The present work was carried out in order to determine whether a decrease in cardiac Na + -Ca 2+ exchanger (NCX) activity observed in diabetes is caused by a reduction in NCX protein and mRNA levels ...and to elucidate the
significance of this decrease in alterations in Ca 2+ i homeostasis in diabetic cardiomyocytes.
The NCX current was significantly reduced in ventricular myocytes freshly isolated from streptozotocin-induced diabetic rat
hearts, and its current density was about 55 % of age-matched controls.
Diabetes resulted in a 30 % decrease in cardiac protein and mRNA levels of NCX1, a NCX isoform which is expressed at high
levels in the heart.
The reduced NCX current and the decreased protein and mRNA levels of NCX1 in diabetes were prevented by insulin therapy.
Although both diastolic and peak systolic Ca 2+ i were not different between the two groups of myocytes, increasing external Ca 2+ concentration to high levels greatly elevated diastolic Ca 2+ i in diabetic myocytes. Inhibition of NCX by reduction in extracellular Na + by 50 % could produce a marked rise in diastolic Ca 2+ i in control myocytes in response to high Ca 2+ , as seen in diabetic myocytes. However, cyclopiazonic acid, an inhibitor of sarcoplasmic reticulum Ca 2+ pump ATPase, did not modify the high Ca 2+ -induced changes in diastolic Ca 2+ i in either control or diabetic myocytes.
Only in papillary muscles from diabetic rats did the addition of high Ca 2+ cause a marked rise in resting tension signifying a partial contracture that was possibly due to an increase in diastolic
Ca 2+ i .
In conclusion, the diminished NCX function in diabetic myocytes shown in this study results in part from the decreased levels
of cardiac NCX protein and mRNA. We suggest that this impaired NCX function may play an important role in alterations in Ca 2+ handling when Ca 2+ i rises to pathological levels.
The aim of this study was to determine whether endothelium‐dependent hyperpolarization and relaxation are altered during experimental diabetes mellitus. Membrane potentials were recorded in ...mesenteric arteries from rats with streptozotocin‐induced diabetes and age‐matched controls. The resting membrane potentials were not significantly different between control and diabetic mesenteric arteries (−55.3±0.5 vs −55.6±0.4 mV). However, endothelium‐dependent hyperpolarization produced by acetylcholine (ACh; 10−8–10−5 M) was significantly diminished in amplitude in diabetic arteries compared with that in controls (maximum −10.4±1.1 vs −17.2±0.8 mV). Furthermore, the hyperpolarizing responses of diabetic arteries were more transient.
ACh‐induced hyperpolarization observed in control and diabetic arteries remained unaltered even after treatment with 3×10−4 M NG‐nitro‐L‐arginine (L‐NOARG), 10−5 M indomethacin or 60 u ml−1 superoxide dismutase.
Endothelium‐dependent hyperpolarization with 10−6 M A23187, a calcium ionophore, was also decreased in diabetic arteries compared to controls (−8.3±1.4 vs −18.0±1.9 mV). However, endothelium‐independent hyperpolarizing responses to 10−6 M pinacidil, a potassium channel opener, were similar in control and diabetic arteries (−20.0±1.4 vs −19.2±1.1 mV).
The altered endothelium‐dependent hyperpolarizations in diabetic arteries were almost completely prevented by insulin therapy. Endothelium‐dependent relaxations by ACh in the presence of 10−4 M L‐NOARG and 10−5 M indomethacin in diabetic arteries were also reduced and more transient compared to controls.
These data indicate that endothelium‐dependent hyperpolarization is reduced by diabetes, and this would, in part, account for the impaired endothelium‐dependent relaxations in mesenteric arteries from diabetic rats.
Vascular abnormalities are a major cause of postoperative complications in irradiated tissues. Endothelial cell dysfunction characterized by diminished endothelium-dependent relaxation may be ...involved. We examined the endothelium-dependent relaxation and morphology of the endothelium in irradiated human cervical arteries.
Irradiated arteries were taken from the neck region of patients who had radiation therapy. Arteries from patients who did not receive radiation therapy were used as controls. Endothelium-dependent relaxation to acetylcholine and A23187 was impaired in irradiated arteries. Norepinephrine-induced contraction and sodium nitroprusside-induced relaxation were unchanged. In control arteries, N(omega)-nitro-L-arginine and indomethacin each caused a partial inhibition of endothelium-dependent relaxation. In irradiated arteries, the impaired endothelium-dependent relaxation was unaffected by these agents, but it was abolished by high K(+). Acetylcholine produced similar degrees of hyperpolarization in control and irradiated arteries. Immunohistochemical examination for endothelial nitric oxide synthase indicated no expression in the endothelium of irradiated arteries. Electron scanning microscopy showed morphologically intact endothelial cells in irradiated arteries.
In irradiated human cervical arteries, the nitric oxide- and prostacyclin-mediated endothelium-dependent relaxation, but not endothelium-derived hyperpolarizing factor-mediated relaxation, are specifically impaired, without significant morphological damage of the endothelium. The impaired nitric oxide-mediated relaxation was associated with a lack of endothelial nitric oxide synthase expression. Our results suggest the importance of impaired endothelial function in irradiated human blood vessels, which may partly explain the development of vascular stenosis and poor surgical wound healing in irradiated tissues.
1
In rat mesenteric artery, acetylcholine (ACh) causes endothelium‐dependent hyperpolarization by releasing endothelium‐derived hyerpolarizing factor (EDHF). Recent evidence suggests that EDHF may be ...a cytochrome P450‐derived arachidonic acid metabolite. The aim of the present study was to investigate whether such a metabolite is indeed contributing to ACh‐induced hyperpolarization observed in rat mesenteric artery.
2
The phospholipase A2 inhibitor quinacrine (30 μm) nearly completely eliminated ACh‐induced hyperpolarization. However, the hyperpolarizing effect of pinacidil was also abolished in the presence of quinacrine.
3
The imidazole antimycotic agents ketoconazole (50 μm), clotrimazole (30 μm) and miconazole (10 μm), which bind to the heme moiety of cytochrome P450, eliminated not only ACh‐induced hyperpolarizations but also those induced by pinacidil. SKF525A (30 μm), a prototype inhibitor of the enzyme, also abolished the hyperpolarizing responses to both agents. In contrast, neither 17‐octadecynoic acid (10 μm), a mechanism‐based inhibitor of cytochrome P450 metabolism of fatty acids, nor eicosatetraynoic acid (20 μm), an inhibitor of all arachidonic acid metabolic pathways, altered ACh‐induced hyperpolarization. Furthermore, the hyperpolarization was unaffected by the preferential inhibitors of specific cytochrome P450 isozymes, α‐naphtofiavone (1 μm), diedthyldithiocarbamate (50 μm), metyrapone (20 μm) and troleandomycin (10 μm).
4
Pretreatment of rats with lipopolysaccharide (2mgkg−1) and exposure to nitroprusside (10 μm), both of which are expected to inhibit cytochrome P450 activity due to nitric oxide overproduction, were without effect on ACh‐induced hyperpolarization. Pretreatment of rats for 3 days with pentobarbitone (80 mg kg−1 day−1), a cytochrome P450 inducer, also did not affect the hyperpolarizing response to ACh.
5
Arachidonic acid in concentrations up to 100 μM had no detectable effect on smooth muscle membrane potential. 11,12‐Epoxyeicosatrienoic acid (EET, 10 μm), one of cytochrome P450‐derived epoxygenase metabolites of arachidonic acid, elicited a small endothelium‐independent membrane hyperpolarization. The hyperpolarizing response to EET was blocked by glibenclamide (30 μm), in contrast to the response to ACh.
6
These results suggest that the contribution of a cytochrome P450‐derived metabolite of arachidonic acid to ACh‐induced hyperpolarization via EDHF release is minimal or absent in rat mesenteric artery.
The aim of the present study was to identify the sources of Ca2+ contributing to acetylcholine (ACh)‐induced release of endothelium‐derived hyperpolarizing factor (EDHF) from endothelial cells of rat ...mesenteric artery and to assess the pathway involved. The changes in membrane potentials of smooth muscles by ACh measured with the microelectrode technique were evaluated as a marker for EDHF release.
ACh elicited membrane hyperpolarization of smooth muscle cells in an endothelium‐dependent manner. The hyperpolarizing response was not affected by treatment with 10 μm indomethacin, 300 μm NG‐nitro‐l‐arginine or 10 μm oxyhaemoglobin, thereby indicating that the hyperpolarization is not mediated by prostanoids or nitric oxide but is presumably by EDHF.
In the presence of extracellular Ca2+, 1 μm ACh generated a hyperpolarization composed of the transient and sustained components. By contrast, in Ca2+‐free medium, ACh produced only transient hyperpolarization.
Pretreatment with 100 nm thapsigargin and 3 μm cyclopiazonic acid, endoplasmic reticulum Ca2+‐ATPase inhibitors, completely abolished ACh‐induced hyperpolarization. Pretreatment with 20 mm caffeine also markedly attenuated ACh‐induced hyperpolarization. However, the overall pattern and peak amplitude of hyperpolarization were unaffected by pretreatment with 1 μm ryanodine.
In the presence of 5 mm Ni2+ or 3 mm Mn2+, the hyperpolarizing response to ACh was transient, and the sustained component of hyperpolarization was not observed. On the other hand, 1 μm nifedipine had no effect on ACh‐induced hyperpolarization.
ACh‐induced hyperpolarization was nearly completely eliminated by 500 nm U‐73122 or 200 μm 2‐nitro‐4‐carboxyphenyl‐N,N‐diphenylcarbamate, inhibitors of phospholipase C, but was unchanged by 500 nm U‐73343, an inactive form of U‐73122. Pretreatment with 20 nm staurosporine, an inhibitor of protein kinase C, did not modify ACh‐induced hyperpolarization.
These results indicate that the ACh‐induced release of EDHF from endothelial cells of rat mesenteric artery is possibly initiated by Ca2+ release from inositol 1,4,5‐trisphosphate (IP3)‐sensitive Ca2+ pool as a consequence of stimulation of phospholipid hydrolysis due to phospholipase C activation, and maintained by Ca2+ influx via a Ni2+‐ and Mn2+‐sensitive pathway distinct from L‐type Ca2+ channels. The Ca2+‐influx mechanism seems to be activated following IP3‐induced depletion of the pool.
British Journal of Pharmacology (1997) 120, 1328–1334; doi:10.1038/sj.bjp.0701027
1
Electrophysiological effects of MS‐551, a new class III antiarrhythmic drug, were examined and compared with those of (+)‐sotalol in rabbit ventricular cells.
2
In rabbit ventricular muscles ...stimulated at 1.0 Hz, MS‐551 (0.1–10 μm) and (+)‐sotalol (3–100 μm) prolonged action potential duration (APD) and effective refractory period without affecting the maximum upstroke velocity of phase 0 depolarization . The class III effect of MS‐551 was approximately 30 times more potent than that of (+)‐sotalol.
3
Class III effects of MS‐551 and (+)‐sotalol showed reverse use‐dependence, i.e., a greater prolongation of APD at a longer cycle length.
4
In rabbit isolated ventricular cells, 3 μm MS‐551 and 100 μm sotalol inhibited the delayed rectifier potassium current (IK) which was activated at more positive potentials than −50 mV and saturated around + 20 mV.
5
MS‐551 at a higher concentration of 10 μm decreased the transient outward current (Ito) and the inward rectifier potassium current (IK1) although 100 μm sotalol failed to inhibit these currents.
6
MS‐551 is a non‐specific class III drug which can inhibit three voltage‐gated K+ channels in rabbit ventricular cells.
Endothelium-dependent relaxation was examined in aortic ring preparations obtained from rats with streptozotocin-induced diabetes. The endothelium-dependent relaxation which was produced by ...acetylcholine and histamine in aortic rings precontracted with norepinephrine was significantly attenuated in aortic rings from diabetic rats when compared with the relaxation in rings from age-matched control animals. However, the relaxation induced by sodium nitroprusside (an endothelium-independent relaxant agent) in diabetic preparations was comparable to the control. These results show that diabetes leads to an impairment of the endothelium-dependent relaxation of aorta.
1
The present study was designed to determine whether putative, selective inhibitors of the Ca2+–pump ATPase of endoplasmic reticulum, thapsigargin (TSG) and cyclopiazonic acid (CPA), induce ...endothelium‐dependent hyperpolarization in the rat isolated mesenteric artery. The membrane potentials of smooth muscle cells of main superior mesenteric arteries were measured by the microelectrode technique.
2
In tissues with endothelium, TSG (10−8–10−5m) caused sustained hyperpolarization in a concentration‐dependent manner. In tissues without endothelium, TSG did not cause any change in membrane potential. CPA (10−5 m) also hyperpolarized the smooth muscle membrane, an effect that was endothelium‐dependent and long‐lasting.
3
The hyperpolarizing responses to these agents were not affected by indomethacin or NG–nitro‐L‐arginine (L‐NOARG).
4
In Ca2+–free medium, neither TSG nor CPA elicited hyperpolarization, in contrast to acetylcholine which generated a transient hyperpolarizing response.
5
In rings of mesenteric artery precontracted with phenylephrine, TSG and CPA produced endothelium‐dependent relaxations. L‐NOARG significantly inhibited the relaxations to these agents, but about 40–60% of the total relaxation was resistant to L‐NOARG. The L‐NOARG‐resistant relaxations were abolished by potassium depolarization.
6
These results indicate that TSG and CPA can cause endothelium‐dependent hyperpolarization in rat mesenteric artery possibly by releasing endothelium‐derived hyperpolarizing factor and that membrane hyperpolarization can contribute to the endothelium‐dependent relaxations to these agents. The mechanism of hyperpolarization may be related to increased Ca2+ influx into endothelial cells triggered by depletion of intracellular Ca2+ stores due to inhibition of endoplasmic reticulum Ca2+–pump ATPase activity.
Deartments of Pharmacology and Cardiovascular Medicine, Hokkaido
University School of Medicine, Sapporo 060-8638; and Department of
Clinical Pharmacology and Therapeutics, Hamamatsu University School ...of
Medicine, Hamamatsu 431-3192, Japan
Elevation of intracellular Ca 2+ concentration
(Ca 2+ i ) in endothelial cells is proposed to
be required for generation of vascular actions of endothelium-derived
hyperpolarizing factor (EDHF). This study was designed to determine the
endothelial Ca 2+ source that is important in development of
EDHF-mediated vascular actions. In porcine coronary artery
precontracted with U-46619, bradykinin (BK) and cyclopiazonic acid
(CPA) caused endothelium-dependent relaxations in the presence of
N G -nitro- L -arginine
( L -NNA). The L -NNA-resistant relaxant responses were inhibited by high K + , indicating an involvement of
EDHF. In the presence of Ni 2+ , which inhibits
Ca 2+ influx through nonselective cation channels, the
BK-induced EDHF relaxant response was greatly diminished and the
CPA-induced response was abolished. BK and CPA elicited membrane
hyperpolarization of smooth muscle cells of porcine coronary artery.
Ni 2+ suppressed the hyperpolarizing responses in a manner
analogous to removal of extracellular Ca 2+ . EDHF-mediated
relaxations and hyperpolarizations evoked by BK and CPA in porcine
coronary artery showed a temporal correlation with the increases in
Ca 2+ i in porcine aortic endothelial cells.
The extracellular Ca 2+ -dependent rises in
Ca 2+ i in endothelial cells stimulated with
BK and CPA were completely blocked by Ni 2+ . These results
suggest that Ca 2+ influx into endothelial cells through
nonselective cation channels plays a crucial role in the regulation of EDHF.
bradykinin; cyclopiazonic acid; vascular relaxation; membrane
hyperpolarization; intracellular calcium concentration
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