Toshihiko Hashimoto

Inositol 1,4,5-trisphosphate releases Ca2+ from intracellular store sites in skinned single cells of porcine coronary artery

Effects of inositol 1,4,5- trisphosphate , extracted from human erythrocyte ghosts, on Ca2+ release from intracellular store sites were studied in saponin-treated single muscle cells of the porcine coronary artery. Application of micromolar concentrations of inositol 1,4,5- trisphosphate released Ca2+ from the intracellular non-mitochondrial store sites, within 1 min. However, when the concentrations of free Ca2+ were over 1.5 X 10(-6) M, the release of Ca2+ by this agent was inhibited. The Ca2+ releasing mechanism differed from that seen with A23187, therefore this release of Ca2+ from store sites was not due to Ca2+ ionophore actions. This agent may play the role of messenger in increasing the cytosolic Ca2+, provoking pharmaco-mechanical coupling, and thus producing the contraction.

Oxidative stress measurement by in vivo electron spin resonance spectroscopy in rats with streptozotocin-induced diabetes.

Summary Enhanced oxidative stress in diabetic patients may contribute to the pathogenesis of diabetic angiopathy. We have recently developed a method to determine the electron spin resonance (ESR, electron paramagnetic resonance; EPR) of reactive oxygen species and free radicals in vivo, using the nitroxide derivative, carbamoyl-PROXYL as a probe. In this study, diabetes was induced in Wistar rats by streptozotocin (STZ) injection (65 mg/kg, body weight, intravenously). Two, 4, and 8 weeks later, the animals received carbamoyl-PROXYL (300 nmol/g, intravenously), and ESR was measured at the upper abdominal level at a frequency of 300 MHz. The intensity of the carbamoyl-PROXYL ESR signal decreased gradually after the injection, and the spin clearance rate was determined over the first 5 min. At all time points, the spin clearance rate was significantly greater in the diabetic rats than in control rats. Moreover, the spin clearance rate in the diabetic rats was significantly correlated with urinary malondialdehyde (MDA) levels, which serve as a marker for lipid peroxidation. Daily treatment with 4 units neutral protamin Hagedorn (NPH) insulin for 4 weeks reduced the spin clearance rate in the diabetic rats. Simultaneous injection of carbamoyl-PROXYL and superoxide dismutase reduced the spin clearance rate in the diabetic rats in a dose-dependent manner. Injection of the antioxidant α-tocopherol (40 mg/kg, intraperitoneally) for 2 weeks restored the spin clearance rate in the diabetic rats without concomitant glycaemic restoration. These results suggest that a diabetic state enhances the generation of free radicals in vivo, and that both glycaemic control and antioxidant treatment can reduce this oxidative stress. Non-invasive in vivo ESR measurement may be useful for evaluating oxidative stress in diabetes. [Diabetologia (1998) 41: 1355–1360]

A role for inositol 1,4,5‐trisphosphate in the initiation of agonist‐induced contractions of dog tracheal smooth muscle

1 To elucidate the role of inositol 1,4,5‐trisphosphate (Ins‐P3) in the initiation of agonist‐induced contraction of the smooth muscle cells of the dog trachea, we investigated the effects of acetylcholine (ACh) on the concentrations of Ins‐P3, phosphatidylinositol‐4,5‐bisphosphate (PI‐P2) or phosphatidic acid (PA). The effects of Ins‐P3 on the Ca2+ stored in the smooth muscle cells were also studied in saponin‐permeabilized smooth muscle cells. 2 A half maximal or maximal Ca2+ accumulation into the cells was observed in the dispersed single, smooth muscle cells treated by saponin, in free Ca2+ concentrations of 4.6 × 10−7 or 5 × 10−5 M, respectively. The ATP‐dependent Ca2+ accumulation was maximal at 0.63 nmol/105 cells. 3 Effects of Ins‐P3 on stored Ca2+ were observed at a free Ca2+ concentration of 3.7 × 10−7 M, which induces about half maximal ATP‐dependent Ca2+‐accumulation. Ins‐P3 released the Ca2+ accumulated by ATP, in a dose‐dependent manner. About 40% of the total Ca2+ was released following application of 3 μM Ins‐P3. 4 The release of stored Ca2+ induced by application of Ins‐P3 was followed by its re‐uptake into the smooth muscle cells. Thus, the stored Ca2+ was repeatedly released with repetitive applications of Ins‐P3. 5 Application of ACh (10−5 M) to the dog trachea stimulated the production of Ins‐P3 in the soluble fraction and 10 s after this application, the relative amount of Ins‐P3 was 290% of the control value. 6 Concomitantly, ACh (10−5 M) either reduced or increased the contents of phosphatidyl inositol 4,5‐biphosphate (PI‐P2) or phosphatidic acid (PA) in the lipid fraction of the smooth muscle cells to 60% or to 350% of the control value, respectively, thereby indicating that ACh stimulates the phosphodiesteric hydrolysis of PI‐P2. 7 5‐Hydroxytryptamine (5‐HT; 10−5 M) also reduced or increased the contents of PI‐P2 or PA to 80 or to 200% of the control values, respectively. However, neither histamine (10−5 M), in the presence or absence of cimetidine (10−5 M), nor prostaglandin F2α (PGF2α 10−7 M) showed any effect on the contents of PI‐P2 or PA in the lipid fraction of the smooth muscle cells. 8 These results indicate that in muscle cells of the dog trachea, Ins‐P3 may play the role of intracellular second messenger in the initiation of ACh or 5‐HT‐induced contraction, but not in the case of histamine or PGF2α‐induced contraction.

Retinal expression, regulation, and functional bioactivity of prostacyclin-stimulating factor

Prostacyclin-stimulating factor (PSF) acts on vascular endothelial cells to stimulate the synthesis of the vasodilatory molecule prostacyclin (PGI2). We have examined the expression, regulation, and hemodynamic bioactivity of PSF both in whole retina and in cultured cells derived from this tissue. PSF was expressed in all retinal cell types examined in vitro, but immunohistochemical analysis revealed PSF mainly associated with retinal vessels. PSF expression was constitutive in retinal pericytes (RPCs) but could be modulated in bovine retinal capillary endothelial cells (RECs) by cell confluency, hypoxia, serum starvation, high glucose concentrations, or inversely by soluble factors present in early vs. late retinopathy, such as TGF-beta, VEGF, or bFGF. In addition, RPC-conditioned media dramatically increased REC PGI2 production, a response inhibited by blocking PSF with a specific antisense oligodeoxynucleotide (ODN). In vivo, PGI2 increased retinal blood flow (RBF) in control and diabetic animals. Furthermore, the early drop in RBF during the initial weeks after inducing diabetes in rats, as well as the later increase in RBF, both correlated with levels of retinal PSF. RBF also responded to treatment with RPC-conditioned media, and this effect could be partially blocked using the antisense PSF ODN. We conclude that PSF expressed by ocular cells can induce PGI2, retinal vascular dilation, and increased retinal blood flow, and that alterations in retinal PSF expression may explain the biphasic changes in RBF observed in diabetes.

Saturated non-esterified fatty acids stimulate de novo diacylglycerol synthesis and protein kinase c activity in cultured aortic smooth muscle cells

Aims/hypothesis. Insulin resistance is linked with a cluster of multiple risk factors and excessive acceleration of atherosclerosis. The underlying mechanism is not, however, fully understood. Methods. To determine the link between insulin resistance and altered vascular function, we focused on the effect of various non-esterified fatty acids on diacylglycerol-protein kinase C pathway and mitogen-activated protein kinase activity in cultured aortic smooth muscle cells. Results.Incubation of the cells with saturated non-esterified fatty acids (200 μmol/l) for 24 h, such as palmitate or stearate, induced a significant increase in diacylglycerol concentrations by about fivefold or eightfold, respectively, whereas oleate induced a slight increase in diacylglycerol concentrations by 1.8-fold and arachidonate induced none. In addition, the increased diacylglycerol concentrations induced by palmitate were completely restored to control concentrations by triacsin C, acyl-CoA synthetase inhibitor. These results suggest that saturated non-esterified fatty acids may increase diacylglycerol concentrations through de novo pathway by stepwise acylation. In parallel with the increased diacylglycerol, incubation of the cells with saturated non-esterified fatty acids significantly induced the activation of protein kinase C and mitogen-activated protein kinase. The palmitate-induced increase in mitogen-activated protein kinase activity was restored to control concentrations by GF109203X (5 · 10–7 mol/l), a specific protein kinase C inhibitor, suggesting a protein kinase C-dependent activation of mitogen-activated protein kinase. Conclusion/interpretation. Saturated non-esterified fatty acids induced an increase in de novo diacylglycerol synthesis and subsequent activation of protein kinase C and mitogen-activated protein kinase in cultured aortic smooth muscle cells. This could contribute to the altered vascular functions in the insulin resistant state. [Diabetologia (2001) 44: 614–620]

Changes in Phosphoinositide Turnover, Ca2+ Mobilization, and Protein Phosphorylation in Platelets From NIDDM Patients

Enhanced platelet functions have been demonstrated in patients with non-insulin-dependent diabetes mellitus (NIDDM). This study evaluated abnormalities in platelet signal transduction in diabetic patients, including turnover of phosphoinositides, mobilization of intracellular Ca2+, and phosphorylation of 20,000- and 47,000-Mr proteins (P20 and P47). Washed platelets were obtained from 6 patients with NIDDM whose platelet aggregation rates were abnormally elevated (DM-A group), 11 NIDDM patients with normal platelet aggregation rates (DM-B group), and 8 age-matched healthy control subjects. The mass and specific radioactivity of phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol (PI), and phosphatidic acid (PA) in 32P-labeled platelets were not different among the three groups. Hydrolysis of PIP2, PIP, and PI; accumulation of PA; and phosphorylation of P20 in platelets stimulated by 0.05 U/ml thrombin were significantly increased in the DM-A group compared with the control or DM-B group. There was no difference in P47 phosphorylation among the three groups. On the contrary, P20 and P47 phosphorylation induced by 50 nM of 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C, was significantly decreased in the DM-A group. Additionally, the intracellular free Ca2+ concentration ([Ca2+]1) was measured with the fluorescent Ca2+ indicator fura 2. Although the basal [Ca2+]1 value was similar in the three groups, the rise in [Ca2+]1 induced by 0.05 U/ml thrombin in the presence and the absence of extracellular Ca2+ was significantly higher in the DM-A group than the other groups. These data suggest that the enhanced platelet aggregation in NIDDM may be closely related to 7) increases in phosphoinositide hydrolysis, intracellular Ca2+ mobilization, and myosin light-chain kinase-mediated P20 phosphorylation and 2) decreases in P20 and P47 phosphorylation by protein kinase C.

Increased intracellular calcium mobilization in platelets from patients with Type 2 (non-insulin-dependent) diabetes mellitus

SummaryEnhanced platelet functions have been reported in patients with diabetes mellitus. Our recent study demonstrated that phosphoinositide turnover is increased in platelets from diabetic patients. In the present study, we evaluated the abnormality in platelet intracellular calcium mobilization in patients with Type 2 (non-insulin-dependent) diabetes mellitus using fura-2, a fluorescent calcium indicator. Washed platelets were prepared from six diabetic patients with increased platelet aggregation rates (DM-A group), seven diabetic patients with normal platelet aggregation rates (DM-B group), and eight age-matched healthy control subjects. The basal intracellular free calcium concentrations in platelets were similar among the three groups. Thrombin (0.025–0.1 U/ml) induced a dose-dependent increase in intracellular calcium in both the presence and the absence of extracellular calcium. This increase in the presence of extracellular calcium, which depends on calcium influx and release, was significantly higher in the DM-A group than in the DM-B and control groups. However, there was no significant difference between the control group and the DM-B group. In the absence of extracellular calcium, thrombin-induced calcium increase, which depends only on calcium release, was also significantly enhanced in the DM-A group. Furthermore, the calcium increase stimulated by platelet-activating factor (10 nmol/l) with and without extracellular calcium was significantly higher in the DM-A group than in the other groups. Additionally, calcium ionophore A23187 (100 nmol/l) caused a significantly higher calcium increase in the DM-A group with extracellular calcium, while the calcium increase without extracellular calcium showed no significant difference among the three groups. These observations suggest that enhanced intracellular calcium mobilization due to increased calcium influx and release may be closely related to platelet hyperfunctions in diabetes mellitus.

Increased Transendothelial Permeation of Albumin by High Glucose Concentration

Vascular endothelial cells, which are polyfunctional, play an important role in the pathogenesis of diabetic complications. The increase in vascular permeability, ie, regulated by vascular endothelial cells, has been reported in patients with diabetes mellitus complicated by angiopathy. To determine the role of hyperglycemia in endothelial cell permeability, we examined the effect of high concentrations of glucose on the permeability of cultured bovine aortic endothelial cells. The permeations of albumin and fluorescein-labeled dextran (FD) across endothelial cell monolayers were increased when cultured with a high concentration of glucose (400 mg/dL). This increased permeation of albumin but not FD was temperature-dependent and was partially reduced by adding 100 mumol/L ponalrestat (ICI 128,436, Statil; ICI, Cheshire, UK), which is an aldose reductase inhibitor. Stimulation or inhibition of Na,K-adenosine triphosphatase (ATPase) in bovine aortic endothelial cells failed to alter their permeability. These findings suggest that high concentrations of glucose enhance transendothelial permeability of albumin in part by activating the polyol pathway, but independently of Na,K-ATPase activity.

Actions of nicorandil on vascular smooth muscles.

The action of nicorandil on vascular smooth muscles has been studied in vitro using the microelectrode, Ca-transient, isometric tension recording methods, and the bioassay methods of cyclic nucleotides and inositol phospholipids. Nicorandil increased Ca-insensitive K conductance and hyperpolarized the membrane and thus prevented the activation of the voltage-dependent Ca channel. The hyperpolarization occurred to a greater extent in venous tissue than in arterial tissue. Nicorandil stimulated the synthesis of cyclic guanosine monophosphate (cGMP) in the polarized and depolarized muscle tissues, as did nitroglycerine. Consequently, nicorandil reduces the concentrations of free Ca in the myoplasm, due to acceleration of the Ca pump at the sarcolemma, and may prevent the phosphorylation of myosin through phosphorylation of myosin light chain kinase. These actions of nicorandil may not contribute to the synthesis of inositol-1,4,5-trisphosphate hydrolyzed from phosphatidylinositol-4,5-bisphosphate. The above actions of nicorandil—hyperpolarization and increase in the cyclic GMP—may cause relaxation of the tissues precontracted by various stimulants.

INCREASED MRNA EXPRESSION OF A NOVEL PROSTACYCLIN-STIMULATING FACTOR IN HUMAN COLON CANCER

Abstract: We recently cloned a prostacyclin (PGl2)-stimulating factor (PSF), which stimulates PGl2 production by cultured vascular endothelial cells. Immunohistochemistry and Northern blot analysis demonstrated that PSF was highly expressed in colon cancer sites compared with normal colon mucosa obtained from the same patient, as well as in cultured adenocarcinoma cell lines compared with cultured normal colon mucosal cell lines. Increased levels of the PSF protein were detected in the culture media of these adenocarcinoma cells compared with levels in the culture media of normal mucosal cells. These results suggest that PSF is closely associated with carcinogenesis of colon mucosa.