2 yr) obtained from
Nebeker Ranch (Lancaster, CA). The ewes were anesthetized and killed
with 100 mg/kg intravenous pentobarbital sodium, after which we
obtained anterior, middle, and posterior cerebral arteries or, in the
case of tension and [Ca2+]i measurements, the
main branch middle cerebral artery (MCA). For comparison with an
extracranial vessel, for some studies, we also obtained segments of
common carotid artery (CCA). We have shown that this method of
death has no significant effect on vessel reactivity compared with the
use of other anesthetic agents (23). Studies were
performed in isolated vessels cleaned of adipose and connective tissue,
as previously described (23, 24). To avoid the
complications of endothelial-mediated effects, we removed the
endothelium by carefully inserting a small wire three times (23,
24).
Measurement of PKC.
Arteries (~30 mg wet wt) were placed in ice-cold 50 mM
Tris · HCl (pH 7.4) and exposed to various treatments according
to the protocol. The reaction was terminated by freezing in liquid N2, and the tissues were homogenized in 2 ml
Tris · HCl buffer containing (in mM) 50 Tris · HCl, 5 EDTA, 10 EGTA, 10 benzamidine, 0.3% -mecaptoethanol, and 50 µg/ml
phenylmethylsulfonyl fluoride (pH 7.5) with a glass grinder. The
homogenate was centrifuged at 100,000 g for 30 min. Assay of
the supernatant was defined as cytoplasmic PKC activity. Membrane-bound
PKC activity was extracted from the pellet by resuspension in 20 mM
Tris · HCl (pH 7.4) containing 1% Triton X-100 and assayed in
the supernatant after recentrifugation. Proteins in both the membrane
and cytosolic fractions were measured by the Bradford method
(4). PKC activity was determined by an assay (Amersham)
based on PKC-catalyzed transfer of the 
-phosphate group of ATP to a
PKC-specific peptide. Samples were incubated with a PKC assay mixture
and 32P-labeled ATP for 20 min at 37°C. Aliquots of the
mixture were spotted on paper discs, washed two times with 75 mM
orthophosphoric acid, and the discs were counted for 2 min in 10 ml
scintillation fluid. Data were expressed as picomoles of
PO4 incorporated per minute per milligram of protein. The
intra- and interassay coefficients of variation for the assay were 4%
and 3%, respectively.
7 to 104 M) for 10 min [in some cases we
repeated the study with the more selective PKC activator indolactam V
(Indo), 107 to 106 M]. The reaction was
terminated by quickly freezing the tissue in liquid N2, and
it was analyzed for PKC activity. To examine the NE dose-PKC response,
arteries were prepared and pretreated 30 min before the study with
107 M yohimbine to block 2-ARs and
106 M propranolol to block -ARs. First, we determined
the NE (107 to 104 M) dose-PKC response
relationship, rapidly freezing the tissue and quantifying PKC after a
given NE dose. We also examined the response to NE plus
104 M PDBu or 106 M Indo concentration
(which we have shown gives peak response). To examine PKC responses in
the presence of the PKC inhibitors staurosporine (Sta;
107 M) or calphostin C (Cal; 107 M)
administered 10 min before the study, we repeated the NE
(107 to 104 M) stimulation in the absence
or presence of these agents.
Immunoblotting of PKC isoenzymes.
Fetal and adult sheep cerebral arteries were first isolated, cleaned in
the Krebs buffer, and then frozen in liquid nitrogen. Frozen samples
were homogenized in liquid N2 with a porcelain mortar and
pestle. Homogenized samples then were incubated for 10 min in the
lysing buffer [20 mM Tris · HCl, 1 mM EDTA, 1 ug/ml pepstatin, 1 ug/ml leupeptin, 1 ug/ml aprotinin, 0.1 mg/ml
benzamidine, and 8 ug/ml calpain inhibitors I and II (pH 7.4)]. Nuclei
and debris were pelleted by centrifugation at 1,000 g for 20 min. The whole cell lysate was then stored at 
20°C. The pellet
(membrane-bound protein) was resuspended in the lysing buffer,
sonicated, and stored at 20°C.
, -I, -II, -, -, etc., and
-tubulin as an internal control and the -tubulin blocking peptide
as a negative control; Santa Cruz Biotechnology, Santa Cruz, CA) in
blotting solution at room temperature (22°C) followed by a wash (3 times for 10 min each with TBS, 0.1% Tween 20, and once for 5 min with
TBS). For each isoform, the controls were as follows: Jurkat whole cell lysate (25 µg/lane), PKC-, -I, -II, -, -
, -µ; rat
heart (25 µg/lane), PKC-; rat brain, PKC-
and -
; 3611-RF
whole cell lysate (25 µg/lane), PKC- and -
(Santa Cruz
Biotechnology). These several antibodies have been shown to
react with the specific PKC isoforms in a number of mammalian tissues.
Incubation with horseradish peroxidase and a conjugated secondary
antibody (Santa Cruz Biotechnology) for 1 h at room temperature at
a 1:2,000 dilution in blotting solution was followed by a wash (3 times
for 10 min each with TBS, 0.1% Tween 20, and once for 5 min with TBS).
The membrane was incubated in chemiluminescence luminol reagent (Santa
Cruz Biotechnology) for 1 min, and the protein band was detected by using Hyperfilm (Amersham Life Science, Arlington Heights, IL). In an
attempt to maintain the immunoblotting labeling conditions constant, we
used the same titer of polyclonal anti-PKC antibodies and the same
protein concentration in all samples. The polyclonal antibody titers
and protein concentrations were chosen on the basis of preliminary
experiments to determine optimal conditions on the linear portion of
the titration curve.
Ins(1,4,5)P3 quantification. From each fetal and adult animal, cleaned cerebral arteries were cut into segments weighing ~20 mg (wet wt) and placed in Krebs buffer and bubbled with 95% O2-5% CO2 for 0.5 h. The Krebs buffer contained (in mM) 115.2 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 22.1 NaHCO3, 7.9 Dextrose, 0.03 EDTA, and 1.8 CaCl2 (pH 7.4). We exposed the arteries to various treatments outlined in Measurement of PKC (PKC stimulation or inhibition) at 37°C according to the protocol. The reactions to the various treatments were terminated by freezing in liquid N2, and frozen samples were homogenized in 2 ml iced 16% trichloracetic acid with a glass grinder. The homogenate was centrifuged for 30 min at 1,500 g, the supernatant transferred, and the pellet resuspended in 1 M NaOH for protein measurements (4). The supernatant was washed for 30 s with H2O-saturated ether (5× volume, 2 times). After ether evaporation, Ins(1,4,5)P3 mass was determined by competitive ligand binding assay in which a radioactive ligand competes with a nonradioactive ligand for a fixed number of receptor binding sites. Quantification was achieved by determining the radioactivity in 5 ml scintillation fluid for 5 min in a scintillation counter. [3H]Ins(1,4,5)P3 assay kits were obtained from DuPont (Boston, MA). Our intra- and interassay coefficients of variation were 7% and 9%, respectively.
PKC-Ins(1,4,5)P3 interaction.
To examine the role of PKC negative feedback on
Ins(1,4,5)P3 response, in arteries from
fetuses and adults prepared as above and 30 min before the study
pretreated with 107 M yohimbine to block
2-ARs, 106 M propranolol to block -ARs,
we quantified both basal PKC activity and
Ins(1,4,5)P3 concentration. In other vessels,
we administered 104 M PDBu 10 min before the study, then
administered 107 to 104 M NE, and, in
45 s terminated the reaction and quantified
Ins(1,4,5)P3. We have shown this to be the
optimal time to quantify the Ins(1,4,5)P3 response in both adult (24) and fetal (23)
arteries. To explore PKC inhibition on
Ins(1,4,5)P3 generation, we repeated the above study in the presence of 107 M Sta or 107 M
Cal administered 30 min before NE stimulation. Then, 45 s after administering NE, we stopped the reaction and measured
Ins(1,4,5)P3.
Simultaneous measurement of tension and [Ca2+]i. We cut the fetal or adult MCAs into rings of 2 mm in length, mounted them on two Tungsten wires (0.13-mm diameter; A-M Systems, Carlsborg, WA), attaching 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 a Jasco CAF-110 Intracellular Ca2+ Analyzer (Jasco, Easton, MD). The tension value along with vessel inside diameter, wall thickness, length, and potassium-induced force enabled calculation of force per unit cross-sectional area, as previously described (32). 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. Mean cytoplasmic [Ca2+]i depends on where fura 2 is located (9). Fura 2 fluorescence and force were measured simultaneously at 38°C, as previously described by Long et al. (22). 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. 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 EC50 or half-maximal concentration for NE and an index of tissue "sensitivity" or "potency") (22). As previously reported, although in absolute terms, the maximal values of fetal MCA K+- and NE-induced tension are 20-30% less than those of the adult (22, 32), expressing these in terms of Kmax helps to normalize the data and does not alter the interpretation of the results.
In arteries used for response to PKC stimulation (PDBu or Indo) or inhibition (Sta or Cal) after the NE dose response, we added the pharmacological agent and repeated the NE dose response. In studies with PKC stimulation, we examined this in two ways. After the unblocked NE-induced response, we repeated the dose response after PKC stimulation. Then, in a second study, we administered 105
M NE to achieve near-maximal response, at which time we gave PDBu in
increasing doses to follow the [Ca2+]i and
tension-inhibition response. In a separate series of studies, we
examined PDBu- and Indo-induced vascular contractility per se.
Role of extracellular Ca2+.
To determine the role of extracellular Ca2+ concentration
([Ca2+]o) in PKC-mediated responses, we
measured [Ca2+]i and tension in response to
NE or PKC stimulation after vessels had been exposed to calcium-free
(<108 M Ca2+ with 5 × 104 M EGTA to chelate Ca2+) Krebs buffer for
2 min (n = 4). Again, we evaluated the contractile response of tension and fluorescence ratio by measuring the maximum peak height and expressing it as percent Kmax
(22).
Statistical analysis.
All values were calculated as means ± SE. In all cases,
n values refer to the number of vessels (which corresponds
to the number of animals and is included in Table
1) for a particular study. Because of the
nature of these studies, several statistical tests were used to test
for significant differences. For testing differences between two
groups, a simple unpaired Student's t-test was used. For
multiple comparisons, one- and two-way ANOVA (vessel, age) coupled with
Duncan's multiple-range test was used. When 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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Basal PKC values.
Table 1 gives the basal levels of PKC activity in adult and fetal
cerebral arteries, both as total absolute value (cytosolic + membrane bound; pmol · min1 · mg
protein1) and as the percent membrane bound (i.e., the
active form). PKC values in both age groups were similar, whether
expressed as total PKC (adult and fetal were 204 ± 17 and
239 ± 31, respectively) or as percent membrane bound (32 ± 4% and 38 ± 4%, respectively; Table 1). By way of comparison,
in contrast to these values that showed no significant difference
between adult and fetus, in CCA, the values were significantly
different; namely, total PKC for adult and fetal CCA were 532 ± 32 and 215 ± 13, respectively (P < 0.01;
n = 12), whereas the percent membrane-bound values were 28 ± 2% and 52 ± 4%, respectively (P < 0.01).
PKC isoenzymes.
To determine the extent to which the relative expression of different
PKC isoenzymes may account for differences in responses, in fetal and
adult cerebral arteries, we measured these isoforms under basal
conditions. Figure 1 illustrates the PKC
isoenzyme profile in whole cell extracts that were subjected to Western analysis using enhanced chemiluminescence detection, as described in
METHODS. As seen in Fig. 1, adult ovine cerebral arteries
appear to express relatively abundant amounts of both the "classic"
or "group A" isoforms [PKC- (2+), -I
(2+), and -II (1+)] and "novel" or "class B"
isoforms [PKC- (3+) and - (1+)]. In contrast, whereas in fetal
cerebral arteries, immunoblotting shows PKC- (2+), -I (2+), -II (1+), and - (2+), these vessels demonstrate
low levels of the PKC- (1+) isoform. Immunoblots of -tubulin are
shown as an internal control.
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PKC and its activation.
Table 1 also presents adult and fetal cerebral artery PKC activity as
percent membrane bound in response to several treatments. For instance,
in response to 104 M NE, whereas total PKC did not change
significantly from control, the percent membrane bound increased to
44 ± 5% and 51 ± 6% for adult and fetus, respectively
(P < 0.01 for each). In adult and fetal cerebral
arteries, 104 M PDBu increased PKC activity to 50 ± 6% and 66 ± 8%, respectively (P < 0.01 for
each). Administration of 104 M NE after 104
M PDBu did not alter the fetal and adult PKC values compared with PDBu
alone (Table 1). Administration of the more selective PKC activator
Indo (106 M) resulted in PKC increases similar to those
seen with 104 M PDBu. Under these conditions of
stimulation, the total PKC values
(pmol · min1 · mg protein1)
in adult or fetal cerebral arteries did not change significantly from
control values. In adult and fetal cerebral arteries, 107
M Sta had no significant effect on PKC activity (34 ± 6% and 29 ± 4%, respectively). The results were similar after
administration of the more selective inhibitor Cal (107
M; Table 1). In contrast, administration of 106 or
105 M Sta decreased total PKC precipitously to 91 ± 14 and 102 ± 20 pmol · min1 · mg
protein1, respectively (P < 0.01 for
each; n = 5).
4 M PDBu
stimulation increased PKC activity 68% (to 47 ± 5% from 28 ± 2%; n = 15; P < 0.01), it had no
effect on the fetal vessel (51 ± 6%, n = 5).
Again, with PKC inhibition by 106 M Sta, the total PKC
value of adult and fetal CCA fell 60-80% to 205 ± 30 and
40 ± 8 pmol · min1 · mg
protein1, respectively from control (P < 0.01 for each; n = 5).
PKC-Ins(1,4,5)P3 interaction.
To examine the role of PKC activation on the NE-induced
Ins(1,4,5)P3 increase, we measured this
response under several conditions. As shown in Fig.
2A, in cerebral arteries of
the adult, NE stimulation resulted in a significant dose-dependent
104% increase in Ins(1,4,5)P3 concentration
from 23 ± 4 to 47 ± 5 pmol/mg protein (n = 7 each point; pD2 = 5.2 ± 0.1). The response to
the selective 1-AR agonist phenylephrine was similar.
With 104 M PDBu pretreatment to stimulate PKC activity,
Ins(1,4,5)P3 concentrations were below basal
control values, e.g., 8-12 pmol/mg protein, and failed to respond
to NE (n = 5; Fig. 2A). In a similar manner after pretreatment with 106 M Indo,
Ins(1,4,5)P3 did not respond to NE
stimulation. In contrast, with 107 M Sta pretreatment to
inhibit PKC activity, NE stimulation resulted in a robust increase of
Ins(1,4,5)P3 concentration as NE dose increased (for instance, to 53 ± 2 pmol/mg protein at 3 × 106 M NE), with an increase of pD2 to
6.1 ± 0.1 (n = 5; Fig. 2A). In a
similar manner, pretreatment with 107 M Cal
resulted in increased sensitivity of
Ins(1,4,5)P3 to NE stimulation that was not
significantly different from that seen in the presence of Sta.
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4 M PDBu
pretreatment, Ins(1,4,5)P3 concentrations were
below the basal control value, e.g., 8-12 pmol/mg protein, with no
increase in response to NE. In contrast to the adult vessels, however, 107 M Sta pretreatment followed by NE stimulation
resulted in an increase of Ins(1,4,5)P3
concentration not significantly different from that of NE alone
(n = 5 each point; Fig. 2B).
By way of comparison with an extracranial vessel, in adult CCA,
Ins(1,4,5)P3 showed a dose-dependent rise in
response to increasing NE concentration from a basal value of 20 ± 2 to 37 ± 3 pmol/mg protein (85% increase at
104 M; n = 6 for each point;
pD2 = 5.2 ± 0.1). As with the cerebral arteries,
when adult CCA was pretreated with 104 M PDBu, basal
Ins(1,4,5)P3 decreased to 12 ± 2 pmol/mg
protein and failed to increase in response to 104 M NE.
As with the adult cerebral arteries, when adult CCA was pretreated with
107 M Sta, Ins(1,4,5)P3
increased 42% above control to 34 ± 3 pmol/mg protein at 3 × 106 M NE (pD2 = 5.6 ± 0.1;
n = 5). In contrast to the cerebral arteries and adult
CCA, fetal CCA failed to show a significant increase in
Ins(1,4,5)P3 in response to NE stimulation
from its basal value of 21 ± 2 pmol/mg protein. However, in a
manner similar to the other vessels, with 104 M PDBu
pretreatment, Ins(1,4,5)P3 concentrations were
below basal values and failed to respond to NE. In contrast to the
cerebral arteries, after 107 M Sta pretreatment of the
fetal CCA, NE stimulation resulted in a robust elevation of
Ins(1,4,5)P3 concentration to 45 ± 6 pmol/mg protein at 3 × 106 M NE (n = 5; pD2 = 5.8 ± 0.1), suggesting release of
PKC-mediated inhibition.
PKC-[Ca2+]i
interaction.
To examine the effect of PKC activation on NE-mediated increase in
[Ca2+]i and tension, we quantified these
responses under several conditions. Figure
3A shows tension as percent
Kmax in adult MCAs in response to increasing NE
concentration (n = 8). The figure also shows the
effects of 107, 106, and 104
M PDBu (e.g., PKC stimulation) on inhibiting the contractile response
(n = 4). As is evident, with 106 and
107 M PDBu, NE responses were attenuated, whereas with
104 M PDBu, there was no response at all. Maximal
tensions, as percent Kmax, at 104 M NE for NE
alone and in the presence of 107, 106, or
104 M PDBu were 91 ± 7, 34 ± 5, 7 ± 1, and 1 ± 0.1%, respectively. Figure 3B shows the adult
MCA fura 2 fluorescence ratio (F340/380), a measure of free
[Ca2+]i, in response to NE. Also shown are
the attenuated [Ca2+]i responses in the
presence of 107 or 106 M PDBu and the lack
of response in the presence of 104 M PDBu. The
F340/380 values as percent Kmax at
104 M NE for NE alone and 107,
106, and 104 M PDBu were 63 ± 4, 38 ± 4, 13 ± 3, and 0.2 ± 0.1%, respectively. The
MCA tension and [Ca2+]i responses to NE in
the presence of 106 M Indo (n = 3) were
similar to those of PDBu.
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7,
106, and 104 M PDBu (n = 4). As is evident, whereas both 106 and 107
M PDBu attenuated the response, 104 M PDBu eliminated it.
Maximal tensions as percent K+-induced tension at
104 M NE for NE alone and in the presence of
107, 106, or 104 M PDBu were
82 ± 7, 38 ± 5, 22 ± 4, and 1 ± 0.1, respectively. Figure 3D shows the fetal MCA fura 2 fluorescence ratio in response to NE. Also shown are attenuated
[Ca2+]i responses in the presence of
107 or 106 M PDBu and the lack of response
in the presence of 104 M PDBu. The F340/380
values as percent Kmax at 104 M NE for NE
alone and 107, 106, and 104 M
PDBu were 76 ± 8, 32 ± 4, 18 ± 3, and 0.2 ± 0.1, respectively. As shown in Fig. 3, the tension (A and
B) and [Ca2+]i (C and
D) responses in fetal MCA did not differ significantly from
those of the adult. As in the adult, fetal MCA responses to NE in the
presence of 106 M Indo (n = 3) were
similar to those of PDBu.
We also examined the role of PKC inhibition on NE-induced increase in
[Ca2+]i and tension in adult and fetal
cerebral arteries. For adult MCA, Fig.
4A shows the NE-induced
tension increase in the presence of NE alone and 108 or
107 M Sta. The markedly enhanced sensitivity is apparent,
i.e., shift to the left of the dose-response curve. The pD2
value in the presence of 107 or 106 M Sta
was 5.9 ± 0.1 for each compared with the control value of
5.5 ± 0.1 (n = 3). Maximal NE-induced tensions of
adult MCA in the presence of Sta were 120 ± 5 and 98 ± 22%
compared with the control value of 91 ± 7%. Figure 4B
shows the adult MCA fluorescence ratio in response to NE alone and in
the presence of 108 or 107 M Sta. In the
presence of Sta, the sensitivity of the NE-induced fluorescence ratio
was increased, with pD2 values of 6.6 ± 0.1 and
6.3 ± 0.1 for 107 or 108 M,
respectively, compared with the control value of 5.8 ± 0.1. The
NE-induced responses in the presence of 107 M Cal
(n = 3) were not significantly different from those of Sta.
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8 or 107 M
Sta. In contrast to responses in the adult, in fetal MCA,
108 M Sta resulted in attenuated NE-induced tension to
~40% Kmax with no shift of the dose-response curve
(n = 5). Pretreatment with 107 M Sta
(n = 3) resulted in essentially no increase in tension in response to NE (Fig. 4C). Figure 4D shows the
fetal MCA fluorescence ratio in response to NE with attenuated response
in the presence of 108 or 107 M Sta. In the
presence of 108 M Sta, the maximal
F340/380 was decreased to 38% Kmax. Again, with 107 M Sta, there was no NE-induced increase in
fluorescence ratio.
PKC-induced contractions.
To explore the role of PKC activation per se in increasing cerebral
artery tone, we examined contractile responses in cerebral arteries to
both PDBu and Indo. In response to 105 M PDBu, adult MCA
tension increased to peak at ~120 s. In contrast, the fluorescence
ratio did not change significantly. Figure
5A summarizes the
dose-response relationship in adult MCA for 108 to
104 M PDBu of both tension and fluorescence ratio as
percent Kmax (n = 4). The role of PDBu in
increasing Ca2+ sensitivity is evident with the
significantly increased tension to ~65% Kmax
(pD2 = 6.0 ± 0.1) with essentially no change in
fluorescence ratio. In response to increasing doses of Indo, tension
also increased similarly to ~50% Kmax with no increase
in [Ca2+]i. Also, after 20 min exposure to
105 M PDBu, neither vascular tension nor
F340/380 increased in response to increasing doses of NE
(109 to 104 M). Again, after ~20 min
steady-state Indo, in response to increasing doses of NE
(109 to 104 M), neither vascular tension
nor fluorescence ratio increased significantly.
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5 M PDBu with an increase in vascular tension similar
to that seen with NE. In contrast to the adult cerebral artery,
however, in the fetus, the [Ca2+]i also
increased significantly. Figure 5C summarizes the fetal MCA
dose-response relationship for 108 to 104 M
PDBu of both tension and fluorescence ratio as percent Kmax (n = 4). Despite the PDBu-induced increase in
[Ca2+]i, the role of PDBu in increasing
Ca2+ sensitivity is evident with the significantly
increased tension to ~65% Kmax (pD2 = 5.8 ± 0.1) with an increased fluorescence ratio to ~30%
Kmax (pD2 = 6.2 ± 0.1). After 20 min
exposure to 105 M PDBu, neither vascular tension
nor F340/380 increased in response to 109 to
104 M NE. Again, after ~20 min steady-state Indo,
increasing doses of NE (109 to 104 M)
failed to increase either vascular tension or fluorescence ratio.
To examine the role of extracellular Ca2+ in
PDBu-stimulated tone, we repeated these studies in the presence of zero
[Ca2+]o. As shown in Fig. 5B, for
the adult MCA, when [Ca2+]o = 0, the
responses of tension and fluorescence ratio (n = 4) were not significantly different from those seen in the presence of 1.8 mM [Ca2+]o (Fig. 5A). In
contrast, in the fetal MCA, the PDBu-stimulated responses in the
presence of zero [Ca2+]o for 2 min, differed
markedly from those in normal Krebs buffer. With
[Ca2+]o equal to zero, the fetal MCA showed
no response of either tension or fluorescence ratio (Fig.
5D; n = 4).
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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 in relation to PKC in cerebral arteries, its role in NE-mediated contraction, and the extent to which this function differs in the fetus compared with the adult. In several respects, the role of PKC in fetal and adult cerebral arteries appears similar, namely in regards to both total PKC and its activity, the increase in PKC activity in response to stimulation by phorbol ester (Table 1), the corresponding decrease in Ins(1,4,5)P3 (Fig. 2), as well as tension and [Ca2+]i (Fig. 3), in response to PKC activation, and PDBu-mediated increase in tension (Fig. 5). In contrast, in other respects, PKC showed significant differences between fetal and adult cerebral arteries, e.g., the several PKC isoenzymes and their relative abundance differed (Fig. 1); with Sta-induced PKC inhibition, fetal cerebral arteries showed no NE-mediated Ins(1,4,5)P3 increase over that seen with NE alone (Fig. 2B), whereas in adult arteries, the Ins(1,4,5)P3 response was markedly augmented with a significant increase in pD2 (Fig. 2A). Also, in fetal MCA, PKC inhibition resulted in profound inhibition of both tension and [Ca2+]i in response to NE (Fig. 4, C and D) compared with the adult MCA, which showed markedly increased sensitivity (e.g., increase in pD2; Fig. 4, A and B). In addition, in adult cerebral arteries, PDBu-induced PKC stimulation significantly increased Ca2+ sensitivity (Fig. 5A), whereas in the fetal artery, although PKC stimulation by PDBu also increased Ca2+ sensitivity somewhat, this was accompanied by a significant increase in [Ca2+]i (Fig. 5C). Finally, although adult cerebral arteries responded to PKC stimulation in the absence of [Ca2+]o (Fig. 5B), arteries of the fetus did not (Fig. 5D).
These results in fetal and adult cerebral arteries are the first to demonstrate the dual role of PKC in modulating the responses of Ins(1,4,5)P3, [Ca2+]i, and tension. For fetal vessels, the data suggest that PKC is more closely linked with modulating Ca2+ flux via plasma membrane Ca2+ channels and perhaps by other non-Ins(1,4,5)P3-related events. The studies also suggest that PKC and its several isoforms play a key role in the regulation of tone in these vessels.
PKC and vascular contraction.
PKC, a phospholipid-dependent serine/threonine protein kinase, is
present in essentially all animal cells including vascular smooth
muscle (18, 29). Molecular cloning and biochemical analysis have shown that the PKC family includes at least 11 isoforms, representing 10 separate gene products in three classes with differing structures of the regulatory domain. "Conventional," classic, or
group A PKCs (, I, II, and
) are activated by cytosolic free Ca2+, diacylglycerol,
and phosphatidylserine (PS), a membrane phospholipid. "Novel,"
"new," or "group B" PKCs (, , 
, and ) are activated by DAG but are Ca2+ independent, whereas "atypical"
PKCs ( and ) require neither Ca2+ nor DAG for
activation but are activated by PS (37, 38). The isoforms
PKC-, -I, -II, -, -, and -
have been reported in vascular smooth muscle (14, 25, 37);
however, their role in modulating contraction is not well defined. In
general, PKC activation has been shown to be associated with its
translocation from the cytoplasm to the plasma membrane (11,
29). DAG binding to the PKC regulatory domain (C1
region) dramatically increases Ca2+ affinity and enzyme
activity. PKC isoforms have been shown to differ considerably among
different tissues (37). We know of only several other
studies of PKC and/or isoform changes with development.
PKC-II expression is prominent in rat kidney glomerulus on postnatal days 1-5, but not thereafter, and
appears to be associated with the immature glomerulus
(36). In the rat pituitary, PKC- decreased as PKC-
increased with developmental age (6 days to 3 mo), whereas in whole
brain, PKC- increased as PKC- decreased (8). Jia and
co-workers (17) have reported that in cat visual cortex
and hippocampus, [3H]PDBu binding (labeled PKC) appears
earlier in postnatal development (0, 10, 40, 75, 120 days, and adult)
than [3H]Ins(1,4,5)P3-binding
[labeled Ins(1,4,5)P3-receptors].
Nonetheless, we know of no previous studies of these isoforms in
cerebral vascular smooth muscle, much less changes with development.
1-AR-mediated contraction of vascular smooth muscle may
occur by the activation of two distinct protein kinase-dependent pathways. The Ins(1,4,5)P3-Ca2+
release-mediated myosin light chain kinase (MLCK) pathway predominates with elevated [Ca2+]i, promoting formation of
Ca2+-calmodulin complex with activation of MLCK and
relatively rapid development of tension (10). In contrast,
the PKC-mediated pathway is believed to predominate during the
prolonged tonic phase of agonist-induced contractions and may occur in
the presence of relatively low [Ca2+]i
(35) and sustained phospholipid hydrolysis. PKC activation is believed to phosphorylate a myofilament regulatory protein in the
mitogen-activated kinase (19, 20) or, by other mechanisms, increase Ca2+ sensitivity and contraction via ras p21 and
related small G proteins (3)
The dual effect of PKC on vascular contractility is well documented.
PKC negative feedback can inhibit Ins(1,4,5)P3
synthesis, [Ca2+]i release, and thus vascular
tension (2, 6, 26). In addition, a number of workers have
demonstrated vascular contraction after PKC activation, which typically
develops slowly, is sustained, and is [Ca2+]i
independent (1, 7, 11, 19, 33, 35). Among its several
isoforms, the Ca2+-independent isoform PKC- appears to
play a key role in vascular contraction (21) by
disinhibition of thin-filament myosin light chains (14,
38) and associated calponin and/or inhibition of myosin light
chain dephosphorylation (25). Thus the current findings of
significantly lower PKC- levels in fetal cerebral arteries, compared
with the adult, may be important in terms of their altered PKC-mediated function.
Although PKC has been quantified in brain, heart, and other tissues,
there are relatively few studies in vascular smooth muscle (19,
20, 28). We are unaware of quantitative PKC measurements or the
determination of isoforms in cerebral arteries. The present results
demonstrate an apparent paradox. On one hand, total PKC levels and
activity were similar in fetal and adult cerebral arteries, as were
agonist-induced increases in PKC activity (Table 1). On the other hand,
PKC stimulation and inhibition had widely differing effects in fetal
and adult cerebral arteries. The significant differences in PKC
function in the fetal and adult cerebral arteries probably reflect the
differences in relative abundance of the several isoenzymes as well as
other differences in the two age groups.
PKC modulation of Ins(1,4,5)P3 response to
inhibit contraction.
The present results demonstrate phorbol ester inhibition of NE-induced
Ins(1,4,5)P3 formation in cerebral arteries of
both adult and fetus (Fig. 2) and agree with other studies. For
instance, several investigators have demonstrated phorbol myristate
acetate (PMA) dose-dependent suppression of NE-induced
Ins(1,4,5)P3 formation in rat aorta (2,
26). In addition, our demonstration that PKC inhibition by Sta
potentiates the NE-induced Ins(1,4,5)P3 response in adult cerebral arteries (Fig. 2) has been shown for other
vascular smooth muscle. For instance, in rat aorta, 107 M
Sta pretreatment increased NE-induced
Ins(1,4,5)P3 levels severalfold (2) and significantly inhibited PMA-induced contraction
(5)
7 and 106 M
PDBu attenuated, whereas 104 M PDBu eliminated,
contractile and [Ca2+]i responses to NE (Fig.
3) with little difference in the response of the two age groups. The
studies also demonstrate the contrasting effects of PKC inhibition by
Sta on NE-induced contraction in adult and fetal MCAs. As is evident,
in the adult, 108 and 107 M Sta resulted in
markedly increased sensitivity to NE with increases in pD2
values of both tension and [Ca2+]i. In the
fetus, however, Sta-induced inhibition of PKC had a strikingly
different effect in the fetus with inhibition of NE-induced increase in
tension and [Ca2+]i (Fig. 4). This supports
the idea that in the fetal cerebral arteries, PKC inhibition reduced
NE-induced Ca2+ flux via plasma membrane Ca2+
channels (see PKC modulation of Ca2+ sensitivity to
increase contractility and Ref. 22).
PKC modulation of Ca2+ sensitivity to increased
contractility.
As noted above, PKC activation can result in smooth muscle contraction
in the presence of little, if any, increase in
[Ca2+]i, although the mechanism of this
effect is not clear (5, 35). In rabbit (12)
and rat (16) aortas, PKC activation may potentiate
1-adrenergic-induced contraction by modulation of plasma
membrane Ca2+-channel activity. In rat cerebral arteries,
both PKC activation and inhibition modified basic myogenic tone and
artery caliber (31).
5 M
PDBu-induced contraction, there was essentially no change in [Ca2+]i (Fig. 5A). The
PDBu-induced increase in Ca2+ sensitivity is less evident
in fetal cerebral arteries. In fact, as shown in Fig. 5C, in
fetal MCA, 105 M PDBu induced a significant increase in
both tone and [Ca2+]i. Nonetheless, the
PDBu-mediated [Ca2+]i increase per increase
in tension was not as great as that seen in response to NE or other
agonists (22). Because in fetal MCA (in contrast to that
of the adult), the PDBu-induced increases in tension and
[Ca2+]i were abolished in the presence of
zero [Ca2+]o (Fig. 5D), this
implies a lack of Ca2+ flux via plasma membrane
Ca2+ channels (22). As noted, several studies
in vascular smooth muscle cells support the idea that PKC can increase
Ca2+ flux via L-type, voltage-gated Ca2+
channels and thus contraction, presumably by phosphorylation and
increasing their probability of open state (13, 30).
Perspectives
In cerebral arteries of the adult, PKC appears to function in the traditional manner, with PKC existing in dual regulating roles, e.g, a membrane-bound component that is receptor activated and exerts negative feedback on PLC and Ins(1,4,5)P3 synthesis, and a second more general component that, on activation, increases Ca2+ sensitivity and vascular contraction. In the adult vessel, inhibition of1-AR-coupled PKC decreases its
negative feedback on PLC, thereby increasing sensitivity to NE and
augmenting NE-induced Ins(1,4,5)P3 responses.
In the fetal cerebral artery, in contrast, perhaps because of
relatively low PKC- levels, but also with other factors (differences
in small GTPases, mitogen-activated protein kinases, tyrosine kinases,
and so forth), PKC coupling to PLC and the 1-AR is poor or nonexistent. Also, in the fetus, but not adult, these responses were eliminated in the presence of zero
[Ca2+]o. This latter finding, in concert with
the Sta-associated decrease in MCA tension and
[Ca2+]i, strongly suggests that in fetal
cerebral arteries, PKC appears to be directly coupled to the plasma
membrane L-type Ca2+ channel per se (or indirectly coupled
to it by inhibiting the Ca2+-activated K+
channel that opens the Ca2+ channel) to increase
Ca2+ flux. PKC may be less coupled to Ca2+
sensitivity mechanisms per se. Of interest in fetal CCA is that, whereas 1-adrenergic stimulation failed to increase
Ins(1,4,5)P3 under control conditions, such
stimulation elicited a robust Ins(1,4,5)P3 response when PKC was inhibited. This supports the idea of
tonic feedback inhibition by PKC in some fetal vessels.
During the course of development from fetus to newborn to adult, the
cerebral blood vessels undergo striking changes in morphology as well
as in pharmacomechanical and electromechanical coupling mechanisms.
These changes are accompanied by changes in 1-AR density
and NE-induced Ins(1,4,5)P3 responses
(23, 24), Ins(1,4,5)P3-receptor density (39), and numerous other factors
(22) including the presently observed differences in PKC
levels, isoenzymes, and PKC-related mechanisms of contraction. On the
basis of the present results, one may speculate that PKC activation by
DAG or other agonists may be important in the modulation of
pharmacomechanical coupling. PKC modulation of NE-induced
cerebrovascular signal-transduction responses thus may play a key role
in the regulation of cerebral blood flow under physiological
conditions. Of potential importance, these PKC-mediated mechanisms,
including the relative lack of PKC- and the abundance of other
isoforms, may also play a role in conditions associated with
dysregulation of cerebral blood flow in the fetus, newborn, and/or
adult. The role of these mechanisms and their change with development
are important avenues for future studies.
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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 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 13 July 1999; accepted in final form 5 June 2000.
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REFERENCES |
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|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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) and in the presence of PKC stimulation by
10
), 10
), or
10
). With 10
