Biomedical subjects

A Alfonso

Publications and source records attributed to A Alfonso.

At least 37 records · Page 2Linked to original sources

PubMed1999

Breathing pattern in highly competitive cyclists during incremental exercise.

The purpose of our investigation was to analyse the breathing patterns of professional cyclists during incremental exercise from submaximal to maximal intensities. A group of 11 elite amateur male road cyclists [E, mean age 23 (SD 2) years, peak oxygen uptake (VO2peak) 73.8 (SD 5.0) ml kg(-1) min(-1)] and 14 professional male road cyclists [P, mean age 26 (SD 2) years, (VO2peak) 73.2 (SD 6.6) ml kg(-1) min(-1)] participated in this study. Each of the subjects performed an exercise test on a cycle ergometer following a ramp protocol (exercise intensity increases of 25 W x min(-1)) until the subject was exhausted. For each subject, the following parameters were recorded during the tests: oxygen consumption (VO2), carbon dioxide output (VCO2), pulmonary ventilation (VE), tidal volume (VT), breathing frequency (fb), ventilatory equivalents for oxygen (VE x VO2(-1)) and carbon dioxide (VE x VCO2(-1)), end-tidal partial pressure of oxygen and partial pressure of carbon dioxide, inspiratory (tI) and expiratory (tE) times, inspiratory duty cycle (tI/tTOT, where tTOT is the time for one respiratory cycle), and mean inspiratory flow rate (VT/tI). Mean values of VE were significantly higher in E at 300, 350 and 400 W (P < 0.05, P < 0.05 and P < 0.01, respectively); fb was also higher in E in most moderate-to-maximal intensities. On the other hand, VT showed a different pattern in both groups at near-to maximal intensities, since no plateau was observed in P. The response of tI and tE was also different. Finally, VT/tI and tI/tTOT showed a similar response in both P and E. It was concluded that the breathing pattern of the two groups differed mainly in two aspects: in the professional cyclists, VE increased at any exercise intensity as a result of increases in both VT and fb, with no evidence of tachypnoeic shift, and tE was prolonged in this group at high exercise intensities. In contrast, neither the central drive nor the timing component of respiration seem to have been significantly altered by the training demands of professional cycling.

Adult↗

PubMed1999

Imaging synaptic activity in intact brain and slices with FM1-43 in C. elegans, lamprey, and rat.

The fluorescent probe FM1-43 has been used extensively for imaging vesicle recycling; however, high nonspecific adsorption resulting in elevated background levels has precluded its use in certain tissues, notably brain slices. We have found that a sulfobutylated derivative of beta-cyclodextrin (ADVASEP-7) has a higher affinity for FM1-43 than the plasma membrane. ADVASEP-7 was used as a carrier to remove FM1-43 nonspecifically bound to the outer leaflet of the plasma membrane or extracellular molecules, significantly reducing background staining. This has enabled us to visualize synaptic vesicle recycling in the nematode C. elegans, intact lamprey spinal cord, and rat brain slices.

Animals↗

PubMed1999

UNC-11, a Caenorhabditis elegans AP180 homologue, regulates the size and protein composition of synaptic vesicles.

The unc-11 gene of Caenorhabditis elegans encodes multiple isoforms of a protein homologous to the mammalian brain-specific clathrin-adaptor protein AP180. The UNC-11 protein is expressed at high levels in the nervous system and at lower levels in other tissues. In neurons, UNC-11 is enriched at presynaptic terminals but is also present in cell bodies. unc-11 mutants are defective in two aspects of synaptic vesicle biogenesis. First, the SNARE protein synaptobrevin is mislocalized, no longer being exclusively localized to synaptic vesicles. The reduction of synaptobrevin at synaptic vesicles is the probable cause of the reduced neurotransmitter release observed in these mutants. Second, unc-11 mutants accumulate large vesicles at synapses. We propose that the UNC-11 protein mediates two functions during synaptic vesicle biogenesis: it recruits synaptobrevin to synaptic vesicle membranes and it regulates the size of the budded vesicle during clathrin coat assembly.

Adaptor Proteins, Vesicular Transport↗

PubMed1999

Characterization of the Na+/Ca2+ exchanger on rat mast cells. Evidence for a functional role on the regulation of the cellular response.

The Na+/Ca2+ exchanger has not been characterized in rat mast cells, although its existence has been previously suggested. In this work, we determine that this exchanger exists on rat mast cells and that it has an important regulatory role on the cellular function. We have studied uptake and release of 45Ca in the presence of different external sodium concentrations and, under the same conditions, the simultaneous uptake of 22Na and 45Ca. The results show that uptake and release of 45Ca in these cells are related to the concentration of sodium in the extracellular medium and that there is also a perfect coupling between 22Na and 45Ca fluxes. In these conditions, we evaluated the intracellular calcium levels in fura-2 loaded cells. When the extracellular sodium concentration was lower than 60 mM, we observed an increase in intracellular calcium, reaching its maximum when the extracellular medium has no sodium. Then we investigated the effect on histamine release. The ionophore A23187 elicits histamine release in rat mast cells, depending on the extracellular calcium concentration. This drug releases more histamine (up to twofold) with sodium concentrations <60 mM. In the presence of 2,4-dichlorobenzamil hydrochloride, a Na+/Ca2+ exchanger inhibitor, the release of histamine induced by the ionophore was lower than in controls in media with low external sodium, and, on the contrary, at extracellular sodium concentrations >60 mM, the histamine release was higher than in controls. In the same conditions, but when the Na+-K+ ATPase was inhibited by ouabain, and as a consequence more sodium was inside the cells, a high increase in histamine release induced by A23187 in a sodium-free medium was observed. Under the same conditions, a high increase in intracellular calcium takes place. The overall data are preliminary evidence suggesting the existence of a Na+/Ca2+ exchanger in rat mast cells with a threshold to get reversed at 60 mM external sodium, lower concentrations of this ion increasing internal calcium and producing higher histamine release.

Amiloride↗

PubMed1998

Synthesis and antiallergic activity of pyridothienopyrimidines.

The synthesis of a series of pyridothienopyrimidines and their evaluation as inhibitors or inducers of the release of histamine from rat mast cells is reported. The activity was measured after immunological stimulation with ovoalbumin and chemical stimulation with polymer 48/80 and the drugs adryamicin and vinorelbine. The experiments were carried out with and without preincubation of the stimulus with the cells before addition of the drug. Several pyridothienopyrimidines show inhibitory IC50 values in the range 2-25 microM, indicating they are up to 100 times more potent than cromoglycate (DSCG) and 10 times greater than Ketotifen. Compound 9l is a potent inhibitor in all the conditions tested and shows IC50 = 9-25 microM. Pyridothienopyrimidines 4l and 9e are very strong inducers of histamine release in the immunological (4l, 170-230%) and chemical (9e, 100-150%) assays, respectively. Compounds 4l and 9i are cytotoxic in vitro (IC50 = 0.1-0.2 microgram/mL) against P-388, A-549, HT-29, and MEL-28 tumor cell lines.

Animals↗

PubMed1998

Construction of a whole-genome radiation hybrid panel for high-resolution gene mapping in pigs.

We have developed a panel of 152 whole-genome radiation hybrids by fusing irradiated diploid pig lymphocytes or fibroblasts with recipient hamster permanent cells. The number and size of the porcine chromosome fragments retained in each hybrid clone were checked by fluorescence in situ hybridization with a SINE probe or by primed in situ labeling (PRINS) with SINE-specific primers. A strategy based on the interspersed repetitive sequence polymerase chain reaction (IRS-PCR) was developed for selected clones to determine if the large fragments painted by the SINE probe corresponded to one pig chromosome or to different fragments of several chromosomes. This strategy was buttressed by a double PRINS approach using primers specific for alpha-satellite sequences of two different groups of swine chromosomes. Genome retention frequency was estimated for each clone by PCR with 32 markers localized on different porcine chromosomes. Of the 152 hybrids produced, 126 were selected on the basis of cytogenetic content and chromosome retention frequency to construct a radiation hybrid map of swine chromosome 8. Our initial results for this chromosome indicate that the resolution of the radiation hybrid map is 18 times higher than that obtained by linkage analysis.

Animals↗

PubMed1998

Sodium, PMA and calcium play an important role on intracellular pH modulation in rat mast cells.

In a series of experiments aimed to understand the signaling pathways that regulate intracellular pH (pHi) in rat mast cells, the effect of different intracellular mechanisms on the activity of the Na+/H+ exchanger was studied. After promoting an artificial acidification with sodium propionate we determined the variations on pHi rate recovery. pHi was measured with the dye 2, 7-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester. We studied the effect that the inhibition of some cellular exchangers with different drugs induced on pHi. When the Na+/H+ exchanger was inhibited in the presence of amiloride, the recovery rate constant was twofold smaller than the control value. After the recovery, the final pH was lower than the initial value when the cells were treated either with amiloride or with 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid (an anionic antiport inhibitor). No effect was observed when the Na+/K+-ATPase or the Na+/Ca2+ exchanger were inhibited. The suppression of intracellular and extracellular calcium did not induced any change in pHi. The addition of thapsigargin, an activator of capacitative calcium influx, or the phorbol esther 12-O-tetradecanoylphorbol-13-acetate (PMA), a protein kinase C (PKC) activator, increased the activity of the antiporter. Both effects were abrogated by inhibition of the Na+/K+-ATPase with ouabain. The increase in cAMP levels did not affect the effect of PMA on pHi recovery, but it blocked the effect of thapsigargin. Our results indicate that rat mast cells regulate pHi by the combination of some anionic exchanger and the Na+/H+ antiporter. And also that the modulation of this exchanger is the consequence of the connection between different intracellular mechanisms, Na+/K+-ATPase-PKC-calcium, among which cAMP seems not to have a direct role.

1-Methyl-3-isobutylxanthine↗

PubMed1997

Recovery of Ca2+ pools and growth in Ca2+ pool-depleted cells is mediated by specific epoxyeicosatrienoic acids derived from arachidonic acid.

Depletion of Ca2+ pools using the irreversible Ca2+ pump blocker, thapsigargin, induces DDT1MF-2 smooth muscle cells to enter a stable nonproliferative state. Reversal of this state can be mediated by high (20%) serum treatment, which induces new Ca2+ pump protein, return of Ca2+ pools, and reentry of cells into the cell cycle; the effect of serum can be mimicked by the essential fatty acids (EFA), arachidonic, linoleic, and alpha-linolenic acids (Graber, M.N., Alfonso, A., and Gill, D.L., (1996) J. Biol. Chem. 271, 883-888). The possible requirement for EFA metabolism in inducing recovery of Ca2+ pool-depleted growth-arrested cells was investigated. Neither cyclooxygenase or lipoxygenase inhibitors had any effect on arachidonic acid-induced growth recovery of thapsigargin-treated cells. In contrast, the cytochrome P-450 epoxygenase inhibitors, SKF525A and metyrapone, substantially reduced arachidonic acid-induced recovery of growth while having minimal effects on control cell growth. Both epoxygenase inhibitors completely prevented the arachidonic acid-induced recovery of bradykinin-releasable Ca2+-pumping pools, whereas cyclooxygenase and lipoxygenase inhibitors had no effect. The effectiveness of the four cytochrome P-450 metabolites of arachidonic acid on recovery of Ca2+ pools were compared; 8,9- and 11,12-epoxyeicosatrienoic acid (EET) at 1.5 microM were completely effective in recovering agonist-sensitive Ca2+ pools, whereas the 5,6- and 14,15-EETs were without effect. SKF525A did not block the action of 8,9- or 11, 12-EET indicating further P-450 metabolism was not required. Hydration of the active EET molecules prevented Ca2+ pool recovery since the dihydroxy-derivatives of both 8,9- and 11,12-EET were ineffective. The specificity of effectiveness among EET molecules for subsequent resumption of growth of thapsigargin-treated cells was the same as for Ca2+ pool recovery. Significantly, the P-450 inhibitors, SKF525A and metyrapone, both prevented the action of 20% serum in inducing recovery of thapsigargin-treated cells, whereas cyclooxygenase and lipoxygenase inhibitors were ineffective, indicating that EFAs are the active component within serum that is responsible for recovery of Ca2+ pool-depleted cells. The specific action of EETs in mediating recovery of Ca2+ pools and growth of thapsigargin-treated cells represents not only a novel action of epoxygenase products from EFAs, but also a potentially significant new signaling pathway that may effect translational control and regulate transition from a stationary to proliferative growth state.

8,11,14-Eicosatrienoic Acid↗

PubMed1997

The antineoplastic drug vinorelbine activates non-immunological histamine release from rat mast cells.

OBJECTIVE AND DESIGN: We explore the mechanism of the antineoplastic drug vinorelbine activation in its rat mast cell exocytosis. MATERIALS: The study was carried out on mast cells obtained from Sprague-Dawley rats. TREATMENT: Vinorelbine (5-100 micrograms/mL), cholera toxin (200 ng/mL), pertussis toxin (100 ng/mL), benzalkonium chloride (10 micrograms/mL), compound 48/80 (1 microgram/mL), okadaic acid (1 microM), 12-tetradecanoate-acetate (50 ng/ml), perphenazine (1 microgram/ml), theophylline (10 mM), IBMX (1 mM), rolipram (15 microM). METHODS: Histamine release was measured fluorimetrically. RESULTS: The drugs that modify G-protein activity, cholera toxin, pertussis toxin or benzalkonium chloride, do not modify the response profile. The exocytosis elicited by compound 48/80 is decreased by Gs or Gi modulation, which suggests that G proteins are not involved in vinorelbine stimulated secretion. The phosphatase inhibitor okadaic acid shows no effect on vinorelbine-stimulated release, nor on the activation or inhibition of protein kinase C with phorbol 12-tetradecanoate-acetate or perphenazine. The unspecific phosphodiesterase inhibitors theophylline and IBMX inhibited histamine release, but not the phosphodiesterase IV inhibitor rolipram. CONCLUSIONS: The overall results show that vinorelbine activates histamine release through a rather selective mechanism that may be mediated by certain phosphodiesterase-dependent transduction pathways.

Animals↗

PubMed1996

A genetic selection for Caenorhabditis elegans synaptic transmission mutants.

We have isolated 165 Caenorhabditis elegans mutants, representing 21 genes, that are resistant to inhibitors of cholinesterase (Ric mutants). Since mutations in 20 of the genes appear not to affect acetylcholine reception, we suggest that reduced acetylcholine release contributes to the Ric phenotype of most Ric mutants. Mutations in 15 of the genes lead to defects in a gamma-aminobutyric acid-dependent behavior; these genes are likely to encode proteins with general, rather than cholinergic-specific, roles in synaptic transmission. Ten of the genes have been cloned. Seven encode homologs of proteins that function in the synaptic vesicle cycle: two encode cholinergic-specific proteins, while five encode general presynaptic proteins. Two other Ric genes encode homologs of G-protein signaling molecules. Our assessment of synaptic function in Ric mutants, combined with the homologies of some Ric mutants to presynaptic proteins, suggests that the analysis of Ric genes will continue to yield insights into the regulation and functioning of synapses.

Acetylcholine↗

PubMed1996

Ca2+ pools and cell growth: arachidonic acid induces recovery of cells growth-arrested by Ca2+ pool depletion.

The intracellular Ca2+ pump blocker, thapsigargin, induces emptying of Ca2+ pools and entry of DDT1MF-2 smooth muscle cells into a quiescent G(0)-like growth state. Although thapsigargin blocks pumps essentially irreversibly, high serum (20%) induces appearance of new pump protein, return of functional pools, and reentry of cells into the cell cycle (Waldron, R. T., Short, A. D., Meadows, J.J., Ghosh, T. K., and Gill, D. L. (1994) J. Biol. Chem. 269, 11927-11933). Through analysis of the effects of defined serum components and growth supplements, we reveal here that the factors in serum responsible for inducing recovery of Ca2+ pools and growth in thapsigargin-arrested DDT1MF-2 cells are exactly mimicked by the three essential fatty acids, arachidonic, linoleic, and alpha-linolenic acids. The EC50 values for arachidonic and linoleic acids on growth induction of thapsigargin-arrested cells were the same, approximately 5 microM. Nonessential fatty acids, including myristic, palmitic, stearic, oleic, and arachidic acids, were without any effect. Although not proven to be the active component of serum, levels of arachidonic and linoleic acids in serum were sufficient to explain serum-induced growth recovery. Significantly, arachidonic or linoleic acids induced complete recovery of bradykinin-sensitive Ca2+ pools within 6 h of treatment of thapsigargin-arrested cells. Protein synthesis inhibitors (cycloheximide or puromycin) completely blocked the appearance of serum-induced or arachidonic acid-induced agonist-sensitive pools. The sensitivity and fatty acid specificity of Ca2+ pool recovery in thapsigargin-arrested cells were almost identical to that for growth recovery. No pool or growth recovery was observed with 5,8,11,14-eicosatetraynoic acid, the nonmetabolizable analogue of arachidonic acid, suggesting that conversion to eicosanoids underlies the pool and growth recovery induced by essential fatty acids. The results provide not only further information on the link between Ca2+ pools and cell growth but also evidence for a potentially important signaling pathway involved in inducing transition from a stationary to a proliferative growth state.

Animals↗

PubMed1996

Calcium pools, calcium entry, and cell growth.

The Ca2+ pump and Ca2+ release functions of intracellular Ca2+ pools have been well characterized. However, the nature and identity of Ca2+ pools as well as the physiological implications of Ca2+ levels within them, have remained elusive. Ca2+ pools appear to be contained within the endoplasmic reticulum (ER); however, ER is a heterogeneous and widely distributed organelle, with numerous other functions than Ca2+ regulation. Studies described here center on trying to determine more about subcellular distribution of Ca2+ pools, the levels of Ca2+ within Ca2+ pools, and how these intraluminal Ca2+ levels may be physiologically related to ER function. Experiments utilizing in situ high resolution subcellular morphological analysis of ER loaded with ratiometric fluorescent Ca2+ dyes, indicate a wide distribution of inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pools within cells, and large changes in the levels of Ca2+ within pools following Insp3-mediated Ca2+ release. Such changes in Ca2+ may be of great significance to the translation, translocation, and folding of proteins in ER, in particular with respect to the function of the now numerously described luminal Ca(2+)-sensitive chaperonin proteins. Studies have also focussed on the physiological role of pool Ca2+ changes with respect to cell growth. Emptying of pools using Ca2+ pump blockers can result in cells entering a stable quiescent G(o)-like growth state. After treatment with the irreversible pump blocker, thapsigargin, cells remain in this state until they are stimulated with essential fatty acids whereupon new pump protein is synthesized, functional Ca2+ pools return, and cells re-enter the cell cycle. During the Ca2+ pool-depleted growth-arrested state, cells express a Ca2+ influx channel that is distinct from the store-operated Ca2+ influx channels activated after short-term depletion of Ca2+ pools. Overall, these studies indicate that significant changes in intraluminal ER Ca2+ do occur and that such changes appear linked to alteration of essential ER functions as well as to the cell cycle-state and the growth of cells.

Animals↗

PubMed1995

A novel Ca2+ entry mechanism is turned on during growth arrest induced by Ca2+ pool depletion.

Ca2+ pool depletion with Ca2+ pump blockers induces growth arrest of rapidly dividing DDT1MF-2 smooth muscle cells and causes cells to enter a stable, quiescent G0-like growth state (Short, A.D., Bian, J., Ghosh, T.K., Waldron, R.T., Rybak, S.L., and Gill, D.L. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 4986-4990). Here we reveal that induction of this quiescent growth state with the Ca2+ pump blocker, thapsigargin, is correlated with the appearance of a novel caffeine-activated Ca2+ influx mechanism. Ca2+ influx through this mechanism is clearly distinct from and additive with Ca2+ entry through store-operated channels (SOCs). Whereas SOC-mediated entry is activated seconds after Ca2+ pool release, caffeine-sensitive influx requires at least 30 min of pool emptying. Although activated in the 1-10 mM caffeine range, this mechanism has clearly distinct methylxanthine specificity from ryanodine receptors and is not modified by ryanodine. It is also unaffected by the Ca2+ channel blockers SKF96365 or verapamil and is independent of modifiers of cyclic nucleotide levels. Growth arrest by thapsigargin-induced Ca2+ pool depletion can be reversed by treatment with 20% serum (Waldron, R.T., Short, A.D., Meadows, J.J., Ghosh, T.K., and Gill, D.L. (1994) J. Biol. Chem. 269, 11927-11933). The serum-induced return of functional Ca2+ pools and reentry of cells into the cell cycle correlates exactly with the disappearance of the caffeine-sensitive Ca2+ influx mechanism. Therefore, appearance and function of this novel Ca2+ entry mechanism are closely tied to Ca2+ pool function and cell growth state and may provide an important means for modifying exit from or entry into the cell cycle.

Animals↗

PubMed1995

Study of the activation mechanism of human GRF(1-29)NH2 on rat mast cell histamine release.

Human growth releasing factor (GRF) (1-29)NH2 releases histamine from pleural and peritoneal rat mast cells by a non cytotoxic and non immunological mechanism. Pretreatment of cells with pertussis toxin markedly inhibits the secretion, suggesting a possible function of a Gi-protein in the activation pathway. In order to determine the role of cAMP on GRF mediated secretion, mast cells were preincubated with isobutylmethylxanthine (IBMX) or cholera toxin, since both drugs greatly and enhance cAMP levels. IBMX inhibits mediator secretion while, in contrast, cholera toxin is ineffective to modify histamine release. The PKC activator TPA amplifies the response of mast cells to human GRF, shifting the dose-response curve to the left. The pretreatment of mast cells with the phosphatase inhibitor okadaic acid exerts no effect on the dose-response function curve to GRF. The response to human GRF does not depend on extracellular calcium, but there is a good correlation between the percent of histamine released and 45calcium uptake. The kinetic of calcium uptake is fast, maximum uptake being reached in 30 seconds.

1-Methyl-3-isobutylxanthine↗

PubMed1995

Determination of phosphodiesterase activity in rat mast cells using the fluorescent cAMP analogue anthraniloyl cAMP.

Cyclic AMP and the isozyme families that control its concentration have an important role in rat mast cells. We have attempted to determine the total phosphodiesterase activity in rat mast cells by means of specific and non-specific inhibitors of phosphodiesterases. We used a fluorescent analogue of cAMP, 2'-O-anthraniloyl cAMP, the fluorescence intensity of which decreases when hydrolysed by phosphadiesterase (PDE), providing a measurement of total activity of PDE. The PDE inhibitors produced a decrease in the fluorescence fall. Therefore, we can establish that at least Type I, III, IV and probably Type V PDE are present in rat mast cells. We have also studied the effect of these PDE inhibitors on histamine release elicited by compound 48/80 and sodium fluoride. Chlorpromazine, a Type I PDE inhibitor, only slightly inhibits the fluoride-evoked response, while, on the other hand, milrinone, a Type III PDE inhibitor, does not modify the response to compound 48/80.

Affinity Labels↗

PubMed1995

Caenorhabditis elegans mutants resistant to inhibitors of acetylcholinesterase.

We characterized 18 genes from Caenorhabditis elegans that, when mutated, confer recessive resistance to inhibitors of acetylcholinesterase. These include previously described genes as well as newly identified genes; they encode essential as well as nonessential functions. In the absence of acetylcholinesterase inhibitors, the different mutants display a wide range of behavioral deficits, from mild uncoordination to almost complete paralysis. Measurements of acetylcholine levels in these mutants suggest that some of the genes are involved in presynaptic functions.

Acetylcholine↗

PubMed1994

Alternative splicing leads to two cholinergic proteins in Caenorhabditis elegans.

The cha-1 gene of Caenorhabditis elegans encodes choline acetyl-transferase (the acetylcholine synthetic enzyme). The C. elegans unc-17 gene encodes a synaptic vesicle-associated acetylcholine transporter. The two genes thus define sequential biochemical steps in the metabolism of the neurotransmitter acetylcholine. Cloning, sequencing, and molecular analysis of the unc-17 region indicate that cha-1 and unc-17 transcripts share a 5' untranslated exon, and the rest of the unc-17 transcript is nested within the long first intron of cha-1. Thus, two proteins with related functions but with no sequences in common are produced as a result of alternative splicing of a common mRNA precursor. The structure of this transcription unit suggests a novel type of coordinate gene expression, and a temporal processing model is proposed for the regulation of cha-1 and unc-17 expression.

Alleles↗

PubMed1994

Effect of signal transduction pathways on the action of thapsigargin on rat mast cells. Crosstalks between cellular signalling and cytosolic pH.

Thapsigargin elicits histamine release on rat mast cells, and this effect is increased if cells are pretreated with thapsigargin before the addition of external calcium. Okadaic acid does not modify the response of mast cells to thapsigargin, while sodium fluoride or the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) increases several fold the sensitivity of cells to thapsigargin. On the other hand, pertussis and cholera toxins inhibit the response to thapsigargin. Thapsigargin increases the activity of the Na(+)-H+ exchanger, this effect being blocked by fluoride and not modified by TPA. The metals cadmium and lanthanum completely block the effect of TPA or thapsigargin on the Na(+)-H+ exchanger. The influx of 45Ca in rat mast cells is not modified by thapsigargin, but if cells are treated with thapsigargin before the addition of calcium, the influx is markedly increased in the first 2 min before returning to normal. Our results indicate that exocytosis is modulated by crosstalks between intracellular calcium, cytosolic pH and external calcium.

Animals↗