Department of Pharmacology, Hokkaido University School of Medicine,
Sapporo, Japan.
The purpose of this study was to determine the mechanism responsible
for alterations in NaF-induced contractions of blood vessels from
streptozotocin-induced diabetic rats. In the presence of
AlCl3, NaF (
7.5 mM) produced significantly greater
contractions in diabetic aorta and mesenteric artery compared with
age-matched controls. Pretreatment with 1 µM nifedipine eliminated
the enhanced contractile responses of diabetic vessels to NaF,
resulting in no difference in the magnitude of NaF-induced contractions
between control and diabetic vessels. In the presence of 100 µM
deferoxamine, an Al3+ chelator, NaF-induced contractions of
diabetic vessels were markedly attenuated, whereas only the responses
to lower concentrations of NaF were reduced in control vessels. No
significant difference was found in the peak amplitude of transient
contractions induced by 10 µM cyclopiazonic acid between control and
diabetic vessels. The addition of 10 µM okadaic acid produced
attenuated contractions in diabetic vessels. These findings indicate no
involvement of the inhibitory effects of NaF on endoplasmic reticular
Ca2+-pump ATPase and protein phosphatases in the genesis of
the enhanced responsiveness of diabetic vessels to NaF. Western blot
analysis showed a 2.5-fold increase in the expression of
Gq
in diabetic aortic membranes. In contrast, the
Gi level was modestly decreased and the
Gs and G
levels were unchanged in
diabetes. The present results suggest that enhanced vascular contractions to NaF in diabetes is attributed predominantly to a G
protein-mediated Ca2+ channel activation that results from
markedly increased Gq expression in vascular tissues
under this pathological state.
 |
Introduction |
It
is well known that NaF produces strong contractions of smooth muscle in
blood vessel preparations (Casteels et al., 1981
; Nguyen-Duong, 1985;
Zeng et al., 1989; Adeagbo and Triggle, 1991). NaF-induced vascular
contractions have been proposed to be attributable to fluoride
complexing with aluminum (which can come from contamination of
glassware) to form fluoroaluminates, which have been shown to be
activators of G proteins (Zeng et al., 1989). The cellular mechanism by
which fluoroaluminates activate G proteins is based on the structural
similarity of AlF4
to
PO43, enabling the former to
interact with guanosine 5'-diphosphate situated on the 
-subunit of
the G proteins where it can mimic GTP (Bigay et al., 1985). However,
vascular contractions induced by NaF cannot be explained totally by the
fluoroaluminate complex formed from contaminating aluminum. It has been
demonstrated that contractions produced by NaF are different from those
produced by fluoroaluminates in rabbit femoral artery (Ratz and
Blackmore, 1990). Furthermore, the possibility has been suggested that
inhibition of phosphatase activity may contribute to NaF-induced
contractions in rat and rabbit aortae (Adeagbo and Triggle, 1991). In
addition, NaF is known to inhibit the Ca2+-pump
ATPase of endoplasmic reticulum (Murphy and Coll, 1992), and
thapsigargin and cyclopiazonic acid (CPA), endoplasmic reticulum Ca2+-pump ATPase inhibitors, can produce vascular
contractions (Low et al., 1991; Naganobu et al., 1994). Therefore, it
seems most likely that NaF could produce vascular contractions through
several different mechanisms. This notion could be supported by
previous reports, which have proposed that NaF-induced vascular
contractions may be due to activation of L-type
Ca2+ channels, release of
Ca2+ from intracellular stores, and/or
enhancement of myofilament Ca2+ sensitivity (Zeng
et al., 1989; Ratz and Blackmore, 1990; Adeagbo and Triggle, 1991;
Fermum et al., 1991; Kawase and van Breemen, 1992; Ratz and Lattanzio,
1992).
In the light of the multiple cellular actions of NaF, it is possible
that the predominant mechanism involved in NaF-induced vascular
contractions may be altered in diseased states. Most studies have shown
that blood vessels from streptozotocin-induced diabetic rats develop
greater contractions in response to norepinephrine and
5-hydroxytryptamine than those from age-matched control rats (Ramanadham et al., 1984; White and Carrier, 1988; Orei and Aloamaka, 1993; Weber and MacLeod, 1994; Hattori et al., 1995b), although the
precise mechanism is not fully defined. These enhanced responses are
not due to a generalized increase in contractility of blood vessels
from diabetic rats because previous studies from this laboratory and
others have demonstrated a decrease in contractions evoked by high
K+ and Bay K 8644 in diabetic rat vessels
(Pfaffman et al., 1980; Head et al., 1987; Orei and Aloamaka, 1993;
Hattori et al., 1996). Recently, Weber et al. (1996) have shown that
NaF-induced contractions are enhanced in blood vessels from diabetic
rats. This raises an important question to us: which plays a dominant
role in the enhanced contractile responsiveness of diabetic vessels to
NaF among several cellular mechanisms by which NaF elicits vascular contractions? Thus, we attempted to elucidate the main cellular mechanism underlying NaF-induced contractions in blood vessels from 8- to 12-week streptozotocin-induced diabetic rats in comparison with
their age-matched controls. By doing so, we expected to find a clue to
the mechanism involved in the enhanced vascular responses to
norepinephrine and 5-hydroxytryptamine in diabetes.
| |
Materials and Methods |
Induction of Diabetes.
Male Wistar rats, 8 weeks old and 180 to 200 g in body weight, were randomly assigned to two groups. One
group of rats (diabetic group) received a single tail-vein injection of
streptozotocin (45 mg/kg) under light anesthesia with diethyl ether.
Streptozotocin was dissolved in a citrate solution (0.1 M citric acid
and 0.2 M sodium phosphate, pH 4.5). Another group (control group)
received an equivalent volume of citrate buffer alone. Control and
diabetic rats were caged separately but housed under similar
conditions. Both groups of animals were fed with the same diet and
water ad libitum until they were used 8 to 12 weeks later. This period of diabetes was chosen because our previous studies have well characterized alterations in vascular responses during this period (Hattori et al., 1991, 1995b, 1996). On the day of the experiments, a
blood sample was collected and the serum glucose level was measured. All animals injected with streptozotocin developed severe diabetes, as
indicated by increased serum glucose levels. Mean serum glucose levels
were 145 ± 4 and 532 ± 16 mg/dl for control
(n = 34) and diabetic rats (n = 37), respectively.
Organ Bath Experiments.
Eight to twelve weeks after
treatment with streptozotocin or buffer, rats were anesthetized with
diethyl ether. The thoracic aorta and the main branch of the superior
mesenteric artery were carefully excised and placed in an oxygenated
bathing medium at room temperature. The blood vessels were then cleaned
of adhering fat and connective tissue under a microscope and cut into
rings of 3 mm (for mesenteric artery) and 4 mm (for aorta) length. The intimal surface of the rings was gently rubbed with a wooden rod to
avoid any inhibitory or stimulatory influence of the vascular endothelium. The effectiveness of endothelium removal was confirmed by
the absence of the characteristic relaxation induced by 1 µM acetylcholine in blood vessels precontracted with 1 µM norepinephrine or 1 µM phenylephrine. Rings from the aorta were placed in a
water-jacketed chamber filled with 25 ml of physiological salt solution
(PSS), whereas rings from the mesenteric artery were placed in a small chamber that was filled with 6 ml of PSS. The composition of PSS (pH
7.4) was: 118.2 mM NaCl; 4.7 mM KCl; 1.2 mM MgCl2; 2.5 mM CaCl2; 25.0 mM NaHCO3; 10.0 mM glucose. The
solution in the chamber was gassed with 95% O2
and 5% CO2, and its temperature was maintained at 37°C. Each ring was suspended by a pair of stainless steel hooks
under a resting tension of 1.5 g for the aorta and 1.0 g for
the mesenteric artery. The resting tension was confirmed to be the
optimal resting preload for both control and diabetic vessels. Isometric tension was monitored with a transducer and recorded on a pen
recorder. The rings were allowed to equilibrate for at least 60 min
before the start of recordings. The rings were repeatedly challenged
with 1 µM norepinephrine or 1 µM phenylephrine until reproducible
contractile response was obtained.
Concentration-response curves for NaF-induced contractions were
constructed by a stepwise increase in the concentration of NaF in the
tissue chamber. The tissue was exposed to each concentration of NaF
until the contractile response reached a plateau, which usually
occurred within 10 min. When AlCl3, nifedipine,
or deferoxamine was used, they were added to the chamber 30 min before
the administration of NaF. In some experiments, the vascular
responsiveness to CPA or okadaic acid was tested, and the peak of
tension development induced by each agent at a fixed concentration was
compared between control and diabetic arteries.
After completion of each procedure, the rings were carefully blotted
dry and weighed. Contractile responses were expressed as milligrams of
developed tension per milligram tissue wet weight to normalize the
differences in the cross-sectional area of the ring preparation.
Western Blot Analysis for G Proteins.
Thoracic aortae were
excised and placed in ice-cold Tris-HCl buffer containing 75 mM
Tris-HCl (pH 7.4) and 5 mM EDTA. Fat and connective tissues were
trimmed from aortae. Endothelium-denuded aortae were minced finely with
scissors and homogenized in Tris-HCl buffer by means of a polytron. The
homogenate was centrifuged at 6000gmax
for 10 min, and the supernatant was retained. The protein concentration
of the supernatant was determined by the methods of Lowry et al.
(1951), with BSA used as standard. The supernatant was stored at
80°C until used.
Samples (20 µg) were subjected to a 14% polyacrylamide SDS gel and
electroblotted onto a polyvinylidene difluoride (PVDF) membrane. The
PVDF was washed in PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM
NH2PO4) and was blocked for
60 min at room temperature in 1% BSA in PBS-Tween buffer (137 mM NaCl,
2.7 mM KCl, 8.1 mM NH2PO4,
0.05% Tween 20) to reduce nonspecific binding. Thereafter, the PVDF
was washed twice in PBS-Tween buffer and incubated overnight at 4°C
with specific rabbit antiserum recognizing Gq
(Gramsch Laboratories, Schwabhausen, Germany) at 1:300 dilution,
Gi (Oncogene Research Product, Cambridge, MA)
at 1:200, Gs (Gramsch Laboratories) at 1:1000,
or G (Calbiochem-Novabiochem Corporation,
San Diego, CA) at 1:2000 in TBS-Tween buffer. The PVDF was washed twice
in PBS-Tween buffer then incubated with horseradish
peroxidase-conjugated anti-rabbit antibody (Bio-Rad Laboratories,
Hercules, CA) diluted at 1:6000 in PBS-Tween buffer at room temperature
for 60 min. After washed twice in PBS-Tween buffer, the blots were
visualized with the enhanced chemiluminescence detection system
(Amersham, Buckghamshire, UK), exposed to X-ray film for 7 min, and
analyzed by the free software NIH Image produced by Wayne Rasband
(National Institutes of Health, Bethesda, MD). To check for protein
loading/transfer variations, all blots were stained with Ponceau red
(washable, before incubation with antibodies) and with Coomassie
brilliant blue (permanent, after the enhanced chemiluminescence
detection system). Intensity of total protein bands per lane was
evaluated by densitometry. Negligible loading/transfer variation was
observed between samples.
Chemicals.
Streptozotocin, l-phenylephrine
hydrochloride, nifedipine, deferoxamine mesylate, and CPA were
purchased from Sigma (St. Louis, MO). NaF was purchased from Nakalai
Tesque (Kyoto, Japan), l-norepinephrine bitartrate and
acetylcholine chloride were obtained from Wako Pure Chemical Industries
(Osaka, Japan), and okadaic acid was obtained from
Calbiochem-Novabiochem Corporation. Other chemicals used in this study
were of the highest purity available from Sigma, Wako, Nakalai, or
Bio-Rad. All drugs except streptozotocin (see above), nifedipine, CPA,
and okadaic acid were dissolved in distilled water. Nifedipine was
prepared in absolute ethanol, and CPA and okadaic acid were in dimethyl
sulfoxide. Further dilutions to the desired concentrations were made
with PSS.
The experiments with nifedipine were performed in the dark, and
solution bottles and tubing were covered with aluminum foil for further
security against degradation.
Statistical Analysis.
All values are presented in terms of
means ± S.E. Comparisons of variables obtained by various
treatments with basal values were made by a one-way ANOVA with a
repeated measures design, and if any significant difference was found
the Scheffé's multiple comparison test was applied. Student's
t test was used to make comparisons between control and
diabetic groups. Nonparametric data were analyzed by the Mann-Whitney
U test or Wilcoxon signed-rank test. A P
value <.05 was considered statistically significant.
| |
Results |
NaF-Induced Contractions.
In the presence of 10 µM
AlCl3, NaF (1-20 mM) caused
concentration-dependent contractions in aortae and mesenteric arteries from both streptozotocin-induced diabetic and age-matched control rats
(Fig. 1). However, in diabetic blood
vessels, the responses to NaF at concentrations of 7.5 mM were
significantly greater than the corresponding values obtained in control
vessels. Diabetes significantly decreased the sensitivity to NaF in
aorta, whereas it increased, apparently but insignificantly, the
sensitivity in mesenteric artery (Table
1). As described later, because this was
lost when nifedipine was used, it may be related to the
Ca2+ entry through sarcolemmal
Ca2+ channels. In the presence of
AlCl3, 20 mM NaCl did not cause any change in
tension of either control or diabetic vessels (n = 4 for each).
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Fig. 1.
Concentration-response curves for NaF-induced
contractions of aortae (A) and mesenteric arteries (B) from control
( ) and diabetic ( ) rats. AlCl3 (10 µM) was present
throughout. Data are expressed as means ± S.E. of 6 to 12 experiments. **P < .01;
***P < .001 versus the corresponding
control values.
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TABLE 1
Characteristics of NaF concentration-response curves in the absence and
presence of 1 µM nifedipine in control and diabetic vessels
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In control aorta, treatment with 1 µM nifedipine did not affect the
maximum response that was obtained with 15 mM NaF, although the
sensitivity to NaF was clearly decreased by this treatment (Table 1).
In contrast, in diabetic aorta, nifedipine treatment caused a decrease
in the tension development over the entire range of NaF concentrations
(compare Fig. 2A with Fig. 1A).
As a result, in the presence of nifedipine, no significant difference
was found in the magnitude of the contractile responses of aortae from
control and diabetic rats to NaF (Fig. 2A). In mesenteric
arteries, nifedipine treatment greatly inhibited the contractile
responses to NaF at all concentrations used in both control and
diabetic groups (compare Fig. 2B with Fig. 1B and Table 1).
However, the inhibition was more marked in diabetic mesenteric artery.
Thus, treatment with nifedipine resulted in elimination of the
difference between control and diabetic mesenteric arteries in the
contractile responses to NaF (Fig. 2B).
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Fig. 2.
Effect of nifedipine on the concentration-response
curves for NaF-induced contractions of aortae (A) and mesenteric
arteries (B) from control () and diabetic () rats. Nifedipine (1 µM) was added to the bath 30 min before construction of the curves.
AlCl3 (10 µM) was present throughout. Data are expressed
as means ± S.E. of 6 to 10 experiments.
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In control aorta and mesenteric artery, 100 µM deferoxamine, an
Al3+ chelator (Ackrill and Day, 1984), abolished
contractions induced by 3 and 5 mM NaF in the presence of 10 µM
AlCl3 (Fig. 3).
However, contractions produced by 10 mM NaF were unaffected by
deferoxamine (Fig. 3). Similar to controls, diabetic aorta and
mesenteric artery failed to exhibit any contraction in response to
lower concentrations (
5 mM) of NaF with 10 µM
AlCl3 in the presence of deferoxamine (Fig.
4). In addition, treatment with
deferoxamine markedly depressed the contractile responses of diabetic
aorta and mesenteric artery to 10 mM NaF in the presence of 10 µM
AlCl3 (Fig. 4). The amplitudes of contractions
induced by 10 mM NaF were reduced by deferoxamine to 62 ± 9%
(n = 4, P < .01) and 56 ± 7%
(n = 4, P < .001) of the prevalue in
diabetic aorta and mesenteric artery, respectively. This suggests that
NaF induced contractions in the wide range of its concentrations
through the action of fluoroaluminates. Indeed, the presence of the
high concentration of AlCl3 (1 mM) reversed the
inhibitory effect of deferoxamine on the NaF responses of diabetic
vessels, although the extent of the recovery was somewhat variable from
preparation to preparation (Fig. 4). Nevertheless, the contractile
responses of diabetic vessels to NaF in the absence of
AlCl3 were not substantially different from those
in the presence of 10 µM AlCl3 (data not
shown). However, this may be explained by considering that the part of
the contractile responses of diabetic vessels to NaF alone was probably
due to fluoroaluminates formed by the complexing of fluoride with
alminium present as a contaminant of glass, because the glassware was
used in the experiments.
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Fig. 3.
Effect of deferoxamine on the concentration-dependent
contractile responses to NaF in control rat aorta (upper traces) and
mesenteric artery (lower traces). Deferoxamine (100 µM) was added to
the bath 30 min before application of NaF. AlCl3 (10 µM)
was present throughout. Recordings for each vessel were from the same
preparation. Similar results were obtained with three other aortae and
mesenteric arteries.
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Fig. 4.
Effect of deferoxamine on contractions induced by 5 and 10 mM NaF in diabetic rat aorta (A) and mesenteric artery (B).
Deferoxamine (100 µM) and AlCl3 (10 µM or 1 mM) were
added to the bath 30 min before application of NaF. Superimposed
records of NaF-induced contractions in the presence of 10 µM
AlCl3 alone (a), presence of 10 µM AlCl3 and
100 µM deferoxamine (b), and presence of 1 mM AlCl3 and
100 µM deferoxamine (c) are depicted. The results shown are
representative of three additional experiments.
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Contractions Induced by CPA and Okadaic Acid.
In both aorta
and mesenteric artery, the addition of 10 µM CPA caused a transient
increase in tension (Fig. 5). The peak of the transient contraction was about 15 to 35% of the contraction induced by 10 µM phenylephrine. In both of the two vessels, the peak
amplitude of the CPA-induced transient contraction was essentially the
same between control and diabetic groups (aorta: 30.6 ± 5.0 versus 30.9 ± 3.7 mg/mg wet weight; mesenteric artery: 143.6 ± 31.4 versus 142.8 ± 30.2 mg/mg wet weight, n = 5 for each).
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Fig. 5.
Contractions induced by 10 µM CPA in aortae and
mesenteric arteries from control (A) and diabetic (B) rats. For
comparison, contractions induced by 1 µM phenylephrine (Phe) are
shown. Similar results were obtained with four other vessels in each
group.
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Okadaic acid at a concentration of 10 µM produced a slowly developing
contraction in control mesenteric artery (Fig.
6A). A plateau was attained 40 to 60 min after the addition, which was 47 ± 11% (n = 3) of the contraction induced by 10 µM phenylephrine. However, the
contractile response to 10 µM okadaic acid was very small in diabetic
mesenteric artery (Fig. 6B). Thus, the peak amplitude of okadaic
acid-induced contractions was significantly (P < .05)
less in diabetic mesenteric artery (47.9 ± 13.0 mg/mg wet weight,
n = 4) compared with that in control (347.2 ± 60.5 mg/mg wet weight, n = 3).
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Fig. 6.
Contractions induced by 10 µM okadaic acid in
mesenteric arteries from control (A) and diabetic (B) rats. For
comparison, contractions induced by 1 µM phenylephrine (Phe) are
shown. Similar results were obtained with two other control and three
other diabetic vessels.
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Assessment of G Proteins.
Western blot analysis of crude
membranes with the Gq antiserum clearly
visualized a major band with a molecular mass of 42 kDa in rat aorta
(Fig. 7A). Quantification of
Gq protein indicated a 2.5-fold increase in
this protein in diabetes when compared with the control (250 ± 8%, n = 5, P < .001). The
Gq protein level of mesenteric artery was also
markedly increased in diabetes (269 ± 6%, n = 4, P < .001).
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Fig. 7.
Immunoblot analysis of Gq (A),
Gi (B), Gs (C), and G
(D) proteins in aortic membranes from control and diabetic rats. The
upper trace of each panel shows representative blots of the respective
protein in control (lane 1) and diabetic (lane 2) membranes. The
experiments were conducted by loading equal amounts of control and
diabetic membrane proteins in each lane. The lower trace of each panel
shows the bar graph summarizing the immunoblot data. Densitometric
results are expressed as a percent of each band obtained in controls.
Data are expressed as means ± S.E. of five experiments.
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The Gi antiserum identified a major protein
band with a molecular mass of 40/41 kDa in rat aorta (Fig. 7B). We
found a modestly lower level of the Gi protein
in diabetes. Quantitative analysis of immunoblots showed a decrease of
19 ± 1% (n = 5, P < .001) when
compared with the control.
The Gs antiserum recognized a 44-kDa band in
rat aorta (Fig. 7C). Densitometric quantification of the signal
showed no difference in the amount of aortic
Gs protein between control or diabetic rats
(95 ± 1% of control, n = 5).
The 
- and 
-subunits should dissociate and migrate on
SDS-polyacrylamide gel electrophoresis with apparent molecular weights of 35~36 and 6~9 kDa (Asano et al., 1993). However, the antibody that specifically reacts with the
G hetrodimeric complex of G proteins, used
in this study, identified one single band with a molecular mass of 45 kDa in rat aorta (Fig. 7D). Thus, we assumed that the protein
expression level of G can be reflected by
this protein band. No change in the protein level was observed in
diabetic aorta (96 ± 1% of control, n = 5).
| |
Discussion |
Being compatible with the concept that fluoroaluminates are an
activator of G proteins (Bigay et al., 1985), NaF with added or
contaminating aluminum, forming aluminum fluorides, has been shown to
interact with G proteins to stimulate a variety of cellular effector
systems (Sternweis and Gilman, 1982; Blackmore et al., 1985; Hall et
al., 1990). Thus, it has been considered that the mechanisms by which
NaF causes vascular contraction may involve interaction with G proteins
(Zeng et al., 1989; Cushing et al., 1990). In the present study, the
contractile responses to NaF at lower concentrations (5 mM) in the
presence of AlCl3 in aorta and mesenteric artery
from control rats were blocked by deferoxamine, a chelator of
Al3+ (Ackrill and Day, 1984). In contrast,
deferoxamine had no effect on contractions induced by 10 mM NaF. At the
concentration employed in this study (100 µM), deferoxamine can
eliminate completely the positive inotropic effect of NaF in the
presence of AlCl3 in rabbit left atrial muscles
(Hattori et al., 1995a). Therefore, the data with deferoxamine suggest
that, in rat blood vessels used in this study, the contractile action
induced by NaF at lower concentrations is a result of the
fluoroaluminate complex activating G proteins, whereas that by NaF at
higher concentrations may be due to the mechanism(s) being independent
of G protein activation.
The present study showed that aortae and mesenteric arteries from
streptozotocin-induced diabetic rats exhibited greater contractions in
response to NaF at higher concentrations (7.5 mM) compared with the
corresponding blood vessels from age-matched control rats. This
observation is consistent with the recent results reported by other
investigators (Weber et al., 1996). Interestingly, we found that the
contractile responses of diabetic vessels to the high concentrations of
NaF were suppressed in the presence of deferoxamine, being in contrast
with the results obtained in control vessels. The depressed NaF
responses of diabetic vessels in the presence of deferoxamine was
reversed by adding the high concentration of
AlCl3 (1 mM). Thus, NaF appears to induce
contractions in diabetic vessels through the action of fluoroaluminates
as a G protein activator in a wide range of NaF concentrations.
Alternatively, increased activation of G proteins is most likely to be
responsible for the enhanced contractile responses of diabetic vessels
to NaF. In the present investigation, assessment of
Gq by immunochemical quantification with
antiserum revealed marked increases in diabetic aorta and mesenteric
artery compared with control vessels. The fluoroaluminate complex
formed from NaF in the presence of AlCl3 could
activate potentially all G proteins. However, its stimulatory effect on
a G protein (perhaps Gq), which triggers
activation of phospholipase C (Abdel-Latif, 1986), is
outstandingly manifested in vascular smooth muscle, because NaF elicits
increases in formation of inositol phosphates in rat aortic myocytes
(Berta et al., 1988), rat tail artery (Zeng et al., 1989; Cheung et
al., 1990), and rabbit femoral artery (Ratz and Blackmore, 1990). Thus,
we suggest that if NaF-induced vascular contractions involve activation
of Gq, the increased level of
Gq expression could contribute to the enhanced
contractile response of diabetic vessels to NaF. This may explain the
reason why NaF-induced contractions in diabetic vessels were much more
sensitive to deferoxamine. Cushing et al. (1990) have shown that
NaF-induced contractions in porcine coronary artery are markedly
attenuated by pretreatment with pertussis toxin and cholera toxin.
However, the present study indicates no involvement of
Gi, which is one of pertussis toxin-sensitive G
proteins, and Gs, which is irreversibly activated
by cholera toxin, in the enhancement of NaF-induced vascular
contractions in diabetes. We found that the Gs
protein level was not changed and the Gi
protein level was significantly decreased rather than increased in
diabetic aorta. This is in good agreement with the results from our
laboratory and others showing unchanged expression of
Gs protein and diminished expression of
Gi protein in myocardium (Wichelhaus et al.,
1994; Gando et al., 1997) and liver (Gawler et al., 1987) from
streptozotocin-induced diabetic rats. In addition, no change in the
G protein level was found in diabetic aorta.
Stimulation of phospholipase C with Gq activation
catalyzes hydrolysis of phosphatidyl inositol-4,5-bisphosphate to form
the two second messengers: inositol-1,4,5-trisphosphate, which causes release of Ca2+ from intracellular stores, and
1,2-diacylglycerol, which activates protein kinase C (Abdel-Latif,
1986). The latter event then results in phosphorylation of
various proteins including L-type Ca2+ channels.
There is evidence that Ca2+ channels are
activated through protein kinase C-dependent phosphorylation in a
vascular smooth muscle cell line (Fish et al., 1988). The present study
showed that in the presence of nifedipine the contractile responses to
NaF of diabetic vessels were lowered to the same levels as those of
control vessels, suggesting that the enhanced responsiveness of
diabetic vessels to NaF was dependent on the entry of
Ca2+ via Ca2+ channels.
Thus, NaF may cause activation of Gq and results
in opening of Ca2+ channels allowing the influx
of extracellular Ca2+ into the cell through a
protein kinase C-mediated phosphorylation mechanism, thereby leading to
increased tension development in diabetic vessels. With respect to this
assumption, Weber et al. (1996) have found a significant reduction in
the enhanced NaF contractile response of diabetic mesenteric artery by
calphostin C, which selectively inhibits protein kinase C.
NaF is known to inhibit the Ca2+-pump ATPase of
endoplasmic reticulum (Murphy and Coll, 1992). However, the
contribution of the inhibitory action of NaF on endoplasmic reticular
Ca2+-pump ATPase activity to its contractile
response of diabetic vessels would be minimal. The time course of
contractions induced by CPA, a selective inhibitor of the
Ca2+-pump ATPase of endoplasmic reticulum
(Seidler et al., 1989), in rat aorta and mesenteric artery was quite
different from that of NaF-induced contraction. CPA elicited a rapid
transient contraction, whereas NaF produced a sustained contraction
with slow time course. Furthermore, the finding that the peak amplitude
of the transient contraction induced by CPA was essentially the same
between control and diabetic vessels indicates that inhibition of
endoplasmic reticular Ca2+-pump ATPase activity
is not responsible for the enhanced NaF contractile responses of
diabetic vessels.
The possibility has been previously suggested that NaF-induced
contractions of rat and rabbit aortae may partly result from inhibition
of phosphatase activity (Adeagbo and Triggle, 1991). In this study,
okadaic acid caused a long-lasting contraction of rat mesenteric
artery. This is most likely due to inhibition of two of the protein
phosphatases, type 1 and 2A, involved in the dephosphorylation of
serine and threonine residues (Takai et al., 1987). NaF is a classical
inhibitor for phosphatases including type 1 and 2A (Shenolikar and
Nairn, 1991). Therefore, we infer that the
deferoxamine-resistant component of NaF-induced contractions in rat
vessels may involve phosphatase inhibition. In aortae from control
rats, the contractile responses to lower concentrations of NaF were
reduced but those to its higher concentrations were marginally
unaffected by nifedipine. This nifedipine-insensitive part of NaF
contractions was apparently identical with the deferoxamine-resistant component. Thus, some part of NaF-induced contractions in rat aorta may
result from the Ca2+ sensitizing action on the
contractile proteins via phosphatase inhibition. The ability of NaF to
enhance Ca2+ sensitivity of the contractile
proteins has been demonstrated in -toxin-permeabilized rabbit
vascular smooth muscle (Kawase and van Breemen, 1992). In contrast,
NaF-induced contractions in mesenteric arteries from control rats were
greatly reduced by nifedipine. This suggests that even the
deferoxamine-resistant component of NaF-induced contractions appears to
be strongly dependent on the Ca2+ influx via
Ca2+ channels in rat mesenteric artery. NaF has
been shown to cause contractions of rabbit femoral artery primarily by
activating Ca2+ channels in the presence of
deferoxamine, possibly due to phosphatase inhibition (Ratz and
Lattanzio, 1992). Accordingly, it seems likely that the mechanisms by
which NaF elicits contractions of rat mesenteric artery in a manner
independent of G protein activation could include an increased
Ca2+ influx via Ca2+
channels in addition to an enhanced myofilament
Ca2+ sensitivity, probably resulting from
inhibition of protein phosphatases. The present study showed that the
contractile response to okadaic acid was markedly less in diabetic
mesenteric artery, suggesting no involvement of phosphatase inhibition
in the enhancement of NaF-induced vascular contractions in diabetes.
However, an alternative explanation is that vascular phosphatases may
be significantly depressed as a result of the induction of diabetes and
thus there may be no additional effect of okadaic acid. Furthermore, we
cannot exclude the possiblity that okadaic acid is relatively specific for type 1 and 2A phosphatases although NaF may affect a much wider array.
In conclusion, aortae and mesenteric arteries from
streptozotocin-induced diabetic rats exhibited greater contractions to NaF in the presence of AlCl3 compared with
age-matched controls. The enhancement of NaF-induced contractions was
entirely dependent on the predominant contribution of a G
protein-mediated mechanism to the response in diabetes. This was
associated with the markedly increased level of vascular
Gq expression, which may also explain, in
part, the enhanced contractile responses of diabetic vessels to the
Gq-coupled receptor agonists including
norepinephrine and 5-hydroxytryptamine.
CPA, cyclopiazonic acid;
PSS, physiological
salt solution;
PVDF, polyvinylidene difluoride.
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Expression
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Abstract
Introduction
