15-HETE影响大鼠脑动脉平滑肌细胞内钙离子浓度的机制

目的 观察15-羟化二十烷四烯酸(15-HETE)对脑动脉平滑肌细胞内钙离子浓度([Ca2+]i)的影响,进一步探讨15-HETE引起[Ca2+]i变化的钙来源,从而明确15-HETE引起脑动脉平滑肌收缩的机制. 方法 酶法分离大鼠脑动脉平滑肌细胞,分为15-HETE组与对照组,15-HETE组添加15-HETE处理;对照组正常培养,不做其他处理.激光共聚焦技术测定15-HETE对[Ca2+]i的影响;进一步通过阻断外钙内流和耗竭内钙,探明15-HETE引起钙动员的来源;应用血管环技术从功能上判定细胞外钙对15-HETE引起的颈内动脉环收缩有无影响. 结果 15-HETE组与对照组相比,[Ca2+]i明显增加,差异有统计学意义(P＜0.05);预先加入硝苯地平、镧离子及改用无钙液阻断外钙内流后,15-HETE组[Ca2+]i仍明显高于对照组,差异有统计学意义(P＜0.05);而预先加入咖啡因耗竭细胞内钙后,15-HETE组[Ca2+]i较对照组差异无统计学意义(P＞0.05);采用无钙液去除细胞外钙后,15-HETE引起的血管环张力增加与有钙液中比较差异无统计学意义(P＞0.05). 结论 15-HETE可通过促使内钙释放而使[Ca2+]i增加,进而引起大鼠脑动脉平滑肌收缩.     

Bloody cerebrospinal fluid from patients with subarachnoid hemorrhage alters intracellular calcium regulation in cultured human vascular endothelial cells

Endothelial cell dysfunction may contribute to cerebral vasospasm and aggravation of ischemic brain damage following subarachnoid hemorrhage (SAH). It has been suggested that oxyhemoglobin derived from subarachnoid blood clots might be a prime candidate for cerebral vasospasm. In this study, cisternal bloody cerebrospinal fluid (bCSF) was collected from SAH patients four and seven days after aneurysmal rupture, and the effects of bCSF on the cell growth and intracellular calcium ion ([Ca2+]i) dynamics were investigated in cultured human umbilical vein endothelial cells. CSF collected from patients undergoing other intracranial surgeries was used as a control. Pre-treatment with bCSF4 significantly facilitated cell proliferation and DNA synthesis in the cultured endothelial cells, and significantly enhanced histamine-induced [Ca2+]i increase, while acute treatment of the bCSF elicited no [Ca2+]i change. Pre-treatment with interleukin-1 beta showed a similar significant enhancement of the histamine-induced [Ca2+]i response, while pre-treatment with high concentrations of serum or interleukin-6 did not change the [Ca2+]i response. It is concluded that bCSF collected from SAH patients contains some substances which enhance endothelial cell proliferation and sensitivity to inflammatory mediator.     

Intracellular Ca2+ changes in cultured vascular smooth muscle cells by treatment with various spasmogens.

In order to evaluate various spasmogens, which are candidates for cerebral vasospasm after subarachnoid haemorrhage, the intracellular calcium mobilizations were examined in cultured vascular smooth muscle cells preloaded with a fluorescent Ca2+ probe fura-2. Endothelin, oxyhaemoglobin, 5-hydroxytryptamine, norepinephrine, prostaglandin F2 alpha, leukotrienes C4 and D4 produced dose-dependent increases in intracellular Ca2+ concentration ([Ca2+]i). However, bilirubin did not induce any significant [Ca2+]i elevation. The maximal levels of [Ca2+]i peak attained by endothelin or oxyhaemoglobin were higher than those of other compounds. Endothelin was the most potent in that it induced a high sustained [Ca2+]i elevation at much lower concentrations compared with others. The combination of oxyhaemoglobin and endothelin induced a transient increase in [Ca2+]i followed by a sustained lower plateau, then the [Ca2+]i level was again increased slowly followed by a sustained higher plateau which lasted for more than 10 min after the exposure. These results suggest that endothelin and/or oxyhaemoglobin may play a crucial role in contraction of vascular smooth muscle after subarachnoid haemorrhage.     

Vasorelaxing effect of the Rho-kinase inhibitor, Y-27632, in isolated canine basilar arteries

OBJECTIVES: Increased calcium sensitization mediated by Rho/Rho-kinase may be important in the pathogenesis of cerebral vasospasm. The effects of a highly selective Rho-kinase inhibitor, Y-27632, were investigated on spasmogen-induced contractions of canine basilar artery. METHODS: Typical spasmogenic substances present after subarachnoid hemorrhage (SAH), including prostaglandin F2a (PGF2a), 12-deoxyphorbol 13-isobutyrate (DPB), sphingosylpho-sphorylcholine (SPC) and high K+, were used in the study. Isometric tension was recorded in canine basilar artery rings in vitro. Intracellular calcium concentration ([Ca2+]i) and contraction force were measured simultaneously in fura-2-loaded canine basilar artery strips. The myosin light chain (MLC) phosphorylation levels were measured by glycerol gel electrophoresis followed by Western blotting. RESULTS: Isometric tension recording revealed that the Rho-kinase inhibitor, Y-27632, dose-dependently inhibited vasocontraction induced by PGF2a and SPC, but not that induced by DPB. Simultaneous recordings of [Ca2+]i and tension revealed that the vasorelaxing effect of Y-27632 was not associated with changes in [Ca2+]i, suggesting that Y-27632 may inhibit calcium sensitization. Vasocontraction induced by DPB was not inhibited by Y-27632, but was inhibited by staurosporine. Phosphorylation of MLC was increased by PGF2a and SPC, and significantly inhibited by Y-27632, whereas such phosphorylation was increased by DPB, but not significantly inhibited by Y-27632. DISCUSSION: Several spasmogenic mediators released after SAH may cause vasospasm through Rho-kinase-mediated increase in calcium sensitization. Rho-kinase inhibitors, including Y-27632, may be effective for the prevention of cerebral vasospasm after SAH.     

Involvement of mitochondrial Na+-Ca2+ exchange in intracellular Ca2+ increase induced by ATP in PC12 cells

The involvement of mitochondrial Na+-Ca2+ exchange in Ca2+ responses to ATP was examined in rat pheochromocytoma (PC) 12 cells. Intracellular Ca2+ ([Ca2+]i) and Na+ concentrations ([Na+]i) were measured using fura-2 and SBFI, respectively. ATP caused concentration-dependent increases in [Ca2+]i and [Na+]i. High concentrations of ATP elicited a Ca2+ transient followed by a slow recovery of [Ca2+]i (a sustained phase) in 77% of PC12 cells. The sustained phase of Ca2+ response appeared only when the peak Ca2+ transient exceeded 500 nM. FCCP, a protonophore, greatly enhanced Ca2+ responses to ATP only in cells with the sustained phase but not without this phase. The sustained phase was decreased by clonazepam and CGP37157, mitochondrial Na+-Ca2+ exchange inhibitors, and extracellular Na+ removal but not by cyclosporin A, an inhibitor of permeability transition pores. The reintroduction of Na+ 3.5 min after ATP stimulation in the absence of Na+ caused Na+ concentration-dependent increases in [Ca2+]i and [Na+]i. The increase in [Na+]i was correlated with that in [Ca2+]i. FCCP caused a great increase in [Ca2+]i 4.5 min after ATP stimulation in the absence of extracellular Na+ but not in its presence, indicating that mitochondria retain Ca2+ in the absence of Na+. These results suggest that ATP causes a large increase in [Ca2+]i which was sequestered in mitochondria and that the sustained phase of Ca2+ response to ATP are mainly due to the release of mitochondrial Ca2+ through Na+-Ca2+ exchangers in PC12 cells.     

Pharmacological characterization of endothelin-stimulated phosphoinositide breakdown and cytosolic free Ca2+ rise in rat C6 glioma cells.

Because increasing evidence indicates that glial cells are a target of endothelin, we have characterized endothelin-induced phosphoinositide (PI) turnover and Ca2+ homeostasis in C6 glioma cells. Endothelin-1 (ET) increased formation of 3H-inositol phosphate (IP) from PI and elicited an increase in cytosolic free Ca2+ ([Ca2+]i) in rat C6 glioma. In the presence of Li+, the increase in 3H-inositol trisphosphate formation was rapid, reaching its peak at 5 min after stimulation. ET also elicited a rapid and sustained increase in [Ca2+]i in a dose-dependent manner (1-100 nM). The rank orders of efficacy for ET-related peptides in increasing [Ca2+]i were ET = ET-2 greater than sarafotoxin greater than ET-3. Both ET-mediated stimulation of IP formation and [Ca2+]i increase were largely inhibited in the absence of external Ca2+ but unaffected by the depletion of external Na+ and the presence of dihydropyridine derivatives or verapamil. Inorganic Ca2+ channel blockers Cd2+, La3+, and Mn2+ at 1 mM inhibited both responses induced by ET. Cross-desensitization and nonadditivity were observed for both events among ET-related peptides tested, but not between ET and ATP. Pretreatment of cells with pertussis toxin (PTX) attenuated the PI response to ET, but had no effect on ET-elicited [Ca2+]i increase. ET-induced Ca2+ mobilization (measured in Ca(2+)-free medium) was only transient and was inhibited by 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate. Moreover, the intracellular Ca2+ pools mobilized by ET and ATP appeared to overlap, as indicated by their partial heterologous desensitization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Secreted dense granule adenine nucleotides promote calcium influx and the maintenance of elevated cytosolic calcium levels in stimulated human platelets

Evidence that secreted dense granule adenine nucleotides mediate part of the agonist-induced cytosolic calcium ([Ca2+]i) responses in human platelets was obtained from comparisons of fura-2-loaded platelets from normal subjects and from patients with a form of platelet storage pool deficiency (SPD) in which the secretory dense granules and their contents are virtually absent. SPD platelets had normal initial [Ca2+]i increases induced by thrombin and the endoperoxide analog U46619, but a significantly enhanced decay of elevated [Ca2+]i levels following the initial increases. With thrombin, this enhanced [Ca2+]i decay was associated with decreased Ca2+ influx, as measured by Mn2+ quench of fura-2 fluorescence. Addition of micromolar concentrations of ADP, alone or together with ATP, after stimulation reversed the enhanced [Ca2+]i decay and increased Mn2+ quench in SPD platelets, but had no effect on these responses in normal platelets, while addition of 100-fold higher concentrations of ATP or apyrase before stimulation increased [Ca2+]i decay and decreased Mn2+ quench in normal platelets, but had little effect in SPD platelets. ATP and alpha,beta-methylene ATP, a specific agonist for P2X1 receptors, at micromolar concentrations also increased Mn2+ quench, but to lesser extents than did ADP, in SPD platelets isolated and loaded with fura-2 in the presence of apyrase. Similar effects of ADP and excess ATP were seen in U46619-stimulated platelets, but decreased Ca2+ influx could not be measured directly in SPD platelets, presumably due to the very transient influx response seen with U46619. These results suggest that secreted dense granule ADP and ATP contribute to the maintenance of elevated [Ca2+]i levels, but not to the initial [Ca2+]i increases, in stimulated human platelets, most likely via a nucleotide-specific component of Ca2+ influx which may be mediated by interactions with both P2X1 and P2Y1 purinoceptors.     

Human alpha-thrombin induces phosphoinositide turnover and Ca2+ movements in cultured human umbilical vein endothelial cells

This report shows that purified human alpha-thrombin was able to stimulate a rapid and transient formation of water-soluble phosphorylated 3H-inositols in cultured human umbilical vein endothelial cells (HUVEC) prelabelled with 3H-inositol. A parallel breakdown and resynthesis of 3H-inositol-containing phospholipids was observed. Simultaneously, thrombin induced a transient increase of intracellular free Ca2+[( Ca2+]i), as measured from increased fluorescence of quin2 loaded cells. Phosphoinositide turnover and Ca2+ mobilization showed a similar dependence on thrombin dose. [Ca2+]i rise resulted from both influx from extracellular medium and redistribution from intracellular storage sites. On the other hand thrombin-induced phosphoinositide hydrolysis was not dependent on [Ca2+]i rise. [Ca2+]i elevation might be, at least partially, a consequence of increased phosphoinositide turnover, as suggested by [Ca2+]-mobilizing activity of inositol-trisphosphate in other cells.     

The effect of ecdysterone on cerebral vasospasm following experimental subarachnoid hemorrhage in vitro and in vivo

OBJECTIVES: Cerebral vasospasm has been the dreaded complication of ruptured intracranial aneurysms. Worldwide effort has led to many promising experimental treatments but none was confirmed to be effective in clinical trials. Ecdysterone is an insect steroid hormone. Our previous study showed that ecdysterone might prevent cerebral vasospasm in vitro. Even after all these works, rare attempts have been made to test the effect of ecdysterone on vascular adventitial fibroblast (VAF) proliferation, a process known to play an important role in various pathogenic vascular conditions. Thus, we tested the hypothesis that ecdysterone could affect VAF characteristics and have an effect on SAH induced cerebral vasospasm. METHODS: OxyHb of 100 microM was used in the in vitro study to mimic the clinical situation. The effect of OxyHb on the cell proliferation and migration of cultured aortic smooth muscle cells was investigated. In the in vivo study, 20 rabbits were equally divided into four groups: control group, SAH group, SAH/nimodipine group and SAH/ecdysterone group. Changes in neurological function and cerebral angiograms were observed after SAH. RESULTS: OxyHb increased the proliferation of vascular adventitial fibroblasts at 24 hours. Ecdysterone co-treatment was apparently similar to the suppression of proliferation. Cell cycle analysis indicated that ecdysterone inhibited the progression of vascular adventitial fibroblasts from G1 to S. The results of the migration assay showed that 100 microM OxyHb obviously prompted vascular adventitial fibroblast migration and that ecdysterone would attenuate this effect. In the SAH/nimodipine and SAH/ecdysterone groups, neurological deficit, cerebral vasospasm and structural changes in basilar artery were alleviated with nimodipine or ecdysterone treatment. CONCLUSION: Ecdysterone could affect vascular adventitial fibroblast characteristics and attenuate vasospasm after SAH.     

Nucleotide-induced Ca2+ signaling in sustentacular supporting cells of the olfactory epithelium

Extracellular purines and pyrimidines are important signaling molecules acting via purinergic cell-surface receptors in neurons, glia, and glia-like cells such as sustentacular supporting cells (SCs) of the olfactory epithelium (OE). Here, we thoroughly characterize ATP-induced responses in SCs of the OE using functional Ca2+ imaging. The initial ATP-induced increase of the intracellular Ca2+ concentration [Ca2+]i always occurred in the apical part of SCs and subsequently propagated toward the basal lamina, indicating the occurrence of purinergic receptors in the apical part of SCs. The mean propagation velocity of the Ca2+ signal within SCs was 17.10 +/- 1.02 microm/s. ATP evoked increases in [Ca2+]i in both the presence and absence of extracellular Ca2+. Depletion of the intracellular Ca2+ stores abolished the responses. This shows that the ATP-induced [Ca2+]i increases were in large part, if not entirely, due to the activation of G protein-coupled receptors followed by Ca2+ mobilization from intracellular stores, suggesting an involvement of P2Y receptors. The order of potency of the applied purinergic agonists was UTP > ATP > ATPgammaS (with all others being only weakly active or inactive). The ATP-induced [Ca2+]i increases could be reduced by the purinergic antagonists PPADS and RB2, but not by suramin. Our findings suggest that extracellular nucleotides in the OE activate SCs via P2Y2/P2Y4-like receptors and initiate a characteristic intraepithelial Ca2+ wave.     

Cell shape change and cytosolic Ca2+ in human umbilical-vein endothelial cells stimulated with thrombin.

We quantified thrombin-induced endothelial cells shape change and investigated the role of Ca2+ in such shape change. We used the fluorescent Ca2+ indicator, fura2, to measure both shape change as cell size and intracellular free Ca2+ ([Ca2+]i), in cultured human umbilical-vein endothelial cells (HUVEC). Thrombin induced concentration-dependent decreases in cell size (percentage of cell size at 6 min after stimulation with 0.01 U/ml, 0.1 U/ml, or 1 U/ml thrombin) was 90.1 +/- 1.5%, 78.1 +/- 2.4%, and 40.9 +/- 2.4%, respectively. Thrombin also increased [Ca2+]i in a concentration-dependent manner. Both depletion of extracellular Ca2+, and also the addition of W5, a calmodulin antagonist, inhibited thrombin-induced size reduction. These results indicate an association between shape change and [Ca2+]i mobilization in human endothelial cells stimulated by thrombin.     

Mechanisms of orexin A-evoked changes of intracellular calcium in primary cultured cortical neurons

We have investigated the effect of orexin A on the intracellular free calcium concentration ([Ca2+]i) in primary cultured cortical neurons and explored the exact mechanisms of orexin A-evoked changes of [Ca2+]i. In the present study, changes of [Ca2+]i induced by orexin A in primary cultured cortical neurons were first detected by confocal laser scanning microscopy using Ca2+-sensitive dye fluo-4 as a novel calcium fluorescent probe. Our results showed that 1-0.1 microM orexin A induced the increase in [Ca2+]i in cortical neurons. The increase in [Ca2+]i by acute application of orexin A occurred in a dose-dependent manner. Orexin A-induced increase in [Ca2+]i was not observed under the condition of Ca2+-free Dulbecco's modified Eagle's medium. Pretreatment on the cells with 1 microM thapsigargin did not block orexin A-evoked response. These findings first illuminated the fact that orexin A-induced increase in [Ca2+]i may be mainly from extracellular calcium influx in cortical neurons.     

Modulation of N-methyl-D-aspartate receptor-mediated increases in cytosolic calcium in cultured rat cerebellar granule cells.

The concentration of intracellular free Ca2+ ([Ca2+]i) was measured in rat cerebellar granule cells using the fluorescent indicator fura-2. Culturing the cells as monolayers on plastic squares which could be placed into cuvettes allowed measurements of [Ca2+]i to be performed on large and homogeneous populations of CNS neurons. Granule cells so cultured maintained low levels of [Ca2+]i (around 90 nM) which increased promptly upon the addition of various excitatory amino acids including N-methyl-D-aspartate (NMDA). Increases in [Ca2+]i elicited by NMDA were inhibited by Mg2+ (1 mM) and often potentiated by glycine (1 microM). The addition of TTX or strychnine (5 microM each) did not alter responses to NMDA or NMDA plus glycine. Cytosolic Ca2+ responses to NMDA/glycine were dependent on the presence of extracellular Ca2+ and were unaffected by concentrations of nifedipine or verapamil that blocked increases in [Ca2+]i elicited by K+ depolarization. Responses elicited by NMDA/glycine were inhibited competitively by 2-amino-5-phosphonovalerate or 3-((+-)-2-carboxypiperazin-4-yl)-propyl-1- phosphonic acid and non-competitively by MK-801 or Mg2+. HA-966 and 7-chlorokynurenate inhibited responses to NMDA alone and blocked competitively the potentiating effects of glycine. The results demonstrate NMDA-mediated increases in [Ca2+]i in cerebellar granule cells that arise solely from influx of extracellular Ca2+ through dihydropyridine-insensitive channels. The strict dependence of the NMDA-evoked response on extracellular Ca2+ provides little evidence for a coupling of NMDA receptors to inositol phosphate metabolism and mobilization of intracellular Ca2+. The effect of various agents on NMDA/glycine-induced increases in [Ca2+]i parallels their effects on ligand binding to or current flow through the NMDA receptor-channel complex. The measurement of cytosolic Ca2+ in this preparation of neuronal cells thus appears especially well suited for assessing, on a functional level, the regulation of NMDA receptors in the CNS.     

Hydroxyfasudil, an active metabolite of fasudil hydrochloride, relaxes the rabbit basilar artery by disinhibition of myosin light chain phosphatase.

Fasudil hydrochloride (AT877, hexahydro-1-(5-isoquinolinesulfonyl)-1H-1,4-diazepine hydrochloride, identical to HA1077) inhibits cerebral vasospasm after subarachnoid hemorrhage in experimental animals and humans. In the current study, the vasorelaxing mechanism of hydroxyfasudil, a hydroxylated metabolite of fasudil hydrochloride, was determined in the rabbit basilar artery. The effects of hydroxyfasudil on tension, intracellular Ca2+ concentration ([Ca2+]i), and phosphorylation of the myosin light chain were examined using the isolated and intact or permeabilized rabbit basilar artery without endothelium in vitro. In the intact rabbit basilar artery, hydroxyfasudil elicited a concentration-dependent relaxation of the artery precontracted with 1 nmol/L endothelin-1 (ET-1) plus 20 mmol/L KCl without any significant decrease in [Ca2+]i as determined by fura-2 microfluorometry (IC50: 5.1 +/- 4.6 micromol/L). The relaxation induced by hydroxyfasudil was accompanied with dephosphorylation of the myosin light chain. In the permeabilized preparation, hydroxyfasudil inhibited the contraction induced by ET-1, guanosine 5'-O-(3-thiotriphosphate), or the catalytic subunit of rho-associated kinase, but it did not inhibit Ca2+-induced contraction under the condition of inhibited myosin light chain phosphatase. Hydroxyfasudil showed a greater relaxant effect under decreased adenosine triphosphate (ATP) levels. The present study indicated that hydroxyfasudil relaxes the rabbit basilar artery mainly by disinhibiting myosin light chain phosphatase through the inhibition of rho-associated kinase and that this effect depends on the intracellular ATP concentration.     

Estrogen suppresses the impact of glucose deprivation on astrocytic calcium levels and signaling independently of the nuclear estrogen receptor

Glucose deprivation of astrocytes results in an elevation of cytosolic calcium concentration ([Ca2+]i) [Kahlert, S., Reiser, G., 2000. Requirement of glycolytic and mitochondrial energy supply for loading of Ca2+ stores and InsP3-mediated Ca2+ signaling in rat hippocampus astrocytes. J. Neurosci. Res. 61, 409-420; Silver, I.A., Deas, J., Erecinska, M., 1997. Ion homeostasis in brain cells: differences in intracellular ion responses to energy limitation between cultured neurons and glial cells. Neuroscience 78, 589-601] equivalent to an impairment of astrocytic energy metabolism and function. Superfusion of fura-2 loaded primary cortical astrocytes with glucose-free solution triggered a slow and progressive, 56-fold increase of the [Ca2+]i from 60 nM up to 3.3 microM within 2 h. Re-addition of glucose led to a rapid drop of [Ca2+]i, yet [Ca2+]i did not fully recover to the low levels recorded prior to glucose deprivation and, moreover, astrocytic Ca2+ signaling was impaired: adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP) were no longer able to trigger a transient Ca2+ response as recorded in controls. 17beta-estradiol protected astrocytes from the glucose deprivation-induced [Ca2+]i increase and the impaired signaling independently of the nuclear estrogen receptor, as the antiestrogen tamoxifen and the protein synthesis inhibitor cycloheximide did not impede the protective effect of 17beta-estradiol.     

Neuroprotection and intracellular Ca2+ modulation with fructose-1,6-bisphosphate during in vitro hypoxia-ischemia involves phospholipase C-dependent signaling.

The neuroprotectant fructose-1,6-bisphosphate (FBP) preserves cellular [ATP] and prevents catastrophic increases in [Ca2+]i during hypoxia. Because FBP does not enter neurons or glia, the mechanism of protection is not clear. In this study, we show that FBP's capacity to protect neurons and stabilize [Ca2+]i during hypoxia derives from signaling by a phospholipase-C-intracellular Ca2+-protein kinases pathway, rather than Ca2+ chelation or glutamate receptor inhibition. FBP reduced [Ca2+]i changes in hypoxic hippocampal neurons, regardless of [Ca2+]e, and preserved cellular integrity as measured by trypan blue or propidium iodide exclusion and [ATP]. FBP also prevented hypoxia-induced increases in [Ca2+]i when glucose was absent and when [Ca2+]e was increased to negate Ca2+ chelation by FBP. These protective effects were observed equally in postnatal day 2 (P2) and P16 neurons. Inhibiting glycolysis with iodoacetate eliminated the protective effects of FBP in P16 neurons. FBP did not alter Ca2+ influx stimulated by brief applications of NMDA or glutamate during normoxia or hypoxia, but did reduce the increase in [Ca2+]i produced by 10 min of glutamate exposure during hypoxia. Because FBP increases basal [Ca2+]i and stimulates membrane lipid hydrolysis, we tested whether FBP's protective action was dependent on phospholipase C signaling. The phospholipase C inhibitor U73122 prevented FBP-induced increases in [Ca2+]i and eliminated FBP's ability to stabilize [Ca2+]i and increase survival during anoxia. Similarly, FBP's protection was eliminated in the presence of the mitogen/extracellular signal protein kinase (MEK) inhibitor U0126. We conclude that FBP may produce neuroprotection via activation of neuroprotective signaling pathways that modulate Ca2+ homeostasis.     

Effects of storage and 37 degrees C-incubation with fresh plasma on platelet calcium distribution, mobilization, influx and aggregability

Aggregability of platelet concentrate decreased progressively during storage at room temperature and recovered when these stored platelets were incubated with fresh plasma at 37 degrees C. During storage, the resting level of cytoplasmic free Ca2+ ([Ca2+]i) which was measured by fluorescence Ca2+ indicator, quin 2, increased. On the other hand, the amount of Ca2+ mobilization and influx after stimulation with ADP or thrombin decreased. After 37 degrees C-incubation of these stored platelets with fresh plasma for 2-3 hours, resting level of [Ca2+]i decreased, and the amount of Ca2+ mobilization and influx increased (recovered). The resting level of [Ca2+]i indicated reverse correlation to aggregability and to the amount of Ca2+ mobilization and influx. These results suggest that the platelet Ca2+ influx in resting or activation state change during storage and by 37 degrees C with fresh plasma. These changes cause the reduction and the restoration of platelet aggregability.     

Hemolysate inhibits cerebral artery relaxation

In helical strips of dog middle cerebral arteries partially contracted with prostaglandin (PG) F2 alpha, relaxations induced by angiotensin-II, possibly mediated by PGI2, and those induced by PGH2 were reversed to a contraction or markedly reduced by treatment with hemolysate, which, however, attenuated the PGI2-induced relaxation only slightly. The relaxant response of human middle cerebral arterial strips to PGH2 was also suppressed by hemolysate. Dog and monkey middle cerebral arteries responded to transmural electrical stimulation and nicotine with transient relaxations, which were quite susceptible to tetrodotoxin and hexamethonium, respectively; the relaxations were abolished almost completely by hemolysate and methylene blue. On the other hand, the relaxant response of dog cerebral arteries to a low concentration of K+ was not influenced by hemolysate or by methylene blue, but was reversed to a contraction by treatment with ouabain. Relaxations induced by substance-P and nitroglycerin were markedly inhibited by hemolysate; removal of endothelium abolished the relaxation by substance-P, but did not influence the nitroglycerin-induced relaxation. Hemolysate may interfere with the biosynthesis of PGI2 in the vascular wall, thereby reversing the relaxation induced by angiotensin-II and PGH2 to a contraction. Relaxations induced by electrical and chemical stimulation of vasodilator nerves innervating cerebral arteries appear to be elicited by a mechanism dependent on cellular cyclic guanosine monophosphate (GMP), like that underlying the substance-P-induced and nitroglycerin-induced relaxation. These actions of hemolysate may be involved in the genesis of cerebral vasospasm after subarachnoid hemorrhage.     

Arg8]-vasopressin-induced increase in intracellular Ca2+ concentration in cultured rat hippocampal neurons.

Changes in intracellular Ca2+ concentration ([Ca2+]i) induced by [Arg8]-vasopressin (AVP) were studied in cultured rat hippocampal neurons by fura-2 fluorometry. AVP (10-1,000 nM) caused a dose-dependent increase in [Ca2+]i. The selective V1 vasopressin receptor agonist [Phe2, Ile3, Orn8]-vasopressin also induced a significant increase in [Ca2+]i, whereas the selective V2 vasopressin receptor agonist [deamino Cys1, D-Arg8]-vasopressin showed no effect. The AVP-induced increase in [Ca2+]i was inhibited by the selective V1 vasopressin receptor antagonist d(CH2)5[Tyr2(Me), Arg8]-vasopressin and nonpeptide V1 antagonist OPC-21268. On the other hand, no antagonistic effects were observed with the V2 vasopressin antagonist desglycinamide-[d(CH2)5, D-Ile2, Ile4, Arg8]-vasopressin and nonpeptide V2 antagonist OPC-31260. The increase in [Ca2+]i induced by AVP was abolished after removal of extracellular Ca2+. In addition, AVP-induced [Ca2+]i elevation was not affected by treatment with verapamil, which blocked the [Ca2+]i increase induced by an isotonic high K(+)-medium (50 mM). However, omega-conotoxin GVIA completely inhibited the effect of AVP. These results suggested that the AVP-induced [Ca2+]i increase in cultured rat hippocampal neurons is due to influx of Ca2+ through V1 VP receptors coupled with N-type calcium channels.     

Low-[Mg2+]-induced Ca2+ fluctuations in organotypic hippocampal slice cultures

We show here by whole field monitoring of free intracellular Ca2+ ([Ca2+]i), locally recorded field potential (fp) and external [Ca2+], that low-[Mg2+] induces seizure like events (SLEs) accompanied by simultaneous fluctuations of [Ca2+]i and [Ca2+]e in cultured hippocampal slices. Within a SLE, complex [Ca2+]e fluctuations are seen throughout phases of Ca2+ depletion (tonic) and Ca2+ recovery (clonic) of the extracellular space. Information theory entropy-based analyses revealed strong asymmetric associations of [Ca2+]i and [Ca2+]e kinetics. By contrast, signal-associations between SLEs were found to be weak and of symmetric nature distinguishing seizure-like and interictal events by extensive coupling and decoupling of [Ca2+]i and [Ca2+]e fluctuations, respectively.