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PubMedDate not supplied

Effects of TMB-8, a putative calcium antagonist, on neuromuscular transmission and muscle contractility in the mouse phrenic nerve-hemidiaphragm preparation.

The effects of TMB-8 [8-(N.N.-diethylamino)octyl-3,4,5-trimethoxybenzoate], a putative calcium antagonist, on directly and indirectly evoked isometric twitches, tetanic contractions and potassium- and caffeine-induced contractures, were investigated in the mouse isolated phrenic nerve-hemidiaphragm preparation. In the lowest concentration tested (10(-6) M), TMB-8 produced an augmentation of both directly and indirectly induced twitches. In higher concentrations (10(-5)-3 x 10(-5) M), this augmentation was followed by twitch reduction. In the highest concentrations (10(-4) M-3 x 10(-4) M), only twitch reduction in a concentration-dependent manner was observed. TMB-8 also depressed both directly and indirectly induced tetanic contractions. However, the drug was more effective in depressing neurotransmission than in reducing muscle contractility. Elevated Ca2+ (4-8 mM) or 3,4-diaminopyridine (10(-4) M) produced a good reversal of neuromuscular blockade but this effect was transient. Pretreatment with 4 mM Ca2+ had no significant effect on the time required to produce a 50% or a 90% inhibition of directly or indirectly induced twitches. However, 8 mM Ca2+ significantly prolonged the inhibitory effects of TMB-8 on indirectly, but not directly induced twitches. On the other hand, neostigmine (3 microM) appeared to hasten the blockade of transmission. Submaximal potassium-induced contractures were markedly depressed while caffeine-induced contractures were only slightly depressed by TMB-8 in the concentration range tested (10(-5)-3 x 10(-4) M). The results are consistent with the hypothesis that TMB-8 inhibits skeletal muscle contractility by a reduction in transmembrane Ca2+ movement, a depression of postsynaptic acetylcholine receptor sensitivity, and a decreased mobilization of sequestered calcium from the sarcoplasmic reticulum.

Animals↗

PubMed1986

Downregulation of cell-to-cell communication by the viral src gene is blocked by TMB-8 and recovery of communication is blocked by vanadate.

The viral src gene downregulates junctional communication, closing cell-to-cell membrane channels presumably by way of the phosphoinositide signal route. We show that TMB-8 [8-N, N-(diethylamino) octyl-3,4,5-trimethoxybenzoate] counteracts this downregulation in cells transformed by temperature-sensitive mutant Rous sarcoma virus: TMB-8 (36-72 microM) raises junctional permeability when applied during activity of src protein kinase, i.e., at steady permissive temperature; and TMB-8 inhibits the fall of junctional permeability, when the activity of src protein kinase gets turned on. TMB-8 also (reversibly) inhibits the growth of the cells at permissive temperature and reverses the morphological changes associated with transformation. The morphological reversal lags several hours behind the junctional-permeability reversal. Communication recovers within a few minutes when the activity of the src protein kinase is turned off (in absence of TMB-8). Sodium orthovanadate (20 microM) prevents this recovery, but it has no major effect on junctional permeability on its own. We discuss possible modes of action of these agents on critical stages of the signal route, related to intracellular Ca2+ and protein kinase C.

Animals↗

PubMedDate not supplied

Influence of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) on cell cycle progression and proliferation of cultured arterial smooth muscle cells.

8-(N,N-Diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), a putative inhibitor of intracellular calcium mobilization, causes a dose-dependent inhibition of serum-induced proliferation of arterial smooth muscle cells in culture. Neither early rise in cytosolic calcium concentration nor induction of early induced cell cycle dependent genes (c-fos, ornithine decarboxylase) are inhibited after serum stimulation in presence of 100 microM TMB-8. In contrast, expression of thymidine kinase, a gene normally induced in late-G1 phase, is entirely inhibited by TMB-8. Taken together with flow cytometry studies, these results indicate that TMB-8 blocks cell cycle progression in mid- or late-G1 phase by a mechanism not directly related to early responses to serum stimulation since TMB-8 is also effective when introduced several hours after serum stimulation.

Animals↗

PubMed1986

TMB-8 can block twitches without blocking high K+ or caffeine induced contractures in frog's skeletal muscle.

TMB-8 [8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate] is known to inhibit calcium ion dependent processes in several tissues by stabilizing some intracellular stores of membrane-bound calcium. TMB-8 was used to study the excitation-contraction (E-C) coupling process in frog's skeletal muscle. TMB-8 (5 X 10(-5) - 10(-4) M) blocked electrically evoked twitches but not high K+ (123 mM)- or caffeine (2.36 mM)-induced contractures in isolated, curarized toe muscles. TMB-8 (10(-4) M) produced a small decrease (16%) in the action potential of frog's sartorius muscle fibres. However, reducing extracellular Na+ to 44.7 mM produced a similar reduction (17%) in action potential amplitude but did not suppress the twitch; i.e. it produced only a small increase (about 10%) in twitch amplitude. It is known that potassium contractures are produced by extracellular Ca++ ions which enter through calcium channels in the t-tubules and that caffeine produces contractures by sensitizing the sarcoplasmic reticulum to Ca++-induced Ca++ release. The present results suggest that TMB-8 blocks twitches by preventing the release of Ca++ ions bound to the intracellular surface of the t-tubular membrane which is often called the store of 'trigger-calcium' ions.

Action Potentials↗

PubMed1987

The intracellular calcium antagonist, TMB-8, inhibits prolactin gene expression in GH3 cells.

We examined the effects of the drug, TMB-8, which promotes sequestration of intracellular Ca2+, on the ability of extracellular Ca2+ to stimulate prolactin gene expression in GH3 cells. TMB-8 inhibited prolactin mRNA levels in a dose-dependent manner in the concentration range of 2.5-10 microM. Prolactin mRNA levels were increased about 18-fold by the addition of 0.1 mM CaCl2, and about 25-fold by the addition of 0.4 mM CaCl2. Addition of 10 microM TMB-8 reduced these levels to about 4-fold and 7-fold, respectively. At 10 microM TMB-8 did not effect total protein synthesis or the Ca2+-induced aggregation of the cells, indicating a selective inhibition by the drug of prolactin gene expression. Both TMB-8 and the calmodulin inhibitor, calmidazolium, reversed the effects of Ca2+ on prolactin mRNA levels in cells that had been pretreated for 2 days with 0.4 mM CaCl2.

Adrenal Gland Neoplasms↗

PubMed1985

A new stabilizing agent for the tetramethyl benzidine (TMB) reaction product in the histochemical detection of horseradish peroxidase (HRP).

In this paper, an alternative procedure for the histochemical detection of HRP using amonium heptamolybdate (AHM) as a stabilizing agent and tetramethyl benzidine (TMB) as a chromogen is reported. This procedure avoids the two main problems that occur in previous methods using sodium nitroferricyanide (SNF) as the stabilizer, namely, the appearance of needle-shaped crystals at non-specific anatomical sites, and intensive tissue shrinkage. A comparative study of both, the TMB-AHM and TMB-SNF methods, was performed in the analysis of cerebral cortex afferent connections of the lizard Podarcis hispanica. This study demonstrates that the two methods are of similar sensitivity. The TMB-AHM reaction can be carried out at physiological pH (from 6 to 8), thus, avoiding tissue contraction. The reaction product is of an intense blue-green colour and, as with the TMB-SNF method, shows granulation. The appearance of non-specific precipitates is completely avoided when the incubation medium is maintained at a pH in excess of 5.

Animals↗

PubMed1993

Effect of TMB-8 on alpha-adrenoceptor agonist and KC1 induced-contractions in isolated rabbit aorta.

1. TMB-8 (10(-6) M-10(-4) M) depresses the contractile effect of the selective alpha 1-adrenoceptor agonists methoxamine and phenylephrine in the isolated rabbit aorta. 2. TMB-8 also depresses contractions evoked by 80 mM KCl in this tissue when used at similar concentrations. 3. The calcium antagonist nifedipine potentiates the inhibitory effect of TMB-8 on the alpha 1-contractions. 4. In preparations mounted in Ca-free solution containing 0.5 mM EGTA, 10(-4) M TMB-8 markedly depressed the contractions caused by both alpha 1-adrenoceptor agonists. 5. The Ca2+ agonist BAY K 8644 (10(-6) M) partially prevented the inhibitory effect of TMB-8 on 80 mM KCl contractions.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1996

A prototypic intracellular calcium antagonist, TMB-8, protects cultured cerebellar granule cells against the delayed, calcium-dependent component of glutamate neurotoxicity.

The effect(s) of a prototypic intracellular Ca2+ antagonist, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), on glutamate-induced neurotoxicity was investigated in primary cultures of mouse cerebellar granule cells. Glutamate evoked an increase in cytosolic free-Ca2+ levels ([Ca2+]i) that was dependent on the extracellular concentration of Ca2+ ([Ca2+]o). In addition, this increase in [Ca2+]i correlated with a decrease in cell viability that was also dependent on [Ca2+]o. Glutamate-induced toxicity, quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining, was shown to comprise two distinct components, an "early" Na+/Cl(-)-dependent component observed within minutes of glutamate exposure, and a "delayed" Ca(2+)-dependent component (ED50 approximately 50 microM) that coincided with progressive degeneration of granule cells 4-24 h after a brief (5-15 min) exposure to 100 microM glutamate. Quantitative analysis of cell viability and morphological observations identify a "window" in which TMB-8 (at > 100 microM) protects granule cells from the Ca(2+)-dependent, but not the Na+/Cl(-) -dependent, component of glutamate-induced neurotoxic damage, and furthermore, where TMB-8 inhibits glutamate-evoked increases in [Ca2+]i. These findings suggest that Ca2+ release from a TMB-8-sensitive intracellular store may be a necessary step in the onset of glutamate-induced excitotoxicity in granule cells. However, these conclusions are compromised by additional observations that show that TMB-8 (1) exhibits intrinsic toxicity and (2) is able to reverse its initial inhibitory action on glutamate-evoked increases in [Ca2+]i and subsequently effect a pronounced time-dependent potentiation of glutamate responses. Dantrolene, another putative intracellular Ca2+ antagonist, was completely without effect in this system with regard to both glutamate-evoked increases in [Ca2+]i and glutamate-induced neurotoxicity.

Animals↗

PubMed1994

Mixed actions of TMB-8 as a Ca2+ antagonist in cultured mouse cortical neurones.

The action of TMB-8 [8-N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate] on glutamate (Glu)- and 55 mM KCl (K+)-induced increases in intracellular free calcium levels ([Ca2+]i) was studied in cultured mouse cerebral cortical neurones. Glu-induced responses were mediated by two mechanisms, one independent of and the other dependent on extracellular Ca2+, both being inhibited by TMB-8 in a dose-dependent manner. TMB-8 had no effect on [3H]dizocilpine binding kinetics in cortical membrane preparations. The transient and sustained phases of K(+)-induced increases in [Ca2+]i were also inhibited by TMB-8 in a dose-dependent manner. The Ca2+ channel antagonist, verapamil, blocked K(+)-induced responses but had no effect on Glu-induced responses. Although TMB-8 displays multiple effects, its action at voltage-gated Ca2+ channels does not appear to contribute significantly to its inhibition of the Glu-induced increase in [Ca2+]i.

Animals↗

PubMed1993

Inhibitory effect of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8) on germination of Bacillus cereus T spores.

Effect of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), a calcium antagonist, on germination of Bacillus cereus T spores induced by L-alanine and inosine was investigated. TMB-8 had no effect on the germination of heat-activated spores, whereas it inhibited that of nonactivated spores. The TMB-8 inhibitory effect was antagonized competitively by inosine, but not by L-alanine. Addition of Ca2+ reversed the inhibitory effect of TMB-8 in a dose-related fashion. Based on the results, a role of inosine and a site(s) for inhibitory action of TMB-8 in the process leading to the germination of nonactivated spores were discussed.

Alanine↗

PubMed2000

Effects of TMB-8 on constriction of pial arteries in rats.

AIM: To study the effects of 8-(N,N-diethylamino)-n-octyl-3,4,5-trimethoxybenzoate (TMB-8) on constriction of pial arteries (PA). METHODS: The change of PA in rats was observed continuously and directly through cranial window using microcircular image-shearing system. RESULTS: Nimodipine (Nim) 1 mumol.L-1 produced dilatation of PA immediately and the maximal response occurred in 2 min. But the diameter was not changed by TMB-8 50 mumol.L-1. PA diameter decreased immediately after the application of CSF containing KCl. TMB-8 25, 50 mumol.L-1 apparently inhibited KCl-induced PA constriction. When persistent constriction was evoked by 5-HT, diameter of PA were increased in a concentration-dependent manner after application of TMB-8, which was inhibited by N omega-nitro-L-arginine methyl ester (L-NAME). CONCLUSION: TMB-8 inhibited the contraction induced by 5-HT or KCl in cerebral artery, probably via its calcium antagonization and nitric oxide release.

Animals↗

PubMed1999

Inhibitory effects of 8-(N,N-diethylamino)-n-octyl-3,4,5-trimethoxybenzoate (TMB-8) on intracellular Ca2+ elevated by neurotransmitters in brain cells.

AIM: To study the effects of TMB-8 on [Ca2+]i elevation induced by neurotransmitters in dissociated brain cells. METHODS: The brain cell suspension was made using a gentle trituration for 1 min with a polished pipette. The changes of [Ca2+]i were detected by the fluorescent indicator, Fura 2-AM. RESULTS: In the presence of extracellular Ca2+ 1.3 mmol.L-1, sodium glutamate (Glu), histamine (His), and serotonin (5-HT) markedly increased the [Ca2+]i which were reduced by TMB-8 30 mumol.L-1. TMB-8 3 mumol.L-1 produced inhibitory effects on the increase of [Ca2+]i by His and 5-HT in a Ca(2+)-free Hanks' solution. The increase of [Ca2+]i by His and 5-HT was reduced to control level by TMB-8 10 mumol.L-1. CONCLUSION: TMB-8 inhibited the [Ca2+]i elevation induced by Glu, 5-HT, and His in brain cells.

Animals↗

PubMed1982

Effects of 8-(N, N-diethylamino)ocytl 3, 4, 5-trimethoxybenzoate HCl (TMB-8) on washed blood platelets with special reference to a specific loss of serotonin.

8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate HCl (TMB-8) has been used to indicate involvement of Ca++ in platelet activities, but possible other effects have not been investigated. This study investigates TMB-8-induced leakage of cytoplasmic content and specific loss of one granule constituent, serotonin from washed platelets. TMB-8 concentrations above 0.5 mM block secretion initially and induce leakage after short time incubation. TMB-8 (0.15 mM) enhances a slow release of serotonin, but inhibits a slow release of low affinity platelet factor 4 antigen which take place when washed platelets are incubated at pH 7.4 and 37 degree C. These effects should be taken into consideration when TMB-8 is used as a calcium antagonist in platelets.

Adenine Nucleotides↗

PubMed1993

An intracellular calcium release inhibitor TMB-8 suppresses renal nerve stimulation-induced antinatriuresis in dogs.

Effects of nifedipine and TMB-8 on antinatriuresis induced by renal nerve stimulation (RNS) were examined in pentobarbital-anesthetized dogs. RNS (1 Hz) decreased urine flow rate, urinary sodium excretion rate and fractional excretion of sodium and increased renal norepinephrine efflux and renal venous plasma renin activity with little changes in renal hemodynamics. Intrarenal arterial infusion of nifedipine (0.1 microgram/kg/min) or TMB-8 (50 and 100 micrograms/kg/min) increased basal urine flow rate, urinary sodium excretion rate and fractional excretion of sodium without affecting renal venous plasma norepinephrine concentration or plasma renin activity. Neither nifedipine nor TMB-8 affected the RNS-induced increases in norepinephrine efflux and plasmin renin activity. The RNS-induced decreases in urinary sodium excretion rate and fractional excretion of sodium were suppressed during the TMB-8 infusion, whereas nifedipine failed to affect these urinary responses. These results raise the possibility that the release of intracellular calcium from TMB-8-sensitive stores, but not the influx of extracellular calcium through dihydropyridine-sensitive calcium channels, participates in neural control of tubular sodium reabsorption in the dog kidney.

Animals↗

PubMed1988

Effect of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), an inhibitor of intracellular Ca2+ release, on autoregulation of renal blood flow in the dog.

To examine whether Ca2+ release from intracellular Ca2+ store sites contributes to autoregulation of renal blood flow, experiments were performed on perfused kidneys of anesthetized dogs. Control observations showed excellent autoregulation of renal blood flow over the perfusion pressure range of 120-200 mm Hg. This autoregulatory response was not influenced by the intra-arterial infusion of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8, 1.0 mg/min), an inhibitor of intracellular Ca2+ release. However, TMB-8 (0.3 and 1.0 mg/min i.a.) suppressed the renal vasoconstriction induced by intra-arterial injection of noradrenaline (0.5-2.0 micrograms). On the other hand, TMB-8 (0.3 and 1.0 mg/min) had no effect on the renal vasoconstriction induced by the Ca channel activator, BAY K 8644 (0.5-2.0 micrograms). These results show that TMB-8 has no effect on renal vasoconstriction induced by the activation of voltage-dependent Ca channels, and does not influence autoregulation of renal blood flow. Thus, Ca2+ release from intracellular stores does not appear to contribute the processes of autoregulation of renal blood flow.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1984

TMB-8 inhibits secretion evoked by phorbol ester at basal cytoplasmic free calcium in quin2-loaded platelets much more effectively than it inhibits thrombin-induced calcium mobilisation.

TMB-8 is widely regarded as an 'intracellular calcium antagonist', supposedly inhibiting the mobilisation of intracellular calcium. Rarely, however, have the effects of this compound on Ca2+ movements been measured. We report here that TMB-8 is not very effective in inhibiting thrombin-induced Ca2+ influx or internal release in human platelets judged from the fluorescent signal of cytoplasmic quin2. Only approx. 40% inhibition was seen at 500 microns TMB-8. Somewhat lower concentrations blocked the secretory response to thrombin and also the secretion evoked at basal [Ca2+]i by phorbol ester and collagen. It is suggested that one target for TMB-8 may be the C-kinase pathway.

Aminoquinolines↗

PubMed1993

Enhancement of cell killing and increase in cytosolic calcium concentration by combined treatments with hyperthermia and TMB-8 in mouse mammary carcinoma FM3A cells.

Mouse mammary carcinoma FM3A cells were exposed 42 degrees C or 44 degrees C hyperthermia for 60 min in a culture medium with or without 8-(N,N-diethylamino) octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) at a concentration of 100 microM. Cell survival, cytosolic calcium ion concentration, [Ca2+]C, and influx of 45Ca2+ were investigated to examine the cardinal role of intracellular Ca2+ in hyperthermic cell killing. The [Ca2+]C in a single cell was measured by a digital image analyzing technique using Fura-2. TMB-8 enhanced cell killing, increase in [Ca2+]C, and influx of 45Ca2+ induced by both hyperthermic treatments. In contrast, TMB-8 abolished the increase in [Ca2+]C induced by hyperthermia at 42 degrees C and 44 degrees C in the absence of extracellular Ca2+. These results suggest that TMB-8, an agent usually called an inhibitor of intracellular calcium mobilization at 37 degrees C or lower, enhances Ca2+ entry from the extracellular space at 42 degrees C or 44 degrees C and, in turn, promotes cell killing under hyperthermic conditions.

Animals↗

PubMed1988

A simple method for combining HRP-TMB histochemistry with tritiated thymidine autoradiography on the same tissue section.

In order to determine the sequence of development of various types of spinal neurons defined by their projection, we have developed a method for combining tritiated thymidine autoradiography for birthdate determination, with the demonstration of retrogradely transported horseradish peroxidase (HRP) using tetramethylbenzidine (TMB) as the chromogen. Because of its greater sensitivity, TMB is the chromogen of choice for the demonstration of HRP. However, the HRP-TMB reaction product is unstable and completely destroyed when the tissue is processed for autoradiography. The present study describes the use of osmium tetroxide as a postreaction stabilizing agent which preserves the HRP-TMB reaction product in the form of a dark brown precipitate which is not destroyed when the tissue is subsequently processed for autoradiography. Background levels of autoradiographs stabilized with the osmium procedure are extremely low.

Animals↗