Intracellular Calcium Antagonist Research Articles

Ryanodine receptor antagonism alleviates skeletal muscle ischemia reperfusion injury by modulating TNF-α and IL-10.

Intracellular calcium overload has been implicated in various pathological conditions including ischemia reperfusion injury. This study aims to explore the effect and probable mechanism of dantrolene, a ryanodine receptor and intracellular calcium antagonist, on the skeletal muscle ischemia reperfusion injury. SD rats were randomly divided into three groups: sham group which underwent anaesthesia and exposure of femoral vein, reperfusion group that received 2 h ischemia and the amount of diluent via femoral vein before 4 h reperfusion, dantrolene group that underwent 2 h ischemia and was given 2 mg/kg dantrolene via femoral vein before 4 h reperfusion. The parameters measured at the end of reperfusion included serum maleic dialdehyde (MDA), tissue myeloperoxidase (MPO) and muscle histology, as well as serum TNF-α and IL-10. Levels of MDA, MPO and TNF-α increased in the reperfusion group, whereas the relevant expressions in the dantrolene group decreased significantly. Histological examination demonstrated significant improvements between the same both groups. IL-10 reflected the protection observed above with a significant up-regulation of expression after dantrolene administration. Ryanodine receptor antagonist dantrolene exerted a significant protective effect against the inflammatory injury of skeletal muscle ischemia reperfusion. The underlying molecular mechanism is probably related to the suppression of TNF-α levels and the increment of IL-10 expression.

Mechanism of interferon-gamma production by monocytes stimulated with myeloperoxidase and neutrophil extracellular traps

Neutrophil extracellular traps (NETs) have an important role in antimicrobial innate immunity and release substances that may modulate the immune response. We investigated the effects of soluble factors from NETs and neutrophil granule proteins on human monocyte function by using the Transwell system to prevent cell-cell contact. NET formation was induced by exposing human neutrophils to phorbol myristate acetate (PMA). When monocytes were incubated with PMA alone, expression of interleukin (IL)-4, IL-6, IL-8, and tumor necrosis factor (TNF)-alpha mRNA was upregulated, but IL-10, IL-12, and interferon (IFN)-gamma mRNA were not detected. Incubation of monocytes with NETs enhanced the expression of IL-10 and IFN-gamma mRNA, but not IL-12 mRNA. Myeloperoxidase stimulated IFN-gamma production by monocytes in a dose-dependent manner. Both a nuclear factor-kappaB inhibitor (PDTC) and an intracellular calcium antagonist (TMB-8) prevented upregulation of IFN-gamma production. Neither a combined p38alpha and p38beta inhibitor (SB203580) nor an extracellular signal-regulated kinase inhibitor (PD98059) suppressed IFN-gamma production. Interestingly, a combined p38gamma and p38delta inhibitor (BIRB796) significantly decreased IFN-gamma production. These findings suggest that myeloperoxidase induces IFN-gamma production by monocytes via p38gamma/delta mitogen-activated protein kinase.

Mechanism of tissue factor production by monocytes stimulated with neutrophil elastase.

Monocytes and neutrophils are activated during disseminated intravascular coagulation. Tissue factor, the main initiator of coagulation, is expressed by monocytes, while elastase is released by neutrophils. This study investigated tissue factor production by peripheral monocytes after stimulation with human neutrophil elastase. Tissue factor mRNA levels were investigated by the reverse transcriptase-polymerase chain reaction and tissue factor protein production was assessed by western blotting when monocytes were exposed to neutrophil elastase with or without preincubation using various inhibitors. Neutrophil elastase upregulated tissue factor mRNA and protein levels in monocytes. Both U73122 (phospholipase C inhibitor) and TMB-8 (intracellular calcium antagonist) prevented the upregulation of tissue factor mRNA. SB203580 (p38 mitogen-activated protein kinase inhibitor) suppressed this response, but PD98059 (extracellular signal-regulated kinase inhibitor) did not. Ro-318425 (ATP-competitive and selective protein kinase C (PKC) inhibitor) and Go 6976 (inhibitor of conventional PKCs and PKCμ) blocked the upregulation of tissue factor mRNA expression. Go 6983 (broad-spectrum PKC inhibitor) and CGP 4125 (staurosporine analog) partially attenuated it, as did a PKC theta/delta inhibitor. Neutrophil elastase mainly enhances tissue factor production by monocytes via the phospholipase C/conventional PKC/p38 MAPK pathway, although a novel PKC is also involved.

Effects of 2-Azafluorenones on Phosphatidyl-Inositol Specific Phospholipase C Activation in C6 Glioma Cells

The purpose of this study was to determine the effects of an extract from Moringa oleifera (MO) on the development of monocrotaline (MCT)-induced pulmonary hypertension (PH) in Wistar rats. An ethanol extraction was performed on dried MO leaves, and HPLC analysis identified niaziridin and niazirin in the extract. PH was induced with a single subcutaneous injection of MCT (60 mg/kg) which resulted in increases in pulmonary arterial blood pressure (Ppa) and in thickening of the pulmonary arterial medial layer in the rats. Three weeks after induction, acute administration of the MO extract to the rats decreased Ppa in a dose-dependent manner that reached statistical significance at a dose of 4.5 mg of freeze-dried extract per kg body weight. The reduction in Ppa suggested that the extract directly relaxed the pulmonary arteries. To assay the effects of chronic administration of the MO extract on PH, control, MCT and MCT+MO groups were designated. Rats in the control group received a saline injection; the MCT and MCT+MO groups received MCT to induce PH. During the third week after MCT treatment, the MCT+MO group received daily i.p. injections of the MO extract (4.5 mg of freeze-dried extract/kg of body weight). Compared to the control group, the MCT group had higher Ppa and thicker medial layers in the pulmonary arteries. Chronic treatments with the MO extract reversed the MCT-induced changes. Additionally, the MCT group had a significant elevation in superoxide dismutase activity when normalized by the MO extract treatments. In conclusion, the MO extract successfully attenuated the development of PH via direct vasodilatation and a potential increase in antioxidant activity.

A Ca2+Threshold for Induction of Spike-Timing-Dependent Depression in the Mouse Striatum

The striatum is the principal input nucleus of the basal ganglia, receiving glutamatergic afferents from the cerebral cortex. There is much interest in mechanisms of synaptic plasticity in the corticostriatal synapses. We used two-photon microscopy and whole-cell recording to measure changes in intracellular calcium concentration ([Ca(2+)](i)) associated with spike-time-dependent plasticity in mouse striatum. Uncaging glutamate adjacent to a dendritic spine caused a postsynaptic potential at the soma and a rise in spine [Ca(2+)](i). Action potentials elicited at the soma raised both dendrite and spine [Ca(2+)](i). Pairing protocols in which glutamate uncaging preceded action potentials by 10 ms (pre-post protocol) produced supralinear increases in spine [Ca(2+)](i) compared with the sum of increases seen with uncaging and action potentials alone, or timing protocols in which the uncaging followed the action potentials (post-pre protocols). The supralinear component of the increases in [Ca(2+)](i) were eliminated by the voltage-sensitive calcium channel blocker nimodipine. In the adjacent parent dendrites, the increases in [Ca(2+)](i) were neither supralinear nor sensitive to the relative pre-post timing. In parallel experiments, we investigated the effects of these pairing protocols on spike-timing-dependent synaptic plasticity. Long-term depression (t-LTD) of corticostriatal inputs was induced by pre-post but not post-pre protocols. Intracellular calcium chelators and calcium antagonists blocked pre-post t-LTD, confirming that elevated calcium entering via voltage-sensitive calcium channels is necessary for t-LTD. These findings confirm a spine [Ca(2+)](i) threshold for induction of t-LTD in the corticostriatal pathway, mediated by the supralinear increase in [Ca(2+)](i) associated with pre-post induction protocols.

Effect of 1,25 (OH)2 Vitamin D3 (Calcitriol) on TRH-Induced Thyrotropin Secretion in Man

Thyrotropin (TSH) secretion was assessed in three groups of healthy volunteers before and after four days administration of 1.25(OH)2 vitamin D3 (calcitriol) by the oral route -1.5 micrograms/d (group A), 3.0 micrograms/d (group B) or 3.0 micrograms/d combined with trifluoperazine (8-12 mg/d by mouth) (group C). The control trials and experiments proper were made in the same subjects within one month. The lower calcitriol dose did not change significantly the TRH-stimulated TSH levels nor the secretory reserve measured as the difference of TSH levels at the rest and TRH-stimulated levels (delta TSH) during the 20th, 30th, 40th and 60th minute following TRH administration. A larger calcitriol dose caused a significant increase of TSH values at rest and TRH-stimulated values during the 20th, 30th, 40th and 60th minute following TRH administration (p less than 0.05, p less than 0.05, p less than 0.05, p less than 0.05 and p less than 0.01, respectively) as well as delta TSH (p less than 0.05 at all time intervals). The intracellular calcium antagonist trifluoperazine interfered only with the stimulating effect of calcitriol on the TSH secretion at rest and on delta TSH during the 60th minute following TRH administration, while the TSH levels during the 20th, 30th, 40th and 60th minute after TRH administration were significantly higher, as compared with the control examination (p less than 0.01, p less than 0.01, p less than 0.05 and p less than 0.05, respectively) and also delta TSH was significantly elevated during 20th, 30th, and 40th minute after TRH administration during combined treatment (p less than 0.05, p less than 0.01, p less than 0.05, respectively). Calcitriol thus causes a dose-dependent increase of TSH secretion, probably partly also by a mechanism which is independent on the change of intracellular calcium homeostasis.

ER Stress Triggers Apoptosis by Activating BH3-Only Protein Bim

Endoplasmic reticulum (ER) stress caused by misfolded proteins or cytotoxic drugs can kill cells and although activation of this pathway has been implicated in the etiology of certain degenerative disorders its mechanism remains unresolved. Bim, a proapoptotic BH3-only member of the Bcl-2 family is required for initiation of apoptosis induced by cytokine deprivation or certain stress stimuli. Its proapoptotic activity can be regulated by several transcriptional or posttranslational mechanisms, such as ERK-mediated phosphorylation, promoting its ubiquitination and proteasomal degradation. We found that Bim is essential for ER stress-induced apoptosis in a diverse range of cell types both in culture and within the whole animal. ER stress activates Bim through two novel pathways, involving protein phosphatase 2A-mediated dephosphorylation, which prevents its ubiquitination and proteasomal degradation and CHOP-C/EBPalpha-mediated direct transcriptional induction. These results define the molecular mechanisms of ER stress-induced apoptosis and identify targets for therapeutic intervention in ER stress-related diseases.

Ryanodine Receptor Antagonism Protects the Ischemic Liver and Modulates TNF-α and IL-10

Dantrolene is a ryanodine receptor and intracellular calcium antagonist. The ryanodine receptor (RyR) Ca(2+) release channel mobilizes Ca(2+) from internal stores to support a variety of cellular functions, including the inflammatory response after ischemia and reperfusion. The pharmacological mechanism of dantrolene is associated with the inhibition of the release of Ca(2+) from the skeletal muscle sarcoplasmic reticulum (SR). We hypothesized that dantrolene could exert a protective effect in our model of liver ischemia and reperfusion by modulating TNF-alpha and IL-10. Mice subjected to 90 min of partial (70 to 80%) hepatic ischemia and 3 h of reperfusion were divided into five groups (n = 6/group): sham, ischemic control, and the dantrolene 1 mg/kg group studied at three times of administration: 15 min before reperfusion (DAN-PRE), at the time of reperfusion (DAN-RP), and 15 min after reperfusion (DAN-POS). The parameters measured at 3 h of reperfusion included serum liver function tests alanine aminotransferase (ALT) and aspartate aminotransferase (AST), TNF-alpha, and IL-10 in serum and liver histology. It was demonstrated that the RyR intracellular calcium antagonist dantrolene offered the most significant protection for the ischemic liver when given before reperfusion and at the time of reperfusion. AST significantly differed between the control group and the DAN-PRE and DAN-RP groups (P < 0.05). ALT showed a statistically significant decrease in the DAN-PRE treated group and a decrease, although not significant, in the DAN-RP. Histological examination demonstrated a significant decrease in vacuolization in the same both groups (P < 0.05). Necrosis was significantly diminished when dantrolene was used at the time of reperfusion; congestion decreased in the same groups but without statistical significant difference. The levels of TNF-alpha were significantly decreased in the DAN-RP group. There was a decrease in TNF-alpha in the DAN-PRE group but not statistically significant. IL-10 reflected the protection observed in necrosis and vacuolization in the histopathology with an increment at the time of reperfusion (P < 0.05). DAN-POS did not exert a protective effect in ALT, AST, liver histology, or cytokine response. For the first time the ryanodine receptor antagonist dantrolene offered significant functional and structural protection of the ischemic liver when given at the time for reperfusion and partial protection when given prereperfusion. RyR inhibition approach down-regulated the expression of TNF-alpha and induced an increment of the protective cytokine IL-10 when administered at the time of reperfusion. There was no protective effect of dantrolene after reperfusion.

Lysophosphatidic Acid Induces Histamine Release from Mast Cells and Skin Fragments

The present study examined whether lysophosphatidic acid (LPA) induces histamine release and the role of Rho-associated protein kinase (ROCK) in histamine release utilizing Y-27632, an inhibitor of ROCK. Rat peritoneal mast cells and mouse skin fragments were challenged with LPA; subsequently, histamine contents were measured by spectrofluorometric assay. LPA-induced histamine release from mast cells and skin fragments occurred in a time- and dose-dependent manner Pretreatment with Y-27632 inhibited LPA-induced histamine release in a dose-dependent fashion. LPA-induced histamine release was not influenced by calcium-free conditions; however, A23187-induced histamine release decreased significantly. TMB-8, an intracellular calcium antagonist, dose-dependently inhibited the histamine release under these conditions. Additionally, Y-27632 scarcely affected A23187-induced histamine release. These findings suggest that LPA-induced histamine release may be attributable to calcium release from intracellular stores. Moreover, ROCK participates in the extracellular calcium-independent process of LPA-induced histamine release.

Ca2+ requirement of prostanoid but not of superoxide production by rat Kupffer cells

The release of the prostanoids prostaglandin D2 (PGD2), prostaglandin E2 (PGE2) and thromboxane induced by zymosan and phorbol ester in cultured rat Kupffer cells was found to depend on the extracellular concentration of Ca2+ to some extent. Prostanoid formation following the addition of the calcium ionophore A 23187 was totally inhibited when calcium ions were withdrawn from the medium whereas the prostanoid synthesis from added arachidonic acid was independent of Ca2+. A half-maximal rate of PGE2 release by cells treated with zymosan, phorbol ester or A23187 was obtained at 0.6–0.7 μM free extracellular Ca2+ and ≥ 100 μM free Ca2+ was required to stimulate PGE2 formation maximally. The calmodulin antagonist R24571 partially inhibited the release of PGE2 elicited by zymosan and A23187 but not by phorbol ester or arachidonic acid. Verapamil and nifedipine, two calcium channel blockers, had no effect on the formation of PGE2 irrespective of the stimulus. TMB 8 [3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester] an intracellular calcium antagonist, inhibited the synthesis of PGE2 induced by zymosan and phorbol ester. The superoxide formation following the addition of zymosan and phorbol ester was not influenced by removal of calcium ions from the medium or by addition of the various calcium antagonists. The data presented here suggest that Ca2+-dependent reactions are involved in the synthesis of prostanoids induced by zymosan and phorbol ester and that both extracellular Ca2+ and mobilization of Ca2+ from intracellular stores are needed to induce maximally the production of prostanoids in cultured rat Kupffer cells.

Norepinephrine-induced inositol 1,4,5-trisphosphate formation in atrial myocytes is regulated by extracellular calcium, protein kinase C, and calmodulin.

We investigated whether alteration of extracellular and intracellular Ca2+ concentrations, protein kinase C, and calmodulin modulate norepinephrine (NE)-induced inositol 1,4,5-trisphosphate (IP3) formation in neonatal rat atrial myocytes. NE-induced IP3 production in atrial myocytes was stimulated by elevation of extracellular Ca2+ in a dose-dependent manner. However, TMB-8 (an intracellular calcium antagonist) and A23187 (an intracellular calcium agonist) did not significantly affect NE-induced IP3 production. PMA (a protein kinase C agonist) significantly decreased and staurosporine (a protein kinase C antagonist) significantly stimulated NE-induced IP3 production. W7 (a calmodulin antagonist) significantly increased the NE-induced IP3. In conclusion, elevation of extracellular Ca2+ concentrations affects NE-induced IP3 formation in atrial myocytes. Protein kinase C and calmodulin may control the IP3 response to NE by a negative feedback mechanism.

Investigations into the mechanisms controlling prostaglandin production by the guinea-pig placenta: roles of calcium and gonadotrophin-releasing hormone

The outputs of PGF2 α, PGE2 and 6-keto-PGF1α were higher from the day 29 guinea-pig placenta than from the sub-placenta in culture, with PGF2αbeing the major prostaglandin produced by the placenta. Lack of extracellular calcium reduced the production of all three prostaglandins by the sub-placenta and 6-keto-PGF1 α production by the placenta, but had no effect on the production of PGF2 α and PGE2 by the placenta. EGTA (a calcium chelator) and a low concentration (30 μM) of TMB-8 (an intracellular calcium antagonist) generally inhibited prostaglandin output from the placenta and sub-placenta at various time points during culture, although EGTA had no effect on PGE2 output from the placenta. Trifluoperazine and W-7 (calmodulin inhibitors) had no inhibitory effect on the outputs of PGF2α and PGE2 from the placenta, nor on the outputs of any prostaglandin from the sub-placenta. However, these two compounds inhibited the output of 6-keto-PGF1 α from the placenta. Nifedipine and verapamil (calcium channel blocking drugs) generally reduced the outputs of prostaglandins from the placenta and sub-placenta, except verapamil had no inhibitory effect on PGF2 α output from the sub-placenta. Gonadotrophin-releasing hormone (GnRH) did not stimulate the output of prostaglandins from the placenta, and tended to have a weak inhibitory action on this tissue. On the sub-placenta, GnRH had an initial inhibitory action on the outputs of PGF2α and 6-keto-PGF1 α, which was then followed by a stimulation of the outputs of PGF2α and, to a lesser extent, of PGE2.

The effects of crustacean cardioactive peptide on locust oviducts are calcium-dependent

The role of calcium as a second messenger in the crustacean cardioactive peptide (CCAP)-induced contractions of the locust oviducts was investigated. Incubation of the oviducts in a calcium-free saline containing, a preferential calcium cation chelator, or an extracellular calcium channel blocker, abolished CCAP-induced contractions, indicating that the effects of CCAP on the oviducts are calcium-dependent. In contrast, sodium free saline did not affect CCAP-induced contractions. Co-application of CCAP to the oviducts with preferential L-type voltage-dependent calcium channel blockers reduced CCAP-induced contractions by 32–54%. Two preferential T-type voltage-dependent calcium channel blockers both inhibited CCAP-induced oviduct contractions although affecting different components of the contractions. Amiloride decreased the tonic component of CCAP-induced contractions by 40–55% and flunarizine dihydrochloride decreased the frequency of CCAP-induced phasic contractions by as much as 65%, without affecting tonus. Flunarizine dihydrochloride did not alter the proctolin-induced contractions of the oviducts. Results suggest that the actions of CCAP are partially mediated by voltage-dependent calcium channels similar to vertebrate L-type and T-type channels. High-potassium saline does not abolish CCAP-induced contractions indicating the presence of receptor-operated calcium channels that mediate the actions of CCAP on the oviducts. The involvement of calcium from intracellular stores in CCAP-induced contractions of the oviducts is likely since, an intracellular calcium antagonist decreased CCAP-induced contractions by 30–35%.

Ketamine suppresses norepinephrine-induced inositol 1,4,5-trisphosphate formation via pathways involving protein kinase C.

Inositol 1,4,5-trisphosphate (IP(3)) is not only involved in the physiologic regulation of excitation-contraction coupling, but could also play a role in cardiac pathophysiology. We investigated the mechanism of ketamine modulation of norepinephrine (NE)-induced IP(3) formation in neonatal rat cardiomyocytes. Ketamine 1 and 10 microM significantly decreased the IP(3) response to 1 microM NE by 27% and 43%, respectively. One micromolar TMB-8 (an intracellular calcium antagonist) produced 42% more decreases in IP(3) production than produced by ketamine alone. One hundred micromolar anthranilic acid (a phospholipase A(2) inhibitor) significantly decreased NE (1 microM)-induced IP(3) formation, and the inhibition was further enhanced by ketamine. Ten micromolar U 73122 (a phospholipase C inhibitor) did not significantly affect NE-induced IP(3) in the presence or absence of ketamine. One micromolar ketamine significantly inhibited staurosporine (a nonselective protein kinase C antagonist)-, bisindolylmaleimide (a selective protein kinase C antagonist)-, and wortmannin (a phosphatidylinositide 3-kinase antagonist)-stimulated IP(3) formation. In conclusion, ketamine suppresses NE-induced IP(3) production, and the inhibition is caused through pathways including protein kinase C and a decrease in intracellular Ca(2+) concentrations. Ketamine inhibits norepinephrine-induced inositol 1,4,5-triphosphate formation in a dose-dependent manner via pathways that involve protein kinase C and a decrease in intracellular Ca(2+) concentrations.

Helicobacter pylori induces pepsinogen secretion by rat gastric cells in culture via a cAMP signal pathway.

Infection with Helicobater pylori (H. pylori) is associated with various stomach diseases such as chronic gastritis, peptic ulcer, and gastric carcinoma. In order to investigate the mechanisms of enhanced production of pepsinogen by H. pylori in cultured rat gastric cells that have the potential to produce pepsinogen, secretion and synthesis of pepsinogen in the cells exposed to H. pylori extract were determined by measuring the hydrolysis of hemoglobin. Various drugs were used to study the mechanisms of effects of H. pylori on the cells. Exposure of the gastric cells to H. pylori extract caused a significant increase in pepsinogen secretion into the culture medium within 30-180 min in a dose-dependent manner, accompanied by a significant increase in pepsinogen synthesis in the gastric cells after 60 min of incubation. Heat treatment of the H. pylori sonicate at 100 degrees C for 10 min completely abolished the stimulatory effect of H. pylori on pepsinogen secretion. 2',3'-Dideoxyadenosine (50 microM), a specific adenylate cyclase inhibitor, abolished the effect of H. pylori-induced pepsinogen secretion. Puromycin (10 microg/ml), a protein synthesis inhibitor, and nicorandil (0.1 mM), a specific intracellular calcium antagonist, reduced the H. pylori-induced pepsinogen secretion by 37% (p<0.01) and 25% (p<0.05), respectively. On the other hand, actinomycin D (1 microg/ml), an RNA synthesis inhibitor, did not affect the H. pylori-induced pepsinogen secretion. Consequently, dibutyryl cAMP potentially stimulated the pepsinogen secretion from gastric epithelial cells in a dose-dependent manner. H. pylori induces pepsinogen secretion and synthesis by gastric epithelial cells through an increase in the intracellular cAMP and mobilization of the intracellular calcium. In addition, H. pylori affects pepsinogen synthesis at the translational level.

Effect of Fasudil HCl, a protein kinase inhibitor, on cerebral vasospasm.

Fasudil HCl (HA1077, AT877, Eril) was first introduced as a unique vasodilator and intracellular calcium antagonist. It dilated spastic basilar artery by the intravenous injection on day 7 in a two-hemorrhage canine model of chronic cerebral vasospasm. Effectiveness of Fasudil on symptomatic and angiographic vasospasm, cerebral infarction by spasm and clinical outcome had been shown by a double blind trial in patients. Now, it is widely used in Japan in patients with aneurysmal subarachnoid hemorrhage. Current and approved use of Fasudil (30 mg iv 3 times a day for 14 days) ameliorates risk of spasm by about 50%.KeywordsCerebral vasospasmcalcium antaponistFasudil HCl

Sevoflurane stimulates inositol 1,4,5-trisphosphate in skeletal muscle.

Inositol 1,4,5-triphosphate (IP(3)) plays an important role in excitation-contraction coupling and malignant hyperthermia in skeletal muscle. We investigated whether sevoflurane affects IP(3) formation in L(6) skeletal muscle cells and studied the mechanisms that modulate IP(3). Sevoflurane stimulated IP(3) production from a basal level of 78.4 +/- 6.1 to 730.0 +/- 53.1 pmol. mg. protein(-1) in 2 mM of sevoflurane in a dose-dependent manner. A dose of 10 microM of U73122 (a phospholipase C antagonist) significantly decreased 0.8 mM of sevoflurane-stimulated IP(3) production from 387. 8 +/- 24.7 to 247.8 +/- 19.8 pmol. mg. protein(-1). A dose of 100 microM of (p-amylcinnamoyl) anthranilic acid (a PLA(2) antagonist) also significantly decreased sevoflurane-stimulated IP(3) production to 282.0 +/- 24.0 pmol. mg. protein(-1). Exposure to 1 microM of genistein and tyrphostin A23 (tyrosine kinase inhibitors) significantly decreased sevoflurane-stimulated IP(3) production to 241.0 +/- 35.3 and 267.4 +/- 32.9 pmol. mg. protein(-1). Sevoflurane-stimulated IP(3) production was significantly decreased by 10 microM of 8-(N,N-diethylamino) octyl-3,4-5-trimathoxybenzoate (an intracellular calcium antagonist) and 100 microM and 1 mM of guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS), a guanosine 5'triphosphate-binding protein inhibitor. Elevation of IP(3) production was significantly higher in halothane than in sevoflurane and isoflurane at the same concentration of 0.8 mM. We conclude that sevoflurane-stimulated IP(3) production involves phospholipase C, phospholipase A(2), tyrosine kinase, and guanosine 5'triphosphate-binding protein and the stimulation is associated with concentration of intracellular ionized calcium. Inhaled anesthetics increase intracellular ionized calcium in the skeletal muscle cell and the ionized calcium increase is partly released from the intracellular store by inositol 1,4,5-triphosphate (IP(3)) formation. IP(3) plays an important role in excitation-contraction coupling and malignant hyperthermia. We studied whether sevoflurane affects IP(3) formation and the mechanisms that modulate IP(3).

Effects of angiotensin-converting enzyme inhibitors on glucose uptake.

We investigated the effect of angiotensin-converting enzyme inhibitors on glucose uptake regulation as well as the effect of bradykinin (BK) on glucose uptake and its regulation by using inhibitors of phospholipase C, BK B2 receptor, protein kinase C, phosphatidylinositol 3-kinase, tyrosine kinase, and intracellular Ca(2+). We measured 2-deoxyglucose uptake by using L(6) skeletal muscle cells. In the presence of 1 nmol/L of insulin, 1 micromol/L of enalaprilat enhanced insulin-induced glucose uptake from 89.2+/-8. 1 to 138.0+/-13.6 pmol/h per mg protein. The stimulation of glucose uptake with enalaprilat was blocked to 92.7+/-7.8 pmol/h per mg protein by 10 micromol/L HOE 140 (a BK B2 receptor antagonist). In the presence of 1 nmol/L of insulin, exposure to 10 micromol/L BK stimulated glucose uptake from 89.2+/-8.1 to 171.6+/-10.1 pmol/h per mg protein. However, in the absence of insulin, BK could not enhance glucose uptake. One hundred nanomoles per liter of tyrphostin A-23 and genistein, which are tyrosine kinase inhibitors, significantly decreased the BK-induced glucose uptake from 142.0+/-8.4 to 87.6+/-6. 4 and 85.2+/-7.3 pmol/h per mg protein, respectively. BK-induced glucose uptake was inhibited significantly by 10 micromol/L U73122 (a phospholipase C antagonist) from 142.0+/-8.4 to 95.7+/-9.5 pmol/h per mg protein. One and 20 micromol/L of TMB-8 (an intracellular calcium antagonist) significantly decreased BK-induced glucose uptake from 142.0+/-8.4 to 108.0+/-9.6 and 100.8+/-11.4 pmol/h per mg protein. Angiotensin-converting enzyme inhibitors enhanced insulin-induced glucose uptake via the BK B2 receptor. BK-stimulated glucose uptake is related to phospholipase C, tyrosine kinase, and an increase in intracellular calcium.

Endothelial cell oxidant production: effect of NADPH oxidase inhibitors.

The effects of known leukocyte NADPH oxidase inhibitors on general cellular oxidant production in cultured human endothelial cells (EC) has been investigated. EC were stimulated with 10 nM phorbol 12-myristate 13-acetate and cellular oxidant production measured in the presence and absence of inhibitors that act on various substituents of the oxidase complex and its activation pathways. The effects of the cytosolic oxidase subunit translocation inhibitors, catechols (3,4-dihydroxybenzaldehyde, caffeic acid, and protocatechuic acid), ortho-methoxy-substituted catechols (apocynin, vanillin, and 4-nitroguaiacol), and quinone, 1,4-naphthoquinone; flavoprotein inhibitors, diphenylene iodonium and quinacrine; haem ligands, imidazole and pyridine; directly acting thiol reagents, disulfiram and penicillamine; NADPH analogue, Cibacron Blue; redox active inhibitors, quercetin and esculetin; intracellular calcium antagonist, TMB-8; and calmodulin antagonists, W-7 and trifluoperazine, were determined. All compounds reduced oxidant production in stimulated EC. These findings add to previous observations suggesting the presence of a functionally active NADPH oxidase in EC. Identifying the major cellular reactive oxygen species source in perturbed EC will provide new insights into our understanding of endothelial dysfunction, which has been hypothesized to be a major contributing factor in the pathogenesis of atherosclerosis.

Intracellular calcium antagonist protects cultured peritoneal macrophages against anthrax lethal toxin-induced cytotoxicity.

The lethal toxin of Bacillus anthracis is central to the pathogenesis of anthrax. Using primary cultures of mouse peritoneal macrophages, we have demonstrated that intracellular calcium release inhibitors protect against anthrax lethal toxin-induced cytotoxicity. The cytolytic effect of anthrax lethal toxin was markedly reduced by dantrolene, an inhibitor of calcium release from intracellular calcium stores. Pretreatment of macrophages with cyclosporin A, which has been shown to be a potent inhibitor of calcium release from mitochondria, also protected cells against cytotoxicity. These results indicate that calcium release from intracellular store may be an essential step for the propagation of anthrax lethal toxin-induced cell damage in macrophages. Thus our findings suggest that dantrolene, cyclosporin A, and possibly other drugs affecting intracellular calcium pools might be effectively preventing the toxicity from anthrax lethal toxin.