Histamine-stimulated production of matrix metalloproteinase 1 by human rheumatoid synovial fibroblasts is mediated by histamine H1-receptors

The purpose of this study was to investigate the role of histamine in human rheumatoid synovial fibroblasts in the production of factors responsible for tissue remodelling and cartilage breakdown in rheumatoid arthritis. We examined the effects of histamine of tritiated thymidine incorporation, production of matrix metalloproteinase-1 (MMP-1), histamine H₁-receptor expression, phosphoinositide metabolism and intracellular calcium ion concentration ([Ca²⁺] _i ) in human rheumatoid synovial fibroblasts. Tritiated thymidine incorporation studies demonstrated that histamine markedly stimulated the proliferation of rheumatoid synovial fibroblasts. Immunofluorescence and Northern blot analyses revealed that proMMP-1 production was also stimulated by histamine. The levels of inositol phosphates and [Ca²⁺] _i in the cells were elevated in response to histamine, indicating that the cells expressed histamine H₁-receptors; and Northern blot analysis indicated that these H₁-receptors were up-regulated by histamine. In in situ hybridization, large amounts of histamine H₁-receptor mRNA were also detected in rheumatoid synovial tissue. These results suggest that the interaction between H₁-receptor expression in rheumatoid synovial fibroblasts and histamine secretion by mast cells and macrophages in the affected sites is an important event responsible for tissue remodelling and joint destruction in rheumatoid arthritis.     

A postsynaptic inhibitory histamine H2-receptor in the mouse isolated vas deferens

The effect of histamine on adrenergic neurotransmission in the mouse isolated vas deferens preparation was investigated. Concentrations of histamine ranging from 0.2 to 650 M depressed, in a dose-related manner, not only the contractile response elicited by field stimulation but also the response caused by the addition of exogenous noradrenaline and acetylcholine. However, the release of [³H]-NA evoked by field stimulation or by high K⁺ remained unchanged in the presence of these concentrations of histamine. The inhibitory effect of histamine on the contractile responses caused by various stimuli was reduced or completely antagonized by cimetidine, a histamine H₂-receptor antagonist but not by mepyramine, a conventional antihistamine. The inhibitory effect of histamine was found to be inversely proportional to both the Ca²⁺ concentration in the bathing medium and to the frequency of field stimulation. Further, the inhibitory effect of histamine was markedly reduced when Mg²⁺ was omitted from the bathing medium. It is concluded that the mouse vas deferens preparation contains a post-junctional inhibitory H₂-receptor. The stimulation of H₂-receptors by histamine inhibits the contractile response of the vas deferens, possibly by decreasing the availability of Ca²⁺ required for contraction by depressing the influx of Ca²⁺.     

Effects of Ca2+ ions on gastric acid secretion by the rat isolated stomach

The role of Ca²⁺ in the stimulatory action of histamine has been evaluated in the isolated gastric fundus from immature rats, by changing the concentration of calcium ions in the bathing solutions. Lowering Ca²⁺ to 1.2 mM greatly enhanced the secretory response to histamine, while leaving unaffected that to the H₂-receptor agonist, dimaprit. The effect of histamine was competitively antagonized by ranitidine (pA₂=6.78) in normal solutions; conversely in 1.2 mM Ca²⁺, the antagonism by ranitidine became unsurmountable. Basal rates of acid secretion did not change in low Ca²⁺ solutions, whereas they were reduced approximately by 50% in Ca²⁺-free media. Finally, the secretory response to theophylline was significantly lower in low Ca²⁺ solutions in comparison with that in control conditions. From the above results it may be concluded that changes in the concentration of Ca²⁺ ions caused different changes in the secretory response of the rat stomach in the various experimental conditions. The marked enhancement of the response to histamine observed in low Ca²⁺ is unlikely to be connected with H₂-receptors, as suggested by the lack of interference in the response to dimaprit, but it could be related to intracellular mechanisms (H⁺/K⁺-ATPase, carbonic anhydrase activation etc.).     

Resolvin D1 and aspirin-triggered resolvin D1 regulate histamine-stimulated conjunctival goblet cell secretion

Resolution of inflammation is an active process mediated by pro-resolution lipid mediators. As resolvin (Rv) D1 is produced in the cornea, pro-resolution mediators could be effective in regulating inflammatory responses to histamine in allergic conjunctivitis. Two key mediators of resolution are the D-series resolvins RvD1 or aspirin-triggered RvD1 (AT-RvD1). We used cultured conjunctival goblet cells to determine whether histamine actions can be terminated during allergic responses. We found cross-talk between two types of G protein-coupled receptors (GPRs), as RvD1 interacts with its receptor GPR32 to block histamine-stimulated H₁ receptor increases in intracellular [Ca²⁺] ([Ca²⁺]_i) preventing H₁ receptor-mediated responses. In human and rat conjunctival goblet cells, RvD1 and AT-RvD1 each block histamine-stimulated secretion by preventing its increase in [Ca²⁺]_i and activation of extracellular regulated–protein kinase (ERK)1/2. We suggest that D-series resolvins regulate histamine responses in the eye and offer new treatment approaches for allergic conjunctivitis or other histamine-dependent pathologies.     

Autoinhibition of histamine release by H3 receptors in rat brain cortex depends on stimulation frequency

Rat cortical slices preloaded with [³H]histidine released [³H]histamine upon electrical stimulation or after depolarization with elevated K⁺ levels. The release was dependent on the presence of Ca²⁺, suggesting a neurosecretory process. Histamine has been shown to inhibit its own release mediated by an autoreceptor belonging to the H₃-receptor subclass. In this study we have investigated the autoinhibition using different electrical field stimulation conditions (1, 10, 20 and 33.3 Hz). Applying electrical stimulation, the inhibition of [³H]histamine release by histamine is decreased when the stimulation frequency is elevated. When stimulated with 1 Hz histamine is able to block [³H]histamine release completely, with ap(EC₅₀) of 8.1±0.1. At higher frequencies histamine still blocks [³H]histamine release completely, but with a lowerp(EC₅₀).     

Mechanisms of vancomycin-induced histamine release from rat peritoneal mast cells

The mechanisms of vancomycin (VCM)-induced histamine release were studied with rat peritoneal mast cells. VCM (>1×10^–3 M) released histamine from the isolated mast cells in a dose-dependent and noncytotoxic manner. In the absence of extracellular Ca²⁺, the histamine release was reduced markedly. When the intracellular Ca²⁺ was depleted, it was further decreased. The Fura-2-loaded single mast cells showed a biphasic increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) by VCM: the first transient and the second sustained components. In the absence of extracellular Ca²⁺, the transient component was unchanged, while the sustained component was eliminated completely. The IP₃ content in the mast cells increased within 10 s after the application of VCM. These results suggest that VCM releases histamine from rat peritoneal mast cells via an IP₃ production and increase in [Ca²⁺]_i.     

Biochemical and pharmacological characterization of histamine-mediated up-regulation of human platelet serotonin uptake. Evidence for a subclass of histamine H2 receptors (H2h) highly sensitive to H2 receptor antagonists

The stimulatory effect of histamine: H (1.2 to 3-fold increase) on serotonin (5-HT) uptake by human platelets was observed after a 5 min incubation period in the presence of 2.5×10^–7 M histamine, followed by subsequent 5 min incubation of the platelets with 10^–7 M [³H] 5-HT. Methyl, ethyl and acetyl substituents in the side chain of H mimiked the stimulatory effect of H. In contrast, H analogs methylated at the position N-1 of the imidazole ring of H, as well as imidazole and histidine inhibited platelet 5-HT uptake. The cAMP-inducing agents forskolin and theophylline have no effect on 5-HT uptake when they are tested alone or in combinations with H. In contrast, the cGMP-inducing agent sodium nitroprusside (10^–7 M–10^–6 M) stimulated and potentiated H-mediated up-regulation of 5-HT uptake. Histamine H₂ receptor agonists and antagonists are more potent than drugs acting on H₁ receptors (H₂>H₁). However, the inhibition constants Ki are not consistent with those determined for typical H₁, H₂, H₃ receptors characterized in other tissues. This findings provide further evidence for the existence of multiple forms of H receptors and suggest the involvement of a subpopulation of H₂ receptors, highly sensitive to H₂ receptors antagonists (H_2h), mediating 5-HT uptake in human platelets.     

Influence of histamine H3-antagonists on human leukocytes

To determine the role of the histamine H₃-receptor on basophils, different specific H₃-antagonists were investigated. Incubation of washed leukocytes with N-acylated histamine-derivatives (N-ahd) induced elevated histamine levels. This process turned out to be dependent on dose, time and temperature, but independent of Ca²⁺ and Mg²⁺ ions. IgE-mediated histamine release was not modulated. [³H]-l-histidine was not decarboxylated into [³H]-histamine in spite of the observed histamine increase. Highly purified basophils did not show any histamine elevation but purified neutrophils and eosinophils were found to have increased histamine levels even after disintegration and subsequent incubation with N-ahd. It seems that the increased histamine levels result from the cleavage of the applied histamine amides. Other potent H₃-antagonists (e.g. thioperamide) neither produced increased histamine levels nor influenced IgE-mediated release from basophil leukocytes. The existence of H₃-receptors on human basophils therefore seems unlikely.This work was supported by Grant No. KI 622/1-1 from the Deutsche Forschungsgemeinschaft, Bonn, FRG.     

Modification by some antagonists of the shape changes of venous endothelial cells in response to inflammatory agentsin vitro

Endothelial cells lining guinea pig inferior venae cavae change shape when exposed to histamine, bradykinin, A23187 or platelet-activating factor (PAF)in vitro. Pre-treatment of the endothelium with isoprenaline or quin 2 significantly reduced the shape changes produced in response to histamine, bradykinin and A23187, but not those to PAF. Since both isoprenaline and quin 2 may reduce the concentration of cytoplasmic Ca⁺⁺, the former by raising cyclic AMP (cAMP) levels and the latter by acting as a Ca⁺⁺ buffer, the results provide further evidence for the involvement of Ca⁺⁺ in the responses of large vein endothelial cells to inflammatory agentsin vitro. The effects of pre-treating the endothelium with the histamine receptor-blockers mepyramine (H₁) or cimetidine (H₂), or the bradykinin receptor-blockers des-arg⁹[leu⁸] bradykinin (B₁) or des-arg[Hyp³, Thi^5,8, D-Phe⁷] bradykinin (B₂) suggest that the response to histamine is both H₁ and H₂ receptor-mediated, while the response to bradykinin is only B₂ receptor-mediated.     

Different Reactions of Aortic and Venular Endothelial Cell Monolayers to Histamine on Macromolecular Permeability: Role of cAMP, Cytosolic Ca2+ and F-actin

Endothelial cells assume a central role in the one process that the permeation of microvessels is accelerated in case of inflammation. We studied the effect of histamine on endothelial permeability, [Ca²⁺]_i, cAMP and F-actin, using same origin aortic and venular cultured endothelial monolayers. When HUVEC were treated with histamine (10^–7–10^–5 M), permeability of FITC-dextran (molecular weight 70,000) and [Ca²⁺]_i were increased, while cAMP content was unchanged, and F-actin content was reduced. When bovine vein-derived endothelial cells were treated with histamine, [Ca²⁺]_i was increased via H1 receptors, but permeability and F-actin content were not altered. When human aorta-derived endothelial cells were, [Ca²⁺]_i was increased via H1 receptors and cAMP content was increased via H2 receptors, while permeability and F-actin content were not changed. When bovine aorta-derived endothelial cells were, cAMP and F-actin content were increased, while permeability was reduced. These findings suggest that endothelial cells derived from different tissues clearly showed the different reactions to histamine, the increase in [Ca²⁺]_i led to the increase in endothelial permeability, while the increase in cAMP levels led to the reduction in permeability, and finally, F-actin regulated endothelial macromolecular permeability.     

The presence of bestrophin-1 modulates the Ca2+ recruitment from Ca2+ stores in the ER

Bestrophin-1, mainly analyzed in overexpression experiments, functions as Ca²⁺-dependent Cl^– channel. Analysis of endogenously expressed bestrophin-1 suggested an influence on intracellular Ca²⁺. The aim of the study is to analyze the influence of endogenously expressed bestrophin-1 on Ca²⁺ homeostasis. Primary cultures of retinal pigment epithelial (RPE) cells were established from wild-type and bestrophin-1-deficient mice. Intracellular free Ca²⁺ ([Ca²⁺]_i) was recorded by Ca²⁺ imaging; through immunocytochemistry and differential centrifugation, subcellular localization of bestrophin-1 was analyzed. RPE cells of bestrophin-1-deficient mice showed higher levels of resting [Ca²⁺]_i than cells from wild-type mice. In cells from knockout mice and wild-type mice, ATP led to increases in [Ca²⁺]_i subsequent to phospholipase C activation. ATP-induced Ca²⁺ in bestrophin-1-deficient mice rose faster and decayed slower. In cells from wild-type mice, ATP led to [Ca²⁺]_i increase via depletion of Ca²⁺ from thapsigargin-sensitive stores. In cells from bestrophin-1-deficient mice, ATP-dependent increase in [Ca²⁺]_i resulted in 40% of cells from depletion of bafilomycin-sensitive and in 60% from thapsigargin-sensitive Ca²⁺ stores. After differential centrifugation, bestrophin-1 was found in fractions enriched of ClC-3 Cl channel and myosin-7A. Co-localization analysis of bestrophin-1, with β-catenin or pan-cadherin, in fresh sections of porcine retina, revealed bestrophin-1 in the basolateral membrane. A portion of endogenously expressed bestrophin-1,localized in the endoplasmic reticulum, influenced uptake of Ca²⁺ into Ca²⁺ stores. Therefore, bestrophin-1 possibly conducts Cl^– as counter ion for Ca²⁺ uptake into cytosolic Ca²⁺ stores.     

The role of a Ca2+/calmodulin dependent plasma membrane Ca2+ channel during Concanavalin A activation of MC9 mast cells

The effects of Con A on free cytoplasmic calcium concentrations in the cloned murine mast cell, MC9, have been measured using the fluorescent calcium indicator quin 2. Con A causes a rapid, small yet sustained rise in free cytosolic calcium (up to 245 nM) followed closely by increased⁴⁵calcium uptake and more slowly by histamine release. The increases in⁴⁵calcium uptake and histamine release require extracellular calcium. However, the Ca²⁺ influx blockers, nifedipine and verapamil inhibit these responses only at concentrations significantly higher than those used in smooth muscle to oppose potential-dependent events, and diltiazem is inactive. These observations suggest that, in these mast cells, other types of channels control Ca²⁺ entry.In contrast, the intracellular Ca²⁺ blocker, TMB-8, inhibits both the Con A-induced histamine release and the Ca²⁺ changes. The calmodulin antagonists calmidazolium, trifluoperazine and W-7 are also highly effective inhibitors of both the Ca²⁺ changes and histamine release in direct proportion to their potency against calmodulin-dependent phosphodiesterase, implicating calmodulin in the regulation of stimulus-secretion in MC9 cells. These data imply that histamine release follows increases in intracellular Ca²⁺ concentration. Free intracellular Ca²⁺ results from rapid release from internal stores and is followed by a slower but more sustained influx of extracellular Ca²⁺.     

Effects of 2,5-di(tert-butyl)-1,4-hydroquinone on intracellular free Ca2+ levels and histamine secretion in RBL-2H3 cells

The effects of 2,5-di(tert-butyl)-1,4-hydroquinone (DTBHQ) on the intracellular free Ca²⁺ level ([Ca²⁺]_i) and histamine secretion of rat basophilic leukemia (RBL-2H3) cells were examined. DTBHQ (0.1–10 µmol/l) alone induced rapid and sustained increases in [Ca²⁺]_i in a concentration-dependent manner. In cells sensitized with anti-dinitrophenyl IgE, DTBHQ (10 µmol/1) further increased the antigen (dinitrophenylated BSA)-induced Ca²⁺ response. In the absence of external Ca²⁺ with addition of 1 mmol/1 EGTA, both DTBHQ (10 µmol/l) and the antigen (10 µg/ml) induced transient increase in [Ca²⁺]_i. In sensitized cells, both DTBHQ (10 µmol/1) and antigen (10 µg/ml) elicited histamine secretion, although the response was far stronger in the latter case. The DTBHQ-induced histamine secretion was markedly enhanced by addition of the protein kinase C activator, phorbol 12-myristate 13-acetate (TPA) (10 ng/ml) whereas TPA alone did not cause any increase. Moreover, DTBHQ enhanced the antigen-induced histamine secretion. The results suggest that DTBHQ increases [Ca²⁺]_i and enhances antigeninduced histamine secretion while DTBHQ alone does not cause as much histamine secretion as antigen, which support the idea that calcium signals are necessary but are not sufficient for maximum histamine secretion in RBL-2H3 cells.     

Mechanisms underlying changes of tetanic [Ca2+]i and force in skeletal muscle

Force development in skeletal muscle is driven by an increase in myoplasmic free [Ca²⁺] ([Ca²⁺]_i) due to Ca²⁺ release from the sarcoplasmic reticulum (SR). The magnitude of [Ca²⁺]_i elevation during stimulation depends on: (a) the rate of Ca²⁺ release from the SR, (b) the rate of Ca²⁺ uptake by the SR, and (c) the myoplasmic Ca²⁺ buffering. We have used fluorescent Ca²⁺ indicators to measure [Ca²⁺]_i in intact, single fibres from mouse and Xenopus muscles under conditions where one or more of the above factors are changed. The following interventions resulted in increased tetanic [Ca²⁺]_i: β-adrenergic stimulation, which potentiates the SR Ca²⁺ release; application of 2,5-di(tert-butyl)-1,4-benzohydroquinone, which inhibits SR Ca²⁺ pumps; application of caffeine, which facilitates SR Ca²⁺ release and inhibits SR Ca²⁺ uptake; early fatigue, where the rate of SR Ca²⁺ uptake is reduced; acidosis, which reduces both the myoplasmic Ca²⁺ buffering and the rate of SR Ca²⁺ uptake. Reduced tetanic [Ca²⁺]_i was observed in late fatigue, due to reduced SR Ca²⁺ release, and in alkalosis, due to increased myoplasmic Ca²⁺ buffering. Force is monotonically related to [Ca²⁺]_i, but depends also on the myofibrillar Ca²⁺ sensitivity and the maximum force cross-bridges can produce. This is clearly illustrated by changes of intracellular pH where, despite a lower tetanic [Ca²⁺]_i, tetanic force is higher in alkalosis than acidosis due to increases of myofibrillar Ca²⁺ sensitivity and maximum cross-bridge force.     

Effect of human defensins on the cytoplasmic Ca2+ content in platelets

The effect of the total fraction of human defensins (HNP-1, HNP-2, and HNP-3) on the cytoplasmic Ca²⁺ content ([Ca²⁺]_i) in the platelets of healthy donors was studied. At concentrations of 0.1–40 μg/ml and an incubation time of 10 min defensins have no effect on [Ca²⁺]_i in platelets labeled with Fura-2AM. However, at higher concentrations (100 μg/ml) they increased platelet [Ca²⁺]_i. In addition, defensins (40 μg/ml) inhibited the Ca²⁺ increase in platelets induced by thrombin, adenosine diphosphate, and the lipopolysaccharide ofS. typhimurium endotoxin. The most pronounced inhibitory effect was observed in a suspension of thrombin-stimulated platelets. It is shown that the effect of human defensins on the functional activity of platelets is due to the alterations in the intracellular Ca²⁺. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 12, pp. 600–603, December, 1994     

The time course of intracellular calcium movements in single human umbilical vein smooth muscle cells

Intracellular Ca²⁺ ([Ca²⁺]_i) was measured in single isolated human umbilical vein smooth muscle cells. Stimulation with histamine, in the absence of external Ca²⁺, mobilised Ca²⁺ from intracellular stores. When repeated brief applications of agonist were used, the time to onset, amplitude and rate of rise of the Ca²⁺ transients were found to change. Two components could often be discerned in the rising phase of the transients, an initial slow pacemaker and a second faster and larger component. Following the first histamine-activated transient the basal level of [Ca²⁺]_i was invariably lower than that prior to stimulation. This lower value was maintained whilst the cell remained in Ca²⁺-free solution, but could be returned to a higher level if the cell was exposed to external Ca²⁺. When the mobilisation of the intracellular store was reduced to undetectable levels, re-exposure to Ca²⁺-containing medium reactivated responses. In the absence of external Ca²⁺, continuous application of histamine activated a series of transient increases in intracellular Ca²⁺, which decreased progressively in amplitude and rate of rise. The interval between transients also increased. These findings are discussed in terms of the activation of inositol trisphosphate-sensitive intracellular Ca²⁺ stores and their sensitivity to cytoplasmic Ca²⁺ and intrasarcoplasmic reticulum Ca²⁺.     

On the mechanism of histamine induced enhancement of the cardiac Ca2+ current

In guinea pig ventricular myocytes, the effect of histamine on the slow Ca²⁺ current (I_Ca) was studied and the following results were obtained: (1) Superfusion of cells with histamine resulted in a dose-dependent enhancement of the amplitude of I_Ca. The threshold concentration of histamine was 10^–8 M, half maximal increase occurred at 3×10^–7 M and maximal enhancement (about 3–4-fold) at 5×10^–6 M. (2) The histamine effect was greatly reduced by the H₂ antagonist cimetidine (10^–5 M) but only slightly by the H₁ antagonist diphenhydramine (10^–5M). (3) Effects of isoprenaline (ISP) and histamine at maximal effective concentrations on I_Ca were not additive, suggesting that both agents use the same intracellular pathway. Intracellular infusion of a blocker of the cAMP-dependent protein kinase, R_p-cAMPS (10^–4 M), prevented the histamine effect. (4) The involvement of GTP-dependent transducer proteins was studied by cell dialysis with several GTP derivatives. Intracellular application of the stable GDP-analogue, GDP--S, reduced the histamine effect on I_Ca, whereas the stable GTP analogue, GTP--S, mimicked the histamine effect.     

Effects of exogenous hydrogen sulphide on calcium signalling,background (TASK) K channel activity and mitochondrial function in chemoreceptor cells

It has been proposed that endogenous H₂S mediates oxygen sensing in chemoreceptors; this study investigates the mechanisms by which H₂S excites carotid body type 1 cells. H₂S caused a rapid reversible increase in intracellular calcium with EC₅₀ ≈ 6 μM. This [Ca²⁺]_i response was abolished in Ca-free Tyrode. In perforated patch current clamp recordings, H₂S depolarised type 1 cells from −59 to −35 mV; this was accompanied by a robust increase in [Ca²⁺]_i. Voltage clamping at the resting membrane potential abolished the H₂S-induced rise in [Ca²⁺]_i. H₂S inhibited background K⁺ current in whole cell perforated patch and reduced background K⁺ channel activity in cell-attached patch recordings. It is concluded that H₂S excites type 1 cells through the inhibition of background (TASK) potassium channels leading to membrane depolarisation and voltage-gated Ca²⁺ entry. These effects mimic those of hypoxia. H₂S also inhibited mitochondrial function over a similar concentration range as assessed by NADH autofluorescence and measurement of intracellular magnesium (an index of decline in MgATP). Cyanide inhibited background K channels to a similar extent to H₂S and prevented H₂S exerting any further influence over channel activity. These data indicate that the effects of H₂S on background K channels are a consequence of inhibition of oxidative phosphorylation. Whilst this does not preclude a role for endogenous H₂S in oxygen sensing via the inhibition of cytochrome oxidase, the levels of H₂S required raise questions as to the viability of such a mechanism.     

Carboxyeosin decreases the rate of decay of the [Ca2+]i transient in uterine smooth muscle cells isolated from pregnant rats

 In myometrial smooth muscle cells the rate of decline of intracellular calcium ([Ca²⁺]_i) is determined by Ca²⁺ extrusion from the cell and uptake into intracellular stores. The relative quantitative contribution of these processes however, has not been established. We therefore examined the effect of the sarcolemmal Ca²⁺ pump inhibitor, carboxyeosin, on the rate of the [Ca²⁺]_i transient decline in myocytes isolated from pregnant rat uterus. Indo-1 was used in conjunction with the whole-cell patch-clamp technique to measure [Ca²⁺]_i simultaneously with transmembrane calcium current (I _Ca). [Ca²⁺]_i transients were elicited by repetitive membrane depolarization to simulate the natural pattern of uterine electrical activity. The rate of [Ca²⁺]_i removal was calculated from the falling phase of the [Ca²⁺]_i transient. Pre-treatment of the cells with 2 μM carboxyeosin led to a marked decrease in the rate of [Ca²⁺]_i transient decay, suggesting that the sarcolemmal Ca²⁺ pump is involved in the calcium extrusion process. Removal of the extracellular Na also decreased the rate of [Ca²⁺]_i decay, indicating an important role for the Na⁺/Ca²⁺ exchange. When both the sarcolemmal Ca²⁺ pump and Na⁺/Ca²⁺ exchange were inhibited the cell failed to restore [Ca²⁺]_i after the stimulation. Comparison of the rate constants of [Ca²⁺]_i decay in control conditions and after carboxyeosin treatment shows that approximately 30% of [Ca²⁺]_i decay is due to the sarcolemmal calcium pump activity. The remaining 70% can be attributed to the activity of Na⁺/Ca²⁺ exchanger and the intracellular calcium stores. Received: 17 July 1998 / Received after revision: 23 September 1998 / Accepted: 25 September 1998     

尼氟灭酸对气道平滑肌细胞增殖的抑制作用

目的:探讨尼氟灭酸(NFA)对气道平滑肌细胞(ASMCs)增殖的影响及机制。方法:采用含胎牛血清的DMEM原代培养BALB／c小鼠ASMCs,[³H]-TdR掺入法检测不同剂量NFA(10和50 μmol／L)对ASMCs增殖活性的影响。激光共聚焦显微镜下观察NFA及硝苯地平对组胺致ASMCs中[Ca²⁺]i变化的影响。间接免疫荧光法观察NFA对ASMCs表达MAPK蛋白的影响。结果:10 μmol／L和50 mol／L NFA组细胞的活性明显低于对照组,并且高浓度50 μmol／L NFA组比低浓度10 mol／L NFA组更显著(P<0.01),表现出剂量依赖性。共聚焦显微镜下,对照组ASMCs胞浆和胞核呈现亮绿色荧光,胞膜周围较淡。加入激动剂组胺后,荧光亮度逐渐增强。相反,当同时存在NFA或硝苯地平干预时,镜下观察荧光强度变化不够明显。间接免疫荧光染色结果表明,培养24 h后,对照组中ASMCs细胞沿梭形胞浆出现大斑片状亮绿色荧光,而NFA干预组细胞表现为弱绿色荧光,胞浆内分布区域局限。结论:NFA能有效抑制培养ASMCs的增殖活性,这种作用可能是通过阻断钙激活性Cl^-通道对 [Ca²⁺]i的正反馈效应,以及降低MAPK的表达来实现的。