Incubation In Ca2 Research Articles

Modulation of agonist-induced Ca 2+ release by SR Ca 2+ load: direct SR and cytosolic Ca 2+ measurements in rat uterine myocytes

Release of Ca 2+ from sarcoplasmic reticulum (SR) is one of the most important mechanisms of smooth muscle stimulation by a variety of physiologically active substances. Agonist-induced Ca 2+ release is considered to be dependent on the Ca 2+ content of the SR, although the mechanism underlying this dependence is unclear. In the present study, the effect of SR Ca 2+ load on the amplitude of [Ca 2+] i transients elicited by application of the purinergic agonist ATP was examined in uterine smooth muscle cells isolated from pregnant rats. Measurement of intraluminal Ca 2+ level ([Ca 2+] L) using a low affinity Ca indicator, mag-fluo-4, revealed that incubation of cells in a high-Ca 2+ (10 mM) extracellular solution leads to a substantial increase in [Ca 2+] L (SR overload). However, despite increased SR Ca 2+ content this did not potentiate ATP-induced [Ca 2+] i transients. Repetitive applications of ATP in the absence of extracellular Ca 2+, as well as prolonged incubation in Ca 2+–free solution without agonist, depleted the [Ca 2+] L (SR overload). In contrast to overload, partial depletion of the SR substantially reduced the amplitude of Ca 2+ release. ATP-induced [Ca 2+] i transients were completely abolished when SR Ca 2+ content was decreased below 80% of its normal value indicating a steep dependence of the IP 3-mediated Ca 2+ release on the Ca 2+ load of the store. Our results suggest that in uterine smooth muscle cells decrease in the SR Ca 2+ load below its normal resting level substantially reduces the IP 3-mediated Ca 2+ release, while Ca 2+ overload of the SR has no impact on such release.

Warming and response to contractile agents in calf cardiac vein: Role of the Ca2+, KCa2+ and Na+ ion channel blockers

The effects of warming (to 41 degrees C) on the serotonin (5-HT, 10(-8)-3 x 10(-3) M)- and carbachol (10(-)9-3 x 10(-4) M)-induced contractions and the role of calcium (Ca2+), potassium (K+), and sodium (Na+) channel blockers, in the warming-induced responses were investigated in the calf cardiac vein. Concentration-response curves to 5-HT and carbachol were isometrically recorded at 37 and 41 degrees C (control). The same procedure was repeated at 41 degrees C in the presence of verapamil (10(-6) M), caffeine (3 x 10(-4) M), tetraethylammonium (TEA, 10(-3)M), flecainide (10(-6) M), and also in the Ca2+-free medium with ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA). During warming, the sensitivity, but not the maximal response, was significantly higher. Warming to 41 degrees C after treatment with verapamil or flecainide decreased the sensitivity, whereas treatment with caffeine increased the sensitivity significantly. Treatment with TEA did not modify the effect of warming. Furthermore, warming to 41 degrees C after incubation in Ca2+-free solutions with EGTA decreased the sensitivity to 5-HT and carbachol. The results of this study suggest the role for Ca2+ and Na+ ions in the warming-induced changes of cardiac vein treated with 5-HT and carbachol.

The role of calcium in the nuclear maturation of Bufo arenarum oocytes.

In Bufo arenarum, progesterone is the physiological maturation inducer. However, in this species, oocytes reinitiate meiosis with no need of an exogenous hormonal stimulus when deprived of their enveloping cell, a phenomenon called spontaneous maturation. We demonstrated that in Bufo arenarum spontaneous maturation occurs only in oocytes obtained during the reproductive period, which can be considered competent to mature spontaneously, in contrast to those in the non-reproductive period, which are incompetent. Interestingly, full-grown Bufo arenarum oocytes always respond to progesterone regardless of the season in which they are obtained. There is a general consensus that both a transient increase in intracellular calcium and a decrease in cAMP-dependent protein kinase activity are the first steps in the mechanisms by which progesterone induces maturation in amphibians. In the present work we analysed the role of calcium in the spontaneous and progesterone-induced maturation of Bufo arenarum oocytes. Results demonstrated that the absence of calcium in the incubation medium or the prevention of Ca(2+) influx by channel blockers such as CdCl2 or NiCl2 did not prevent meiosis reinitiation in either type of maturation. The inhibition of the Ca(2+)-calmodulin complex in no case affected the maturation of the treated oocytes. However, when the oocytes were deprived of calcium by incubation in Ca(2+)-free AR + A23187, meiosis resumption was inhibited. In brief, we demonstrated that in Bufo arenarum the reinitiation of meiosis is a process independent of extracellular calcium at any period of the year and that oocytes require adequate levels of intracellular calcium for germinal vesicle breakdown to occur.

ACTH stimulates insulin secretion from MIN6 cells and primary mouse and human islets of Langerhans.

It has previously been suggested that ACTH and ACTH-related peptides may act as paracrine modulators of insulin secretion in the islets of Langerhans. We have, therefore, examined the expression and function of the ACTH receptor (the melanocortin 2 receptor, MC2-R) in human and mouse primary islet tIssue and in the MIN6 mouse insulinoma cell line. Mouse MC2-R mRNA was detected in both MIN6 cells and mouse islet tIssue by PCR amplification of cDNA. In perifusion experiments with MIN6 pseudo-islets, a small, transient increase in insulin secretion was obtained when ACTH(1-24) (1 nM) was added to medium containing 2 mM glucose (control) but not when the medium glucose content was increased to 8 mM. Further investigations were performed using static incubations of MIN6 cell monolayers; ACTH(1-24) (1 pM-10 nM) provoked a concentration-dependent increase in insulin secretion from MIN6 monolayer cells that achieved statistical significance at concentrations of 1 and 10 nM (150 +/- 13.6% basal secretion; 187 +/- 14.9% basal secretion, P<0.01). Similar responses were obtained with ACTH(1-39). The phosphodiesterase inhibitor IBMX (100 microM) potentiated the responses to sub-maximal doses of ACTH(1-24). Two inhibitors of the protein kinase A (PKA) signaling pathway, Rp-cAMPS (500 microM) and H-89 (10 microM), abolished the insulin secretory response to ACTH(1-24) (0.5-10 nM). Treatment with 1 nM ACTH(1-24) caused a small, statistically significant increase in intracellular cAMP levels. Secretory responses of MIN6 cells to ACTH(1-24) were also influenced by changes in extracellular Ca2+ levels. Incubation in Ca2+-free buffer supplemented with 0.1 mM EGTA blocked the MIN6 cells' secretory response to 1 and 10 nM ACTH(1-24). Similar results were obtained when a Ca2+ channel blocker (nitrendipine, 10 microM) was added to the Ca2+-containing buffer. ACTH(1-24) also evoked an insulin secretory response from primary tIssues. The addition of ACTH(1-24) (0.5 nM) to perifusions of mouse islets induced a transient increase in insulin secretion at 8 mM glucose. Perifused human primary islets also showed a secretory response to ACTH(1-24) at basal glucose concentration (2 mM) with a rapid initial spike in insulin secretion followed by a decline to basal levels. Overall the results demonstrate that the MC2-R is expressed in beta-cells and suggest that activation of the receptor by ACTH initiates insulin secretion through the activation of PKA in association with Ca2+ influx into beta-cells.

In Vitro Mineralisation of Chitosan Membranes Carrying Phosphate Functionalities

Squid chitosan membranes were phosphorylated through the H3PO 4/P 2O5/Et 3PO 4/butanol method. P-chitosan membranes were immersed in Ca(OH) 2 or NaOH solutions, in order to obtain the Na or the Ca salts, respectively. These materials were investigated regarding their ability to nucleate calcium phosphates, under simulated physiologic conditions. SEM-EDS studies revealed the presence of a calcium phosphate mineral layer all over the sur face of P-chitosan membranes, after incubation in Ca(OH) 2 solution. The release of ionically bound phosphate functionalities under alkaline conditions, possibly contributed to the formation of calcium phosphate precursor sites, due to the chelation of calcium ions from solution. During the immersion in Simulated Body Fluid (SBF), a multilayer porous mineral structure composed of poorly crys talline carbonated apatite was formed on the surface of these membranes, as shown by EDS and ATR-FTIR analysis. Unmodified membranes and P-chitosan membranes pre-incubated in NaOH solution did not mineralise.

Hydrogen peroxide contributes to motor dysfunction in ulcerative colitis

Ulcerative colitis (UC) affects colonic motor function, but the mechanism responsible for this motor dysfunction is not well understood. We have shown that neurokinin A (NKA) may be an endogenous neurotransmitter mediating contraction of human sigmoid colonic circular muscle (HSCCM). To elucidate factors responsible for UC motor dysfunction, we examined the role of hydrogen peroxide (H(2)O(2)) in the decrease of NKA-induced response of HSCCM. As previously demonstrated, NKA-induced contraction or Ca(2+) increase of normal muscle cells is mediated by release of Ca(2+) from intracellular stores, because it was not affected by incubation in Ca(2+)-free medium (CFM) containing 200 microM BAPTA. In UC, however, CFM reduced both cell contraction and NKA-induced Ca(2+) increase, suggesting reduced Ca(2+) release from intracellular stores. In normal Ca(2+) medium, NKA and KCl caused normal Ca(2+) signal in UC cells but reduced cell shortening. The decreased Ca(2+) signal and contraction in response to NKA or thapsigargin were partly recovered in the presence of H(2)O(2) scavenger catalase, suggesting involvement of H(2)O(2) in UC-induced dysmotility. H(2)O(2) levels were higher in UC than in normal HSCCM, and enzymatically isolated UC muscle cells contained much higher levels of H(2)O(2) than normal cells, which were significantly reduced by catalase. H(2)O(2) treatment of normal cells in CFM reproduced the reduction of NKA-induced Ca(2+) release observed in UC cells. In addition, H(2)O(2) caused a measurable, direct release of Ca(2+) from intracellular stores. We conclude that H(2)O(2) may contribute to reduction of NKA-induced Ca(2+) release from intracellular Ca(2+) stores in UC and contribute to the observed colonic motor dysfunction.

Dopamine D 2 receptor-induced COX-2-mediated production of prostaglandin E 2 in D 2-transfected Chinese hamster ovary cells without simultaneous administration of a Ca 2+-mobilizing agent

We have earlier demonstrated that dopamine stimulates the liberation of the prostaglandin E 2 (PGE 2) precursor, arachidonic acid, in Chinese hamster ovary cells transfected with the rat dopamine D 2 receptor (long isoform), also without concomitant administration of a Ca 2+-releasing agent [Nilsson et al., Br J Pharmacol 1998;124:1651–8]. In the present report, we show that dopamine, under the same conditions, also induces a concentration-dependent increase in the production of PGE 2, with a maximal effect of 235% at ∼100 μM, and with an ec 50 of 794 nM. The effect was counteracted by the D 2 antagonist eticlopride, pertussis toxin, the inhibitor of intracellular Ca 2+ release TMB-8, incubation in Ca 2+-free experimental medium, and PKC desensitization obtained by chronic pretreatment with the phorbol ester TPA. It was also antagonized by the non-specific cyclooxygenase (COX) inhibitor, indomethacin, and by the selective COX-2 inhibitor, NS-398, but not by the specific COX-1 inhibitor, valeryl salicylate. Both the non-specific phospholipase A 2 inhibitor, quinacrine, and an inhibitor of cPLA 2 and iPLA 2, AACOF3, counteracted the effect; in contrast, a selective iPLA 2 inhibitor, BEL, and a selective sPLA 2 inhibitor, TAPC, were ineffective. No effects of dopamine were obtained in control cells mock-transfected with the p3C vector only. The results reinforce previous assumptions that dopamine may interact with eicosanoid metabolism by means of D 2 receptor activation, and implicate an involvement of cPLA 2 and COX-2 in this effect. It is suggested that measurement of dopamine-induced PGE 2 production may serve as a convenient way to study D 2 receptor function in vitro.

Glucose-stimulated oscillations in free cytosolic ATP concentration imaged in single islet beta-cells: evidence for a Ca2+-dependent mechanism.

Normal glucose-stimulated insulin secretion is pulsatile, but the molecular mechanisms underlying this pulsatility are poorly understood. Oscillations in the intracellular free [ATP]/[ADP] ratio represent one possible mechanism because they would be expected to cause fluctuations in ATP-sensitive K(+) channel activity and hence oscillatory Ca(2+) influx. After imaging recombinant firefly luciferase, expressed via an adenoviral vector in single human or mouse islet beta-cells, we report here that cytosolic free ATP concentrations oscillate and that these oscillations are affected by glucose. In human beta-cells, oscillations were observed at both 3 and 15 mmol/l glucose, but the oscillations were of a longer wavelength at the higher glucose concentration (167 vs. 66 s). Mouse beta-cells displayed oscillations in both cytosolic free [Ca(2+)] and [ATP] only at elevated glucose concentrations, both with a period of 120 s. To explore the causal relationship between [Ca(2+)] and [ATP] oscillations, the regulation of each was further investigated in populations of MIN6 beta-cells. Incubation in Ca(2+)-free medium lowered cytosolic [Ca(2+)] but increased [ATP] in MIN6 cells at both 3 and 30 mmol/l glucose. Removal of external Ca(2+) increased [ATP], possibly by decreasing ATP consumption by endoplasmic reticulum Ca(2+)-ATPases. These results allow a model to be constructed of the beta-cell metabolic oscillator that drives nutrient-induced insulin secretion.

Calcium-dependent smooth muscle excitatory effect elicited by the venom of the hydrocoral Millepora complanata

In the present paper, we describe the results obtained from a preliminary pharmacological and biochemical study of the fire coral Millepora complanata, a regular component of coral reefs in the Mexican Caribbean. The protein-containing crude extract obtained from M. complanata (tested from 0.001 to 1000 μg protein/ml) caused a concentration-dependent stimulation of spontaneous contractions of the guinea pig ileum. The extract (EC 50=11.55±2.36 μg/ml) was approximately 12-fold less potent than ionomycin (EC 50=0.876±0.25 μg/ml) and its maximum induced contraction (1 mg protein/ml) was equivalent to 68% of the response to 60 mM KCl. FPLC size exclusion chromatography of the M. complanta extract afforded 12 primary fractions, of which only FV (containing proteins with molecular weights ranging from 17 to 44 kDa) and FVIII (consisting of peptides with molecular weights lesser than 1.8 kDa) elicited an excitatory effect when tested at the EC 50 of the original extract. After incubation in Ca 2+-free medium, the ileal response to FV and FVIII was significantly reduced. Blockage of L-type Ca 2+ channels with nifedipine (1 μM) inhibited FV and FVIII-evoked contractions. Cd 2+ (10 μM), an unspecific blocker of voltage-activated calcium channels, also antagonized FV and FVIII-induced effects, whereas the Na + channel blocker tetrodotoxin (10 nM) did not significantly affect FV and FVIII responses. These results suggest that the contractions induced by the bioactive fractions obtained from the crude extract of M. complanata are caused mainly by a direct action on smooth muscle cells, via an increase in Ca 2+ permeability that occurs, at least partly, through L-type voltage-dependent Ca 2+ channels found in the cell membrane of smooth muscle.

Cooling-induced gastrointestinal smooth muscle contractions in the rat.

The aim of this study was to assess the effect of cooling on smooth muscle contraction in various parts of the gastrointestinal tract (esophagus, stomach, duodenum, jejunum and colon) and to investigate the basic mechanism underlying cooling-induced (CIC) tonic and rhythmic contractions. Recordings of isometric tension from smooth muscle strips of different parts of the rat gastrointestinal tract were performed using organ-bath techniques, and stepwise cooling was applied. Cooling was tested before and after the addition of various standard agents interfering with known neurogenic (autonomic blockers, tetrodotoxin, capsaicin) and myogenic mechanisms of contraction (calcium channel blockers, Sarcoplasmatic and Ca2+-ATPase pump inhibitors). Step-wise cooling (37 degrees C to 5 degrees C) of all gastrointestinal smooth muscle preparations induced reproducible graded tonic contractions, inversely proportional to temperature. CIC was most pronounced in the jejunum. Cooling abolished rhythmic smooth muscle activity. CIC was not dependent on a neural mechanism nor the release of neurotransmitters, but linked to translocation of calcium. It was reduced by incubation in Ca2+-free solution. Blockage of the Ca2+-ATPase pump, which inhibits the extrusion of calcium, plays a significant role in the process and enhances CIC. Cooling of gastrointestinal smooth muscle preparations induces graded myogenic contractions inversely proportional to the temperature. The mechanism is not dependent on local nervous control but related to a temperature-sensitive process of calcium translocation.

Calmodulin effect on purified rat cortical plasma membrane Ca 2+-ATPase in different phosphorylation states

The plasma membrane Ca 2+-ATPase in neuronal tissue plays an important role in fine tuning of the intracellular Ca 2+ concentration. The enzyme exhibits a high degree of tissue specificity and is regulated by several mechanisms. Here we analysed the relationship between separate modes of Ca 2+-ATPase regulation, i.e., reversible phosphorylation processes mediated by protein kinases A and C, protein phosphatases PP1 and PP2A, and stimulation by calmodulin. The activity of PKA- or PKC-phosphorylated Ca 2+-ATPase was influenced by the further addition of calmodulin, and this effect was more pronounced for PKC-phosphorylated calcium pump. In both cases the fluorescence study revealed the increased calmodulin binding, and for PKA-mediated phosphorylation it was correlated with a higher affinity of calcium pump for calmodulin. The incubation of Ca 2+-ATPase with CaM prior to protein kinases action revealed that CaM presence counteracts the stimulatory effect of PKA and PKC. Under the in vitro assay cortical Ca 2+-ATPase was a substrate for PP1 and PP2A. Protein phosphatases decreased both the basal activity of Ca 2+-ATPase and its affinity for calmodulin. Fluorescence analysis confirmed the lowered ability of dephosphorylated Ca 2+-ATPase for calmodulin binding. These results may suggest that interaction of CaM with calcium pump and its stimulatory action could be a partly separate phenomenon that is dependent on the phosphorylation state of Ca 2+-ATPase.

Intracellular survival of Brucella spp. in human monocytes involves conventional uptake but special phagosomes.

Brucella spp. are facultative intracellular parasites of various mammals, including humans, typically infecting lymphoid as well as reproductive organs. We have investigated how B. suis and B. melitensis enter human monocytes and in which compartment they survive. Peripheral blood monocytes readily internalized nonopsonized brucellae and killed most of them within 12 to 18 h. The presence of Brucella-specific antibodies (but not complement) increased the uptake of bacteria without increasing their intracellular survival, whereas adherence of the monocytes or incubation in Ca(2+)- and Mg(2+)-free medium reduced the uptake. Engulfment of all Brucella organisms (regardless of bacterial viability or virulence) initially resulted in phagosomes with tightly apposed walls (TP). Most TP were fully fusiogenic and matured to spacious phagolysosomes containing degraded bacteria, whereas some TP (more in monocyte-derived macrophages, HeLa cells, and CHO cells than in monocytes) remained tightly apposed to intact bacteria. Immediate treatment of infected host cells with the lysosomotropic base ammonium chloride caused a swelling of all phagosomes and a rise in the intraphagosomal pH, abolishing the intracellular survival of Brucella. These results indicate that (i) human monocytes readily internalize Brucella in a conventional way using various phagocytosis-promoting receptors, (ii) the maturation of some Brucella phagosomes is passively arrested between the steps of acidification and phagosome-lysosome fusion, (iii) brucellae are killed in maturing but not in arrested phagosomes, and (iv) survival of internalized Brucella depends on an acidic intraphagosomal pH and/or close contact with the phagosomal wall.

Cellular mechanisms involved in iso-osmotic high K + solutions-induced contraction of the estrogen-primed rat myometrium

The aim of the present study was to investigate the mechanisms involved in the contraction evoked by iso-osmotic high K + solutions in the estrogen-primed rat uterus. In Ca 2+-containing solution, iso-osmotic addition of KCl (30, 60 or 90 mM K +) induced a rapid, phasic contraction followed by a prolonged sustained plateau (tonic component) of smaller amplitude. The KCl (60 mM)-induced contraction was unaffected by tetrodotoxin (3 μM), ω-conotoxin MVIIC (1 μM), GF 109203X (1 μM) or calphostin C (3 μM) but was markedly reduced by tissue treatment with neomycin (1 mM), mepacrine (10 μM) or U-73122 (10 μM). Nifedipine (0.01–0.1 μM) was significantly more effective as an inhibitor of the tonic component than of the phasic component. After 60 min incubation in Ca 2+-free solution containing 3 mM EGTA, iso-osmotic KCl did not cause any increase in tension but potentiated contractions evoked by oxytocin (1 μM), sodium orthovanadate (160 μM) or okadaic acid (20 μM) in these experimental conditions. In freshly dispersed myometrial cells maintained in Ca 2+-containing solution and loaded with indo 1, iso-osmotic KCl (60 mM) caused a biphasic increase in the intracellular Ca 2+ concentration ([Ca 2+] i). In cells superfused for 60 min in Ca 2+-free solution containing EGTA (1 mM), KCl did not increase [Ca 2+] i. In Ca 2+-containing solution, KCl (60 mM) produced a 76.0 ± 16.2% increase in total [ 3H]inositol phosphates above basal levels and increased the intracellular levels of free arachidonic acid. These results suggest that, in the estrogen-primed rat uterus, iso-osmotic high K + solutions, in addition to their well known effect on Ca 2+ influx, activate other cellular processes leading to an increase in the Ca 2+ sensitivity of the contractile machinery by a mechanism independent of extracellular Ca 2+.

Stimulatory effect of enkephalins on calcium efflux from bovine adrenal chromaffin cells in culture

The effects of leucine- and methionine-enkephalin, opiate peptides, on Ca 2+ efflux from cultured bovine adrenal chromaffin cells were examined. These enkephalins stimulated the efflux of 45Ca 2+ from cells in a concentration-dependent manner (10 −8M − 10 −6M). Leucine-enkephalin did not increase the intracellular free Ca 2+ level, 45Ca 2+ uptake, catecholamine secretion, cAMP level or cGMP level. The peptide-stimulated 45Ca 2+ efflux was not inhibited by incubation in Ca 2+-free medium, but was inhibited by incubation in Na +-free medium. These results indicate that enkephalins stimulate extracellular Na +-dependent 45Ca 2+ efflux from cultured bovine adrenal chromaffin cells, probably by stimulating membrane Na +/Ca 2+ exchange.

Fusobacterium necrophorum haemolysin stimulates motility of ileal longitudinal smooth muscle of the guinea-pig.

Fusobacterium necrophorum haemolysin (0.5-3.1 mg protein/mL) dose-dependently induced contractions of the isolated ileal longitudinal smooth muscle of the guinea-pig. The haemolysin (3.1 mg protein/mL) -induced maximum contraction of 75% of the response to 60 mM K+ declined within 17 min and the muscles then demonstrated rhythmic contractions. Tetrodotoxin (3.1 x 10(-6) M) had no effect on the contraction due to the haemolysin. After incubation in Ca(2+)-free medium, the ileal response to the haemolysin was lost. Verapamil, a Ca2+ channel blocker, dose-dependently inhibited the contraction to the haemolysin. The rabbit anti-serum against F. necrophorum haemolysin inhibited the haemolysin-induced contraction of ileal muscle. The bacterial haemagglutinin and the lipopolysaccharide had no effect on the response of ileal muscle. These findings suggest that the haemolysin-induced direct stimulation of ileal motility dependant on Ca2+ influx will increase the probability of contact of F. necrophorum and ileal mucosa and could increase the chances of colonization for F. necrophorum.

Thimerosal increases the responsiveness of the calcium receptor in human parathyroid and rMTC6–23 cells

Parathyroid cells express a plasma membrane calcium receptor (CaR), which is stimulated by a rise in extracellular calcium concentration ([Ca2+]ext). A decreased sensitivity to [Ca2+]ext occurs in adenomatous parathyroid cells in patients with primary hyperparathyroidism, but the underlying functional mechanism is not yet fully understood. This study explored whether CaR responsiveness is influenced by increasing the affinity of IP3receptors – a major signalling component of other G-protein-coupled receptors.The sulphydryl reagent thimerosal was used to increase the responsiveness of IP3-receptors. Quantitative fluorescence microscopy in Fura-2-loaded cells was used to investigate the effects of thimerosal on the cytoplasmic calcium concentrations ([Ca2+]i) in human parathyroid cells and to compare its effects in a rat medullary thyroid carcinoma cell line (rMTC6-23) also expressing CaR.During incubation in Ca2+-free medium, thimerosal 5 μM induced a rapid sustained rise in [Ca2+]iin human parathyroid cells and no further [Ca2+]iincrease appeared in response to the CaR agonist Gd3+(100 μM). Thimerosal 1 μM induced only slow and minimal changes of basal [Ca2+]iand allowed a rapid response to Gd3+20 nM (a concentration without effect in control cells). The slope of the thimerosal-induced [Ca2+]iresponses was steeper following exposure to CaR agonists. In the presence of 1 mM [Ca2+]ext, thimerosal (0.5 μM) induced a sharp increase in [Ca2+]ito a peak (within 60 s), followed either by return to basal [Ca2+]ior by a plateau of slightly higher amplitude. Similar results were obtained using rMTC6–23 cells.Thimerosal increases the responsiveness to CaR agonists through modulation of the sensitivity of the IP3receptor in both parathyroid and rMTC6–23 cells.

Strontium supports human sperm capacitation but not follicular fluid-induced acrosome reaction.

The ability of strontium (Sr(2+)) to replace calcium (Ca(2+)) in maintaining human sperm function has still not been completely characterized. In the present study, acrosome reaction (AR) inducibility in response to human follicular fluid (hFF) was compared in spermatozoa incubated in either Ca(2+)- or Sr(2+)-containing media. Other events related to sperm capacitation, such as protein tyrosine phosphorylation and hyperactivation as well as zona pellucida (ZP) recognition under both conditions, were also analyzed. Spermatozoa incubated overnight in the presence of Sr(2+) were unable to undergo the AR when exposed to hFF. Nevertheless, when spermatozoa were incubated under this condition and then transferred to medium with Ca(2+), sperm response to hFF was similar to that of cells incubated throughout in the presence of Ca(2+). The sperm protein tyrosine phosphorylation patterns and the percentages of sperm motility and hyperactivation were similar after incubation in Ca(2+)- or Sr(2+)-containing media. Under both conditions, the same binding capacity to homologous ZP was observed. Similar results were obtained when EGTA was added in order to chelate traces of Ca(2+) present in Sr(2+) medium. From these results, it can be concluded that Sr(2+) can replace Ca(2+) in supporting capacitation-related events and ZP binding, but not hFF-induced AR of human spermatozoa.

Endothelin receptors and calcium translocation pathways in human airways.

Tension and phosphatidyl inositol (PI) turnover experiments were conducted to investigate the receptors and signal transduction pathways responsible for contractions elicited by endothelin (ET) ligands in human bronchus. Nicardipine (1 microM), the L-type calcium channel inhibitor, or incubation in Ca2+-free medium, produced marked inhibition of contractions to the ET(B) receptor-selective agonist, sarafotoxin S6c, and especially those induced by KCl. In contrast, Ca2+-free medium was without appreciable effect against contraction produced by endothelin-1 (ET-1), the non-selective ET(A) and ET(B) receptor agonist. In Ca2+-free medium, ryanodine (10 microM), which inhibits intracellular calcium mobilization, reduced sarafotoxin S6c- and ET-1-induced responses, but was without effect on responses to KCl. Similarly, nickel chloride (Ni2+; 1 mM) caused marked inhibition of contractions induced by sarafotoxin S6c or ET-1, but had no significant effect on KCI concentration-response curves. The mixed ET(A)/ET(B) receptor antagonist SB 209670 (3 microM) inhibited responses to sarafotoxin S6c and ET-1 such that concentration-response curves were shifted rightward, at the 30% maximum response level, by 10.0- and 3.8-fold, respectively, whereas BQ-123 (3 microM), the ET(A) receptor antagonist, was without effect on responses induced by either agonist. ET-1 (1 nM-0.3 microM) caused a concentration-dependent stimulation of PI turnover, whereas sarafotoxin S6c (0.3 nM-0.1 microM) induced only small and variable increases, except at the highest concentration. The increase in PI turnover evoked by ET-1 was inhibited by SB 209670 (3 microM), and also by BQ-123 (3 microM). This is consistent with linkage of ET(A) receptors to activation of inositol phosphate generation in human bronchial smooth muscle cells. Collectively, the data suggest that differences exist in the relative contributions of intracellular and extracellular Ca2+ mobilization mechanisms elicited by ET(A) and ET(B) receptor activation. Thus, sarafotoxin S6c-induced, ET(B) receptor-mediated contraction in human bronchial smooth muscle appears to be dependent, in part, upon extracellular Ca2+, although a significant component of the response was also mediated by intracellular Ca2+ release, including from ryanodine-sensitive stores. ET(A) receptor-mediated contraction of human airway smooth muscle was activated largely via the release of intracellular Ca2+.

COOLING-INDUCED CONTRACTION IN OVINE AIRWAYS SMOOTH MUSCLE

The mechanism of cold-induced bronchoconstriction is poorly understood. This prompted the present study whose aim was to determine the step-wise direct effect of cooling on smooth muscle of isolated ovine airways and analyse the role of calcium in the mechanisms involved. Isolated tracheal strips and bronchial segments were suspended in organ baths containing Krebs' solution for isometric tension recording. Tissue responses during stepwise cooling from 37 to 5°C were examined. Cooling induced a rapid and reproducible contraction proportional to cooling temperature in ovine tracheal and bronchial preparations which was epithelium-independent. On readjustment to 37°C the tone returned rapidly to basal level. Maximum contraction was achieved at a temperature of 5°C for trachea and 15°C for bronchiole. Cooling-induced contractions (CIC) was resistant to tetrodotoxin (1; 10 μm), and not affected by the muscarinic antagonist atropine (1 μm) or the α-adrenergic antagonist phentolamine (1 μm), or the histamine H1-antagonist mepyramine (1 μm) or indomethacin (1 μm). Ca2+antagonists (nifedipine and verapamil) and Mn2+raised tracheal but not bronchiolar tone and augmented CIC. Incubation in Ca2+-free, EGTA-containing Krebs' solution for 5 min had no effect on CIC, although it significantly reduced KCl-induced contraction by up to 75%. Cooling inhibited Ca2+influx measured using45Ca2+uptake. Caffeine (100 μm) significantly inhibited CIC. The results show that cooling-induced contractions do not appear to involve activation of nerve endings, all surface reception systems or Ca2+influx. However, CIC is mainly dependent on release of intracellular Ca2+.

Measurements of ooplasmic pCa in fertilized eggs of the locust, Locusta migratoria, and a role for external calcium in experimental activation of unfertilized eggs

Summary The role of Ca2+ in activation and early development of locust eggs was examined through measurement of ooplasmic Ca2+ levels before and after fertilization, and through experimental activation of unfertilized eggs. Ooplasmic pCa (i.e. the negative logarithm of Ca2+ activity) measured in intact eggs decreased from 5.35 before fertilization, to 4.77 and 3.00 by 1 day and 3 days after fertilization, respectively. pCa was also determined for samples of ooplasm collected by rupturing eggs under paraffin oil. The pCa was 5.10 in ooplasm isolated from unfertilized eggs, and 3.84 in ooplasm collected from eggs within 4 h of fertilization. Ooplasmic pCa remained between 3.97 and 3.12 from 1–6 days after fertilization. Since a decline in pCa indicates an increase in ooplasmic Ca2+ activity, the data suggest that regulation of ooplasmic Ca2+ during post-fertilization development involves release of Ca2+ from internal stores. Experimental egg activation was examined in eggs dissected from the oviducts before fertilization and incubated on moist filter paper. Some eggs were first immersed in experimental solutions for 30–60 minutes before incubation. The presence of an embryo 2 or 4 days after fertilization or experimental treatment was used as an indicator of egg activation. Activation occurred in 92% and 12% of fertilized and untreated eggs, respectively. The percentage of unfertilized eggs which activated increased to 47% if eggs were soaked 30–60 minutes in physiological saline, and to as much as 65%-68% if eggs were injected with Ca2+ buffers or if a Ca2+ action potential was evoked. Up to 36% and 42% of unfertilized eggs activated after incubation in Ca2+-free salines or in the presence of the Ca2+-channel blocker Cd2+, respectively. Taken together, the results suggest that entry of external Ca2+ through voltage dependent channels increases the proportion of eggs which activate, but is not an absolute requirement for activation.