Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “TMB”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 901 records · Page 50Linked to original sources
PubMed1987

Tectal transplants into the occipital cortex of the newborn rat.

Embryonic day 15 rostral tectum (presumptive superior colliculus) was transplanted into the occipital cortex of newborn rats. One to two months later, the transplants were visualized and injected with either horseradish peroxidase (HRP) or wheat germ agglutinin conjugated with HRP (WGA/HRP). After the appropriate survival time and processing with either diaminobenzidine (DAB) or tetramethylbenzidine tetrahydrochloride (TMB), HRP-labelled pyramidal cells were found in layer V of the host ipsilateral occipital cortex. Thus, the occipitotectal connections are formed between host and graft despite the fact that the fibers must grow in a direction opposite to their normal course to reach the aberrantly positioned tectal graft.

Animals↗
PubMed1995

An activated mutant of the alpha subunit of G(o) increases neurite outgrowth via protein kinase C.

The GTP-binding protein, G(o), is present at very high concentration in the neuronal growth cone membrane. The expression of activated mutants of the a subunit of G(o) increases neurite outgrowth. To determine the intracellular mechanism for this outgrowth, we have examined activated alpha o-dependent outgrowth in the presence of agents which modulate different signal transduction cascades. Activation of protein kinase C with phorbol esters or with diacylglycerol prevents the alpha o-dependent increase in neurite extension. Inhibition of protein kinase C with staurosporine, with H7, or with long-term, high dose phorbol ester treatment resulted in greater neurite elongation, and no further increase after activated alpha o transfection. The protein phosphatase inhibitor, okadaic acid, also blocked the effect of activated alpha o. In contrast, tyrosine kinase inhibitors and agents which alter cAMP levels did not alter activated alpha o-dependent neurite extension. We tested a number of compounds which alter intracellular calcium levels. TMB-8 and thapsigargin prevented an increase in outgrowth by activated alpha o, but diltiazem, Bay K8644 and dantrolene had no effect on activated alpha o-dependent outgrowth. These studies suggest that activated alpha o increases neurite outgrowth by inhibiting protein kinase C and by modulating intracellular calcium release.

Alkaloids↗
PubMed1992

Blocking ELISA for distinguishing infectious bovine rhinotracheitis virus (IBRV)-infected animals from those vaccinated with a gene-deleted marker vaccine.

A sensitive and specific blocking enzyme-linked immunosorbent assay (ELISA) was developed to distinguish infectious bovine rhinotracheitis virus (IBRV)-infected animals from those immunized with a glycoprotein gIII deletion mutant, IBRV(NG)dltkdlgIII. For this ELISA, undiluted test sera are used to block the binding of an anti-IBRV gIII monoclonal antibody (mAbgIII)-horseradish peroxidase (HRPO) conjugate to gIII antigen. TMB substrate is used for color development. Negative S/N values (defined as the absorbance at 650 nm of test sera/absorbance at 650 nm of negative control sera) of > 0.80 were obtained with immune sera from gnotobiotic cattle immunized with several bovine viruses, with bovine antisera to bovine herpesvirus-2, and vesicular stomatitis virus, with porcine antisera to pseudorabies virus and parvovirus, and with normal sera from heterologous species. Negative S/N values were also obtained with sera from rabbits twice vaccinated with IBRV(NG)dltkdlgIII. However, the S/N values became positive (S/N < 0.8) 10 to 17 days after the rabbits were challenge exposed to virulent IBRV(Cooper). Most of 116 sera (84%) from feedlot cattle with virus neutralization (VN) titers of < 1:2 or < 1:4 had negative S/N values > 0.8, but 18 sera with negative VN titers had positive S/N values, consistent with observations indicating that an IBRV outbreak was occurring in one of the feedlot herds. Thirty nine sera (98%) from feedlot cattle with VN titers of 1:2 to 1:128 had positive S/N values (< 0.8). One serum with a VN titer of 1:2 had a borderline (+/-) S/N value of 0.81. After immunization with a commercial gIII-positive IBRV vaccine, 115/116 sera with VN titers of 1:2 to 1:256 had positive S/N values (< 0.8). One serum with a VN titer of 1:2 had a negative S/N value of 0.83. Serum from one vaccinated animal that failed to seroconvert after vaccination (VN < 1:4) showed a strongly positive ELISA S/N of 0.48.

Animals↗
PubMed1990

Possible roles of Ca2+ and cGMP as mediators of the exflagellation of Plasmodium berghei and Plasmodium falciparum.

The roles of Ca2+ and cyclic nucleotides as secondary, intracellular messengers for exflagellation of Plasmodium berghei and Plasmodium falciparum were investigated. Treatment with Ca2+ antagonists such as TMB-8 (an inhibitor of intracellular Ca2+ release) or W-7 (a calmodulin inhibitor) strongly inhibited exflagellation induced by alkaline medium at pH 8.0 whereas EGTA (a Ca2+ chelator) or nicardipine and nifedipine (Ca2+ channel inhibitors) had no effect. These results may indicate that mobilization of parasites' internal resources of Ca2+ is a prerequisite for exflagellation. Agents which increase cAMP levels did not induce exflagellation at the non-permissive pH of 7.3, and had no significant inhibitory effect at the permissive pH of 8.0. IBMX (cAMP/cGMP-phosphodiesterase inhibitor), however, enhanced exflagellation at pH 7.3, indicating the possibility that cGMP, but not cAMP, may be involved in the induction of exflagellation. Furthermore, cGMP or agents which increase cGMP levels such as nitroprusside (a potent activator of guanylate cyclase), enhanced exflagellation at pH 7.3, whereas N-methyl-hydroxylamine (guanylate cyclase inhibitor) inhibited the exflagellation at pH 8.0. From these results, it may be concluded that the induction of exflagellation requires both Ca2+ mobilization and an increase in cGMP levels.

Animals↗
PubMed1988

Effect of different calcium modulators on motilin-induced contractions of the rabbit duodenum. Comparison with acetylcholine.

Motilin and acetylcholine (ACh) have a direct contractile effect on rabbit small intestinal smooth muscle. To explore the role of calcium influx in these contractions, we studied the effect of extracellular calcium concentration and of calcium antagonists on the response of longitudinal muscle preparations from rabbit duodenum. Motilin- (10(-7) M) and ACh- (10(-4) M)-induced contractions were abolished in Ca2+-depleted medium. ACh (10(-4) M) or motilin (10(-8) and 10(-7) M) increased the contractile response to added Ca2+ to 130 +/- 6%, 129 +/- 10% and 145 +/- 5% of the maximal response to Ca2+ added alone (10 mM in a cumulative concentration response curve). The sensitivity to Ca2+ was greater in the presence of ACh and motilin (EC50 = 1.0 and 1.1 mM Ca2+) than in the absence of any agonist (1.7 mM). In cumulative concentration response (CCR) curves for motilin and ACh, pD2'-values were 7.0 and 6.6 for diltiazem, 8.4 and 7.8 for verapamil (two calcium entry blockers), 5.6 and 5.2 for TMB-8 (an inhibitor of intracellular calcium), 5.3 and 5.2 for TFP (a calmodulin-antagonist). All CCR-curves showed metactoid-like action of the antagonistic drugs. We conclude that ACh and motilin cause calcium to enter the smooth muscle cell. They are probably operating via separate channels, and use a mechanism which differs from K+-induced influx. Intracellular calcium stores appear to play a minor role in these contractions.

Acetylcholine↗
PubMed1982

The effect of various stimuli and calcium antagonists on the fluorescence response of chlorotetracycline-loaded human neutrophils.

Chlorotetracycline has been used in neutrophils and other cells as probe of the state of membrane-bound calcium. We report here that human neutrophils treated with chlorotetracycline response to soluble secretagogues by a prompt decrease in chlorotetracycline fluorescence. This response was observed within 2-5 s, making it one of the most immediate reactions in neutrophils to stimulation, and was obtained with three secretagogues studied: a chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine, a tumor promotor (phorbol myristate acetate) and a lectin (concanavalin A). The responses of neutrophils to the three stimuli differed both quantitatively and qualitatively. The calcium EGTA, did not effect the onset of the decrease in chlorotetracycline fluorescence, suggesting that the probe was measuring changes in intracellular calcium pools. The intracellular calcium antagonists, TMb-8, W-7 and trifluoperazine, did not block, but actually augmented, the fluorescence response. All four of these calcium antagonists blocked the recovery of chlorotetracycline fluorescence which was usually observed several minutes after stimulation with N-formyl-methionyl-leucyl-phenylalanine. This suggests that recovery was dependent upon both extracellular calcium and active calmodulin. The results are consistent with the hypothesis that changes in chlorotetracycline fluorescence reflect changes in a pool of membrane-bound 'trigger calcium', the release of which is an essential first step in stimulus-response coupling in human neutrophils.

Calcium↗
PubMed1986

Calcium regulation of tissue plasminogen activator and prostaglandin biosynthesis in HeLa cells.

Phorbol 12-myristate 13-acetate, 1-20 nM, induced the synthesis in HeLa cells of a 65 200 Mr tissue-type plasminogen activator, and of prostaglandin E2. Omission of Ca2+ from the incubation medium inhibited the induction of plasminogen activator synthesis by 40-60% and abolished the induction of prostaglandin E2 synthesis. Maximal plasminogen activator synthesis could be maintained at extracellular Ca2+ concentrations of approx. 0.1 mM, while maximal prostaglandin synthesis required at least 0.45-0.9 mM Ca2+. The induction of each factor was inhibited by 10-100 microM 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), an inhibitor of intracellular C2+ mobilization. Prostaglandin synthesis, but not plasminogen activator synthesis, was also inhibited by 10-100 microM verapamil and nifedipine, which inhibit intracellular Ca2+ uptake via the so-called 'slow-channels' and by 0.5-10 microM trifluoperazine, an inhibitor of calmodulin. Neither plasminogen activator synthesis nor prostaglandin synthesis were stimulated by 5-50 microM 1-oleoyl-2-acetylglycerol or 1-250 microM 1,2-dioctanoylglycerol, alone and in combination with 50 nM-1 microM ionophore A23187. These results indicate that the synthesis of plasminogen activator and prostaglandins in HeLa cells is Ca2+-dependent, and that the Ca2+ requirements for each process are not identical. Thus, Ca2+ regulation of the production of tissue plasminogen activator and prostaglandin E2 occurs at multiple points in their biosynthetic pathways.

Calcimycin↗
PubMed1988

Does protein kinase C activation mediate thrombin-induced arachidonate release in human platelets?

Thrombin stimulated rapid formation of diacylglycerol, inositol 1,4,5-trisphosphate (IP3) and thromboxane B2 (TXB2) in human platelets. Formation of diacylglycerol and IP3 appeared to precede that of TXB2. Activation of protein kinase C by diacylglycerol combining with Ca+2 mobilization by IP3 has been implicated in mediating arachidonate release. However, addition of the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) to platelet suspension did not inhibit thrombin-stimulated arachidonate release and TXB2 synthesis, whereas addition of the Ca+2 antagonist, 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (TMB-8) or the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) abolished arachidonate release. The correlation of IP3 production with arachidonate release on increasing the concentrations of thrombin was further examined. IP3 production reached near maximum at 0.2 U/ml, whereas TXB2 synthesis continued to increase at 1 U/ml. These results suggest that protein kinase C activation may not mediate arachidonate release and that Ca+2 mobilization by IP3 may only partially account for arachidonate release in platelets stimulated with relatively high concentrations of thrombin.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1988

Leukotriene B4 triggers highly characteristic and specific functional responses in neutrophils: studies of stimulus specific mechanisms.

By using human neutrophils we studied the on-off phenomenon for leukotriene B4 (LTB4) -induced functional responses compared with fMetLeuPhe (fMLP). LTB4 induced rapidly appearing and disappearing neutrophil chemiluminescent (CL), superoxide anion formation, aggregatory and membrane depolarizing responses, whereas fMLP responses were slower both in onset and termination. Increases of intracellular calcium concentrations (as reflected by quin2 and fura-2 fluorescence) were of similar magnitude for both stimuli; however, LTB4 responses were more rapidly terminated and fMLP responses were biphasic. When intracellular calcium fluxes, calmodulin or protein kinase C activities were inhibited by quin2, trifluoperazine, verapamil or 3,4,5-trimethoxybenzoic acid 8-diethylamino)octyl ester (TMB-8), profound changes were noted for chemiluminescent and aggregation kinetics induced by fMLP, whereas kinetics of LTB4 responses were less affected. When drugs were used to modulate cAMP levels, or to inhibit cyclo- and lipoxygenase metabolites of arachidonic acid, no effects on response kinetics were observed. Cytochalasin B both amplified and delayed responses although chemiluminescent responses to fMLP were amplified more than those to LTB4. Despite those effects cytochalasin B did not enhance peak fura-2 or quin2 responses to either fMLP or LTB4. Thus, LTB4 rapidly initiates functional responses in neutrophils, and stimulus-specific response patterns are already discernable during the mobilization of calcium, and can be modulated by interference with calcium-dependent reactions.

Aminoquinolines↗
PubMed1992

Platelet-activating factor-mediated synthesis of prostaglandins in rat Kupffer cells.

Synthesis of prostaglandins was stimulated in rat Kupffer cells upon challenge with platelet-activating factor (PAF). PAF-mediated synthesis of prostaglandins was inhibited by the Ca2+ ion chelator (EGTA), the Ca2+ channel antagonist (nifedipine) and U66985, a structural analogue and antagonist of the biological effects of PAF in other cellular systems. Inhibitors of protein kinase C, staurosporine and polymixin B, did not affect PAF-induced prostaglandin synthesis. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, stimulated synthesis of prostaglandins in Kupffer cells; PAF and PMA exerted additive actions on this process. Both PAF- and PMA-stimulated prostaglandin production was inhibited by TMB-8. PAF-stimulated synthesis of prostaglandins also was inhibited upon treatment of Kupffer cells with pertussis toxin. Cholera toxin, in contrast, stimulated the production of prostaglandins in a concentration-dependent manner; cholera toxin and PAF together had an additive effect. These results suggest that PAF-induced synthesis of prostaglandins is stimulated via a specific receptor coupled to a pertussis toxin-sensitive G-protein, is dependent upon extracellular Ca2+ and is not influenced by protein Kinase C activation. Since PAF and prostaglandins are produced in the liver under conditions such as endotoxemia, PAF-mediated synthesis of these lipid autacoids may be of importance in the regulation of hepatic function during pathophysiological episodes.

Animals↗
PubMed1994

Taurine efflux and the regulation of cell volume in incubated slices of rat cerebral cortex.

The kinetics of efflux [3H]taurine have been examined in pre-loaded slices of rat cerebral cortex incubated in media of variable osmolality. Alterations in the rate of the slowest phase of efflux, considered to represent cellular loss, have been correlated with cell volumes, provisionally identified as the slice non-inulin space. Efflux was stimulated by reduction in medium osmolality, and impaired in hyperosmotic media; these variations were accompanied by moderate (non-osmometric) cell swelling and shrinkage, respectively. The rates of taurine efflux into media in which NaCl was partly replaced by sucrose, and measurement of the corresponding cell volumes, suggest that ionic strength, rather than osmolality or cell volume per se, may be a significant controlling factor. In both isosmolal and hyposmolal media efflux was significantly impaired by the anion transport inhibitor niflumic acid, with accompanying cell swelling, or by replacement of chloride by gluconate. In hyposmotic, but not isosmotic, media efflux was impaired, and cell volumes increased, in the presence of trifluoperazine or TMB-8, a reported blocker of intracellular calcium release, and the effects of niflumic acid and trifluoperazine on both variables were strongly additive. It is suggested that in both isosmotic and hyposmotic media taurine, efflux occurs by anionic transport, mainly through exchange with external chloride, whereas in hyposmotic media a second pathway is present, probably a volume-activated calmodulin-dependent channel.

Animals↗
PubMed1995

The morphologic change of endothelial cells by ancrod-generated fibrin is triggered by alpha v beta 3 integrin binding and the subsequent activation of a G-protein coupled phospholipase C.

The mechanism of morphologic change of human cultured umbilical vein endothelial cells (HUVECs) caused by fibrin was investigated. Ancrod, a thrombin-like enzyme, did not cause morphologic alteration of HUVEC by itself at concentrations ranging from 0.01 to 10 U/ml. However, when 0.02 U/ml of ancrod was added to cultured HUVEC monolayers in the presence of citrated plasma, it caused pronounced morphologic change of HUVEC after 6-10 h incubation period. Gly-Pro-Arg-Pro (4 mg/ml), an inhibitor of fibrin polymerization, prevented the morphologic alteration, indicating that the morphologic alteration was caused by the polymerized fibrin. The morphologic change of HUVEC caused by ancrod-generated fibrin was not observed in the presence of an intracellular calcium mobilization inhibitor TMB-8 (50 microM), and the morphologic alteration was also less pronounced with BAPTA(15 microM)-loaded HUVECs and HUVECs pretreated with EGTA (1.2 mM). Ancrod (in Medium 199) itself did not stimulate phosphoinositide breakdown of HUVEC. However, when ancrod was present in plasma, it caused an increase of [3H]IP1 of HUVECs preloaded with [3H]myoinositol. This IP1 increment was inhibited by Gly-Pro-Arg-Pro. The increase of IP1 was significantly inhibited by the pretreatment of monoclonal antibodies 23C6 and 7E3 directed against alpha v beta 3 integrin. Neomycin (1 mM) and pertussis toxin (100 ng/ml), but not aspirin or mepacrine, blocked this enhanced phosphoinositide breakdown. The morphologic change was also prevented by the monoclonal antibodies, 23C6 and 7E3. These results suggest that both intra- and extra-cellular calcium participate in the event of morphologic change of HUVEC caused by ancrod-generated fibrin, and the morphologic change is mediated, at least in part, by fibrin binding to integrin alpha v beta 3 on HUVECs, causing the subsequent activation of the endogenous G-protein coupled phospholipase C.

Ancrod↗
PubMed1996

Vitamin D increases expression of the tyrosine hydroxylase gene in adrenal medullary cells.

We examined expression of the 1,25-dihydroxyvitamin D3 [1,25-(OH)2 D3] receptors in chromaffin cells of the adrenal medulla and the effects of 1,25(OH)2 D3 on expression of the tyrosine hydroxylase (TH) gene. Accumulation of 1,25(OH)2 D3 in the nuclei of adrenal medullary cells, but not in the adrenal cortex, was observed in mice intravenously injected with radioactively labeled hormone. 1,25(OH)2 D3 produced concentration-dependent increases in the TH mRNA levels in cultured bovine adrenal medullary cells (BAMC). The maximal increases (2-3-fold) occurred at 10(-8) M 1,25(OH)2 D3. Combined treatment with 1,25(OH)2 D3 and 20 microM nicotine had no additive effect on TH mRNA levels suggesting that transsynaptic (nicotinic) and vitamin D (hormonal) stimulation of TH gene expression are mediated through converging mechanisms. Induction of TH mRNA by 1,25(OH)2 D3 was not affected by calcium antagonist TMB-8. By increasing expression of the rate limiting enzyme in the catecholamine biosynthetic pathway, 1,25-(OH)2 D3 may participate in the regulation of catecholamine production in adrenal chromaffin cells. This regulation provides mechanisms through which 1,25(OH)2 D3 may control response and adaptation to stress.

Adrenal Medulla↗
PubMed1982

Effect of inhibitors of arachidonic acid metabolism upon IgE and non-IgE-mediated histamine release.

Inhibitors of arachidonic acid metabolism were tested for their ability to block histamine release from human basophils. Eighteen inhibitors of lipoxygenases, cyclo-oxygenases, prostaglandin isomerases and thromboxane synthetases were tested. Agents inhibitory to the activity of lipoxygenases were effective blockers of IgE and non-IgE-mediated histamine release; agents antagonistic to cyclo-oxygenases, isomerases and thromboxane synthetases were not. These findings indicate that a functioning lipoxygenase pathway is essential for basophil activation and secretion and that the cyclo-oxygenase, isomerase and thromboxane synthetase pathways are not. Compounds antagonistic to phospholipase A also block histamine release, as does the intracellular Ca2+ antagonist TMB-8. The data are consistent with the idea that mast-cell and basophil activation and secretion involve phospholipase A generation of arachidonic acid, which is metabolized via the lipoxygenase pathway.

Arachidonic Acid↗
PubMed1986

Interleukin-1 stimulates granule exocytosis from human neutrophils.

The interaction of human polymorphonuclear neutrophilic leukocytes (neutrophils) with interleukin-1 (IL-1) resulted in a time- and concentration-dependent, selective, release of azurophil (myeloperoxidase, lysozyme) and specific (lysozyme, vitamin B12-binding protein) granule constituents. Myeloperoxidase (MPO) and lysozyme secretion was markedly attenuated if neutrophils were not exposed to cytochalasin B (CB) prior to contact with IL-1. Degranulation was significantly enhanced in the presence of extracellular calcium. IL-1-elicited granule exocytosis was inhibited by the intracellular calcium antagonist, 8-(N,N-diethylamino)-octyl-(3,4,5-trimethoxy) benzoate hydrochloride (TMB-8), a calmodulin antagonist, trifluoperazine (TFP), and an anion channel blocker, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS). An evaluation of the role of arachidonic acid metabolites in IL-1-induced neutrophil activation revealed a suppressive effect on enzyme release exerted by the lipoxygenase inhibitors, piriprost potassium (6,9,deepoxy-6,9-(phenylimino)-delta 6,8 -prostaglandin I1, U-60,257B) and NDGA (nordihydroguaiaretic acid), and a cyclooxygenase/lipoxygenase inhibitor, ETYA (5,8,11,14-eicosatetraynoic acid). These data describe the characteristics of IL-1 as a human neutrophil secretagogue, and enhance our insight into the mechanism of inflammatory cell activation with this monokine.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1991

The involvement of protein kinase C, calcium, and 5-lipoxygenase in the production of tumor necrosis factor by a cloned interleukin-3 dependent cell line with natural cytotoxic activity.

The cloned interleukin-3 dependent cell line, M1-A5 was studied to determine whether protein kinase C, calcium mobilization, and 5-lipoxygenase activity were involved in the signal transduction pathways required for the production of TNF. TNF release was stimulated by 10 ng/ml phorbol myristate acetate (PMA), 2 microM calcium ionophore A23187, and 1 microgram/ml lipopolysaccharide (LPS) with synergism seen between PMA and A23187. All signals were blocked by phloretin and the PMA signal was blocked by H-7, both drugs acting as protein kinase C inhibitors. Desensitization of protein kinase C by PMA (1 microgram/ml for 24 h) provided evidence that both PMA- and LPS-stimulated TNF production were protein kinase C-dependent while A23187-stimulated TNF production was not. Both the calcium chelator, EGTA, and the intracellular calcium antagonist, TMB-8, inhibited TNF production stimulated by all agents, indicating that TNF stimulation by all agents was calcium dependent. Finally, the 5-lipoxygenase inhibitors, ketoconazole and L-656,224, but not the cyclo-oxygenase inhibitor ASA, inhibited TNF stimulated by all agents. These findings indicate that, although TNF production by M1-A5 cells can be stimulated either by a calcium/protein kinase C- or by a calcium-dependent signal, there is a convergence of signals at the level of 5-lipoxygenase activation.

Animals↗
PubMed1992

Involvement of Ca2+ entry and inositol trisphosphate-induced internal Ca2+ mobilization in muscarinic receptor-mediated catecholamine release in dog adrenal chromaffin cells.

Catecholamine (CA) release from adrenal medulla evoked by muscarinic receptor stimulation has been studied using isolated perfused adrenal gland and cultured chromaffin cells from dogs. Muscarine and oxotremorine (1-100 microM), and bethanechol (0.1-1 mM) dose-dependently stimulated CA release. Muscarine-evoked CA release was antagonized with M1-antagonist, pirenzepine and, to a lesser extent, with atropine; and was reduced either by removal of extracellular Ca2+ or treatment with Ca2+ channel blockers. Muscarine caused an increase of 45Ca uptake and 22Na uptake. Tetrodotoxin (TTX) did not affect muscarine-evoked increase of 22Na uptake and CA release. Under the absence of extracellular Ca2+, muscarine stimulated a 45Ca efflux. Muscarine-induced CA release was attenuated by treating the cells with 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate-HCl (TMB-8) which blocks Ca2+ release from the intracellular store. A phospholipase C inhibitor, neomycin, markedly reduced muscarine-induced CA release but not nicotine- and high K(+)-evoked release. Cinnarizine, a Ca2+ channel blocker, attenuated muscarine-evoked but not caffeine-induced CA release and 45Ca efflux in the absence of extracellular Ca2+. Muscarine caused an increase in intracellular free Ca2+ concentration ([Ca2+]i) in the presence of extracellular Ca2+. It caused a similar increase, but to a lesser extent, in the absence of extracellular Ca2+. The increase of [Ca2+]i induced by muscarine without extracellular Ca2+ was reduced by neomycin and cinnarizine. Polymixin B and retinal, which reduced 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced CA release, had little effect on muscarine-induced CA release. Muscarine increased cellular Ins(1,4,5)P3 production, and atropine inhibited this increase.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1994

A polymerase chain reaction-sequence-specific oligonucleotide procedure for HLA class II typing using biotin- and digoxigenin-labeled probes simultaneously in hybridization.

To simplify PCR-SSO HLA-DRB generic typing, we labeled eight of 15 oligonucleotide probes with DIG and the others with biotin, and hybridized each dot blot with both a biotin-labeled probe and a DIG-labeled probe simultaneously. We chose oligonucleotide pairs which require the same hybridization and stringent washing conditions and do not compete with each other during hybridization. After incubation with a mixture of anti-DIG Fab fragment-alkaline phosphatase and streptavidin-peroxidase conjugates, specific binding of the DIG-labeled probe was revealed by a chemiluminescent substrate (CSPD) and specific binding of the biotin-labeled probe was subsequently visualized by a chromogenic substrate (TMB). The sensitivity of both probes was similar and gave comparable hybridization signals. Using this simplified procedure, the number of hybridizations or dot blots can be reduced to half the usual amount and the labor involved in PCR-SSO typing significantly reduced.

Base Sequence↗