Biomedical subjects

L M Botana

Publications and source records attributed to L M Botana.

At least 73 records · Page 4Linked to original sources

PubMed1994

Effect of ion composition on the changes in membrane potential induced with several stimuli in rat mast cells.

We studied, in different ionic conditions, the effect of various agents on the membrane potential of rat peritoneal mast cells using the fluorescent probe bisoxonol. Ouabain and ionophore A23187 lead to a fast depolarization of the plasma membrane of mast cells, while compound 48/80 and thapsigargin induced membrane hyperpolarization, which was more pronounced in the case of compound 48/80. When using compound 48/80, the amount of gramicidin necessary to depolarize the cells was twice the amount required in resting cells, which indicates that compound 48/80 increases considerably the activity of the Na+/K+ pump. On the other hand, the ionophore A23187 elicited a clear depolarization which was oblated in the absence of intracellular calcium. The increase in the osmolarity of the medium causes a depolarization in the plasma membrane of mast cells. Hypertonicity-stimulated depolarization is inhibited by removing sodium and potassium.

Animals↗

PubMed1994

Differential effects of cAMP-elevating drugs on stimulus-induced cytosolic calcium changes in human basophils.

Drugs that elevate cAMP levels in human basophils are known to inhibit immunoglobulin E (IgE)-mediated histamine release. We have examined whether cAMP-active agents inhibit the cytosolic Ca2+, [Ca2+]i, response that normally accompanies activation of basophils. As previously described, this [Ca2+]i response is biphasic, one phase dependent on internal sources of calcium and a second later phase dependent on extracellular calcium, as observed in many cell types. Forskolin and rolipram or their combination had no effect on the initial elevation of cytosolic calcium that follows stimulation with anti-IgE antibody. In contrast, the second phase of the IgE-mediated calcium response was inhibited by these agents. For IgE-mediated responses, the relative efficacy of various cAMP active agents (rolipram approximately forskolin < dibutyryl cAMP < forskolin + rolipram) for the inhibition of histamine release and the second-phase calcium response was similar and roughly paralleled the measured increase in basophil cAMP. In contrast, neither the first nor the second phase of the f-Met-Leu-Phe (fMLP)-induced calcium response was inhibited by any of the cAMP-active agents tested. Indeed, at low concentrations of fMLP, a combination of forskolin and rolipram caused slight enhancement of the calcium response. This result was consistent with the observations that these agents had no effect or caused slight enhancement of histamine or leukotriene released induced by fMLP. Similarly, cAMP-active agents caused no inhibition of C5a or phorbol ester (phorbol myristate acetate)-induced histamine release. These observations suggest that inhibition of the phase of the calcium response that is dependent on extracellular calcium could account for the inhibition of histamine release by these agents. However, these studies also suggested that (1) this is effect is not exerted at the level of the inositol trisphosphate (InsP3) receptor or InsP3 metabolism and (2) the mechanisms that maintain the second-phase calcium response are possibly distinct for IgE- and fMLP-mediated reactions because cAMP-active agents inhibited the second-phase response of only one stimulus.

1-Methyl-3-isobutylxanthine↗

PubMed1994

Study of the activation mechanism of adriamycin on rat mast cells.

Adriamycin (ADR) induces nonimmunological and noncytotoxic histamine release from peritoneal and pleural rat mast cells. This secretion is unaffected by the pretreatment with pertussis toxin, cholera toxin and benzalkonium chloride. Histamine release induced by compound 48/80, was markedly inhibited by pertussis toxin, cholera toxin, benzalkonium chloride and neuraminidase. The ADR dose-response curve is significantly shifted to the right when cells were preincubated with the unspecific phosphodiesterase inhibitor IBMX. The activation of protein kinase C (PKC) with the phorbol esther TPA increases the response to ADR, while PKC inhibition with trifluoperazine decreases histamine release. The pretreatment of mast cells with okadaic acid did not modify the response to ADR. These results suggest that ADR elicits histamine release with a mechanism notably different from compound 48/80.

1-Methyl-3-isobutylxanthine↗

PubMed1994

Effect of lyophilization on the stability of gonyautoxins obtained from contaminated mussels.

This study describes the stability of gonyautoxins (GTX) and C toxins obtained from contaminated mussels and stored at different temperatures in lyophilized samples. Analyses of extracts from mussels contaminated with paralytic shellfish poison (PSP) indicated the presence of gonyautoxins as the major component in red tides of the North-West coast of Spain. These GTX and C toxins were extracted from contaminated mussels (Mytilus galloprovincialis Lmk) and partially purified by chromatography on Bio-Gel P-2 and Bio-Rex 70. The stability of these toxins was analysed by high performance liquid chromatography. GTX 4 and GTX 6 are the most stable toxins among GTX. We conclude that the lyophilization procedure is not the safest way to process most of the gonyautoxins. However, the lyophilization procedure made the C toxins unstable, so clearly this procedure must be rejected.

Animals↗

PubMed1994

Comparative study of the stability of saxitoxin and neosaxitoxin in acidic solutions and lyophilized samples.

Paralytic shellfish poison (PSP) has historically been a problem for the shellfish industry. In order to prevent the marketing of contaminated seafood products, governments have implemented monitoring programs where standards of toxins are necessary. The stability of these standard toxins is very important. In this paper we analysed the stability of saxitoxin (STX) and neosaxitoxin in acidic solution and lyophilized samples. Individual toxins were determined in each sample using a high-performance liquid chromatographic procedure employing post-column oxidation of the toxins to form fluorescent derivatives. Our results demonstrate that STX is very stable in solution samples and could be adopted as a reference standard. This toxin can be kept in dilute acidic solutions for 18 months without loss of potency. However, neosaxitoxin is unstable, possibly due to transformation to other toxins.

Animals↗

PubMed1993

Effect of okadaic acid on human basophil secretion.

We examined the effects of a phosphatase inhibitor, okadaic acid, on mediator secretion from human basophils. These cells are known to respond to a number of stimuli that appear to utilize distinct biochemical pathways converging on mediator release. Okadaic acid was found to inhibit IgE-mediated release (histamine release inhibited 80 +/- 12% and leukotriene release inhibited 100% following a 10-min preincubation with okadaic acid and stimulation with an optimal concentration of anti-IgE antibody) at a concentration of 1 microM. The concentration-response curve to okadaic acid was steep, with 0.1 microM yielding only 20 +/- 10% inhibition of either mediator. Secretion following stimulation with the univalent stimulus, fMet-Leu-Phe peptide, was not inhibited by okadaic acid. Unlike cAMP-active agents that inhibit cytosolic Ca2+ elevations following IgE-mediated stimulation, the increased state of cellular protein phosphorylation, which presumably results from treatment with 1 microM okadaic acid, had no effect on the elevations in free cytosolic Ca2+ that follow stimulation with anti-IgE antibody of fMet peptide.

Basophils↗

PubMed1993

Solid-phase radioreceptor assay for paralytic shellfish toxins.

Sodium channels obtained from rat brain membrane preparations were coated onto microtiter plates and used to develop a direct solid-phase binding assay. The tritiated sodium channel blocker saxitoxin ([3H]-saxitoxin; STX) was used to detect toxins in paralytic shellfish poisoning (PSP) by measuring the competitive displacement of other toxins. With this assay the amount of STX and tetrodotoxin needed to displace 50% of bound [3H]STX was 1.7 and 1.76 ng/ml for buffer samples, respectively. In the direct solid-phase binding assays, the PSP toxins were effectively bound to the rat brain membranes. The IC50 of this assay for different PSP toxin solutions obtained from mussels contaminated in red tides ranged from 0.03 to 0.30 ng/ml. Therefore, this assay represents a potentially useful method for the detection of toxin-contaminated mussels.

Animals↗

PubMed1993

Biphasic Ca2+ responses in human basophils. Evidence that the initial transient elevation associated with the mobilization of intracellular calcium is an insufficient signal for degranulation.

Human basophils exhibited biphasic cytosolic Ca2+ responses after stimulation with both the bacterial peptide, FMLP and IgE cross-linking agents such as goat polyclonal anti-IgE PS myeloma (anti-IgE) antibody and Ag. The biphasic response to anti-IgE antibody was not apparent unless supraoptimal concentrations of anti-IgE antibody were used to stimulate the cells. Biphasic response characteristics were also observed in single cells. As in many cell types, the first phase of the Ca2+ response appeared independent of extracellular Ca2+. Challenging basophils with anti-IgE in the presence of EGTA, added simultaneously or 3 to 5 min before the stimulus, had little effect on the first transient Ca2+ elevation while completely eliminating the second phase of the response. We noted that while simultaneous addition of EGTA had little effect on the histamine release after challenge with FMLP, there was no histamine release after the simultaneous addition of anti-IgE and EGTA. An examination of the kinetics of histamine release after anti-IgE also demonstrated that no histamine release occurs during the initial transient response. Since the initial Ca2+ transient was intact after both stimuli in the presence of EGTA, we concluded that the initial transient was not sufficient to induce histamine release. The hypothesis that distinct sources of calcium were responsible for the initial Ca2+ transient that followed challenge with FMLP peptide or anti-IgE antibody was also tested. We found that prior treatment of basophils with thapsigargin inhibited the initial Ca2+ response that followed both FMLP and anti-IgE antibody. Furthermore, stimulation with FMLP, in the presence of EGTA, depleted the calcium source responsible for the anti-IgE response. The experiment conducted in reverse order showed that anti-IgE antibody could discharge the calcium source responsible for the initial FMLP Ca2+ response. These experiments indicated that FMLP and anti-IgE antibody use the same internal source of calcium to produce the initial Ca2+ response. Taken together with previous studies on FMLP, anaphylatoxin split product of the C component, C5; and platelet activating factor-induced release in human basophils, these results suggest that the initial transient elevation, which is relatively independent of extracellular Ca2+, is not sufficient to induce degranulation.

Basophils↗

PubMed1993

Amiloride-dependent transport is the main mechanism implicated in sodium influx regulation in rat mast cells.

Mast cell sodium regulation is a largely unknown field. In our effort to study the mechanisms by which mast cells regulate sodium levels, we have examined the effect of amiloride and ouabain on 22Na entry in rat mast cells in isotonic and hypertonic conditions. Ouabain (0.5 mM) enhances sodium uptake by 32% in isotonic conditions. Hypertonicity increases by 400% the uptake of sodium through an amiloride (1 mM) dependent mechanism. Ouabain has no appreciable effect on the entry of 22Na in hypertonic conditions.

Amiloride↗

PubMed1993

Histamine release on rat pleural and peritoneal mast cells elicited by human GRF(1-29)NH2.

We have studied the release of histamine elicited by human GRF(1-29)NH2 on rat peritoneal and pleural mast cells. The secretion is temperature dependent, the release being optimal at 37 degrees C, and it can be inhibited with antimycin A. The activation of pleural and peritoneal mast cells is similar, but there is a slight inhibition for both populations after purification with Percoll.

Animals↗

PubMed1993

Rat pleural and peritoneal mast cells stimulated at different cellular levels: difference in and influence of purification media.

We checked the effect that purification through different media can produce on rat mast cell response, considering different populations of pleural and peritoneal mast cells. We compared the media Ficoll, Percoll, bovine serum albumin and sucrose with the seldom used Nycodenz. Histamine release was elicited with compound 48/80, polymyxin B, ionophore A23187 and calcium on cells preincubated with FNa. High osmolarity media induced the most important changes on the cell response as compared to low osmolarity media. Our results show that Ficoll and sucrose induced the most notable changes in the response. Also, the more sensitive stimulus to the purification procedure is sodium fluoride-calcium. The response to ionophore A23187 did not show any important alteration.

Animals↗

PubMed1992

Effect of purification, theophylline and sodium fluoride on histamine release produced by antineoplastic drugs on rat mast cells. A distinctive mechanism of action for carboplatin.

The antineoplastic drugs, cytarabine, ifosfamide, vinorelbine, doxorubicin, asparaginase and carboplatin, elicit histamine release from rat pleural and peritoneal mast cells. Both cell populations do not show heterogeneity in their response to stimulation by any of these drugs, with the exception of cytarabine, which activates pleural mast cells at lower concentrations. When these cells are purified with Percoll, L-asparaginase, vinorelbine and cytarabine completely lose their capability of inducing histamine release in both cell populations, while the other drugs still show the same pattern of response as with unpurified cells. These results indicate that some membrane component crucial for the action of vinorelbine, L-asparaginase and cytarabine is lost during the purification procedure. Pretreatment of the cells with theophylline completely inhibits the response to cytarabine, ifosfamide, vinorelbine and asparaginase, and inhibits the response to doxorubicin by up to 75% only. Theophylline does not change the response to carboplatin. Sodium fluoride does not change the response to any of the drugs tested, with the exception of carboplatin, in which case a complete inhibition is observed. In conclusion, carboplatin activates rat mast cells through a completely different mechanism of action with respect to the other drugs studied.

Animals↗

PubMed1992

The human recombinant c-kit receptor ligand, rhSCF, induces mediator release from human cutaneous mast cells and enhances IgE-dependent mediator release from both skin mast cells and peripheral blood basophils.

The gene product of the steel locus of the mouse represents a growth factor for murine mast cells and a ligand for the c-kit proto-oncogene receptor, a member of the tyrosine kinase receptor class of oncogenes (for review, see O. N. Witte. 1990. Cell 63:5). We have studied the effect of the human recombinant c-kit receptor ligand stem cell factor (rhSCF) on the release of inflammatory mediators from human skin mast cells and peripheral blood basophils and compared its activity to that of rhIL-3, rhSCF (1 ng/ml to 1 microgram/ml) activated the release of histamine and PGD2 from mast cells isolated from human skin. Analysis by digital video microscopy indicated that purified human skin mast cells (84 +/- 5% pure) responded to rhSCF (0.1 to 1 microgram/ml) challenge with a rapid, sustained rise in intracellular Ca2+ levels that was accompanied by secretion of histamine. A brief preincubation (10 min) of mast cells with rhSCF (0.1 pg/ml to 1 ng/ml) significantly enhanced (100 +/- 35%) the release of histamine induced by anti-IgE (3 micrograms/ml), but was much less effective on IgE-mediated release of PGD2. In contrast, a short term incubation with rhSCF did not potentiate the secretion of histamine activated by substance P (5 microM). A 24-h incubation of mast cells with rhSCF did not affect the release of mediators induced by anti-IgE (3 micrograms/ml), probably due to receptor desensitization, rhSCF (1 ng/ml to 3 micrograms/ml) neither caused release of histamine or leukotriene C4 (LTC4) release from leukocytes of 14 donors, nor induced a rise in intracellular Ca2+ levels in purified (greater than 70%) basophils. Brief preincubation (10 min) of leukocytes with rhSCF (1 ng/ml to 3 micrograms/ml) caused an enhancement (69 +/- 11%) of anti-IgE-induced release of histamine that was significant at concentrations as low as 3 ng/ml (p less than 0.05), whereas it appeared less effective in potentiating IgE-mediated LTC4 release. In contrast, a prolonged incubation (24 h) with rhSCF (0.1 pg/ml to 100 ng/ml) did not enhance the release of histamine or LTC4 induced by anti-IgE (0.1 microgram/ml), whereas rhIL-3 (3 ng/ml) significantly potentiated the release of both mediators.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Monoclonal↗

PubMed1992

Non-immunological release of histamine from rat mast cells elicited by antineoplastic agents.

We studied the histamine-releasing activity of several antineoplastic drugs on rat pleural and peritoneal mast cells. The drugs tested included the nitrogen mustards cyclophosphamide and ifosfamide, the nitrosourea carmustine, the triazene dacarbazine, the folic acid analogue methotrexate, the pyrimidine analogue cytarabine and fluorouracil, the vinca alkaloids vinblastine, vincristine and Vinorelbine, the epipodophyllotoxins etoposide and teniposide, and the enzyme L-asparaginase. Methotrexate, carmustine, fluorouracil, vinblastine and vincristine failed to elicit histamine release on rat mast cells. All of the other drugs evoked histamine release in both the presence and the absence of extracellular calcium, but ifosfamide, cytarabine and asparaginase induced a much lower release in the absence of this cation. The response elicited by cytarabine and etoposide was much higher in pleural than in peritoneal mast cells. These results indicate that some antineoplastic drugs may directly activate the release of histamine, which could contribute to some of their secondary effects.

Animals↗

PubMed1992

Nonimmunological release of histamine from rat mast cells elicited by antineoplastic agents: effect of drug combinations.

We studied the release of histamine elicited by some antineoplastic drugs in rat pleural and peritoneal mast cells. The drugs tested included the antibiotic mitomycin; the anthracyclines daunorubicin, doxorubicin, and epirubicin; the glycopeptide bleomycin; the chromopeptide dactinomycin; the platinum coordination complexes carboplatin and cisplatin; and the anthracenedione mitoxantrone. Mitomycin and bleomycin failed to elicit any release of histamine, whereas all of the other drugs induced histamine release from either pleural or peritoneal mast cells, the release being independent of extracellular calcium. The results indicate that antineoplastic drugs activate histamine release in a manner similar to that shown by compound 48/80, which may contribute to some of their secondary effects.

Animals↗

PubMed1992

Influence of protein kinase C, cAMP and phosphatase activity on histamine release produced by compound 48/80 and sodium fluoride on rat mast cells.

We have studied the effect of protein kinase C and protein kinase A activation, and phosphatase inhibition on two different stimuli with distinct mechanisms of action. The first stimulus is compound 48/80, and its action is mediated probably by a Gi-protein, while the other is sodium fluoride, which unspecifically activates G-proteins. We established a comparative study because the action of compound 48/80 is calcium-independent, while fluoride is strictly calcium-dependent. The activation of protein kinase C was attained with the phorbol esther 12-O-tetradecanoylphorbol-13-acetate, protein kinase A was activated by increasing cAMP levels with forskolin or rolipram, and the phosphatase activity was inhibited with okadaic acid (OA), which inhibits phosphatases type 1 and 2A. Our results show that OA enhances the response to fluoride and compound 48/80 in the absence of calcium, and we conclude that calcium has a negative feedback role on the cell response. Protein kinase A activation strongly inhibits the response to fluoride, and the results show a positive regulation of protein kinase C and a negative regulation of protein kinase A over fluoride response. As previously reported by other authors for the ionophore A23187, TPA notably potentiates the response to fluoride, which supports its possible modulatory role on extracellular calcium-dependent stimuli.

Animals↗

PubMed1991

Lack of specific saxitoxin binding to rat mast cells.

We studied whether or not mast cells are endowed with specific sodium channels, by using tritiated saxitoxin which binds to site 1 of sodium channels on excitable tissues. Our results suggest that rat pleural and peritoneal mast cells lack specific sodium channels.

Animals↗

PubMed1991

Changes in rat mast cell responses in sodium-free media. Lack of demonstrable sodium channel activity.

Exposure of rat mast cells to isotonic sucrose (employed as a sodium free medium) increased several-fold the sensitivity to calcium, which itself became a stimulus for exocytosis. Concentrations of the cation as low as 25 microM permitted maximal histamine release. Preincubation of cells in sucrose to allow sodium efflux before adding the ionophore A23187 led to a slower release of histamine. We postulate that sodium efflux can generate a membrane potential that causes the increased sensitivity to calcium and the delay in response after preincubation. The response to A23187 is somewhat unspecific since the ionophore can release histamine from internal calcium reservoirs. Saxitoxin or veratridine did not affect cell responses, so that sodium activity is not mediated through defined sodium channels.

Animals↗