Clinical and Experimental Pharmacology and Physiology (2008) 35, 568–573
Original
Blackwell
2+ Articles
L
SR
Ma
Caet
mobilization
al.PublishinginAsia
diabetes
doi: 10.1111/j.1440-1681.2007.04832.x
ABNORMALITIES OF SARCOPLASMIC RETICULUM Ca2+
MOBILIZATION IN AORTIC SMOOTH MUSCLE CELLS FROM
STREPTOZOTOCIN-INDUCED DIABETIC RATS
Li Ma,* Banghao Zhu,* Xiangping Chen,* Jie Liu,* Yongyuan Guan* and Jun Ren †
*Department of Pharmacology, Cardiac and Cerebral Vascular Research Center, Zhongshan Medical College,
Sun Yat-Sen University, Guangzhou, China and †Center for Cardiovascular Research and Alternative Medicine,
Division of Pharmaceutical Sciences, University of Wyoming, Laramie, Wyoming, USA
SUMMARY
1. Previously, we found that contractions in response to receptordependent (i.e. a1-adrenoceptor agonist phenylephrine) and
-independent (i.e. cyclopiazonic acid) stimuli are decreased in rat
aorta during late diabetes. The aim of the present study was to
further investigate the changes of intracellular Ca2+ homeostasis
in diabetic aortic smooth muscle cells. Functional changes of inositol
1,4,5-trisphosphate (IP3)- and ryanodine-sensitive Ca2+ stores of the
sarcoplasmic reticulum (SR) were evaluated using Fluo-3 acetoxymethyl ester fluorescence, western blot and organ bath techniques.
2. In aortic smooth muscle cells from diabetic rats, the Ca2+
release and Ca2+ influx caused by both 10 mmol/L phenylephrine
(depletion of IP3-sensitive Ca2+ stores) and 1 mmol/L ryanodine
(depletion of ryanodine-sensitive Ca2+ stores) were both significantly decreased compared with control. Moreover, protein expression levels of IP3 (260 kDa) and ryanodine receptors (500 kDa)
were reduced by 31.8 ± 7.7 and 69.2 ± 8.4%, respectively, in aortas
from diabetic rats compared with those from control rats.
3. In diabetic rat aorta, phenylephrine-induced contractility
was decreased to approximately two-thirds of that in controls,
whereas ryanodine alone did not cause obvious contraction in
aortas from either control or diabetic rats.
4. The present results suggest that the hyporeactivity of aortic
smooth muscle to vasoconstrictors in diabetes results mainly
from changes to the IP3-sensitive Ca2+ release pathway. The SR
Ca2+ signalling pathway plays a crucial role in the development
of diabetic vascular complications.
Key words: aortic smooth muscle cell, diabetes, inositol 1,4,5trisphosphate-sensitive Ca2+ store, ryanodine-sensitive Ca2+ store.
INTRODUCTION
are thought to be caused, at least in part, by the decreased contractility of vascular smooth muscle to vasoconstrictors.2–4 The contraction of smooth muscle is directly dependent on the increase in
intracellular calcium ([Ca2+]i). Regulation of [Ca2+]i is a complicated
process that includes Ca2+ release and reuptake by the endoplasmic
reticulum (ER) or sarcoplasmic reticulum (SR) and Ca2+ influx and
efflux across the plasma membrane. In vascular smooth muscle cells,
the underlying mechanisms of the increases in [Ca2+]i involve Ca2+
entry through voltage-dependent Ca2+ channels, as well as through
store-operated Ca2+ channels, which are activated by depletion of
Ca2+ stores following agonist stimulation.5 The changes in voltagedependent Ca2+ influx in diabetes have been well characterized.6,7
In recent years, increasing evidence suggests that the store-operated
Ca2+ channels are also altered in diabetes and contribute to the
hyporesponsiveness of vascular smooth muscle.8–11
In smooth muscle cells, two types of intracellular Ca2+ stores have
been identified, namely inositol 1,4,5-trisphosphate (IP3)-sensitive
Ca2+ stores and ryanodine-sensitive Ca2+ stores.12–14 The release of Ca2+
from these stores relies on the activation of IP3 and ryanodine receptors,
respectively. Whether the IP3 and ryanodine receptors are colocalized
or distant from each other on the same store or whether they exist
on separate stores remains unresolved.
In a previous study, we found that contractions of aortic rings from
diabetic rats induced by KCl, phenylephrine and cyclopiazonic acid
(CPA) were decreased in the late phase of diabetes.15 However, the
underlying mechanisms are ill-defined. The aims of the present study
were: (i) to determine whether Ca2+ release from IP3- and ryanodinesensitive Ca2+ stores is altered in aortic smooth muscle cells from
diabetic rats; (ii) if so, whether changes in Ca2+ release are due to
decreased expression levels of the IP3 and ryanodine receptors; and
(iii) whether the changes to IP3- and ryanodine-sensitive Ca2+ stores are
involved in the dysfunction of vascular smooth muscle contractility.
Vascular complications of diabetes are the leading cause of morbidity and mortality in patients with diabetes1 and these complications
METHODS
Animal preparations
Correspondence: Dr Bang-Hao Zhu, Department of Pharmacology,
Cardiac and Cerebral Vascular Research Center, Zhongshan Medical College,
Sun Yat-Sen University, No. 74 Zhongshan Road 2, Guangzhou, Guangdong
510080, China. Email: [email protected]
Received 11 June 2007; revision 21 August 2007; accepted 19 September
2007.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Asia Pty Ltd
Male Wistar rats were supplied by the Experiment Animal Center of Sun
Yat-Sen University (Guangzhou, China). All procedures complied with the
standards for the care and use of animals, as stated in the Guide for the Care
and Use of Laboratory Animals (issued by the ministry of Science and
Technology of China, Beijing; http://www.most.gov.cn/kytj/kytjzcwj/200411/
t20041108_32465.htm). Male rats, weighing 200–250 g, were used and were
randomized into two groups (diabetic and control groups). Diabetes was
SR Ca2+ mobilization in diabetes
induced by an intraperitoneal injection of streptozotocin (STZ; 60 mg/kg)
dissolved in sodium citrate buffer (pH 4.5); age-matched control rats were
treated with an injection of equal volume of vehicle. Blood samples for
glucose measurements were taken from the tail vein 72 h after STZ injection
and the day before the animals were killed (i.e. 16 weeks after induction of
diabetes). Hyperglycaemia was determined using a glucometer (Roche
Diagnostics, Indianapolis, IN, USA) and rats with blood glucose levels
> 16.7 mmol/L were considered diabetic.
Cell preparations
Smooth muscle cells were obtained enzymatically. Rats were anaesthetized
by an intraperitoneal injection of pentobarbital sodium (60 mg/kg). The arteries
were isolated, cut open longitudinally and then immersed in a Tyrodes–
HEPES solution (composition (in mmol/L): NaCl 137; KCl 2.7; MgCl2 1;
CaCl2 0.18; glucose 5.6; HEPES 4.2, pH 7.4) aerated with 95% O2 and 5%
CO2 for 2 h. Then, the endothelium and adventitia were removed and smooth
muscle layers were processed in the Tyrodes–HEPES solution containing
collagenase (1.0 mg/mL), elastase (0.5 mg/mL), papain (1.0 mg/mL), trypsin
inhibitor (1.0 mg/mL) and bovine serum albumin (BSA; 2.0 mg/mL) at 37∞C
for 60 min. Samples were then transferred to a solution containing 1.0 mg/mL
collagenase, elastase (0.5 mg/mL) and trypsin inhibitor (1.0 mg/mL) at
37∞C for a further 40 min. After treatment, the samples were transferred to
a dissociation solution (composition (in mmol/L): NaCl 137; KCl 2.7; MgCl2
1; CaCl2 0.18; glucose 5.6; HEPES 4.2, pH 7.4) at room temperature. The
cell-containing solution was then placed in a plastic culture dish and stored
at 4∞C. Freshly isolated smooth muscle cells were used within 8 h. Vascular
smooth muscle cells were identified by morphology and positive immunocytochemical staining against a-smooth muscle actin, as described previously.16
Ca2+ imaging
Smooth muscle cells were incubated in a HEPES balanced sodium (HBS)
buffer solution (composition (in mmol/L): NaCl 145; KCl 3.0; MgCl2 1.0;
CaCl2 2.0; glucose 10; HEPES 10; 1% BSA, pH 7.4) containing 4 mmol/L
Fluo-3AM at room temperature in the dark for 30 min. Extracellular Fluo-3
acetoxymethyl ester (Fluo-3AM) was then removed by washing the cells
twice with HBS buffer solution. The [Ca2+]i was monitored by a confocal
laser scanning system (Olympus, Tokyo, Japan) with excitation at 488 nm
and emission at 530 nm. Fluorescence images were recorded every 5.0 s. At
least five cells were examined in every test. For the Ca2+-free test, Ca2+ was
omitted from the HBS buffer solution and 50 mmol/L EGTA was added.17
Intracellular calcium is expressed as a ratio of fluorescence intensity relative
to basal fluorescence (i.e. DF = F – F0, where F represents the peak value of
fluorescence intensity after being activated by an agonist and F0 represents
the value of fluorescence intensity under resting conditions). The increase
in fluorescence intensity of Fluo-3 is proportional to the rise in [Ca2+]i.18
Western blot analysis
Rat arterial smooth muscle layers were washed with phospahte-buffered
saline (PBS) and lysis buffer (Tris-HCl 50 mmol/L, NaCl 150 mmol/L, NaN3
0.02%, Nonidet P-40 1%, sodium dodecyl sulphate (SDS) 0.1%, sodium
deoxycholate 0.5%, 5.0 mg/mL leupeptin and 1.0 mg/mL aprotinin). Protein
concentrations were determined by the Coomassie brilliant blue method.
Fifteen microgram protein was loaded in each lane and subjected to
SDS–polyacrylamide gel electrophoresis (PAGE) and then transferred to
polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) for
immunoblotting. In addition, HiMarkTM Pre-stained HMW Protein Standard
(Invitrogen, Carlsbad, CA, USA) was loaded on the gels to mark molecular
weight. The membranes were blocked at room temperature (24 –26∞C) for
1 h in PBS + Tween solution (PBST; composition (in mmol/L): NaCl 130;
KCl 2.5; Na2HPO4 10; KH2PO4 1.5; 0.1% Tween 20; 5% BSA, pH 7.4),
incubated initially with antibodies against either the IP3 receptor (1 : 2000
dilution, monoclonal antibody (mAb); Chemicon, Temecula, CA, USA) or
the ryanodine receptor (1 : 400, mAb; Chemicon) overnight at 4∞C and then
569
with horseradish peroxidase (HRP)-labelled anti-mouse IgG (1 : 2500) for
1 h at room temperature. Final detection was performed with the LumiGLO
chemiluminescent reagent (Cell Signalling, Danvers, MA, USA) according
to the manufacturer’s instructions. The densities of target bands were determined
accurately using a computer-aided one-dimensional gel analysis system.
Measurement of isometric contraction
The contractility of aortic rings was determined as described previously.15
Briefly, the aorta was placed into modified Krebs’ solution (containing (in
mmol/L): NaCl 137; KCl 5.4; MgCl2 1.1; CaCl2 2.0; glucose 5.6; NaHCO3
11.9; NaH2PO4 0.4, pH 7.2) at 37∞C, which was gassed continuously with
95% O2/5% CO2. The aortic segments were then cut into 3 mm rings. At most,
two rings were obtained from each aorta. The endothelium was removed by
gently rubbing with the fingers and removal was confirmed by the lack of a
relaxation response to 10 mmol/L acetylcholine. Isolated rings were suspended
in an organ bath containing 4 mL modified Krebs’ solution at 37∞C and gassed
continuously with 95% O2/5% CO2. Contractile responses were measured
using isometric force transducers connected to a data-collection system. At
the end of the experiment, the rings were blotted dry and weighed. Contractile
forces were calculated as g/g wet tissue. For the Ca2+-free test, Ca2+ was omitted
from the modified Krebs’ solution and 50 mmol/L EGTA was added.17
Materials
Phenylephrine, ryanodine and CPA were purchased from Sigma (St Louis,
MO, USA) and were dissolved in water or dimethyl sulphoxide (DMSO),
then stored at –20∞C until use. The final concentration of DMSO that tissues
were exposed to was less than 0.1%. All solutions were made fresh each day.
Fluo-3 acetoxymethyl ester, STZ, papain, collagenase (type I), elastase (type
II), papain, trypsin inhibitor and BSA were also obtained from Sigma. Both
mouse anti-IP3 receptor mAb and mouse anti-ryanodine receptor antibodies
were purchased from Chemicon. All other chemicals and reagents were of
analytical grade.
Statistical analysis
All values are expressed as the mean±SEM. Data analysis was performed
using one-way anova in spss 11.5 (SPSS, Chicago, IL, USA). P < 0.05 was
considered statistically significant.
RESULTS
Bodyweight and blood glucose concentrations
At the time of the experiment (16 weeks after the induction of
diabetes), plasma glucose levels were increased fivefold in STZdiabetic rats compared with age-matched controls (26.1 ± 0.6 vs
5.0 ± 0.06 mmol/L, respectively; n = 45; P < 0.01). However, the
bodyweight of STZ-diabetic rats was significantly less than that of
the corresponding controls (302 ± 6 vs 481 ± 4 g, respectively; n = 45
and 40, respectively; P < 0.01).
Effects of hyperglycaemia on CPA-evoked Ca2+ release
and Ca2+ influx
Cyclopiazonic acid, a specific Ca2+ SR Ca2+-ATPase inhibitor,19 caused
direct depletion of SR Ca2+ stores. In Ca2+-free medium, 10 mmol/L
CPA significantly caused a transient increase in [Ca2+]i (Ca2+ release)
that then declined to basal levels; subsequent addition of 2.0 mmol/L
Ca2+ to the medium evoked a marked, sustained increase that was
considered to be due to Ca2+ influx through store-operated Ca2+ channels
(Fig. 1a).
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Asia Pty Ltd
570
L Ma et al.
Fig. 1 Comparison of Ca2+ release and the Ca2+ influx induced by cyclopiazonic
acid (CPA) in control and diabetic rats 16 weeks after the induction of
diabetes (control = 50 cells from six rats, diabetes = 46 cells from six rats).
(a) In Ca2+-free medium, CPA (10 mmol/L) evoked a transient increase in
[Ca2+]i; restoration of extracellular Ca2+ (the final concentration in the
medium was 2.0 mmol/L) induced a sustained increase in [Ca2+]i. (–––),
control; (- - -), diabetes. (b) Histograms showing mean Ca2+ release and Ca2+
influx in smooth muscle cells from control () and diabetic () rats. Data
are expressed as the mean±SEM. *P < 0.05 compared with the control group.
Intracellular calcium is expressed as a ratio of fluorescence intensity relative
to basal fluorescence (i.e. DF = F – F0, where F represents the peak value of
fluorescence intensity after being activated by an agonist and F0 represents
the value of fluorescence intensity under resting conditions).
In aortic smooth muscle cells from age-matched controls, the Ca2+
release and Ca2+ influx induced by 10 mmol/L CPA were 0.45 ± 0.04
and 0.57 ± 0.05, respectively. In diabetic rats, the Ca2+ release and
Ca2+ influx were 0.28 ± 0.01 and 0.37 ± 0.01, respectively, both
values being significantly lower than those in controls (P < 0.05;
n = 6; Fig. 1).
Effects of hyperglycaemia on Ca2+ release from
IP3-sensitive and ryanodine-sensitive stores
In Ca2+-free HBS, 10 mmol/L phenylephrine induced a transient
increase in [Ca2+]i due to the depletion of IP3-sensitive Ca2+ stores.
Fig. 2 Comparison of Ca2+ release and Ca2+ influx induced by phenylephrine
(10 mmol/L) in control and diabetic rats 16 weeks after the induction of
diabetes (control = 47 cells from seven rats; diabetes = 40 cells from six rats).
(a) In Ca2+-free medium, phenylephrine evoked a transient increase in [Ca2+]i;
restoration of extracellular Ca2+ (the final concentration in the medium
was 2.0 mmol/L) induced a sustained increase in [Ca 2+]i. (–––), control;
(- - -), diabetes. (b) Histograms showing mean Ca2+ release and Ca2+ influx
in smooth muscle cells from control () and diabetic () rats. Data are
expressed as the mean±SEM. *P < 0.05 compared with the control group.
Intracellular calcium is expressed as a ratio of fluorescence intensity relative
to basal fluorescence (i.e. DF = F – F0, where F represents the peak value of
fluorescence intensity after being activated by an agonist and F0 represents
the value of fluorescence intensity under resting conditions).
The restoration of extracellular Ca2+ evoked a sustained increase in
[Ca2+]i. Figure 2 shows phenylephrine-induced Ca2+ release and Ca2+
influx in aortic smooth muscle cells from control rats (0.42 ± 0.04
and 0.53 ± 0.04, respectively; n = 7). In diabetic rats, Ca2+ release
and Ca2+ influx were decreased to 0.24 ± 0.04 and 0.34 ± 0.07,
respectively (P < 0.05; n = 6).
Previous studies have shown that 1 mmol/L ryanodine is able to
induce an increase in Ca2+ from ryanodine-sensitive Ca2+ stores by
activating ryanodine receptors.20 In the present study, we found
that 1 mmol/L ryanodine induced Ca2+ release (0.26 ± 0.07; n = 9)
in aortic smooth muscle cells in Ca2+-free medium; subsequent
restoration of extracellular Ca2+ also evoked a sustained increase in
[Ca2+]i (0.27 ± 0.03; n = 9). In diabetic rats, the ryanodine-induced
Ca2+ release (0.06 ± 0.01; n = 5) and Ca2+ influx (0.15 ± 0.02; n = 5)
were both significantly (P < 0.01) lower compared with controls
(Fig. 3).
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Asia Pty Ltd
SR Ca2+ mobilization in diabetes
Fig. 3 Comparison of Ca2+ release and Ca2+ influx induced by ryanodine
(1 mmol/L) in control and diabetic rats 16 weeks after the induction of
diabetes (control = 76 cells from nine rats; diabetes = 56 cells from five rats).
(a) In Ca2+-free medium, ryanodine evoked a transient increase in [Ca2+]i;
restoration of extracellular Ca2+ (the final concentration in the medium
was 2.0 mmol/L) induced a sustained increase in [Ca 2+]i. (–––), control;
(- - -), diabetes. (b) Histograms showing mean Ca2+ release and Ca2+ influx
in smooth muscle cells from control () and diabetic () rats. Data are
expressed as the mean±SEM. *P < 0.01 compared with the control group.
Intracellular calcium is expressed as a ratio of fluorescence intensity relative
to basal fluorescence (i.e. DF = F – F0, where F represents the peak value of
fluorescence intensity after being activated by an agonist and F0 represents
the value of fluorescence intensity under resting conditions).
Effects of hyperglycaemia on protein expression of IP3
and ryanodine receptors
First, we used HiMarkTM Pre-stained HMW Protein Standard
(Invitrogen) to mark locations of around 260 and 500 kDa. The
protein expression of IP3 and ryanodine receptors was determined
by immunoblot analysis. In aortic smooth muscle, protein levels of
the IP3 receptor (260 kDa) decreased by 31.8 ± 7.7% in diabetic rats
compared controls (P < 0.05; n = 6). Expression of ryanodine receptors
was also detected in aortic smooth muscle, with a molecular mass
of 500 kDa, and expression was decreased by 69.2 ± 8.4% in diabetic
rats compared with controls (P < 0.01; n = 7; Fig. 4).
Contractile response to phenylephrine,
CPA and ryanodine
In Ca2+-free solution, the maximal contractile responses induced by
phenylephrine (10 mmol/L) were decreased by 40.9% in diabetic rats
(108.4 ± 33.2 g/g wet tissue) compared with controls (183.3 ± 17.5 g/g
571
Fig. 4 Western blot analysis of inositol 1,4,5-trisphosphate (IP3) and
ryanodine receptor expression in vascular smooth muscle from 16 week
diabetic rats and age-matched control rats. (a) Representative western blots.
(b,c) Densitometric analysis showed that expression levels of the IP3 (b) and
ryanodine (c) receptors in smooth muscle were reduced in diabetic rats
compared with controls (n = 6 –7; *P < 0.05, **P < 0.01 vs the control group).
wet tissue). In Ca2+-containing solution, the contractile responses
to phenylephrine were also reduced in diabetic rats, by 35.9% (203.8
± 44.3 g/g wet tissue) compared with controls (318.1 ± 33.2 g/g wet
tissue; Fig. 5a,b). Furthermore, the contractile responses induced
by 10 mmol/L CPA were significantly decreased in diabetic rats
compared with controls in both Ca2+-free solution (48.7 ± 6.6 vs 75.3
± 6.1 g/g wet tissue, respectively) and after restoration of 2.0 mmol/L
Ca2+ (85.0 ± 18.1 vs 146.2 ± 22.5 g/g wet tissue, respectively; Fig. 5c,d).
However, ryanodine alone did not cause any contraction in rings
from either control or diabetic rats in the Ca2+-free solution. In Ca2+containing solution (2.0 mmol/L Ca2+), only one of 16 rings (combined diabetic and control groups) developed a slight contraction
(0.1 g) in the presence of 1 mmol/L ryanodine. In addition, maximal
contractile responses to phenylephrine were not affected by further
addition of ryanodine. These results suggest that the Ca2+ release and
Ca2+ influx evoked by ryanodine contribute less to the contractile
function of vascular smooth muscle in rats.
DISCUSSION
Many studies have suggested that abnormal [Ca2+]i homeostasis in
vascular smooth muscle cells may contribute to the pathogenesis of
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Asia Pty Ltd
572
L Ma et al.
Fig. 5 Contractile responses to phenylephrine (10 mmol/L; n = 7) and
cyclopiazonic acid (CPA; 10 mmol/L; n = 6) in aortic rings from both
diabetic () and control () rats at 16 weeks. (a,c) In Ca2+-free medium,
phenylephrine and CPA evoked a transient contraction; restoration of
extracellular Ca2+ (the final concentration in the medium was 2.0 mmol/L)
induced a sustained contraction. (b,d) Histograms showing mean contraction
induced by phenylephrine (b) and CPA (d) and restoration of Ca 2+. Data
are expressed as the mean±SEM. *P < 0.05, **P < 0.01 compared with
the control group.
diabetic vascular complications. A previous study has revealed that
receptor-dependent (i.e. a1-adrenoceptor agonist phenylephrine)and receptor-independent (i.e. CPA)-induced changes in [Ca2+]i and
contractiles responses are decreased in vascular smooth muscle cells
in diabetes.15 In the present study, we found that the Ca2+ release and
influx caused by CPA were attenuated in aortic smooth muscle cells
from diabetic rats; this is consistent with results from a previous
study in aortic rings in which the contractile response to CPA was
also attenuated in 12 week diabetic rats.15 Cyclopiazonic acid is a
specific inhibitor of Ca2+-ATPase in the SR / ER.19 It has been reported
that CPA can deplete Ca2+ stores and subsequently activate Ca2+ influx
by opening membrane store-operated Ca2+ channels.21 Thus, the
attenuation of Ca2+ release and Ca2+ influx induced by CPA suggests
a change in the Ca2+ pump and/or the size of the functional Ca2+
stores in diabetes.
In smooth muscle cells, the balance of SR Ca2+ stores is regulated
by Ca2+ uptake and Ca2+ release. Uptake of Ca2+ is achieved by Ca2+ATPase, which pumps Ca2+ back into the SR, whereas Ca2+ release
is regulated by the IP3- and ryanodine-sensitive Ca2+ release channels.
Some G-protein-coupled receptor agonists, including phenylephrine
and angiotensin II, increase [Ca2+]i by activating the phospholipase
C–IP3 pathway, subsequently causing both Ca2+ release from intracellular IP3-sensitive Ca2+ stores and Ca2+ influx across the plasma
membranes through store-operated Ca2+ channels.22,23 Our results
show that IP3-sensitive Ca2+ release and the subsequent Ca2+ influx
induced by phenylephrine, an a1-adrenoceptor agonist, were both
decreased in aortic smooth muscle cells from diabetic rats. In
addition to the IP3-sensitive Ca2+ store, recent studies have also
identified a ryanodine-sensitive Ca2+ store in smooth muscle cells.12–14
We found ryanodine-sensitive Ca2+ release and subsequent Ca2+
influx were also decreased in diabetic rats. To further understand the
molecular mechanisms of the decrease in IP3- and ryanodine-sensitive
Ca2+ release, we tested the protein expression levels of both receptors
in aortic smooth muscle from diabetic rats. We found that expression
of both the IP3 and ryanodine receptors was decreased in diabetic
rats. Our findings are consistent with a study that reported that
the renal Type I IP3 receptor is reduced in STZ-induced diabetic rats
and mice.24 Furthermore, previous studies have reported that, in mice
with disruption of the Type I IP3 receptor gene, IP3-induced calcium
release in the brain was almost completely abolished25 and the lack
of ryanodine receptor resulted in a decrease in the frequency of
release of calcium units in diaphragm muscles.26 These results suggest
that downregulation of the IP3 and ryanodine receptors contributes,
at least in part, to the attenuation of the corresponding Ca2+ release
in diabetes.
Impaired vascular contraction has been recognized in previous
studies on diabetes. Some of the results show that vascular smooth
muscle from diabetic animals exhibits increased contraction to vasoconstrictors.27,28 However, other studies have demonstrated that the
contraction is decreased in diabetes.29–31 These apparently contradictory
results may be due to different animal models used, different vascular
beds and different durations of diabetes in these experiments. Previously, we found that aortic smooth muscle exhibits hyperreactivity
to KCl, phenylephrine and CPA at an early stage of diabetes and
hyporeactivity at a late stage.15 The contraction of smooth muscle
cells is regulated by [Ca2+]i; Ca2+ release from SR stores and extracellular Ca2+ influx contribute to the regulation of [Ca2+]i. It has been
reported that hyperglycaemia or high glucose can inhibit storeoperated Ca2+ influx in neonatal cardiomyocytes, renal mesangial cells
and retinal microvascular smooth muscle cells.3,5,16,21 In the present
study, we found that Ca2+ release and Ca2+ influx due to depletion
of IP3-sensitive Ca2+ stores were reduced. Moreover, the contractile
responses in response to phenylephrine and CPA were both decreased
in aortas from diabetic rats; these results are consistent those of a
previous study15 in rat aortic rings from 12 week diabetic rats and
suggest that changes to the Ca2+ store contribute, at least in part, to
the hypocontractility of the aorta in the diabetic disease state. However, although Ca2+ release and Ca2+ influx induced by depletion of
ryanodine-sensitive Ca2+ stores were also reduced, ryanodine alone
did not produce any contraction in rings from either diabetic and
control rats, except in one sample from the control group. These
results suggest that ryanodine-sensitive Ca2+ stores may play different
roles from IP3-sensitive Ca2+ stores in aortic vascular smooth muscle
cells and contribute less to vascular contractility. Several studies
have reported that the ryanodine-sensitive Ca2+ store is involved in
apoptosis and secretion in Chinese hamster ovary cells and islet
b-cells.32,33 In rat basilar artery, ryanodine-sensitive Ca2+ stores were
documented not to be involved in the initiation of vasoconstriction,
but were involved in the activation of a hyperpolarizing current.10
Further studies in arterial smooth muscle cells have shown that the
caffeine/ryanodine-sensitive Ca2+ store is decreased when the cells
enter the cell cycle and this change is associated with the downregulation of the ryanodine RyR3 receptor and sarcoplasmic/endoplasmic
reticulum calcium ATPase (SERCA) 2a.34 However, the role of
changes in ryanodine-sensitive Ca2+ stores in diabetes in vascular
smooth muscles needs to be investigated further.
In summary, the results of the present study suggest that the hyporeactivity of aortic smooth muscle to vasoconstrictors in diabetes
results mainly from changes in the IP3-sensitive Ca2+ release
pathway. The SR Ca2+ signalling pathway plays a crucial role in
the development of diabetic vascular complications.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Asia Pty Ltd
SR Ca2+ mobilization in diabetes
ACKNOWLEDGEMENTS
This study was supported by grants from the National Natural Science Foundation of China (No. 30470698) and the emphasis project
of Natural Science Foundation of GuangDong province (No. 36625).
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© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Asia Pty Ltd