Intracellular Calcium Antagonist Research Articles

The Cellular Regulation of Vesicle Exocytosis by Entamoeba histolytica1,2

We studied the cellular regulation of vesicle exocytosis by Entamoeba histolytica utilizing release of endocytosed 125iodine (125I) labeled tyrosine conjugated dextran; 125I-dextran entered the acid pH vesicles of the amebae and was not degraded during these studies. Exocytosis was temperature dependent with 74%, 36%, 4%, and 0% of 125I-dextran released after 120 min at 37 degrees C, 31 degrees C, 25 degrees C, and 4 degrees C, respectively (P less than 0.01 for each). Exocytosis at 37 degrees C was inhibited by cytochalasin D (10 micrograms/ml), EDTA (10 mM), or the putative intracellular calcium antagonist TMB-8 (250 microM) (P less than 0.01 for each at greater than or equal to 60 min). Calcium ionophore A23187 (1 microM) enhanced exocytosis at 5 and 15 min (P less than 0.01). Elevation of vesicle pH with NH4Cl (10 mM) had no effect on release of 125I-dextran; phorbol myristate acetate (10(-6) M) increased exocytosis by 46% at 30 min (P less than 0.01). Centrifugation of amebae with target Chinese hamster ovary cells resulted in decreased 125I-dextran release into the cell supernatant after 30 and 60 min at 37 degrees C (by 40% and 42%, respectively, P less than 0.01); release of 125I-dextran returned to control values with addition of 1.0 g% galactose or GalNac but not with mannose or N-acetyl-D-glucosamine. Amebic phagocytosis of serum-exposed latex beads had no effect on release of dextran by amebae (n = 16). Exocytosis of acid pH vesicles by E. histolytica is temperature-, microfilament-, and calcium-dependent, and stimulated by phorbol esters.

Effect of the Intracellular Calcium Antagonist HA1004 on Cerebral Blood Flow in Rats

The effect of a novel intracellular calcium antagonist, HA1004, on local cerebral blood flow (LCBF) was examined in rats. LCBF was measured in the striatum by the hydrogen clearance method. In the control rats, HA1004 (10 mg/kg, intravenously: IV) increased striatal LCBF by a maximum of 26% despite a transient drop in mean arterial blood pressure (MABP). The LCBF increased for a period of over 100 minutes after the infusion of HA1004. Nicardipine, a calcium entry blocker (0.1 mg/kg, IV), caused a decrease in MABP similar to that induced by HA1004 and increased LCBF by a maximum of 13% for up to 90 minutes after injection. In a second group of rats, the middle cerebral artery (MCA) was occluded by direct ligature of its proximal portion. After MCA occlusion, the LCBF rapidly dropped to 30% of the control value and gradually recovered to the control level during the next 60 minutes. However, in rats prophylactically treated with a small dose of HA1004 (1 mg/kg, IV), LCBF recovered more quickly than in untreated rats. The results of this study suggest that HA1004 increases LCBF by enhancing collateral blood flow to ischemic regions and may be useful for the treatment of ischemia.

Hemodynamic and Electrocardiographic Actions of the New Intracellular Calcium Antagonist, KT-362, in the Conscious Dog

Effects of the intracellular calcium antagonist, KT-362, on systemic and coronary hemodynamics and the electrocardiogram were evaluated in chronically instrumented, awake dogs during intravenous infusion or bolus administration. Both methods of administration resulted in systemic hypotension, tachycardia and decreases in left ventricular pressure and peak positive left ventricular dP/dt. KT-362 produced a transient increase in mean coronary blood flow velocity only during bolus injection, while subendocardial segment shortening was depressed only by drug infusion. Prolongation of QRS duration and the QT interval (corrected for change in heart rate, QTc) also occurred during drug infusion. The results suggest that the hemodynamic effects of intracellular calcium antagonism by KT-362 are qualitatively similar to those of other calcium channel blocking agents which inhibit extracellular calcium influx. In contrast, the actions of KT-362 on cardiac conduction may be different from those of other slow channel calcium blocking agents.

Differential effects of calcium channel blockade and intracellular calcium antagonism on endothelium-dependent responses of the rat aorta to drugs.

The actions of many vasoactive drugs are mediated through, or modified by, the endothelium-derived relaxing (EDRF) and constricting (EDCF) factors. While EDRF appears to be nitric oxide, EDCF is a peptide or cyclooxygenase product. Using verapamil (a calcium channel blocker), propyl methylenedioxyindene (pr-MDI; an intracellular calcium antagonist), and sodium nitroprusside (which liberates nitric oxide from its molecular structure) as EDRF-independent pharmacological probes in rat aortic rings with and without endothelium, we attempted to provide additional insight into the role of extracellular [( Ca]o) and intracellular [( Ca]i) calcium in EDRF and EDCF release and action, and to explain some mechanisms underlying the modulatory effects of these endothelial factors on the actions of vasoactive drugs. The findings suggest that (1) the [Ca]o required for evoked EDRF release does not enter endothelial cells through verapamil-sensitive calcium channels; (2) mobilization of endoplasmic reticular [Ca]i by [Ca]o entering the endothelial cell may be the trigger for evoked EDRF release; (3) spontaneous release of EDRF appears to depend more on mobilization of [Ca]i than on influx of [Ca]o; (4) the action of EDRF on smooth muscle either does not require Ca or does not involve the mobilization of [Ca]i by [Ca]o; (5) Both EDRF and EDCF can modulate the actions of vasoactive drugs; (6) the EDCF of the rat aorta is not a cyclooxygenase product, and (7) the action of EDCF on vascular smooth muscle, and possibly its release from endothelial cells, are Ca-dependent.

The intracellular calcium antagonist, TMB-8, inhibits prolactin gene expression in GH 3 cells

We examined the effects of the drug, TMB-8, which promotes sequestration of intracellular Ca 2+, on the ability of extracellular Ca 2+ to stimulate prolactin gene expression in GH 3 cells. TMB-8 inhibited prolactin mRNA levels in a dose-dependent manner in the concentration range of 2.5–10 μM. Prolactin mRNA levels were increased about 18-fold by the addition of 0.1 mM CaCl 2, and about 25-fold by the addition of 0.4 mM CaCl 2. Addition of 10 μM TMB-8 reduced these levels to about 4-fold and 7-fold, respectively. At 10 μM TMB-8 did not effect total protein synthesis or the Ca 2+-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 Ca 2+ on prolactin mRNA levels in cells that had been pretreated for 2 days with 0.4 mM CaCl 2.

Calcium dependency of massive postmortem bronchoconstriction in guinea pig lungs

Calcium antagonists were employed to examine whether or not guinea pig's postmortem bronchoconstriction is calcium dependent. First, a group of 17 young (3–4-week-old) animals were divided in three subgroups: control, nifedipine, and 8-( N, N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8, an intracellular calcium antagonist). A second group of 12 mature (5–7-week-old) animals were divided into two subgroups: control and nifedipine. Baseline pressure-volume (P-V) curves were performed prior to cannulation of the pulmonary artery in the anesthetized, opened chested animal. The lungs of the animal were then perfused and P-V curves were obtained again at intervals for up to 60 min. Nifedipine (10 −6 M) or TMB-8 (5 × 10 −4 M) was added to the perfusate in experimental groups. A temporal decrease in inflation volume (IV, the lung volume between transpulmonary pressure of 0 and 30 cm H 2O during inflation) was used as an indicator of bronchoconstriction. In control subgroups, the decrease in IV was significantly greater ( P < 0.05) in the young (29.5 ± 12.9% baseline at 20 min) than in the mature animals (75.0 ± 7.0% baseline at 20 min). Nifedipine significantly alleviated ( P < 0.05) the decrease in IV in young animals at 15–30 min (58.6 ± 8.4% baseline at 20 min) while TMB-8 (90.6 ± 2.1% baseline at 20 min) prevented the decrease even further. Nifedipine did not have a significant effect on IV in mature animals. These data suggest that the postmortem bronchospasm is much more severe in young animals and that this severe constriction is calcium dependent.

Effects of intracellular and extracellular calcium blockers on the pulmonary vascular responses to PGF2 alpha and U46619.

In this study, TMB-8, an intracellular calcium antagonist, and verapamil, an extracellular calcium antagonist, were used simultaneously to elucidate the role of calcium in the pulmonary vasopressor response induced by PGF2 alpha and U46619. The pulmonary vasoconstrictor action of these two agonists was evaluated in the canine isolated lung lobe preparation. Lobar arterial pressure was constantly monitored and changes in arterial pressure were recorded as a percentage from baseline. Control responses to PGF2 alpha (42.0 +/- 8.2%) and U46619 (47.2 +/- 7.0%) were obtained prior to the administration of TMB-8 and verapamil. After administration of TMB-8 and verapamil, the PGF2 alpha (7.4 +/- 3.1%) and U46619 (28.8 +/- 6.2%) responses were significantly attenuated. We conclude that the PGF2 alpha pressor response is dependent on a TMB-8-sensitive intracellular calcium pool and a verapamil-sensitive slow-channel calcium influx. In contrast, the degree of attenuation of the U46619 response was similar to the vasopressor response in the presence of verapamil alone, as described previously. This indicates a direct dependence on extracellular calcium. An additional source of calcium insensitive to verapamil and TMB-8 may also be activated and contribute to the pulmonary vasoconstrictor action. These results suggest that each agonist possesses a mechanism of action distinctly different from the other.

Inhibition of Antigen-Induced Contraction of Guinea Pig Isolated Tracheal Muscle with 2-n-butyl-3-dimethylamino-5,6-methylenedioxy indene (MDI-A), indane (MDI-B) and 8-(diethylamino)octyl-3,4,5-trimethoxy benzoate hydrochloride (TMB-8)

The effects of three intracellular calcium antagonists on antigen-induced contraction of sensitized guinea pig tracheal muscle were investigated. The agents employed were: 2-n-butyl-3-dimethylamino-5,6-methylenedioxy indene hydrochloride (MDI-A), 2-n-butyl-3-dimethylamino-5,6-methylenedioxy indane hydrochloride (MDI-B) and 8-(diethylamino)octyl-3,4,5-trimethoxy benzoate hydrochloride (TMB-8). Each of these agents caused a concentration-dependent inhibition of the antigen-induced contraction of sensitized tracheal smooth muscle. Moreover, in histamine and leukotriene D4 (LTD4) incited contractions of guinea pig tracheal muscle, they showed antagonistic action. The induced tracheal muscle contraction, achieved through the addition of compound 48/80 to a solution of ethylene glycol bis (beta-aminoethylether)-N,N,N1, N1-tetraacetic acid (EGTA) in a contained Ca-free medium, was completely inhibited by individual pretreatment with MDI-A, MDI-B and TMB-8. In the case of tracheal muscle contraction induced by CaCl2 in a high potassium, Ca-free medium, only TMB-8 inhibited contraction. Lastly, none of these three intracellular calcium antagonists affected the antigen-induced release of histamine and slow reacting substance of anaphylaxis (SRS-A). These results suggest that MDI-A and MDI-B inhibit the antigen-induced contraction of sensitized guinea pig tracheal muscle by interfering with the contractile responses caused by histamine and LTD4.

Calcium Channels and Histamine Release from Mast Cells

Using isolated peritoneal mast cells from two types of genetically different rats, histamine release induced by allergic stimuli was inhibited, in all cases except one, by significantly lower concentrations of an intracellular calcium antagonist than those required to inhibit release induced by a calcium ionophore. The exception related to inhibition of histamine release induced by clinical dextran in NR rats (which are relatively less sensitive to dextran than are control rats) as the concentrations required were of a similar order as those needed to inhibit release by ionophores. The data further support the suggestion that there are different calcium channels involved in the different release processes.

Calcium antagonists and intrarenal regulation of glomerular filtration rate.

The intrarenal tubuloglomerular feedback mechanism operates at the site of contact between the thick ascending limb and glomerulus where specialized macula densa cells detect changes in tubular fluid composition and transmit information to the smooth muscle cells of the afferent arteriole. Increases in tubular fluid osmolality result in the transmission of vasoconstrictor signals and decreases in the rate of filtrate formation. Recent studies have identified two sites at which cytosolic calcium may play important roles. First, studies indicate that tubuloglomerular mediated vasoconstriction involves calcium mediated excitation contraction coupling of smooth muscle cells of the afferent arteriole. This calcium mediated event is sensitive to calcium channel blockade. Second, recent studies suggest that the macula densa cells may detect changes in tubular fluid osmolality through a cytosolic calcium system. The use of intracellular calcium antagonists further suggests that intracellular calcium mobilization is the primary mechanism responsible for increases in macula densa cytosolic calcium with increases in tubular fluid osmolality. Calmodulin and cyclic AMP may serve as modulators of this cytosolic calcium system. These studies suggest that calcium plays important roles in the regulation of renal hemodynamics.

Effect of serotonin on prostaglandin synthesis in rat cultured mesangial cells.

Serotonin (5-hydroxytryptamine) (5-HT) is a potent vasoactive amine that reduces renal blood flow and glomerular filtration rate. Vasodilator prostaglandins (PGs) modulate the effects of several vasoconstrictors on the renal circulation. Since mesangial cells are smooth muscle-like cells that may regulate glomerular hemodynamics, we studied the effect of 5-HT on PGs synthesis in rat cultured mesangial cells. 5-HT (10(-6)-10(-3) M) resulted in progressive stimulation of prostaglandin E2 (PGE2) synthesis. Significant stimulation in response to 10(-4) M 5-HT started after 2 min of incubation and progressively increased for at least 30 min. This effect was structurally specific for the 5-HT receptor since indole-containing precursors and metabolites of 5-HT as well as the aminergic compounds, adenosine, and dopamine were without effect. Moreover, 5-HT receptor antagonists, but not histaminergic or beta-adrenergic antagonists, abolished 5-HT-stimulated PGE2 synthesis. 5-HT also stimulated prostacyclin (measured as 6-ketoprostaglandin F1 alpha) but not thromboxane synthesis in the same cell cultures. 5-HT-stimulated PGE2 synthesis was not affected by extracellular calcium depletion but was abolished by preincubating the cells with the intracellular calcium antagonist 8-(N,N-diethylamine)-octyl-3,4-5 trimethoxybenzoate (10(-5) M). These studies show that 5-HT stimulates PGE2 and prostacyclin (PGI2) synthesis in mesangial cells via a mechanism dependent on intracellular calcium. These vasodilator PGs may modulate the effect of 5-HT on renal and specifically glomerular hemodynamics.

Stimulatory and inhibitory effects of TMB-8 on pancreatic enzyme secretion

The putative intracellular calcium antagonist 3,4,5-trimethoxybenzoate 8-(diethylamino)-octyl ester (TMB-8) affects carbachol-induced enzyme secretion from rabbit pancreatic acini in a different way than it does that induced by either the C-terminal octapeptide of cholecystokinin (CCK-8), the phorbol ester, 12- O-tetradecanoylphorbol 13-acetate (TPA) or the calcium ionophore, A23187. In the presence of TMB-8 the dose-response curve for carbachol-induced amylase release shifts to the right, suggesting competitive antagonism at the muscarinic receptor. The hypothesis that TMB-8 acts as a muscarinic receptor antagonist is supported by the observation that TMB-8 dose-dependently inhibits the carbachol-, but not CCK-8-induced increases in cytosolic free calcium, measured in acinar cells by means of the fluorescent calcium indicator quin2. At a concentration of 100 μM, TMB-8 maximally potentiates the secretory response to suboptimal, but not (supra)optimal, concentrations of CCK-8. At the same concentration the drug also potentiates TPA- and A23187-induced enzyme secretion. Cytosolic free calcium levels and CCK-8-induced increases in cytosolic free calcium remain unaffected by 100 μM TMB-8. The above results strongly suggest that potentiation occurs at or beyond the site of interaction between the diacylglycerol- and the Ca 2+-activated pathways. At concentrations beyond 100 μM the potentiating effect of TMB-8 declines and, finally, at a concentration of 500 μM the drug completely abolishes the secretory response to CCK-8 and TPA. Basal enzyme secretion, however, remains unaffected. At 500 μM severe side effects are observed as is shown by Trypan blue uptake, lactic dehydrogenase release and release of trapped quin2. It is concluded that at lower concentrations TMB-8 does not act as a specific intracellular calcium antagonist in pancreatic enzyme secretion and that inhibitory effects obtained with rather high concentrations of this drug should be treated with caution.

The effect of calcium antagonists on histamine and leukotriene-induced tracheal microvascular permeability in the guine pig

The effects of several calcium antagonists, i.e., nifedipine, verapamil adn 8-[N,N-diethylamino]-octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8), were evaluated in situ on agonist-induced increases in permeability of the airway microvasculature in anesthetized guinea pigs. Vascular permeability was measured as tracheal extravascular albumin content by using 125I-bovine serum albumin and the utilization of 51Cr labelled-erythrocytes to correct for blood volume. Intratracheal injections of histamine (1, 10 and 100 μg) or leukotriene (LT) D 4 (1, 10 and 100 ng) produced dose-dependent increases in extravasated radiolabelled albumin in the trachea. Although histamine produced a greater maximal response than LTD 4, the latter provocation was tent times more potent than the former. Nifedipine, a dihydropyridine calcium slow channel blocker, exhibited dose-dependent (30, 100 and 300 μg/kg) inhibitory activity against histamine-induced increases in extravascular albumin, while another calcium slow channel blocker, verapamil (100, 300 and 1000 μg/kg), exhibited much less activity. TMB-8, a purported intracellular calcium antagonist (1 and 10 mg/kg), was observed to have some inhibitory activity versus histamine. Similar doses of all three calcium antagonists failed to significantly inhibit increases in tracheal microvascular permeability evoked by LTD 4. These results suggest that differences in mediator-induced microvascular permeability in the guinea pig trachea are evident depending upon the agonist selected and the pool of calcium utilized.

The effects of HA compound calcium antagonists on delayed cerebral vasospasm in dogs.

The authors have examined the effects of the HA compounds HA1004(N-(2-guanidinoethyl)-5-isoquinolinesulfonamide) and HA 1077(1-(5-isoquinolinesulfonyl)homopiperazine), which are intracellular calcium antagonists, on delayed cerebral vasospasm from subarachnoid hemorrhage (SAH). The modes of action of these compounds were compared with those of the more commonly used calcium entry blockers. Calcium ionophore A23187 (4.8 X 10(-6) M)-induced contraction of a canine basilar artery strip was completely antagonized by the HA compounds (10(-5) M) but not by the entry-blocking calcium antagonists nicardipine, diltiazem, and verapamil (10(-5) M), suggesting that the HA compounds act differently. Delayed cerebral vasospasm was induced by a "two-hemorrhage" canine model. The magnitude of the vasospasm and the effects of the HA compounds were determined angiographically. On SAH Day 7, a significant vasospasm was observed in every dog. The diameter of the basilar artery had diminished to 59% +/- 2% (mean +/- standard error) of the control value obtained before SAH (on Day 1). The intravenous administration of HA 1004 caused a mild dilation of the basilar artery of 10% and 11% at doses of 3 and 10 mg/kg, respectively; however, HA 1077 produced a more marked dilation of 19% and 27%, respectively, at the same doses. Both of these drugs lowered mean arterial blood pressure to about 80% and 50% at doses of 3 and 10 mg/kg, respectively. Intracisternal administration of the HA compounds (6 mg) completely reversed cerebral vasospasm without much effect on the blood pressure. The intracellular calcium antagonists of the HA compound group appear to be promising agents for the treatment of intractable cerebral vasospasm.

Implications of a rise in cytosolic free calcium in the activation of RAW-264 macrophages for tumor cell killing

The concentration of cytosolic-free calcium (Ca i 2+) was determined with aequorin in RAW-264 macrophage-like cells activated in vitro for tumor cell killing with lymphokine (LK) and lipopolysaccharide (LPS). Treatments of these cells with optimal doses of stimulants, which evoked the development of cytolytic activity, also induced a rise in their Ca i 2+. No rise in Ca i 2+ could be observed under treatments which failed to activate cells. The presence of both stimulants was an absolute requirement for evoking cytolytic activity and also a rise in Ca i 2+. There was an apparent parallelism between the rate of activation and the rate of rise in Ca i 2+. Cells which slowly developed their cytolytic activity exhibited a slow rise in Ca i 2+, while macrophages which acquired their cytolytic activity at the faster rate also showed a more rapid increase in Ca i 2+. The development of cytolytic activity in RAW-264 macrophages was inhibited by two intracellular calcium antagonists, TMB-8 and ruthenium red. This inhibition could be reversed by high concentrations of extracellular calcium. TMB-8, at the concentrations which were effective in inhibiting the activation process, also completely blocked the associated rise in Ca i 2+. These results suggested that Ca i 2+ might play a role in the mechanism of tumoricidal transformation of RAW-264 macrophages.

Characteristics of aggregated immunoglobulin G as an immunologic phagocytic stimulus for granule enzyme release from human neutrophils.

Aggregated immunoglobulin G (AggIgG) induced a time- and concentration-dependent phagocytic release of granule-associated lysozyme and myeloperoxidase (MPO) from human neutrophils. Degranulation was significantly enhanced in the presence of calcium or magnesium, and maximum granule exocytosis was observed when both divalent cations were present. AggIgG-stimulated enzyme release was inhibited with the intracellular calcium antagonist, TMB-8[8-(N,N-diethylamino)-octyl-(3,4,5-trimethoxy)benzoate] in the absence of extracellular calcium. DIDS (4,4'-diisothiocyano-2,2'-disulfonic acid stilbene), a permeant anion channel blocker, also suppressed AggIgG-induced degranulation. Cycloheximide, an inhibitor of protein synthesis, enhanced granule exocytosis from AggIgG-treated neutrophils. Two inhibitors of transmethylation reactions, 3-deazaadenosine (3-DZA) and homocysteine thiolactone (HCTL) in combination, suppressed AggIgG-elicited granule enzyme release. These data indicate that AggIgG is a useful probe for investigating the requirements for phagocytic enzyme release from human neutrophils.

The calcium dependence of histamine, carbachol and potassium chloride-induced contraction in human airways in vitro

The effect of depletion of calcium from the Krebs Henseleit (KH) solution and the addition of EDTA (1 mM) on contraction of human isolated bronchus by histamine, carbachol and potassium chloride (KCl) was examined. Contractions to histamine and carbachol were almost totally abolished in calcium free KH in the presence of 1 mM EDTA which only reduced KCl responses to 76% of control values. Verapamil (10 −6, 10 −5, 10 −4 M) decreased histamine and KCl contractile responses in a dose-related manner while having no significant effect on carbachol-induced tension. The intracellular calcium antagonist TMB8 10 −4 M caused a slight but significant decrease in histamine contraction but was without effect on the maximal response of the other two agonists. TMB8 decreased the potency of both histamine and carbachol. These results suggest that histamine, carbachol and KCl utilise different pools of calcium for contraction, that only part of the calcium entry for all agonists occurs via voltage-dependent calcium channels and that intracellular calcium stores may play a small role in contraction to these agonists.

Polyphosphoinositide hydrolysis in endothelial cells and carotid artery segments. Bradykinin-2 receptor stimulation is calcium-independent.

Bovine aortic and cerebral microvascular endothelial cells and cultured segments of canine common carotid artery possess functional receptors for the nonapeptide bradykinin which mediate a rapid increase in the formation of [3H]inositol 1-phosphate, [3H]inositol 1,4-bisphosphate, and [3H]inositol 1,4,5-trisphosphate from cell membranes containing isotopically labeled myo-inositol. Bradykinin stimulated the formation of [3H]inositol phosphates from cells in culture or tissues at threshold concentrations of 0.1 nM and 1 nM, and with a half-maximal effective concentration of 0.6-1.0 nM and 30 nM, respectively. In cultured cells, the formation of [3H]inositol trisphosphate and [3H]inositol bisphosphate preceded the formation of [3H]inositol monophosphate. Similarly, [3H]inositol phosphate formation was not inhibited by addition of calcium channel blockers, a calcium chelator, or an intracellular calcium antagonist. Calcium ionophore A23187 did not promote [3H]inositol phosphate accumulation. The receptor selectivity of the bradykinin response in cultured cells was most compatible with a type-2 mediated response. Kallidin stimulated with the same potency as bradykinin but was more potent than methionyl-lysyl-bradykinin or des-Arg9-bradykinin. The B1 receptor antagonists des-Arg9-[Leu8]-bradykinin and des-Arg10-[Leu9]-kallidin were without effect. The rapidity of the inositol phosphate response as well as the close correspondence between the bradykinin type-2 receptor mediated hydrolysis of polyphosphoinositides and changes in prostacyclin synthesis, vessel dilation, and permeability suggests that breakdown products of inositol lipids serve as second messengers mediating the effects of bradykinin on the vascular endothelium.

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 B 12-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)- Δ 6,8 -prostaglandin I 1, 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.

Calcium and Histamine Release from Mast Cells

The role of calcium in the process of histamine release from isolated rat peritoneal mast cells and in the pathophysiology of immediate hypersensitivity reactions has been further studied using the intracellular calcium antagonist, TMB-8. Inhibition by TMB-8 of histamine release from mast cells induced by allergic stimuli was found but higher concentrations were needed for reducing release induced by ionophores and activators of calcium gates. This suggests that there are different calcium channels involved in the different release processes.