Mechanism underlying histamine-induced intracellular Ca2+ movement in PC3 human prostate cancer cells.

The effect of histamine on intracellular free Ca2+ levels ([Ca2+]i) in PC3 human prostate cancer cells and the underlying mechanism were evaluated using fura-2 as a Ca2+ dye. Histamine at concentrations between 0.1 and 50 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 1 microM. The [Ca2+]i response comprised an initial rise and a slow decay, which returned to baseline within 3 min. Extracellular Ca2+ removal inhibited 50% of the [Ca2+]i signal. In the absence of extracellular Ca2+, after cells were treated with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 10 microM histamine did not increase [Ca2+]i. After pretreatment with 10 microM histamine in a Ca2+-free medium for several minutes, addition of 3 mM Ca2+ induced [Ca2+]i increases. Histamine (10 microM)-induced intracellular Ca2+ release was abolished by inhibiting phospholipase C with 2 microM 1-(6-((17 beta-3- methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), and by 10 microM pyrilamine but was not altered by 50 microM cimetidine. Collectively, the present study shows that histamine induced [Ca2+]i transients in PC3 human prostate cancer cells by stimulating H1 histamine receptors leading to Ca2+ release from the endoplasmic reticulum in an inositol 1,4,5-trisphosphate-dependent manner, and by inducing Ca2+ entry.     

Group 1 mGlu receptors elevate [Ca2+]i in rat cultured cortical type 2 astrocytes: [Ca2+]i synergy with adenosine A1 receptors.

Brain macroglia are known to express a diverse array of neurotransmitter receptors whose signal transduction pathways may be subject to heteroreceptor 'cross-talk'. In the current study we have examined group 1 mGlu receptor-evoked [Ca2+]i signalling, and possible heteroreceptor cross-talk, in cultured type 2 astrocytes. The selective group 1 metabotropic glutamate (mGlu) receptor agonist (S)-3,5-dihydroxyphenylglycine (DHPG) elevated [Ca2+]i (EC50 = 1.7 +/- 0.6 microM); an effect reversed by the selective mGlu receptor antagonist (S)-alpha-methyl-4-carboxyphenylglycine (IC50 = 52.7 +/- 8.7 microM). DHPG-evoked [Ca2+]i responses were abolished by (1) thapsigargin (100 nM), implicating the involvement of internal Ca2+ stores in group 1 mGlu [Ca2+]i responses and (2) the removal of extracellular Ca2+. When applied alone, the selective adenosine A1 receptor agonist, N6-cyclopentyladenosine (CPA, 100 nM) failed to influence [Ca2+]i. However, in the presence of 1 microM DHPG, CPA potently (EC50 = 12.3 +/- 1.9 nM) increased [Ca2+]i responses. In the presence of 100 nM CPA, the efficacy of DHPG was doubled without any significant change in the DHPG EC50 value. This effect was reversed by either the selective adenosine A1 receptor antagonist, 8-cyclopentyltheophylline (IC50 = 50.3 +/- 19.9 nM) or overnight incubation with Pertussis toxin (100 ng/ml). We conclude that (1) type 2 astrocytes contain group 1 mGlu receptors coupled to [Ca2+]i signalling and (2) co-activation of adenosine A1 receptors enhances group 1 mGlu-evoked [Ca2+]i responses in these cells via a Gi/o G protein-mediated mechanism.     

Histamine increases cytosolic Ca2+ in dibutyryl-cAMP-differentiated HL-60 cells via H1 receptors and is an incomplete secretagogue.

Human neutrophils and dibutyryl-cAMP (Bt2cAMP)-differentiated HL-60 cells possess receptors for the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), which mediate activation of phospholipase C, with subsequent increase in cytosolic Ca2+ concentration ([Ca2+]i) and activation of specific cell functions. In many cell types, histamine, via H1 receptors, activates phospholipase C, but it is unknown whether neutrophilic cells possess functional H1 receptors. We compared the effects of histamine with those of fMet-Leu-Phe on activation of these cells. In Bt2cAMP-differentiated HL-60 cells, substances increased [Ca2+]i in the effectiveness order fMet-Leu-Phe greater than histamine greater than betahistine. Pertussis toxin diminished fMet-Leu-Phe-induced rises in [Ca2+]i to a greater extent than those induced by histamine. H1 but not H2 antagonists inhibited histamine- and betahistine-induced rises in [Ca2+]i. fMet-Leu-Phe and histamine activated phospholipase C and increased [Ca2+]i through release of Ca2+ from intracellular stores and sustained influx of Ca2+ from the extracellular space. The substances also induced Mn2+ influx. Ca2+ and Mn2+ influxes were inhibited by 1-(beta-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl)-1H-imida zole hydrochloride (SK&F 96365). The stimulatory effects of histamine on [Ca2+]i were more sensitive to inhibition by 4 beta-phorbol 12-myristate 13-acetate than were those of fMet-Leu-Phe. Unlike fMet-Leu-Phe, histamine did not activate superoxide anion formation, release of beta-glucuronidase, and tyrosine phosphorylation. In neutrophils, histamine and betahistine did not induce rises in [Ca2+]i. Our data show that (i) in Bt2cAMP-differentiated HL-60 cells, histamine increases [Ca2+]i via H1 receptors coupled to pertussis toxin-sensitive and possibly, pertussis toxin-insensitive heterotrimeric regulatory guanine nucleotide-binding proteins, (ii) histamine activates nonselective cation channels, and (iii) unlike fMet-Leu-Phe, histamine is an incomplete secretagogue.     

Intracellular cross-talk between receptors coupled to phospholipase C via pertussis toxin-sensitive and insensitive G-proteins in DDT1MF-2 cells.

1. The effect on intracellular free calcium concentration ([Ca2+]i) of simultaneous activation of receptors coupled to phospholipase C via pertussis toxin (PTX)-sensitive and -insensitive G-proteins has been investigated in the hamster vas deferens smooth muscle cell line, DDT1MF-2. 2. In fura-2-loaded DDT1MF-2 cells, activation of adenosine A1-receptors (which are linked to PTX-sensitive G-proteins) with a maximal concentration of N6-cyclopentyladenosine (CPA; 300 nM) increased [Ca2+]i from 121 +/- 5 nM to 254 +/- 20 nM (n = 8). These experiments were performed in the presence of extracellular Ca2+. Stimulation of histamine H1-receptors (which are linked to PTX-insensitive G-proteins) with a low concentration of histamine (1 microM) increased [Ca2+]i from 128 +/- 8 nM to 150 +/- 13 nM (n = 8). When combined, CPA (300 nM) and histamine (1 microM) synergistically raised [Ca2+]i from 134 +/- 6 nM to 607 +/- 61 nM (n = 8). 3. Removal of extracellular Ca2+ (experiments performed in Ca(2+)-free buffer containing 0.1 mM EGTA) had no effect on the synergistic interaction between CPA (300 nM) and histamine (1 microM). 4. The addition of maximal concentrations of CPA (300 nM) and histamine (100 microM) resulted in a rise in [Ca2+]i which was additive when compared to the Ca2+ responses obtained with the two agonists alone. Low (30 nM) and subthreshold (3 nM) concentrations of CPA did not alter the Ca2+ response elicited by maximal concentrations of histamine (100 microM). 5. Subthreshold concentrations of CPA (3 nM) and low concentrations of histamine (1 microM) elicited synergistic rises in [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of semotiadil fumarate, a novel Ca2+ antagonist, on cytosolic Ca2+ level and force of contraction in porcine coronary arteries.

1. The mechanisms of action of semotiadil fumarate, a novel Ca2+ antagonist, were examined by measuring the cytosolic Ca2+ level ([Ca2+]i) and force of contraction in porcine coronary arteries, and by determining [3H]-pyrilamine binding to bovine cerebellar membranes. 2. Semotiadil or verapamil (0.1 and 1 microM) inhibited both the high KCl-induced increases in [Ca2+]i and force in a concentration-dependent manner. 3. Histamine (30 microM) produced transient increases followed by sustained increases in [Ca2+]i and force, which were inhibited by semotiadil and verapamil (1 and 10 microM). The agents were different in that semotiadil reduced the maximum [Ca2+]i and force responses to histamine, but not pD2 values, whereas verapamil did reduce the pD2 values for histamine, but not the maximum responses. 4. Verapamil (10 microM), but not semotiadil, inhibited histamine-induced increases in [Ca2+]i and force in Ca(2+)-free solution. Neither semotiadil nor verapamil affected the increases in [Ca2+]i and force induced by caffeine. Semotiadil even at the higher concentration (10 microM) did not displace specific binding of [3H]-pyrilamine to bovine cerebellar membranes. 5. These results suggest that semotiadil inhibits both KCl- and histamine-induced contractions mainly by blocking voltage-dependent L-type Ca2+ channels.     

Short-term desensitization of the histamine H1 receptor in human HeLa cells: involvement of protein kinase C dependent and independent pathways.

1. In this study we have investigated the effects of short-term exposure of cells to histamine on the subsequent H1 receptor responsiveness in HeLa cells, using Ca2+ fluorescence microscopy and video digital imaging. 2. In HeLa cells, histamine (100 microM) induces an immediate H1 receptor-mediated biphasic elevation of the intracellular Ca2+ concentration ([Ca2+]i) (basal [Ca2+]i: 81 +/- 30 nM, histamine-induced Ca2+ response: first phase: 1135 +/- 79 nM; second phase: 601 +/- 52 nM, n = 11). 3. The histamine H1 receptors on HeLa cells are readily susceptible to desensitization since repetitive exposure of the same group of cells to histamine (100 microM) markedly affected the release and influx component of the induced Ca2+ response (second application of histamine: first phase: 590 +/- 92 nM, second phase: 279 +/- 47 nM; third application of histamine: first phase: 454 +/- 127 nM, second phase: 240 +/- 45 nM, n = 6). Video digital imaging revealed an increase in the lag time between stimulation and monitoring of the Ca2+ response and a reduced increase in [Ca2+]i after desensitization with histamine. 4. Neither the release component of the ATP response (50 microM) nor the caffeine (3 mM)-induced Ca2+ release were found to be affected by desensitization with 100 microM histamine. However, the second phase of the ATP response was significantly reduced after desensitization with histamine (control cells: 516 +/- 33 nM; desensitized cells: 331 +/- 96 nM, n = 4, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory action of dilazep on histamine-stimulated cytosolic Ca2+ increase in cultured human endothelial cells.

Using a fluorescent Ca(2+)-sensitive dye, fura-2, and photometric fluorescence microscopy, we measured changes in cytosolic Ca2+ concentration ([Ca2+]i) in cultured human endothelial cells and studied the effect of dilazep on [Ca2+]i elevation induced by histamine. Histamine (1 microM) caused a rapid transient peak in the average [Ca2+]i of a group of cells (approximately 10(2) cells), followed by a decrease to a sustained elevation. Dilazep as well as diltiazem (1.0 to 100 microM) concentration-dependently inhibited the latter sustained elevation, which was eliminated by removal of extracellular Ca2+, while the initial transient response was not changed by dilazep at concentrations up to 100 microM. The IC50 values of dilazep and diltiazem were 16 and 58 microM, respectively. The patterns of the [Ca2+]i elevation responses to histamine were variable among individual cells. Some single cells showed a transient peak and a sustained elevation as observed in a group of cells. Some single cells caused a repetitive spikelike elevation of [Ca2+]i. Dilazep lowered the sustained elevation to the resting level and in some single cells, changed the sustained elevation to the spikelike elevation. The frequency of the spikelike [Ca2+]i elevation was also decreased by dilazep. Decrease in extracellular [Ca2+] showed the same pattern of inhibitory actions as dilazep did. These results indicate that dilazep inhibits the extracellular Ca2+ influx in endothelial cells.     

Effects of inhibitors of protein kinase C on the release and synthesis of histamine in rat basophilic leukemia cells (2H3).

In rat basophilic leukemia cells (2H3), a tumor analog of mast cells, the aggregation of IgE receptors results in histamine secretion and the increase in histidine decarboxylase activity which synthesizes histamine. Using inhibitors of protein kinases C, we studied the relationships between these events and protein kinase C which is activated by antigens. Histamine release is suppressed by inhibitors of protein kinase C, staurosporine, K252-a and H-7, in this decreasing order of effectiveness; and the IC50 values are 1.5 nM, 29.9 nM and 3.8 microM, respectively. The changes in the intracellular Ca concentration monitored by fura-2 fluorescence is not modified by staurosporine, although the histamine response is suppressed. Meanwhile, the increase of histidine decarboxylase was abolished by inhibitors of protein kinase C; staurosporine was the strongest, K-252a of moderate activity and H-7, the weakest, having IC50 values of 0.8 nM, 100 nM and 11.5 microM, respectively. The inhibitors of protein kinase C suppress both histamine secretion and synthesis. Therefore, the histamine synthesis may be stimulated via activation of protein kinase C to supplement the released histamine.     

Adenosine A1 receptor-mediated changes in basal and histamine-stimulated levels of intracellular calcium in primary rat astrocytes.

1. The effects of adenosine A1 receptor stimulation on basal and histamine-stimulated levels of intracellular free calcium ion concentration ([Ca2+]i) have been investigated in primary astrocyte cultures derived from neonatal rat forebrains. 2. Histamine (0.1 microM-1 mM) caused rapid, concentration-dependent increases in [Ca2+]i over basal levels in single type-2 astrocytes in the presence of extracellular calcium. A maximum mean increase of 1,468 +/- 94 nM over basal levels was recorded in 90% of type-2 cells treated with 1 mM histamine (n = 49). The percentage of type-2 cells exhibiting calcium increases in response to histamine appeared to vary in a concentration-dependent manner. However, the application of 1 mM histamine to type-1 astrocytes had less effect, eliciting a mean increase in [Ca2+]i of 805 +/- 197 nM over basal levels in only 30% of the cells observed (n = 24). 3. In the presence of extracellular calcium, the A1 receptor-selective agonist, N6-cyclopentyladenosine (CPA, 10 microM), caused a maximum mean increase in [Ca2+]i of 1,110 +/- 181 nM over basal levels in 30% of type-2 astrocytes observed (n = 53). The size of this response was concentration-dependent; however, the percentage of type-2 cells exhibiting calcium increases in response to CPA did not appear to vary in a concentration-dependent manner. A mean calcium increase of 605 +/- 89 nM over basal levels was also recorded in 23% of type-1 astrocytes treated with 10 microM CPA (n = 30). 4. In the absence of extracellular calcium, in medium containing 0.1 mM EGTA, a mean increase in [Ca2+]i of 504 +/- 67 nM over basal levels was recorded in 41% of type-2 astrocytes observed (n = 41) after stimulation with 1 microM CPA. However, in the presence of extracellular calcium, pretreatment with the A1 receptor-selective antagonist, 8-cyclopentyl-1,3-dipropylxanthine, for 5-10 min before stimulation with 1 microM CPA, completely antagonized the response in 100% of the cells observed. 5. In type-2 astrocytes, prestimulation with 10 nM CPA significantly increased the size of the calcium response produced by 0.1 microM histamine and the percentage of responding cells. Treatment with 0.1 microM histamine alone caused a mean calcium increase of 268 +/- 34 nM in 41% of the cells observed (n = 34). After treatment with 10 nM CPA, mean calcium increase of 543 +/- 97 nM was recorded in 100% of the cells observed (n = 33).(ABSTRACT TRUNCATED AT 400 WORDS)     

Histamine stimulates glycogen breakdown and increases 45Ca2+ permeability in rat astrocytes in primary culture

In astrocyte-enriched cultures from rat brain hemispheres prelabeled with [3H]glucose, histamine stimulates [3H]glycogen breakdown in a concentration-dependent manner, with an EC50 of 0.6 microM. This effect can be induced by activation of both H1 and H2 receptors independently. Thus, neither 1 microM promethazine, an H1 antagonist, or 100 microM metiamide, an H2 antagonist, inhibited the glycogenolytic response to histamine unless they were present together. In addition, the maximal effect of histamine (55% decrease in [3H]glycogen) was also elicited by 300 microM 2-thiazolylethylamine, an H1 agonist, and by 1 mM dimaprit, an H2 agonist. These agonist effects were inhibited by promethazine and metiamide, respectively, and were not additive, indicating that the same glycogen pool was affected. Histamine was more potent in eliciting glycogenolysis through H1 (EC50 of 0.4 microM in the presence of 100 microM metiamide) than through H2 (EC50 of 3.3 microM in the presence of 1 microM promethazine) receptors, as also shown previously for the H1-mediated phosphoinositide hydrolysis compared with the H2-mediated cAMP formation in the same cells. Both dibutyryl cyclic AMP and the Ca2+ ionophore A23187 could independently mimic the glycogenolytic effect of histamine, whereas the absence of extracellular Ca2+ abolished the H1 component of the response. Histamine also stimulated rapid transmembrane 45Ca2+ influx (maximum, 48% of basal at 15 sec) and efflux (maximum, 25% of basal at 1 min) in astrocytes by activation of H1 receptors. This histamine-increased 45Ca2+ entry was abolished by the nonspecific Ca2+ channel blocker lanthanum but not by the voltage-operated Ca2+ channel inhibitor nifedipine. The enhanced 45Ca2+ release was more a consequence of the histamine-increased Ca2+ permeability than intracellular Ca2+ mobilization, because it was largely diminished when Ca2+ entry was prevented and was little affected by pretreatment of the cells with 12-O-tetradecanoyl-phorbol-13-acetate. Thus, the histamine-induced glycogen breakdown in astrocytes may involve increases in cAMP formation and in intracellular Ca2+ levels, this latter resulting mainly from H1-mediated extracellular Ca2+ uptake.     

The histamine H1 receptor in GT1-7 neuronal cells is regulated by calcium influx and KN-62, a putative inhibitor of calcium/calmodulin protein kinase II.

1. In GT1-7 cells, histamine stimulated the initial [Ca2+]i transient in a dose-dependent manner with a best-fit EC50 value of 4.2 +/- 4.2 microM (mean +/- s.e.mean, n = 4) and a best-fit maximal effect of 138 +/- 56 nM (n = 4) increase above basal calcium levels. 2. Pretreatment of cells with 30 microM histamine for 30 min desensitized the population mean peak calcium signal by 53% to 75 +/- 9 nM, (n = 3, P < 0.04). Analysis of the individual cells revealed that 39 +/- 7% (n = 94 cells from 8 experiments) of pretreated cells exhibited desensitized histamine-stimulated [Ca2+]i transients of < or = 1 standard deviation below the control cells mean calcium transient level. 3. The desensitization induced by histamine was prevented (P < 0.01) by KN-62 (10 microM), a putative inhibitor of the calcium/calmodulin-dependent protein kinase II (CaMKII). KN-62 (10 microM) alone did not induce [Ca2+]i mobilization, nor did it antagonize the histamine-stimulated [Ca2+]i signal. In addition, KN-62 did not appear to have its effect by hastening the rate of recovery from desensitization. 4. Histamine pretreatment in nominal (zero calcium + 0.2 mM EGTA) or in low (0.3 mM) extracellular calcium did not induce histamine receptor desensitization, supporting a role for extracellular calcium in the homologous H1 receptor desensitization process. 5. Histamine (30 microM) stimulated at least four different types of [Ca2+]i signals in GT1-7 cells. The majority (61%) were of single spikes with the remaining cells showing some form of calcium oscillatory behaviour. The proportion of GT1-7 cells showing histamine-induced calcium oscillations was histamine concentration-dependent and significantly reduced after acute desensitization. KN-62, when present during histamine pretreatment, prevented this fall in calcium oscillation. Under the conditions of nominal or 0.3 mM extracellular calcium the proportion of cells exhibiting histamine-stimulated calcium oscillations was not significantly different from the controls. 6. Bradykinin stimulated a [Ca2+]i transient in GT1-7 cells with a population mean peak response of 147 +/- 8 nM (n = 5) over basal levels. The bradykinin-induced [Ca2+]i signal was without any calcium oscillatory activity. Histamine pretreatment caused the heterologous desensitization of the bradykinin [Ca2+]i signal (44% reduction, P < 0.007), which was unaffected by KN-62. 7. The results presented here suggest that the histamine-mediated homologous H1 receptor desensitization process involves extracellular calcium and can be blocked by KN-62, a putative inhibitor of CaMKII. In contrast, KN-62 does not appear to prevent the histamine-mediated heterologous desensitization cascade. These findings suggest fundamental differences in the mechanisms underlying homologous and heterologous H1 receptor desensitization pathways in GT1-7 neuronal cells.     

Effectiveness of GABAB antagonists in inhibiting baclofen-induced reductions in cytosolic free Ca concentration in isolated melanotrophs of rat.

1. The purpose of the present experiments was to assess the activities of GABAB receptor antagonists in mammalian isolated melanotrophs. 2. Cytosolic free Ca concentration ([Ca2+]i) in rat melanotrophs in primary culture was monitored with the fluorescent probe, fura-2. 3. (-)-Baclofen lowered [Ca2+]i in a concentration-dependent manner with an EC50 of 0.96 microM. The reduction in [Ca2+]i produced by (-)-baclofen at a maximally effective concentration (100 microM) was similar to that produced by the classic transmitter inhibitory to melanotroph secretion, dopamine, at a corresponding concentration (100 nM), or by perifusion with a nominally Ca-free solution. 4. The GABAB receptor antagonists, 3-aminopropyl(diethoxymethyl)phosphinic acid (CGP 35348), 2-hydroxy saclofen, phaclofen and 4-amino-3-(5-methoxybenzo[b]furan-2-yl) butanoic acid (9H), had inhibitory effects on the reduction in [Ca2+]i produced by (-)-baclofen (3 microM). Of the antagonists tested, CGP 35348 was the most potent with an IC50 of 60 microM, compared to 120 to 400 microM for the others. CGP 35348 acted competitively. 5. CGP 35348 alone had no effect on basal [Ca2+]i, or on the changes in [Ca2+]i produced by dopamine (10 nM) or the specific GABAA receptor agonist, muscimol (10 microM). 6. The evidence indicates that of the antagonists tested, CGP 35348 offers the greatest promise for pharmacological analysis of the functional significance of the GABAB receptors in melanotrophs.     

Discrimination of histamine H1 and muscarinic receptor-mediated signalling pathways by phorbol ester in human astrocytoma cells

1. Histamine H1 receptor-mediated signalling was compared with muscarinic receptor-mediated signalling in 1321N1 human astrocytoma cells. 2. Short-term (2 min) treatment of cells with phorbol 12-myristate 13-acetate (PMA) resulted in a reduction of increases in intracellular Ca2+ ([Ca2+]i) induced by carbachol or histamine. 3. Carbachol-induced increases in [Ca2+]i were 10-fold more sensitive to PMA than the histamine-induced increases. 4. When cells were treated with PMA for 48 or 72 h (long-term treatment), protein kinase C (PKC) was down-regulated and PMA did not inhibit carbachol-induced increases in [Ca2+]i. 5. Histamine-induced increases in [Ca2+]i were significantly reduced by long-term treatment with PMA. 6. These findings suggest that the signalling pathways mediated by histamine H1 and muscarinic receptors can be distinguished by using PKC in 1321N1 human astrocytoma cells.     

Vasorelaxation and inhibition of the voltage-operated Ca2+ channels by FK506 in the porcine coronary artery

Using fura-2 fluorometry, the effects of FK506, an immunosuppressant, on changes in cytosolic Ca2+ concentrations ([Ca2+]i) and tension were investigated in porcine coronary arterial strips. The effects of FK506 on the activity of voltage-operated Ca2+ channels were examined by applying a whole cell patch clamp to the isolated smooth muscle cells of porcine coronary artery. FK506 inhibited the sustained increases in both [Ca2+]i and tension induced by 118 mM K+ depolarization and 100 nM U46619 in a concentration-dependent manner (1-30 microM). The extent of inhibition of the K+-induced contraction was greater than that of the U46619-induced contraction. The increases in [Ca2+]i and tension induced by histamine and endothelin- in the presence of extracellular Ca2+ were also inhibited by 10 microM FK506. FK506 (10 microM) had no effect on Ca2+ release induced by caffeine or by histamine in the Ca2+-free solution. FK506 (10 microM) had no effect on the [Ca2+]i-tension relationships of the contractions induced by cumulative increases of extracellular Ca2+ during K+ depolarization or stimulation with U46619. In the patch clamp experiments, FK506 (30 microM) partially inhibited the inward current induced by depolarization pulse from -80 mV to 0 mV. In conclusion, FK506 induces arterial relaxation by decreasing [Ca2+]i mainly due to the inhibition of the L-type Ca2+ channels, with no effect on the Ca2+ sensitivity of the contractile apparatus.     

Pneumadin-evoked intracellular free Ca2+ responses in rat aortic smooth muscle cells: effect of dexamethasone.

The direct vascular effect of pneumadin (PN) was determined by studying the changes in intracellular free calcium ([Ca2+]i) levels in cultured rat aortic smooth muscle cells maintained between the second and fifth passages. PN evoked a rapid, concentration-dependent, biphasic increase in [Ca2+]i. The [Ca2+]i level rose from a basal value of 108 nM to a maximum increase in peak value of 170 nM. Although the level of maximal [Ca2+]i response evoked by PN was less than with other vasoactive agonists, it was more potent (EC50 0.5 nM) than even endothelin-1 (EC50 3.1 nM). At concentrations > 100 nM, [Ca2+]i elevations induced by PN above basal levels were no longer observed. Pretreatment with dexamethasone (100 nM for 24 hr) resulted in a significant increase (P < 0.01) in the peak [Ca2+]i response (310 nM) to PN. However, the biphasic pattern in the peak [Ca2+]i responses encountered with increasing concentrations of PN remained unaffected. The exaggerated [Ca2+]i response to PN was abolished by preincubation of cells with either the glucocorticoid antagonist mifepristone (RU 486) or the protein synthesis inhibitor cycloheximide. Inclusion of either an AT1 antagonist (losartan), a V1 selective vasopressin antagonist (d(Ch2)5 Tyr (Me) AVP), or an alpha-adrenoceptor antagonist (phentolamine) failed to affect the increases in [Ca2+]i induced by PN. PN-evoked increases in inositol 1,4,5-trisphosphate levels paralleled the [Ca2+]i changes. These data suggest that PN increases Ca2+ mobilization in rat aortic smooth muscle cells via activation of phospholipase C coupled receptors. This effect is up-regulated by dexamethasone.     

delta- and mu-opioid receptor mobilization of intracellular calcium in SH-SY5Y human neuroblastoma cells.

1. In this study we have investigated delta and mu opioid receptor-mediated elevation of intracellular Ca2+ concentration ([Ca2+]i) in the human neuroblastoma cell line, SH-SY5Y. 2. The Ca(2+)-sensitive dye, fura-2, was used to measure [Ca2+]i in confluent monolayers of SH-SY5Y cells. Neither the delta-opioid agonist, DPDPE ([D-Pen2,5]-enkephalin) nor the mu-opioid agonist, DAMGO (Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol enkephalin) elevated [Ca2+]i when applied alone. However, when either DPDPE or DAMGO was applied in the presence of the cholinoceptor agonist, carbachol (100 nM-1 mM) they evoked an elevation of [Ca2+]i above that caused by carbachol alone. 3. In the presence of 1 microM or 100 microM carbachol, DPDPE elevated [Ca2+]i with an EC50 of 10 nM. The elevation of [Ca2+]i was independent of the concentration of carbachol. The EC50 for DAMGO elevating [Ca2+]i in the presence of 1 microM and 100 microM carbachol was 270 nM and 145 nM respectively. 4. The delta-receptor antagonist, naltrindole (30 nM), blocked the elevations of [Ca2+]i by DPDPE (100 nM) without affecting those caused by DAMGO while the mu-receptor antagonist, CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Pen-Thr-NH2) (100 nM-1 microM) blocked the elevations of [Ca2+]i caused by DAMGO (1 microM) without affecting those caused by DPDPE. 5. Block of carbachol activation of muscarinic receptors with atropine (10 microM) abolished the elevation of [Ca2+]i by the opioids. The nicotinic receptor antagonist, mecamylamine (10 microM), did not affect the elevations of [Ca2+]i caused by opioids in the presence of carbachol. 6. Muscarinic receptor activation, not a rise in [Ca2+]i, was required to reveal the opioid response. The Ca2+ channel activator, maitotoxin (3 ng ml-1), also elevated [Ca2+]i but subsequent application of opioid in the presence of maitotoxin caused no further changes in [Ca2+]i. 7. The elevations of [Ca2+]i by DPDPE and DAMGO were abolished by pretreatment of the cells with pertussis toxin (200 ng ml-1, 16 h). This treatment did not significantly affect the response of the cells to carbachol. 8. The opioids appeared to elevate [Ca2+]i by mobilizing Ca2+ from intracellular stores. Both DPDPE and DAMGO continued to elevate [Ca2+]i when applied in nominally Ca(2+)-free external buffer or when applied in a buffer containing a cocktail of Ca2+ entry inhibitors. Thapsigargin (100 nM), an agent which discharges intracellular Ca2+ stores, also blocked the opioid elevations of [Ca2+]i. 9. delta and mu Opioids did not appear to mobilize intracellular Ca2+ by modulating the activity of protein kinases. The application of H-89 (10 microM), an inhibitor of protein kinase A, H-7 (100 microM), an inhibitor of protein kinase C, protein kinase A and cyclic GMP-dependent protein kinase, or Bis I, an inhibitor of protein kinase C, did not alter the opioid mobilization of [Ca2+]i. 10. Thus, in SH-SY5Y cells, opioids can mobilize Ca2+ from intracellular stores but they require ongoing muscarinic receptor activation. Opioids do not elevate [Ca2+]i when applied alone.     

P2-purinoceptor-mediated formation of inositol phosphates and intracellular Ca2+ transients in human coronary artery smooth muscle cells.

1. The effects of extracellular adenosine 5''-triphosphate (ATP) on smooth muscles are mediated by a variety of purinoceptors. In this study we addressed the identity of the purinoceptors on smooth muscle cells (SMC) cultured from human large coronary arteries. Purinoceptor-mediated increases in [Ca2+]i were measured in single fura-2 loaded cells by applying a digital imaging technique, and the formation of inositol phosphate compounds was quantified after separation on an anion exchange column. 2. Stimulation of the human coronary artery SMC (HCASMC) with extracellular ATP at concentrations of 0.1-100 microM induced a transient increase in [Ca2+]i from a resting level of 49 +/- 21 nM to a maximum of 436 +/- 19 nM. The effect was dose-dependent with an EC50 value for ATP of 2.2 microM. 3. The rise in [Ca2+]i was independent of the presence of external Ca2+, but was abolished after depletion of intracellular stores by incubation with 100 nM thapsigargin. 4. [Ca2+]i was measured upon stimulation of the cells with 0.1-100 microM of the more specific P2-purinoceptor agonists alpha, beta-methyleneadenosine 5''-triphosphate (alpha,beta-MeATP), 2-methylthioadenosine 5''-triphosphate (2MeSATP) and uridine 5''-triphosphate (UTP). alpha, beta-MeATP was without effect, whereas 2MeSATP and UTP induced release of Ca2+ from internal stores with 2MeSATP being the most potent agonist (EC50 = 0.17 microM), and UTP having a potency similar to ATP. The P1 purinoceptor agonist adenosine (100 microM) did not induce any changes in [Ca2+]i. 5. Stimulation with a submaximal concentration of UTP (10 microM) abolished a subsequent ATP-induced increase in [Ca2+]i, whereas an increase was induced by ATP after stimulation with 10 microM 2MeSATP. 6. The phospholipase C (PLC) inhibitor U73122 (5 microM) abolished the purinoceptor-activated rise in [Ca2+]i, whereas pretreatment with the Gi protein inhibitor pertussis toxin (PTX, 500 ng ml-1) was without effect on ATP-evoked [Ca2+]i increases. 7. Receptor activation with UTP and ATP resulted in formation of inositol phosphates with peak levels of inositol 1, 4, 5-trisphosphate (Ins(1, 4, 5)P3) observed 5-20 s after stimulation. 8. These findings show, that cultured HCASMC express G protein-coupled purinoceptors, which upon stimulation activate PLC to induce enhanced Ins(1, 4, 5)P3 production causing release of Ca2+ from internal stores. Since a release of Ca2+ was induced by 2MeSATP as well as by UTP, the data indicate that P2y- as well as P2U-purinoceptors are expressed by the HCASMC.     

Characterization of prostanoid receptors on rat neutrophils.

1. The effects of various prostanoid agonists have been compared on the increase in intracellular free calcium ([Ca2+]i) and the aggregation reaction of rat peritoneal neutrophils induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP). 2. Prostaglandin E2 (PGE2) and the specific IP-receptor agonist, cicaprost, both inhibited the FMLP-induced increase in [Ca2+]i (IC50 33 nM and 18 nM respectively) and the FMLP-induced aggregation reaction (IC50 5.6 nM and 7.9 nM respectively). PGD2, PGF2 alpha, and the TP-receptor agonist, U 46619, were inactive at the highest concentration tested (1 microM). 3. The EP1-receptor agonist, 17-phenyl-omega-trinor PGE2, and the EP3-receptor agonists, GR 63799X and sulprostone, had no inhibitory effect on FMLP-stimulated rat neutrophils. 4. PGE1 (EP/IP-receptor agonist) and iloprost (IP-receptor agonist) inhibited the FMLP-induced increase in [Ca2+]i with IC50 values of 34 nM and 38 nM respectively. The EP2-receptor agonists, butaprost and misoprostol (1 microM), inhibited both FMLP-stimulated [Ca2+]i and aggregation. However another EP2-receptor agonist, AH 13205, was inactive in both assays. 5. Prostanoid receptors present on rat neutrophils were further characterized by measuring [3H]-adenosine 3':5'-cyclic monophosphate ([3H]-cyclic AMP) accumulation. Only those agonists capable of stimulating [3H]-cyclic AMP accumulation were able to inhibit both FMLP-stimulated [Ca2+]i and aggregation. 6. These results indicate that rat neutrophils possess inhibitory IP and EP-receptors; the relative potencies of PGE2, misoprostol and butaprost are those expected for the EP2-receptor subtype. No evidence for DP, FP, TP or EP1 and EP3-receptors was obtained.     

Homologous and heterologous desensitization of histamine H1- and ATP-receptors in the smooth muscle cell line, DDT1MF-2: the role of protein kinase C.

1. The possible role of protein kinase C (PKC) in homologous and heterologous desensitization of histamine H1- and ATP-receptors has been studied in monolayers of cultured vas deferens smooth muscle cells (DDT1MF-2). Cells were loaded with the calcium-sensitive fluorescent dye fura-2 and increases in intracellular free Ca2+ concentration ([Ca2+]i) monitored in response to histamine H1- or ATP-receptor activation. 2. Histamine and ATP stimulated the release of Ca2+ from intracellular Ca2+ stores and Ca2+ influx across the plasma membrane. Activation of PKC with the phorbol ester beta-phorbol-12,13 dibutyrate (PDBu; 1 microM) attenuated histamine (100 microM) and ATP (10 microM)-induced release of intracellular Ca2+ and Ca2+ influx. 3. The selective PKC inhibitor, Ro 31-8220 (10 microM), reversed the PDBu-induced attenuation of histamine (100 microM)-stimulated Ca2+ responses. 4. Histamine H1- and ATP-receptors are readily susceptible to homologous desensitization since short-term exposure to histamine or ATP (450 s) attenuated the Ca2+ responses elicited by a second application of the same agonist. Furthermore, H1-receptor activation-induced heterologous desensitization of ATP stimulated Ca2+ responses and vice versa. 5. Homologous and heterologous desensitization of histamine and ATP Ca2+ responses still occurred in the presence of the PKC inhibitor, Ro 31-8220 (10 microM). 6. These data suggest that PKC activation can attenuate histamine H1- and ATP-receptor mediated Ca2+ responses. However, based on our experimental data, PKC-independent mechanisms appear to be involved in the homologous and heterologous desensitization of histamine H1- and ATP-receptor mediated Ca2+ responses in DDT1MF-2 cells.     

Effect of nortriptyline on intracellular Ca2+ handling and proliferation in human osteosarcoma cells

The effect of the antidepressant nortriptyline, on bone cells is unknown. In human osteosarcoma MG63 cells, the effect of nortriptyline on intracellular Ca2+ concentration ([Ca2+]i) and proliferation was measured by using fura-2 and tetrazolium, respectively. Nortriptyline (> or = 10 microM) caused a [Ca2+]i rise in a concentration-dependent manner (EC50 = 200 microM). Nortriptyline-induced [Ca2+]i rise was prevented by 60% by removal of extracellular Ca2+ but was not altered by voltage-gated Ca2+ channel blockers. In Ca2+ -free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ -ATPase, caused a monophasic [Ca2+]i rise, after which the increasing effect of nortriptyline on [Ca2+]i was abolished; also, pretreatment with nortriptyline abolished thapsigargin-induced [Ca2+]i increase. U73122, an inhibitor of phospholipase C, did not affect nortriptyline-induced [Ca2+]i rise; however, activation of protein kinase C decrease nortriptyline-induced [Ca2+]i rise by 32%. Overnight incubation with 50 and 100 microM nortriptyline killed 78% and 97% of cells, respectively; while 10 microM nortriptyline had no effect. These data suggest that nortriptyline rapidly increases [Ca2+]i in human osteosarcoma cells by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release, and is cytotoxic at high concentrations.