Effects Of TMB-8 Research Articles

Involvement of Cyclic GMP in Nitric-Oxide-induced Gastric Relaxation: Comparison of the Actions of Cyclic GMP and Cyclic AMP

Background: Smooth muscle relaxation induced by various agents that increase the cellular levels of cyclic nucleotides (cAMP and cGMP) is accompanied by a decrease in intracellular Ca2+ concentration. However, little is known about the differences between the inhibitory effects of cAMP and cGMP on the contraction of smooth muscle. Objective: To compare the effects and underlying mechanisms of cAMP and cGMP on the inhibition of gastric smooth muscle contraction, cyclic nucleotide promoting agents, as well as cell membrane permeable cyclic nucleotides were used. Methods: Isometric contraction was measured from circular muscle strips prepared from the fundus of cat stomach in a cylinder-shaped chamber filled with Krebs-Ringer solution (pH 7.4, temperature 36°C) bubbled with 5% CO2 in O2. The level of inositol phosphates (IPs) was measured. Results: Forskolin and sodium nitroprusside significantly inhibited acetylcholine (ACh)-induced gastric smooth muscle contraction and increased the cellular levels of cAMP and cGMP, respectively. Direct application of 8-Br-cAMP and 8-Br-cGMP also significantly inhibited ACh-induced contraction. Both verapamil and TMB-8 inhibited ACh-induced contraction. The combined inhibitory effect of verapamil and TMB-8 was significantly greater than the effect of either one, separately. Forskolin or sodium nitroprusside similarly augmented the effect of verapamil. However, the inhibitory effect of TMB-8 was augmented only by 8-Br-cGMP or sodium nitroprusside but not by 8-BrcAMP or forskolin. Forskolin and 8-Br-cAMP significantly inhibited the formation of inositol phosphates stimulated by ACh. Conclusions: cAMP inhibits the contraction mechanism associated with intracellular Ca2+ mobilization as well as extracellular Ca2+ influx, while cGMP inhibits contraction by inhibiting the mechanism associated with extracellular Ca2+ influx.

A Ca2+-induced Ca2+ release mechanism involved in asynchronous exocytosis at frog motor nerve terminals.

The extent to which Ca2+-induced Ca2+ release (CICR) affects transmitter release is unknown. Continuous nerve stimulation (20-50 Hz) caused slow transient increases in miniature end-plate potential (MEPP) frequency (MEPP-hump) and intracellular free Ca2+ ([Ca2+]i) in presynaptic terminals (Ca2+-hump) in frog skeletal muscles over a period of minutes in a low Ca2+, high Mg2+ solution. Mn2+ quenched Indo-1 and Fura-2 fluorescence, thus indicating that stimulation was accompanied by opening of voltage-dependent Ca2+ channels. MEPP-hump depended on extracellular Ca2+ (0.05-0.2 mM) and stimulation frequency. Both the Ca2+- and MEPP-humps were blocked by 8-(N, N-diethylamino)octyl3,4,5-trimethoxybenzoate hydrochloride (TMB-8), ryanodine, and thapsigargin, but enhanced by CN-. Thus, Ca2+-hump is generated by the activation of CICR via ryanodine receptors by Ca2+ entry, producing MEPP-hump. A short interruption of tetanus (<1 min) during MEPP-hump quickly reduced MEPP frequency to a level attained under the effect of TMB-8 or thapsigargin, while resuming tetanus swiftly raised MEPP frequency to the previous or higher level. Thus, the steady/equilibrium condition balancing CICR and Ca2+ clearance occurs in nerve terminals with slow changes toward a greater activation of CICR (priming) during the rising phase of MEPP-hump and toward a smaller activation during the decay phase. A short pause applied after the end of MEPP- or Ca2+-hump affected little MEPP frequency or [Ca2+]i, but caused a quick increase (faster than MEPP- or Ca2+-hump) after the pause, whose magnitude increased with an increase in pause duration (<1 min), suggesting that Ca2+ entry-dependent inactivation, but not depriming process, explains the decay of the humps. The depriming process was seen by giving a much longer pause (>1 min). Thus, ryanodine receptors in frog motor nerve terminals are endowed with Ca2+ entry-dependent slow priming and fast inactivation mechanisms, as well as Ca2+ entry-dependent activation, and involved in asynchronous exocytosis. Physiological significance of CICR in presynaptic terminals was discussed.

Volume regulation and the efflux of amino acids from cells in incubated slices of rat cerebral cortex. II. Dependence on Ca 2+ ions

The efflux of γ-amino isobutyric acid (GABA) and l-glutamate from pre-loaded cells in rat cerebral cortical slices has been studied during interventions designed to affect the availability of intracellular Ca 2+ during hyposmotic swelling and membrane depolarization due to raised extracellular K +. Calmodulin-dependent acceleration of amino acid efflux in hyposmotic media, with cell swelling less than would be predicted on the basis of perfect osmometric behaviour (see Ref. [1]), was unaffected by Ca-ionophore in the presence of external Ca 2+ or by the omission of external Ca 2+, but was suppressed by pre-exposure of slices to thapsigargin (2 μM), which is reported to deplete cytosolic Ca 2+, and by TMB-8 (0.5 mM), which blocks release of Ca 2+ from internal stores. TMB-8 also led to significant cell swelling. The effects of TMB-8 were reversed by Ca-ionophore. Raised external K + (54 mM) led to accelerated amino acid efflux which required calmodulin activation and was blocked by (i) omission of external Ca 2+, (ii) the voltage-sensitive Ca 2+ channel blocker nifedipine (1 μM), (iii) the anion transport inhibitor DIDS (25 μM for GABA, 100 μM for l-glutamate, see Ref. [1]), and (iv) the -SH group acetylator N-ethylmaleimide. TMB-8 was without effect in high K + media. These results suggest that while enhanced amino acids efflux probably occurs through the same population of Ca/calmodulin-dependent, DIDS-sensitive pathways following hyposmotic shock or membrane depolarization, the source of Ca 2+ ions required for the activation of these pathways may depend upon which of these acceleratory stimuli is applied.

Vasodilation Effects and Action Mechanisms of TMB-8 on Basilar Artery in Rabbits.

BACKGROUND: 8-(N,N'-diethylamino)-n-octyl-3,4,5-trimethoxybenzoate (TMB-8) is a potent Ca(2+)-antagonist that can prevent/treat ischemic stroke and inhibit the contractility of smooth, skeletal, and cardiac muscles. Further studies are warranted to elucidate the efficacy of TMB-8 on rabbit basilar artery preparation and its action mechanisms on vascular smooth muscle cell cultures. METHODS AND RESULTS: Effects of TMB-8 on the contractility of rabbit's basilar artery in vitro and those on intracellular free Ca(2+) concentrations, [Ca(2+)](i), were studies with isolated organ bath and Fura-2 methods. Histamine-induced concentration-response curves were shifted by TMB-8 in a mixed manner whereas those of norepinephrine and KCl were shifted in a non-competitive manner. In the presence of nifedipine or in a Ca(2+)-free medium, 2,5-di(tert-butyl)-1,4-benzohydroquinone (BHQ) (10 µM) induced an immediate transient contraction in rabbit basilar artery, whereas ryanodine showed a slow, weak, sustained contraction, followed by a weak, sustained relaxation. TMB-8 (30 µM) significantly inhibited these contractions of BHQ and ryanodine. Further, aminophylline enhanced the inhibitory action of TMB-8 on vasocontractions, suggesting that TMB-8's inhibitory actions may be related to the increase of cAMP level. The muscle contraction induced by BHQ was enhanced by pretreatment of the artery ring with TMB-8 for 15 minutes and then TMB-8 was rinsed out. These results indicate that TMB-8 pretreatment can increase Ca(2+) sequestration into sarcoplasmic reticulum, which leads to a larger subsequent Ca(2+) release by BHQ. KCl-induced increase of [Ca(2+)](i) in vascular smooth muscle cells was reduced when the cells were bathed in the medium containing nifedipine. TMB-8 made further reduction on KCl-induced [Ca(2+)](i) increase in nifedipine-containing solution, which had already blocked the voltage-operated Ca(2+) entry. CONCLUSION: These results indicate that (a) TMB-8 can enhance Ca(2+) sequestration into sarcoplasmic reticulum, which leads to a larger amount of Ca(2+) that can be released by BHQ; (b) TMB-8 can inhibit KCl-induced muscle contraction caused by the reduction of [Ca(2+)](i) through saturation of Ca(2+) inside the sarcoplasmic reticulum rather than a direct blockade of Ca(2+)-influx at cell membrane site; and (c) TMB-8 increases cAMP, which enhances Ca(2+) uptake into the sarcoplasmic reticulum.

Influence of TMB-8 on secretion of catecholamines from the perfused rat adrenal glands

An attempt was made to investigate the effect of TMB-8 [3,4,5-trimethoxybenzoate-8 (N,N-diethylamino) octyl ester], which is known to be an inhibitor of intracellular Ca2+ release, on catecholamines (CA) secretion evoked by Ach, excess K+, DMPP, McN-A-343 and caffeine from the isolated perfused rat adrenal glands and to clearify its mechanism of action. The pretreatment with a low dose of TMB-8 (10 μM) for 20 min led to marked inhibition in CA secretion evoked by Ach (5.32 mM), excess K+ (56 mM), DMPP (100 μM), McN-A-343 (100 μM) and BAY-K 8644 (10−5M). Caffeine-induced CA secretion was similar to that of control only during the first periods (0–3 min) but thereafter marked inhibition in CA secretion evoked by caffeine was observed during the rest periods up to 30 min. The increased moderate concentration of TMB-8 (30 μM) caused the result similar to that of 10 μM TMB-8. However, in adrenal glands preloaded with a high dose of TMB-8 (100 μM), CA releases evoked by Ach, excess K+, DMPP, McN-A-343 and caffeine were almost completely blocked by the drug. These experimental data demonstrate that TMB-8 may inhibit cholinergic receptor-mediated and also depolarization-dependent CA secretion, suggesting that these TMB-8 effects seem to be mediated through inhibiting influx of extracellular calcium into the rat adrenal medullary chromaffin cells as well as reducing the release of calcium from intracellular sources.

Effects of TMB-8 on the ischemia-reperfusion induced changes in myocardial cation and adenine nucleotides contents

Effects of TMB-8 on the ischemia-reperfusion induced changes in myocardial cation and adenine nucleotides contents

TMB-8 prevents the hydroosmotic response to ADH in rabbit cortical collecting tubules

Both AVP and dDAVP effect a transient increase in cytosolic free calcium (iCa2+) in cortical collecting tubule (CCT) cells. To investigate the physiological role of this increase in iCa2+, we examined the effect of TMB-8, a putative inhibitor of iCa2+ release, on the initial and sustained phase of AVP- and dDAVP-stimulated water permeability (Pf) in isolated, perfused CCTs. Pretreatment of tubules with TMB-8, 50 microM, suppressed the increase in osmotic water permeability (Pf) induced by 10 microU/ml AVP and dDAVP, but had no effect on the sustained phase of the response. When increased to 100 microM. TMB-8 inhibited the sustained phase of AVP action. A similar pattern was observed on AVP-stimulated adenyly cyclase activity in rabbit renal membranes. Pretreatment of tubules with 50 microM TMB-8 attenuated the initial increase in Pf in response to cholera toxin but not to 8-Br-cAMP or forskolin. There was no effect of this concentration of TMB-8 on the sustained phase of these agonists. These studies suggest that, in lower concentrations, TMB-8 inhibits the mobilization of iCa2+, which is important for the interaction of Gs with the catalytic unit of adenylyl cyclase and the initial increase in AVP-stimulated Pf. In higher concentrations, TMB-8 inhibits adenylyl cyclase activity directly.

8-Diethylamino-octyl-3,4,5-trimethoxybenzoate, a calcium store blocker, increases calcium influx, inhibits alpha-1 adrenergic receptor calcium mobilization, and alters iodide transport in FRTL-5 rat thyroid cells.

8-Diethylamino-octyl-3,4,5-trimethoxybenzoate (TMB-8) is known to inhibit mobilization of calcium from intracellular stores but, more recently, other cellular effects have been described. In the present study, the effects of TMB-8 on cytosolic free calcium [Ca2+]i levels in FRTL-5 rat thyroid cells were determined using the fluorescent dye, Indo-1. TMB-8 increased [Ca2+]i in a dose-dependent manner, with a maximum rise from 120 +/- 7 nM to 229 +/- 16 nM (90 +/- 5% increase) at 5 x 10(-4) M. This effect was considerably reduced in Ca2+ free buffer, demonstrating a dependency upon extracellular calcium influx but not upon membrane potential and which did not involve the Na+/Ca2+ exchanger. In Ca2+ free buffer TMB-8, at a dose which did not affect [Ca2+]i, completely prevented norepinephrine (10(-5) M) from mobilizing intracellular Ca2+. To determine whether TMB-8 affected differentiated functions, iodide uptake and efflux studies were performed with 125I. TMB-8 (10(-4) M) inhibited iodide uptake by approximately 40% without affecting efflux. At 10(-3) M TMB-8, efflux was also enhanced. These studies demonstrate that TMB-8 has at least two effects on [Ca2+]i, promoting calcium influx and preventing alpha-1 adrenergic mobilization from intracellular stores. TMB-8 also has multiple effects on 125I transport, both inhibiting influx and increasing efflux. The results emphasize the importance of characterizing the behavior of this compound in any cell system before using it as a biological probe.

Influence of TMB-8 and calmidazolium on phorbol ester promoted histamine release from isolated rat mast cells

The effects of TMB-8 and calmidazolium were investigated on mast cell responses believed to be mediated by protein kinase C, i.e. histamine release induced by TPA (tetradecanoyl-phorbol-acetate) in combination with sub-threshold concentrations of the ionophore A23187 and with antigen. Inhibition with both drugs was found in the same concentration range as observed earlier and could be counteracted by glucose, indicating an impaired oxidative energy production. Hence, the test drugs do not reveal protein kinase C selectivity.

The effects of TMB-8 on acetylcholine release from frog motor nerve: interactions with adenosine

The putative intracellular calcium (Ca) antagonist TMB-8 was shown to reduce postjunctional sensitivity and quantal acetylcholine (ACh) release at low micromolar concentrations. At 10-fold higher concentrations, TMB-8 also blocked caffeine-induced Ca release (as monitored electrophysiologically by changes in ACh release) but did not impair the ability of adenosine to inhibit quantal ACh release. This last result implies that TMB-8 and adenosine exert their inhibitory actions at different steps in the depolarization-secretion coupling sequence.

Differential effects of ca2+on proliferation of stomach, colonic, and pancreatic cancer cell linesin vitro

Calcium intake inhibits growth of colon cancer in vivo, the mechanisms of which are not fully elucidated. The objective of this study was to determine whether Ca2+ directly affects the growth of colon cancer cells in vitro and to compare the effects of Ca2+ on the growth of several gastroenteropancreatic cancer cells, including mouse colon cancer (MC-26), human colon cancer (LoVo and WIDR), human gastric cancer (AGS and SII), and human pancreatic cancer (PANC-1 and MIA) cells. All tumor cell lines tested grew in medium containing low concentration (approx 0.16 mM) of Ca2+. Higher concentrations of Ca2+ significantly inhibited the growth of all three colon cancer cell lines tested but had no significant effect on proliferation of the stomach and pancreatic cancer cell lines. Growth of AGS cells, in the presence of 0.1 or 0.5 mM EGTA (resulting in the loss of the extracellular Ca2+) was similar to that observed in the absence of EGTA, indicating that AGS cells were relatively insensitive to loss of extracellular Ca2+. In the presence of TMB-8, an inhibitor of intracellular Ca2+ release, the growth of colonic cancer cell lines was inhibited in a dose-dependent manner, indicating that a minimum basal level of intracellular Ca2+ was required for continued proliferation of colon cancer cells. The stomach cancer cell lines (AGS) was once again less sensitive to the effects of TMB-8 than were the colon cancer cells, indicating an inherent difference in Ca2+ requirements and sensitivity to Ca2+ for growth of different gastroenteropancreatic cancer cells in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of TMB-8 on the shape changes of vascular endothelial cells resulting from exposure to various inflammatory agents

Pre-treatment of the endothelial cells lining the guinea pig inferior vena cava with 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8) (10 microM) in vitro significantly reduced the shape changes resulting from subsequent exposure to platelet activating factor (PAF) (0.1 microM), calcium ionophore A23187 (10 microM), histamine (300 microM), bradykinin (2 microM), prostaglandin E2 (PGE2) (30 microM), or leukotrienes (LT) C4, D4 or E4 (1 microM). Since TMB-8 is an intracellular calcium antagonist, this provides evidence to support the suggestion that these inflammatory agents increase the concentration of intracellular calcium which brings about a contraction of the actin-myosin complex resulting in endothelial cell shape changes, and the formation of interendothelial cell gaps.

Mobilization of Ca2+ from intracellular stores of pancreatic beta-cells by the calcium store blocker TMB-8.

TMB-8 has been used experimentally in many cell types, including endocrine cells, because of its ability to block the efflux of Ca2+ from intracellular stores without affecting influx. Unexpectedly, TMB-8 potentiates stimulated insulin release from pancreatic islets, a process believed to be dependent on the level of cytosolic Ca2+. In the present study, while having no effect on basal insulin release (in the presence of 2.8 mM glucose), TMB-8 (10, 30, and 100 microM) caused a concentration-dependent increase in 45Ca2+ efflux from 45Ca2+-preloaded islets. TMB-8 (100 microM) stimulated 45Ca2+ efflux even in the absence of extracellular Ca2+. In the presence of 5.6 mM glucose, TMB-8 (30 and 100 microM) potentiated insulin release and again increased 45Ca2+ efflux in a concentration-dependent manner. Similarly, insulin release stimulated by isobutylmethylxanthine (IBMX) was potentiated significantly, and IBMX-stimulated 45Ca2+ efflux was increased by the simultaneous introduction of 30 microM TMB-8. Thus, in pancreatic islets, TMB-8 appears to mobilize Ca2+ from intracellular stores, rather than inhibit the efflux as has been commonly accepted. In further studies, using insulin-secreting beta-cells of the RINm5F cell line, TMB-8 was shown to increase the cytosolic Ca2+ concentration in the presence and absence of extracellular Ca2+. This confirmed that mobilization of intracellular Ca2+ was occurring in the pancreatic beta-cell in response to TMB-8. Furthermore, a rise in cytosolic Ca2+ of not more than 10 nM (as induced with KCl) was found to mimick the effect of TMB-8 in conjunction with IBMX. No additional effect of TMB-8 to alter Ca2+ handling at the plasma membrane was found when 45Ca2+ uptake experiments were performed. Therefore, the paradoxical mobilization of beta-cell Ca2+ by TMB-8 appears to be a sufficient explanation for its potentiating effect on the rate of insulin secretion.

Effect of TMB-8 on calcium transport in mast cells in relation to histamine secretion

We have previously reported an inhibition of histamine release by TMB-8 both in the presence and absence of calcium and with glucose in the medium. In the present investigation we have studied the effect of TMB-8 on calcium transport. The observations show that TMB-8 inhibits calcium uptake and enhances calcium efflux in mast cells. As antigen-induced histamine release from sensitized mast cells is primarily dependent on extracellular calcium, the inhibition of anaphylactic histamine release by TMB-8 is probably mainly due to an inhibition of calcium influx into the mast cells. We have shown an increased calcium efflux during histamine release from mast cells induced by compound 48/80 in the absence of calcium in the medium, suggesting the release of intracellular calcium stores. The increased calcium efflux was not inhibited by TMB-8. On the contrary, the enhanced calcium efflux caused by compound 48/80, was added to that by TMB-8. TMB-8 thus had no effect on the calcium release from intracellular stores by compound 48/80 but the enhanced calcium efflux by TMB-8 would tend to inhibit histamine release.

The effects of TMB-8, an intracellular Ca2+ antagonist, on impaired left ventricular relaxation by global ischemia in dog

The effects of TMB-8, an inhibitor of Ca2+ release from the intracellular store site, on impaired left ventricular relaxation caused by global ischemia were examined in 45 anesthetized dogs. In the non-ischemic group, the intracoronary infusion of 100 micrograms/min TMB-8 suppressed the decrease in T, an index of left ventricular relaxation, by increasing the atrial pacing rate. In the ischemic group, the intracoronary infusion of 30 and 100 micrograms/min TMB-8 suppressed the increases of T and left ventricular end-diastolic pressure caused by ischemia and increase of pacing rate. The results indicate that TMB-8 suppresses the ability of relaxation in non-ischemic myocardium and prevents the impairment of left ventricular relaxation induced by left ventricular global ischemia.

Effects of TMB-8, a calcium antagonist, on transport properties of the isolated canine tracheal epithelium

The effects of the putative intracellular Ca 2+ antagonist, TMB-8 (8- N, N-diethylamino)-octyl-3,4,5-trimethoxybenzoate), on the canine tracheal epithelium were examined. Luminal addition reduced rapidly, but reversibly, the transmucosal potential difference and increased the resistance across the open-circuited epithelium. Under short-circuit conditions, the drug reduced stimulation by prostaglandin E 2, forskolin, 8-bromo cyclic AMP, prostaglandin F 2α and A23187. Inhibition of prostaglandin E 2 responses were accompanied by reversal of net Cl − fluxes produced by the agonist. The effect of TMB-8 were unaffected by increasing Ca 2+ in the bathing solutions, and were not mimicked by procaine, nitrendipine, calmidazolium, compound 48 80 or trifluoperazine. W7 did, do a limited extent, produce similar responses, though the drug was more toxic, and the effects were irreversible.

Effects of TMB-8 and dantrolene on ACTH- and angiotensin-induced steroidogenesis by frog interrenal gland: Evidence for a role of intracellular calcium in angiotensin action

The influence of intracellular calcium on the steroidogenic response of adrenocortical tissue to ACTH and angiotensin has been studied in the frog, using a perifusion system technique. The release of corticosterone, aldosterone and prostaglandins in the effluent medium was monitored by specific radioimmunoassays. TMB-8 and dantrolene, two potential blockers of calcium mobilization from intracellular pool(s), were tested. Dantrolene (5 × 10 −5 M) significantly reduced basal and angiotensin-induced corticosterone and aldosterone production but had little effect on ACTH-evoked steroid release. Conversely TMB-8 (10 −4 M) profoundly depressed spontaneous as well as ACTH- and angiotensin II-induced corticosteroid secretion, suggesting that this compound may affect not only calcium mobilization from the endoplasmic reticulum pool but also calcium influx. Adrenal glands perifused with both dantrolene and calcium-free medium showed no response to angiotensin II. Conversely, in calcium-free conditions and in the presence of dantrolene, angiotensin II still caused an increase in prostaglandin synthesis. Taken together, these results indicate that 1) dantrolene is a more specific agent than TMB-8 in inhibiting calcium mobilization from intracellular pool(s); 2) ACTH increases corticosteroidogenesis without inducing mobilization of intracellular calcium; 3) angiotensin II stimulates both the efflux of calcium from the endoplasmic reticulum and the influx of calcium through the plasma membrane; 4) calcium is required after prostaglandin production in the steroidogenic response of frog interrenal gland to angiotensin II.

Ca 2+ and phorbol ester activation of protein kinase C at intracellular Ca 2+ concentrations and the effect of TMB-8

A calcium-activated, phospholipid-dependent protein kinase (protein kinase C) was purified to near homogeneity from human polymorphonuclear leukocytes and shown to be identical to bovine protein kinase C. The Ca 2+ activation of the enzyme was studied and the Ca 2+ concentrations required to activate the enzyme were compared to free cytosolic Ca 2+ concentrations in resting and activated polymorphonuclear leukocytes The free calcium concentrations in the cytosol and in the enzyme assay mixture were determined using the calcium indicator quin 2. The enzyme activity was almost totally dependent upon phosphotadylserine and could be strongly activated by Ca 2+ concentrations in the micromolar range, but was not activated by phosphatidylserine at Ca 2+ concentrations corresponding to the intracellular free Ca 2+ concentration under resting conditions. However, at similar Ca 2+ concentration (<2.5·10 −7 M) the enzyme was highly activated by phorbol 12-myristate 13-acetate (PMA) or diolein in the presence of phosphatidylserine. It was demonstrated that PMA stimulation of human polymorphonuclear leukocytes did not induce any increase in the level of the intracellular free calcium concentration. It was concluded that PMA activation of protein kinase C occurred independently of a rise in the intracellular Ca 2+ concentration. K 0.5 (half-maximal activation) for the PMA activation of purified protein kinase C was shown to be equivalent to the K 0.5 for PMA stimulation of superoxide (O 2 −) production in human polymorphonuclear leukocytes, suggesting that protein kinase C is involved in activation of the NADPH oxidase. The presumed intracellular Ca 2+ antagonist TMB-8 inhibited the PMA-induced superoxide production, but neither by an intracellular Ca 2+ antagonism nor by a direct inhibition of protein kinase C activity.

Effect of TMB-8 on Histamine Release from Isolated Rat Mast Cells

TMB-8 was capable of complete inhibition of the histamine release induced by antigen, compound 48/80, and the ionophore A23187 (2 microM). The effective concentrations for 50% inhibition (IC50) were 1.2 X 10(-4) M for antigen, 1.6 X 10(-4) M for compound 48/80 both in the absence and in the presence of calcium, and 0.7 X 10(-4) M for the ionophore. The inhibitory action was not affected by increased calcium concentration in the medium from 1 to 2m M. The presence of glucose in the medium counteracted the inhibition by TMB-8 and, for low concentrations of the ionophore (0.25 microM), TMB-8 caused a pronounced enhancement of the histamine release. Our results indicate that TMB-8 primarily exerts its effects by interference with oxidative metabolism rather than by affecting the intracellular calcium availability.

Muscarinic receptor-mediated increase in cytoplasmic free Ca 2+in isolated bovine adrenal medullary cells : Effects of TMB-8 and phorbol ester TPA

The change in cytoplasmic free calcium, [Ca 2+] iin isolated bovine adrenal medullary cells during stimulation by acetylcholine (ACh) in Ca 2+-free incubation medium was measured using the fluorescent Ca 2+ indicator quin2. ACh (1–100 μM) caused an increase in [Ca 2+] i by mobilization of Ca 2+ from the intracellular pool. Nicotine (10 μM) did not increase [Ca 2+] iin the absence of extracellular Ca 2+. Pretreatment of the cells with atropine (10 μM) completely inhibited ACh-induced increase in [Ca 2+] i, whereas pretreatment with hexamethonium (100 μM) did not. The intracellular Ca 2+ antagonist 8-( N, N-diethylamino)octyl-3,4,5trimethoxybenzoate (TMB-8), inhibited ACh-induced increase in [Ca 2+] i. The activator of protein kinase C 12- O-tetradecanoylphorbol-13-acetate (TPA), but not its ‘inactive’ analog 4α-phorbol-12,13-didecanoate (PDD), also inhibited ACh-induced increase in [Ca 2+] i. These findings suggest that in bovine adrenal medullary cells, stimulation of muscarinic ACh receptor causes an increase in [Ca 2+] i by mobilizing Ca 2+ from the intracellular pool and that protein kinase C is involved in ‘termination’ or ‘down regulation’ of this response.