Stimulation of renin secretion by 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8)

The intracellular calcium antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) prevents the release of Ca2+ from cell storage sites. The effect of this compound on renin secretion from rat renal cortical slices in vitro was investigated. TMB-8 was a potent stimulant of renin secretion within the concentration range 10(-5)M to 5 X 10(-4)M with an optimum concentration of 2 X 10(-4)M. TMB-8 overcame the inhibition of renin secretion by angiotensin II, ouabain, 60 mM KCl and A23187. The results add to the existing evidence that Ca2+ is a common inhibitory messenger for a number of compounds which affect renin release and suggest a role for intracellular calcium stores in the regulation of juxtaglomerular cell Ca2+ levels.     

TMB-8 (8-(N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate) inhibits the ATP-sensitive K+ channel.

Whole-cell current clamp, single-channel recordings and 86Rb+ flux techniques have been used to show that 8-(N,N-diethyl-amino)octyl-3,4,5-trimethoxybenzoate (TMB-8) inhibits ATP-sensitive K+ channels in HIT-T15 beta-cells. TMB-8 inhibition is observed when KATP channels are activated by ATP depletion or by the K+ channel opener, diazoxide.     

Inhibitory effect of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) in vascular smooth muscle

The inhibitory effects of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) on vascular smooth muscle contraction and cytosolic Ca2+ level ([Ca2+]i) were examined using isolated rabbit aorta loaded with a fluorescent Ca2+ indicator, fura-2. TMB-8 (100 microM) decreased the high K(+)-induced increase in muscle tension, and [Ca2+]i and 45Ca2+ influx to their respective resting levels. TMB-8 (100 microM) almost completely inhibited the increase in [Ca2+]i and 45Ca2+ influx due to norepinephrine although muscle tension was only partially decreased. A higher concentration of TMB-8 (300 microM) inhibited the remaining portion of the contraction without additional decrease in [Ca2+]i. The inhibitory effect of TMB-8 on high K(+)-induced contraction, but not on the norepinephrine-induced contraction, was antagonized by the increase in external Ca2+ concentrations or by the Ca2+ channel activators, CGP 28,392 and by Bay K8644. In Ca(2+)-free solution, norepinephrine-induced transient increases in [Ca2+]i and muscle tension and 100 microM TMB-8 inhibited these changes. The caffeine-induced transient increases in [Ca2+]i and muscle tension were also inhibited by TMB-8 at concentrations higher than those needed to inhibit the norepinephrine-induced transient changes. In permeabilized smooth muscle, TMB-8 (300 microM) did not inhibit the Ca(2+)-induced contraction. These results suggest that TMB-8 inhibits vascular smooth muscle contractility by inhibiting Ca2+ influx, Ca2+ release and Ca2+ sensitization of contractile elements.     

Effects of 5-(N,N-diethylamino)-n-pentyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-5) on cardiovascular systems

The effects of 5-(N,N-diethylamino)-pentyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-5) were studied pharmacologically on smooth muscle, skeletal muscle, blood vessel and cardiac preparations. In all cases, TMB-5 inhibited muscle contractions induced by muscle stimulants such as acetylcholine, norepinephrine, KCl and BaCl₂, indicating that the muscle inhibition induced by TMB-5 is unrelated to specific receptors. TMB-5 was found to be most potent in inhibiting skeletal muscles (at 10^?6 –10^?5 M level) and least effective inhibiting smooth muscles (at 10^?4 –10^?3 M level). The potency of vascular inhibition was in between these two levels (at 10^?4 M level). The ability of TMB-5 to raise the threshold of cardiac arrhythmias was quite good at 7 × 10^?7 –7 × 10^?6 M. It is concluded that TMB-5 could be a good antiarrhythmic agent with some skeletal muscle relaxation action.     

Effect of 3,4,5-trimethoxybenzoic acid 8-diethylamino-octyl ester (TMB-8) on neuronal calcium homeostasis, protein synthesis, and energy metabolism.

It has been suggested recently that disturbances of endoplasmic reticulum calcium homeostasis plays a major role in ischaemic cell injury of the brain. Depletion of endoplasmic reticulum calcium stores induces suppression of the initiation process of protein synthesis, a prominent feature of ischaemic cell damage. The benzoic acid derivative 3,4,5-trimethoxybenzoic acid 8-diethylamino-octyl ester (TMB-8), an established inhibitor of calcium release from endoplasmic reticulum, would be an ideal tool for elucidating the role of endoplasmic reticulum dysfunction in this pathological process. The present investigation was performed to study the effects of TMB-8 on neuronal metabolism (cytoplasmic calcium activity, ATP levels and protein synthesis) using hippocampal slices and primary neuronal cell cultures. In addition, we investigated whether the rise in cytoplasmic calcium activity and the suppression of protein synthesis induced by endoplasmic reticulum calcium pool depletion, is reversed by this agent. Exposure of neurones to TMB-8 (100 microM) induced a small transient increase in cytoplasmic calcium activity ([Ca2+]i), whereas a second dose of TMB-8 (200 microM) produced a marked and sustained rise in [Ca2+]i. The increase in [Ca2+]i evoked by blocking endoplasmic reticulum Ca(2+)-ATPase was only transiently suppressed and then aggravated by TMB-8. The dose-dependent suppression of protein synthesis by TMB-8, observed both in neuronal cultures and hippocampal slices, indicates that TMB-8 has a pathological effect on neuronal metabolism. This inhibition was not reversed after washing-off of the drug. TMB-8 did not reverse the inhibition of protein synthesis evoked by caffeine, which depletes endoplasmic reticulum calcium stores by activating the ryanodine receptor. The results indicate that TMB-8 is not a suitable investigative tool for blocking in neuronal cell cultures the depletion of endoplasmic reticulum calcium stores and the suppression of protein synthesis induced by endoplasmic reticulum calcium pool depletion.     

TMB—8抑制神经递质引起的单个脑细胞内游离钙的升高

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.     

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.     

8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8) acts as a muscarinic receptor antagonist in the epithelial cell line HT29

8-(N, N-diethyl amino) octyl-3,4,5-trimethoxybenzoate (TMB-8) is a widely used pharmacological tool to investigate the involvement of intracellular Ca²⁺ stores in cellular responses. In this study we investigate the effect of TMB-8 as a putative inhibitor of Ca²⁺ signalling in single fura-2 loaded HT₂₉ coIonic epithelial cells stimulated by ATP, carbachol (CCH) and neurotensin (NT). TMB-8 effectively inhibited the CCH-induced (100 mol/l intracellular Ca²⁺ ([Ca²⁺]_i) transient with an IC₅₀ of 20 mol/l. However, [Ca²⁺]_i transients induced by other phospholipase C coupled agonists ATP (10 mol/l, n = 4) and NT (10 nmol/l, n = 4) remained unaffected by TMB-8 (50 mol/l). The agonist-induced [Ca²⁺]_i transients remained equally unaffected by 100 mol/l TMB-8 when the stimulatory concentration was reduced to 0.5 mol/I for ATP (n = 4) or 1 nmol/l for NT (n = 4). The competitive nature of the TMB-8-induced inhibition of the CCH-induced [Ca²⁺]_i transient was demonstrated by examining the agonist at various concentrations in absence and presence of the antagonist. High TMB-8 concentrations (100 mol/l) alone induced a small [Ca²⁺]_i increase ([Ca²⁺]_i: 40 ± 5 nmol/l, n = 7). We assume that this increase is a consequence of a TMB-8 induced intracellular alkalinization ( pH: 0.1 ± 0.02, n = 7) occurring simultaneously with the increase in [Ca ⁺]_i. From these results we draw the following conclusions: (1) In sharp contrast to a large number of other studies, but in agreement with studies in other types of cells, these results substantially challenge the value of the tool TMB-8 as an intracellular Ca²⁺ antagonist; (2) TMB-8 acts a muscarinic receptor antagonist at the M₃ receptor; (3) TMB-8 does not influence the release of Ca²⁺ from intracellular stores when IP₃ signal transduction is activated by ATP or NT; (4) TMB-8 as a weak organic base alkalinizes the cytosol at high concentrations; and (5) TMB-8 induces small [Ca²⁺]_i transients at higher concentrations.     

Effects of 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB8) on rat atrial muscle.

Rat atria loaded in vitro with the dye INDO-1 produced fluorescence signals indicative of changes in cytoplasmic calcium ion concentration ([Ca2+]c). Such atria showed systolic/diastolic fluctuations in fluorescence indicative of a systolic rise and a diastolic fall in [Ca2+] while being superfused with a solution containing a normal Ca2+ concentration. Some atria were then exposed to a low [Ca2+] in the superfusate. This caused negative inotropism and fluorescence changes indicative of a decline in [Ca2+]c. Both of these responses were reversed by adding 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB8; 2 microM) to the superfusate. Some atria were exposed instead either to a low [K+] in the superfusate or to an ouabain-containing superfusate. These atria developed a contracture, associated with fluorescence changes indicative of a rise in [Ca2+]c. The addition of TMB8 (2 microM) now relaxed the contracture, and this was associated with fluorescence changes indicative of a decline in [Ca2+]c. Atria that were exposed for 15 min to a low [Na+] in the superfusate developed a period of positive inotropism, followed by a brief period of negative inotropism on return to the normal superfusate. The period of positive inotropism was associated with fluorescence changes indicative of a rise in [Ca2+]c and the period of negative inotropism with a decline in [Ca2+]c to below baseline levels. All of these responses were less marked in atria exposed throughout to superfusates containing TMB8 (2 microM). Some atria were loaded with the dye SNARF-1. This emits fluorescence signals indicative of changes in cytoplasmic pH (pHc). These atria showed no systolic/diastolic fluctuation of fluorescence, but when superfused with a bicarbonate-free solution they displayed a change in fluorescence indicative of a decline in pHc in response to the addition of either ouabain or TMB8. Similarities were found between the effects produced by TMB8 and those produced by amiloride or dichlorobenzyl amiloride, suggesting that all three agents inhibit plasmalemmal Na+/Ca2+ and Na+/H+ exchange.     

Effects of 8-(N-N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8) on skinned myocardial fibres of the rat: reversible inhibition of calcium release from the sarcoplasmic reticulum

The effects of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), which is reported to inhibit the release of intracellularly stored Ca²⁺ in skeletal and smooth muscles, were examined in ventricular myocardia of the adult rat. In skinned papillary muscle fibres with functional sarcoplasmic reticulum (SR) preserved, application of 100 or 300 M TMB-8 during the Ca²⁺ loading period had no significant effect on the peak tension of subsequent caffeine-induced contraction, but when applied during exposure to caffeine, concentration-dependent reduction of the peak tension was observed. At 1000 M, TMB-8 reduced the peak tension of caffeine-induced contraction when applied either during Ca²⁺ loading or during exposure to caffeine. TMB-8 had no substantial influence on the Ca⁺-tension of skinned fibres without functional SR. In isolated papillary muscle preparations, TMB-8 prolonged the action potential duration and decreased the maximum rate of rise of potential, leading to abolition of contraction at 100 M. In conclusion, TMB-8 may be a useful pharmacological tool for inhibiting Ca²⁺ release from SR, but only in skinned myocardial preparations.     

Pharmacological evaluation of a new Ca2+ antagonist, 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8): studies in smooth muscles

The pharmacology of 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8) has been studied using guinea pig ileum and vas deferens preparations. TMB-8 inhibited responses to drugs that excite specific receptors (acetylcholine and norepinephrine) as well as to agents whose actions are not mediated via specific receptors (KCl and BaCl₂) with ID₅₀'s of 3.8 × 10^?6 to 1 × 10^?4 M. TMB-8 inhibited responses to acetylcholine, norepinephrine, nicotine, dimethylphenylpiperazinium and KCl in an insurmountable manner in the guinea pig ileum, while responses to BaCl₂ were inhibited in a competitive manner. Increasing Ca²⁺ concentrations of the bathing medium from 1.35 to 5.40 mM effectively antagonized the TMB-8 inhibition of responses to KCl in the guinea pig ileum and vas deferens preparations. These results indicate that TMB-8 may produce its inhibitory effects in smooth muscle by interfering with the availability of Ca²⁺ for muscle contraction by blocking the Ca²⁺ release from intracellular bound stores.     

Inhibition of calcium uptake and catecholamine release by 8-(n,n-diethylamino)- octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8) In cultured bovine adrenal chromaffin cells

Effects of intracellular calcium antagonists, 8-(f,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8) and 1-(5-(p-nitrophenyl)-furfurylidene-amino) hydantoin sodium hydrate (dantrolene sodium), on catecholamine release and ⁴⁵Ca²⁺ uptake were studied using cultured bovine adrenal chromaffin cells. TMB-8 inhibited carbamylcholine-evoked catecholamine release and ⁴⁵Ca²⁺ uptake in a concentration-dependent manner with a similar potency. On the contrary, dantrolene sodium did not show obvious inhibitory effects of catecholamine release and ⁴⁵Ca²⁺ uptake. Although TMB-8 inhibited the high K⁺-evoked catecholamine release and ⁴⁵Ca²⁺ uptake, the potency of the drug was approximately 100-fold less than when used to inhibit the carbamylcholine-evoked catecholamine release and ⁴⁵Ca²⁺ uptake. The inhibitory effect of TMB-8 on the carbamylcholine-evoked catecholamine release was not overcome by an increase in an extracellular calcium concentration, and was not due to competitive antagonism at the nicotinic receptor site. Moreover, TMB-8 inhibited the carbamylcholine-stimulated ⁴⁵Ca²⁺ efflux, but dantrolene sodium failed to affect it. These results suggest that TMB-8, a well-known intracellular calcium antagonist, prevents the cellular calcium uptake in cultured adrenal chromaffin cells, and thus prevents catecholamine release.     

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

Summary 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 (0.5–2.0g). 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 g). 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. Send offprint requests to: N. Ogawa at the above address     

8-(N,N-二乙铵)-n-辛基-3,4,5-三甲氧基苯甲酸酯对谷氨酸诱导神经细胞DNA损伤的影响

目的研究 8 (N ,N 二乙铵 ) n 辛基 3,4 ,5 三甲氧基苯甲酸酯 (TMB 8)对谷氨酸诱导神经细胞DNA损伤的影响。方法用谷氨酸诱导培养的大鼠皮层神经细胞DNA断裂 ,观察TMB 8对神经细胞DNA断裂和线粒体功能的影响。结果TMB 8能明显减轻谷氨酸诱导的大鼠皮层神经细胞DNA断裂并能改善线粒体功能。结论TMB 8具有抑制神经细胞DNA损伤的作用 ,其机制可能与其降低细胞内游离钙、改善线粒体功能和减轻谷氨酸的氧化毒性有关     

Actions of 8-(N,N-diethylamino)-n-octyl-3,4,5-trimethoxybenzoate in vascular smooth muscle cell cultures

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TMB-8抑制神经递质引起的单个脑细胞内游离钙的升高(英文)

目的:研究TMB-8对神经递质引起的单个脑细胞内游离钙升高的作用。方法:应用AR-CM-MIC阳离子测定系统测定游离大鼠单个脑细胞内钙离子浓度。结果:当细胞外液Ca~(2 )浓度为1.3mmol·L~(-1)时,TMB-8 30μmol·L~(-1)能降低谷氨酸,组织胺,5-羟色胺引起的脑[Ca~(2 )]_i浓度的升高。而当细胞外液无钙时,TMB-8能降低细胞内静息[Ca~(2 )]_i;TMB-8 10μmol·L~(-1)则几乎完全抑制了组织胺和5-羟色胺引起的脑[Ca~(2 )]_i升高作用。结论:TMB-8能降低谷氨酸,组织胺,5-羟色胺引起的脑[Ca~(2 )]_i升高。     

Effect of 3,4,5-Trimethoxybenzoic Acid 8-Diethylamino-octyl ester (TMB-8) on Neuronal Calcium Homeostasis,Protein Synthesis,and Energy Metabolism

Abstract: It has been suggested recently that disturbances of endoplasmic reticulum calcium homeostasis plays a major role in ischaemic cell injury of the brain. Depletion of endoplasmic reticulum calcium stores induces suppression of the initiation process of protein synthesis, a prominent feature of ischaemic cell damage. The benzoic acid derivative 3,4,5-trimethoxybenzoic acid 8-diethylamino-octyl ester (TMB-8), an established inhibitor of calcium release from endoplasmic reticulum, would be an ideal tool for elucidating the role of endoplasmic reticulum dysfunction in this pathological process. The present investigation was performed to study the effects of TMB-8 on neuronal metabolism (cytoplasmic calcium activity, ATP levels and protein synthesis) using hippocampal slices and primary neuronal cell cultures. In addition, we investigated whether the rise in cytoplasmic calcium activity and the suppression of protein synthesis induced by endoplasmic reticulum calcium pool depletion, is reversed by this agent. Exposure of neurones to TMB-8 (100 μM) induced a small transient increase in cytoplasmic calcium activity ([Ca²⁺]_i), whereas a second dose of TMB-8 (200 μM) produced a marked and sustained rise in [Ca²⁺]_i. The increase in [Ca²⁺]_i evoked by blocking endoplasmic reticulum Ca²⁺-ATPase was only transiently suppressed and then aggravated by TMB-8. The dose-dependent suppression of protein synthesis by TMB-8, observed both in neuronal cultures and hippocampal slices, indicates that TMB-8 has a pathological effect on neuronal metabolism. This inhibition was not reversed after washing-off of the drug. TMB-8 did not reverse the inhibition of protein synthesis evoked by caffeine, which depletes endoplasmic reticulum calcium stores by activating the ryanodine receptor. The results indicate that TMB-8 is not a suitable investigative tool for blocking in neuronal cell cultures the depletion of endoplasmic reticulum calcium stores and the suppression of protein synthesis induced by endoplasmic reticulum calcium pool depletion.