Ca-free Medium Research Articles

Mechanisms of calcium transport in human endothelial cells subjected to hyperthermia

We have probed the mechanisms by which severe heat alters cytosolic calcium ion concentrations (Cai) in individual cultured human endothelial cells (ATCC ♯1998). Cells adhering to glass coverslips were heated to as high as 50°C and Cai determined by means of a fluorescence laser imaging system using the calcium-sensitive dye, indo-1, in the presence of thapsigargin, and in Na-free and Ca-free media. Baseline Cai varied between 175–225 nM. When cells were heated to 50°C in a complete Ca-containing medium, there was first a transient fall in Cai, then a rapid rise of 50–100 nM in Cai, followed by a slower, secondary rise of 50–75 nM. Depleting the intracellular calcium stores with thapsigargin blocked both the transient fall and the secondary rise in Cai. Placement of the cells into a Ca-free medium blocked both the transient fall and the initial rapid rise, while use of a Na-free buffer prevented the initial rapid rise only. These data suggest that in human endothelial cells, extreme heat accelerates the CaATPase pumps of the intracellular Ca stores causing the transient fall in Cai which is soon followed by activation of the reverse mode of the Na/Ca exchanger to cause the initial rapid rise in Cai. The Ca-release channels of the intracellular stores become activated by heat to cause the secondary, slow rise in Cai. This preliminary work indicates that the application of heat to cultured cells can be a useful probe to examine the kinetics and unmask mechanisms of intracellular Ca fluctuations.

Are salivary glands cell lines in culture a good model for purinergic receptors in salivary glands?

A major obstacle in studying the physiological and biochemical processes of salivary secretion is the lack of a good ductal cell line model. HSY, an immortalised cell line originating from human parotid gland intercalated ducts, provides a possible model for purinergic, mechanisms in ductal cells. Unlike the biphasic dose response to ATP of isolated submandibular ductal cells, the rise in [Ca2+]i in MY cells shows single Michaelis-Menten kinetics with an apparent K a of 0.8 μM. Pre-incubation with thapsigargin inhibited the ATP induced [Ca 2+ ] i rise. Both ATP (10 μM) and carbachol (100 μM) increased IP 3 production. Intercalated duct cells may differentiate into acinar or ductal cells in response to appropriate stimuli from extracellular matrix. We therefore attempted to induce a duct-like phenotype in the striated duct-derived HSY cells by growing them on microcarrier beads coated with type I collagen. In Ca-containing medium cells grown on all substrates showed similar responses to ATP. In contrast, in Ca-free medium, [Ca2+]i rose only slightly in cells grown on beads relative to those on glass. This probably resulted from reduced IP 3 Production. Carbachol also induced a much smaller increase in [Ca2+]i and less IP 3 production in cells grown on Cytodex-3. The HSY response to purinergic stimuli by an increase in [Ca 2+ ] i and IP 3 means that they can be used to study the metabotropic purinergic pathway. The impairment in the HSY responses grown on Cytodex-3 can be used to probe phosposinositol signal transduction in salivary cells.

Are salivary glands cell lines in culture a good model for purinergic receptors in salivary glands?

A major obstacle in studying the physiological and biochemical processes of salivary secretion is the lack of a good ductal cell line model. HSY, an immortalised cell line originating from human parotid gland intercalated ducts, provides a possible model for purinergic, mechanisms in ductal cells. Unlike the biphasic dose response to ATP of isolated submandibular ductal cells, the rise in [Ca2+]i in MY cells shows single Michaelis-Menten kinetics with an apparent K a of 0.8 μM. Pre-incubation with thapsigargin inhibited the ATP induced [Ca 2+] i rise. Both ATP (10 μM) and carbachol (100 μM) increased IP 3 production. Intercalated duct cells may differentiate into acinar or ductal cells in response to appropriate stimuli from extracellular matrix. We therefore attempted to induce a duct-like phenotype in the striated duct-derived HSY cells by growing them on microcarrier beads coated with type I collagen. In Ca-containing medium cells grown on all substrates showed similar responses to ATP. In contrast, in Ca-free medium, [Ca2+]i rose only slightly in cells grown on beads relative to those on glass. This probably resulted from reduced IP 3 Production. Carbachol also induced a much smaller increase in [Ca2+]i and less IP 3 production in cells grown on Cytodex-3. The HSY response to purinergic stimuli by an increase in [Ca 2+] i and IP 3 means that they can be used to study the metabotropic purinergic pathway. The impairment in the HSY responses grown on Cytodex-3 can be used to probe phosposinositol signal transduction in salivary cells.

Imipramine inhibits histamine and serotonin secretion induced by various secretagogues.

Tricyclic antidepressants have been reported to reduce inflammation by inhibiting the formation or release of inflammatory mediators [1]. Theoharides et al. reported that amitriptyline inhibited histamine and serotonin release induced by compound 48/80 from rat mast cells [2]. Our previous results also showed that many tricyclic antidepressants have an inhibitory effect on histamine and serotonin secretion induced by compound 48/80 [3, 4]. The inhibition was more pronounced in Ca-free medium [4], suggesting that Ca may play a role in this inhibitory action of the tricyclic antidepressants. In this study, we further investigated the action of the antidepressant drug imipramine on the secretion of histamine and serotonin from rat isolated peritoneal mast cells. The mast cells were activated with various secretagogues, utilizing either extracellular Ca ions (concanavalin A, ionophore A23187) or Ca ions from intracellular stores (compound 48/80).

Pharmacological characterisation of metabotropic glutamatergic and purinergic receptors linked to Ca 2+ signalling in hippocampal astrocytes

Intracellular Ca 2+ ([Ca 2+] i) signals induced by metabotropic glutamate receptor (mGluR) agonists and by purinergic agonists in cultured hippocampal astrocytes were investigated using [Ca 2+]-sensitive fluorophores. The mGluR agonists (1 S,3 R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) and ( R, S)-3,5-dihydroxyphenylglycine (DHPG) induced [Ca 2+] i responses in 76 and 93% of the cells, respectively. The broad-spectrum mGluR antagonist (+)- α-methyl-4-carboxyphenylglycine (MCPG) and the mGluR1 antagonists ( S)-4-carboxy-3-hydroxyphenylglycine (4C3HPG) and ( S)-4-carboxyphenylglycine (4CPG) suppressed the agonist-evoked [Ca 2+] i response in about 25% of the cells completely and in about 60% partially, depending on the agonist concentration employed. Together with immunohistochemical receptor localisations these results suggest the presence of at least two subpopulations of class I mGluRs recruited from the truncated splice variants of mGluR1 (mGluR 1b, 1c, 1d) and/or hitherto unknown glial-specific class I mGluRs. Of the hippocampal astrocytes 88, 92 or 83% of the cells responded with a [Ca 2+] i elevation (mostly oscillations) to application of ATP, ADP, or 2-methylthio-ATP (2-MeS-ATP), respectively, whereas only 14 and 5% responded to AMP and adenosine, respectively, indicating the predominance of P2 receptors. The ATP-induced [Ca 2+] i signal was suppressed by suramin. Release of Ca 2+ from intracellular stores was involved in the response to ATP because the cells also exhibited [Ca 2+] i elevations in Ca 2+-free medium. Cells did not respond to 10 μM UTP. We conclude that the P2Y subtype represents the main [Ca 2+] i-linked purinoceptor in hippocampal astrocytes. Sequential application of ATP and DHPG in Ca-free medium showed that metabotropic glutamate and purinergic receptors initiate release of Ca 2+ from subsets of cyclopiazonic acid-sensitive Ca 2+ stores which are partly independent.

Poor correlation between postsynaptic [Ca] i increases and long-term potentiation induced by 2-deoxyglucose inCA1 hippocampal neurons

2-Deoxyglucose (2-DG; 10 mM) replacing glucose for ∼15 min regularly causes a very sustained enhancement of synaptic transmission in the CA1 hippocampal zone (2-DG-induced long-term potentiation, 2-DG-LTP). This LTP is prevented by dantrolene (10μM) or by the NMDA antagonist aminophosphonovalerate (APV; 50–100 μM); it is Ca-dependent [1, 2], and is not depressed by adenosine antagonists (DPCPX and 8-SPT), which prevent the initial block of EPSP caused by 2-DG, but is not observed if glucose is replaced by sucrose instead of 2-DG. To clarify the role of [Ca]i, in 2-DG-LTP, we monitored fluo-3 fluorescence inCA1 neurons, in submerged slices kept at 30°C. 2-DG applied as above consistently led to a rapid, but reversible rise in fluorescece. Isomodar sucrose (for 15–40 min) elicited smaller, but significant increases in fluorescence. Dantrolene and APV greatly reduced 2-DG's effect, but Ca-free medium did not abolish it. Paradoxically, it was fully suppressed by 8-SPT, but not at all by DPCPX. The discrepancies between the effects of sucrose and 8-SPT on the fluorescence changes and on 2-DG-LTP suggest that the observed increases in the postsynaptic [Ca] i are not essential for 2-DG-LTP. In further experiments, intracellular recordings showed that 2-DG applications consistently hyperpolarizeCA1 neurons, probably owing to adenosine-mediated activation of K+ conductance (which could be prevented by DPCPX or 8-SPT, but not by a KATP antagonist, glyburide). The 2-DG-LTP of EPSP was not suppressed by EGTA leakage from electrodes, in keeping with the above evidence that the [Ca] i changes needed for the induction of 2-DG-LTP are not those observed postsynaptically. In this respect, as well as in the absence of postsynaptic depolarization, 2-DG-LTP offers a marked contrast to more conventional forms of LTP.

Use of La 3+ to distinguish activity of the plasmalemmal Ca 2+ pump from Na +/Ca 2+ exchange in arterial myocytes

La 3+ was tested for its ability to distinguish external Na + (Na o)-independent Ca 2+ efflux via the plasma membrane (PM) Ca 2+ pump from Na o-dependent Ca 2+ efflux via Na +/Ca 2+ exchange. Fura-2 loaded cultured rat aortic myocytes were used with digital imaging to measure the cytosolic free Ca 2+ concentration ([Ca 2+] cyt) and to monitor La 3+ entry. At a La 3+ concentration ([La 3+] o) of 0.25 mM, but not at lower concentrations, La 3+ entered the cells; 0.01 mM verapamil blocked this entry. Transient increases in [Ca 2+] cyt were evoked by unloading the sarcoplasmic reticulum with cyclopiazonic acid (CPA) + caffeine (CAF) in Na,Ca-free medium (to inhibit Ca 2+ extrusion via Na +/Ca 2+ exchange and Ca 2+ influx). La 3+ (0.03–0.25 mM with verapamil) augmented the Ca 2+ transients and slowed Na o-independent [Ca 2+] cyt recovery in a dose-dependent manner (IC 50 ∼ 0.01 mM La 3+). This La 3+-sensitive recovery was apparently mediated by the PM Ca 2+ pump. The effects of La 3+ were reversible: [Ca 2+] cyt returned promptly toward base line when La 3+ was washed out in Na,Ca-free medium containing CPA + CAF. Reintroduction of extracellular Na + during [Ca 2+] cyt recovery ([La 3+] o = 0.06–0.25 mM) significantly speeded recovery, indicating that the Na +/Ca 2+ exchanger was not inhibited by [La 3+] o ≤ 0.25 mM. The La 3+-sensitive (Na o-independent) and Na o-dependent [Ca 2+] cyt recovery rates were comparable. In Na +-loaded cells, ≤ 0.25 mM La 3+ also did not affect Na +/Ca 2+ exchange mediated Ca 2+ influx. In medium containing Na + and Ca 2+, 0.125 mM La 3+ abolished the serotonin (5-HT) evoked plateau responses that resulted from Ca 2+ entry via Ca 2+ channels. In Na,Ca-free medium, but not Ca-free medium, however, La 3+ converted 5-HT evoked Ca 2+ transients into sustained responses. We conclude that low [La 3+] o (0.06–0.25 mM) inhibits the PM Ca 2+ pump, but spares Na +/Ca 2+ exchanger mediated Ca 2+ influx and efflux in arterial myocytes.

In vitro response of encapsulated somatic embryos of camellia

Somatic embryos of Camellia japonica were hydrogel encapsulated using 3% sodium alginate and 0.1 M calcium chloride to produce synthetic seeds. Both germinability and repetitive embryogenesis capacity of the encapsulated embryos were investigated. The frequency of in vitro germination into plants of artificial seeds was affected by various nutrient additives included in the encapsulating matrix. The addition of Ca-free Murashige and Skoog basal medium plus 3% sucrose plus 14.4 µM gibberellic acid and 28.5 µM indole-3-acetic acid to the alginate capsule resulted in 63% plant recovery rate, which was similar to that of non-encapsulated embryos. Encapsulation of somatic embryos did not negatively affect the maintenance of their embryogenic competence, the mean number of secondary embryos being significantly increased when the alginate beads included the growth regulators of the secondary embryogenesis medium (4.44 µM 6-benzyladenine and 0.41 µM indole-3-butyric acid). Storage at 4°C significantly reduced the survival and germination into plants frequencies of both encapsulated and non-encapsulated embryos, but the reduction was much greater for non-encapsulated embryos. Plant recovery of encapsulated embryos was 40% and 30% following storage for 30 and 60 days, respectively.

Novel ipriflavone receptors coupled to calcium influx regulate osteoclast differentiation and function.

Ipriflavone (7-isopropoxyisoflavone) is an effective antiresorptive agent used to treat osteoporosis. However, the mechanism of its action on osteoclasts and their precursor cells is not well understood. To determine whether the mechanism involves direct effects on osteoclasts or their precursors, we examined the effects of ipriflavone on cytosolic free calcium ([Ca2+]i) in osteoclasts and their precursors and measured specific binding of 3H-labeled ipriflavone. Highly purified chicken osteoclast precursors, which spontaneously differentiate into multinucleated osteoclasts in 3-6 days, were loaded with fura-2, and the subcellular [Ca2+]i distribution was monitored by videoimaging. Ipriflavone induced a rapid increase in [Ca2+]i followed by a sustained elevation [EC50 = 5 x 10(-7) M, 263 +/- 74 nM (SE) (n = 8) above basal levels, by 10(-6) M ipriflavone, sustained phase]. The responses were the same in differentiated chicken osteoclasts and isolated rabbit osteoclasts. An influx of extracellular Ca2+ is likely to be responsible for the ipriflavone-induced change in [Ca2+]i because the response was abolished by 0.5 mM LaCl3, or by Ca-free medium containing EGTA. Moreover, high [Ca2+]i levels were detected adjacent to the cell membrane after ipriflavone addition. Ipriflavone induced Ca influx mainly through dihydropyridine-insensitive Ca2+ channels, because nicardipine (10(-7)M) and verapamil (10(-7)M) had no effects on ipriflavone-induced [Ca2+]i responses. [3H]Ipriflavone binding studies indicated the presence of specific ipriflavone binding sites (two classes), both in precursor cells [dissociation constant (Kd), 7.60 x 10(-8)M, 2.67 x 10(-6)M] and in mature osteoclasts (Kd, 4.98 x 10(-8)M, 3.70 x 10(-6)M). Specific ipriflavone binding was not displaced by various modulators of avian osteoclast function, such as estradiol (10(-8)M) or retinoic acid (10(-6)M), indicating that ipriflavone receptors differ from the receptors for these Ca-regulating hormones. The fusion of osteoclast precursor cells was significantly inhibited by ipriflavone, which led to dose-dependent inhibition of bone resorption and tartrate-resistant acid phosphatase activity. Novel specific ipriflavone receptors that are coupled to Ca2+ influx were demonstrated in osteoclasts and their precursor cells. These ipriflavone receptors may provide a mechanism to regulate osteoclast differentiation and function.

Thrombin-mediated events implicated in mast cell activation.

It has been suggested that mast cells contain receptors for thrombin because binding of thrombin to peritoneal mast cells (PMCs) results in heparin release. Peritoneal mast cell responsiveness to different thrombin forms was examined by measuring ion conductance, intracellular pH, the concentration of cyclic guanosine monophosphate (cGMP), and release of histamine. Several types of receptors for thrombin are suggested by the results, which demonstrate that: (1) PMCs responded to alpha-thrombin and diisopryopyl-phosphoryl-alpha-thrombin (DIP-alpha-thrombin), but not to gamma-thrombin, by activation of Na/H exchange in reactions involving protein kinase C and by a simultaneous elevation in cell conductance and capacitance; (2) the initial 1-nmol/L alpha-thrombin-induced acidification of PMC cytoplasm was absent in Ca-free medium, and higher doses of alpha-thrombin induced a biphasic reaction (acidification preceeded alkalinization); and (3) PMC stimulation by alpha-thrombin at low concentrations (< 1 nmol/L) resulted in increase of cGMP and simultaneous decrease of histamine release, whereas thrombin concentrations > 1 mumol/L induced the acceleration of histamine release.

Immature properties of large-conductance calcium-activated potassium channels in rat neuroepithelium.

The pharmacological and biophysical properties of large-conductance Ca-activated K (BK) channels from embryonic rat telencephalic neuroepithelium were investigated with in situ patch-clamp techniques. A fraction of these channels exhibited properties characteristic of BK channels recorded in well differentiated cells, including normal gating mode (BKN channels). The vast majority of BK channels expressed distinctive properties, the most conspicuous being their buzz gating mode (BKB channels). BKB channels were insensitive to a concentration of charybdotoxin that completely and reversibly blocked BKN channels. In contrast with the strict dependence of BKN channel activation on cytoplasmic Ca, BKB channels displayed substantially high open probability (Po) after inside-out patch excision in a Ca-free medium. Intracellular trypsin down-regulated the Po of BKB channels, which then exhibited a greater sensitivity to cytoplasmic Ca, mainly in the positive direction (increased Po with increased Ca). This suggested a modulatory role for Ca as opposed to its gating role in BKN channels. Ca ions also reduced current amplitude of both types of channels. BKB channels were less voltage sensitive than BKN channels, but this was not correlated with their lower Ca sensitivity. We speculate that BKB channels may represent immature forms in the developmental expression of BK channels.

Independent external calcium entry and cellular calcium mobilization in Xenopus oocytes

We studied cellular calcium (Ca) mobilization and Ca entry from the medium following injection of various inositol phosphates (IPs) or activation of thyrotropin-releasing hormone receptors (TRH-Rs) in oocytes injected with TRH-R cRNA. We determined the order of potency of various IPs for evoking the rapid depolarizing current in Ca-free medium, which reflects the mobilization of cellular Ca. The most potent compound was inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3), followed by inositol 1,2,4,5-tetrakisphosphate (Ins(1,2,4,5y P 4), which displayed 91% of the activity of Ins(1,4,5)P3, while inositol 1,3,4,6-tetrakisphosphate (Ins(1,3,4,6)P 4) had only 29% effect. All other IPs used in the present study exhibited responses that were 40% or less than those elicited by Ins(1,4,5)P 3. Cellular Ca mobilization was confirmed by 45Ca 2+ efflux for lns(1,4,5)P 3, lns(1,2,4,5)P 4 and lns(1,3,4,6)P 4, or by Fura-2 ratio imaging studies for the latter. In parallel, we assayed the ability of these compounds to promote Ca entry into the cell, as reflected by Ca-evoked depolarizing current or Fura-2 imaging. These assays revealed a different order of potency, where Ins(1,4,5)P 3 > inositol 4,5-bisphosphate (Ins(4,5)P 2) > Ins(1,3,4,6)P 4 = Ins(1,2,4,5)P 4. All other inositol phosphates were largely ineffective. Heparin inhibited the response to TRH by 67% while Ca entry was inhibited only by 22%. The latency of the response to TRH was significantly shorter in the presence of extracellular Ca, suggesting Ca entry preceded the response, i.e. major depletion of Ca stores. These results strongly suggest that the activation of Ca entry is largely independent of cellular Ca mobilization and may be mediated by a receptor for an unidentified phosphorylated compound, different from that for Ins(1,4,5)P 3 on the endoplasmic reticulum.

Characterization of regulatory volume decrease in the THP-1 and HL-60 human myelocytic cell lines.

Exposure to hypotonic stress produces a transient increase in cell volume followed by a regulatory volume decrease (RVD) in both THP-1 and HL-60 cells. In contrast, cells exposed to hypotonic stress in a high K/low Na Hanks' solution not only failed to volume regulate, but displayed a secondary swelling. Thus, while an outward K gradient was required for RVD, the secondary swelling indicated that hypotonic stress increased permeability in the absence of a negative membrane potential. The K channel blocker quinine (1-4 mM) blocked RVD in both cell types. Gramicidin's ability to overcome the quinine block of RVD indicated that RVD is mediated by a quinine-sensitive cation transport mechanism that is independent of the swelling-induced anion transport mechanism. Barium (1-4 mM), another K channel blocker, slowed the rate of RVD, while 4-aminopyridine, charybdotoxin, tetraethylammonium chloride, tetrabutylammonium chloride, and gadolinium had no effect on RVD. Furthermore, RVD was not mediated by calcium-activated conductances, since it occurred normally in Ca-free medium, in medium containing cadmium, and in BAPTA-loaded cells. Gramicidin produced little or no volume change in isotonic medium, suggesting that basal C1 permeability of both THP-1 and HL-60 cells is low. However, swelling induced an anion efflux pathway that is permeable to both chloride and bromide, but is impermeable to methanesulfonate and glutamate. The anion channel blocker 3,5-diiodosalicylic acid (DISA) antagonized RVD in both cell types. In conclusion, RVD in THP-1 and HL-60 cells is mediated by independent anion and cation transport mechanisms that involve both a DISA-sensitive anion pathway and a quinine-inhibitable K efflux pathway, neither of which requires increases in intracellular calcium to be activated.

The role of cytoplasmic [Ca 2+] in glucose-induced inhibition of respiration and oxidative phosphorylation in Ehrlich ascites tumour cells: a novel mechanism of the Crabtree effect

The effect of Ca 2+ on energy-coupling parameters of Ehrlich ascites carcinoma was studied in digitonin-permeabilized cells. In nominally Ca-free medium the permeabilized cells respond to the addition of ADP by increased oxygen uptake with externally added respiratory substrates (succinate or pyruvate), decrease of the mitochondrial membrane potential (Δψ) and alkalinization of the medium. This typical behaviour is drastically changed if Ca 2+ is added. The subsequent addition of ADP induces neither State 3 respiration, nor decrease of Δψ, nor alkalinization of the medium, indicating a complete block of ATP synthesis. These effects are produced by both a single pulse of 100 μM Ca 2+ and a preincubation for 2 min with 0.4–1.0 μM Ca 2+. Preincubation of the cells with glucose or deoxyglucose prior to permeabilization makes them sensitive to Ca 2+ concentrations as low as 0.3 μM. In view of the previous finding that glucose and deoxyglucose produce an increase of cytoplasmic [Ca 2+] in Ehrlich ascites cells [Teplova VV. Bogucka K. Czyż A. Evtodienko YuV. Duszyński J. Wojtczak L. (1993) Biochem. Biophys. Res. Commun., 196, 1148–1154; Czyż A. Teplova VV. Sabała P. Czarny M. Evtodienko YuV. Wojtczak L. (1993) Acta Biochim. Polon., 40, 539–544], the present results suggest that cytoplasmic Ca 2+ plays a crucial role in the Crabtree effect.

In vitro and in vivo evidence of neurotensin release from preganglionic axon terminals in the stellate ganglion of the cat

We have previously shown that the neurotensin (NT) store in preganglionic axon terminals of the cat stellate ganglion (SG) is reversibly depleted by prolonged preganglionic stimulation. The present study addresses the questions of whether the preganglionic axon terminals release NT in response to depolarizing stimuli in vitro and whether in vivo NT is released by the tonic firing of the sympathetic preganglionic neurons. Slices of the SG of the anaesthetized cat, maintained in oxygenated Ringer solution, released NT. The efflux increased when the K concentration was increased from 5 to 25 or 45 mM or when veratridine was added to the medium. In Ca-free medium, efflux was suppressed. The effect of veratridine was blocked by tetrodotoxin (TTX). In awake, freely moving cats, in which TTX was applied for 4 days to the preganglionic input of the right SG, the NT content of this ganglion doubled by comparison with the left SG. Since NT accumulates proximal to a ligature on the preganglionic input of the SG, the increased NT content is likely to result from suppression of action potential-dependent release while influx into the terminals persists. This result suggests that the steady state of the NT store in sympathetic preganglionic terminals is the result of a steady influx from the soma balanced by action potential-dependent loss, presumably release.

Development of the research in the field of histamine release

Histamine release from mast cells is intimately related with degranulation. When basic histamine releasers such as compound 48/80 were applied extracellularly to isolated rat mast cells by means of microelectrophoresis, localized degranulation was evoked near the tip of micropipet in a few seconds. In response to the second electrophoretic application at the opposite side of the membrane of the same mast cells, similar local degranulation was induced. This fact clearly indicates that local degranulation does not damage mast cells to the extent of blocking following degranulation. As intracellular electrophoretic application of compound 48/80 caused a swelling of mast cell, although no degranulation was elicited. When antigen-antibody reaction was induced in a single rat mesentery mast cell by means of microelectrophoresis, the application of antigen was made extracellularly or intracellularly. At the site of extracellular application, localized degranulation and histamine release were evoked. Histamine release was evidenced by the disappearance of histamine fluorescence in the degranulated area. Neither degranulation nor histamine release was induced by intracellular application of antigen. In freeze-fracture electronmicroscopy of the resting rat mast cells, intra-membrane particles (IMPs) were randomly distributed on the plasma membrane. When sensitized cells were exposed to antigen, IMPs were markedly dispersed so as to surround bulging regions of the membrane elicited by swollen granules. As the particles gathered at the periphery of the bulges, actually no particle was seen on the protuberant region. When rat mast cells loaded with quin 2 were exposed compound 48/80 in a Ca-free medium, a marked increase of quin 2 fluorescence was noticed, indicating that Ca2+ was released from intracellular Ca store. The binding of 45Ca was at its peak in the fractions where the highest activity of glucose-6-phosphatase, a marker enzyme for the endoplasmic reticulum, when organelles of mast cells were fractionated. This may indicate that intracellular Ca store is endoplasmic reticulum. It has been shown that microfilaments, and microtubules play some important roles in histamine release from rat mast cells. When permeabilized mast cells were stimulated with Ca2+, a translocation of protein kinase C from cytosol to membrane fraction was observed. This leads to phosphorylation of vimentin, one of intermediate filaments. In membrane skeletons of rat mast cells, alpha- and beta-fodrin, ankyrin and actin were found by means of western blotting analysis. It was supposed that membrane skeleton may be useful as a barrier between the plasma membrane and the granule membrane.

Contractions of the filariid Acanthocheilonema viteae induced by potassium chloride.

The effects of K+ on muscle contractility were explored in the filarial nematode Acanthocheilonema viteae (Dipetalonema viteae). The parasite was slit open longitudinally and mounted in a smooth muscle chamber that was filled with aerated (95% N2-5% CO2) physiological solution at 37 degrees C. KCl at concentrations ranging from 20 to 100 mM induced a rapid isotonic contraction of the filarial muscle. The maximal response from KCl was similar to the maximal response to acetylcholine chloride (ACh). When KCl was applied for several minutes, tolerance frequently occurred. Contractions were also induced by K2SO4 but not by NaCl, Na2SO4 or sucrose. Nifedipine was more than 10 times as potent in reducing the KCl-induced contraction as in reducing that caused by ACh. The KCl-induced contraction was abolished in a Ca-free physiological medium containing ethyleneglycol-bis-(beta-aminoethyl ether) N, N, N', N'-tetraacetic acid (EGTA, 10(-4) M). Low [Ca2+]/[Mg2+] solutions blocked the spontaneous activity, the KCl-induced contractions, and the ACh-induced contractions. KCl also induced contractions in denervated muscle strips, supporting the hypothesis that K+ acts directly on the muscle cells. These results indicate that K+ can depolarize the muscle membrane and induce a muscle contraction that is dependent on extracellular calcium ions.

The effect of EDTA on contractile responses of guinea-pig trachea in calcium-free medium and on the recovery of the contractile responses in calcium-containing solution

1. 1. Acetylcholine (0.1 mmol/l) and KCl (80 mmol/l) induce contractile responses in guinea-pig trachea. 2. 2. In the absence of extracellular Ca, contractile responses to ACh or KCl decrease as the preincubation time or the EDTA concentration increases. 3. 3. Exposure of the tissue to Ca-free medium decreases the basal tone. 4. 4. Prolonged incubation in the presence of EDTA (1 mmol/l) promotes a delay in the recovery of the tonic component of the contraction elicited subsequently by acetylcholine and KCl in physiological salt solution, and abolishes the phasic one.

Actions of alpha-adrenoceptor blocking agents on two types of intracellular calcium stores mobilized by noradrenaline in rat aorta.

In isolated rat aortic strips noradrenaline induces a biphasic contractile response in Ca-free medium, associated with two different intracellular calcium pools, one of which is common to caffeine. We analyzed the mechanisms involved in the depletion and repletion of both intracellular Ca pools sensitive to noradrenaline in different experimental procedures in presence of prazosin, phentolamine and yohimbine. At 37 degrees C the alpha-adrenergic blocking agents inhibited contractile responses to noradrenaline in Ca-free medium, with prazosin being highly selective. alpha 2-adrenoceptors probably do not participate in the release of Ca from internal stores, as no contractile response was observed after addition of clonidine in Ca-free medium. This indicates that noradrenaline-induced Ca-release from internal stores is mainly due to activation of alpha 1-adrenoceptors. At 25 degrees C, these compounds failed to inhibit caffeine-induced contraction in Ca-free medium, but abolished the release of Ca from an intracellular store only sensitive to noradrenaline. This effect is attributable to a blockade of alpha 1-adrenoceptors and/or inhibition of receptor-mediated signal transduction.

Parathyroid hormone-related peptide production and action in a myoepithelial cell line derived from normal human breast.

Physiological roles for PTH-related peptide (PTHrP) appear varied, but remain to be clarified. The peptide is present in large amounts in milk, and PTHrP mRNA has been shown to be present in high amounts in lactating mammary gland. Because PTHrP can cause smooth muscle relaxation, we hypothesized that the peptide might affect the contractility of the breast myoepithelial cell and thereby affect milk ejection. To test this idea, we asked whether PTHrP might affect second messenger responses in a human mammary gland myoepithelial-cell line (Hs578Bst) derived originally from normal breast tissue. To verify the presumed origin of these cells, we also examined the effects of oxytocin. Cells were grown in culture in multiwell plates and exposed to test peptides for 15 min in buffer containing 1 mM isobutylmethylxanthine, and intracellular cAMP was measured by RIA. Both PTHrP-(1-34) and PTH-(1-34) increased cAMP in a dose-related fashion (ED50, 5 nM), with a maximal effect (3-fold) occurring at 100 nM. The ability of PTHrP to stimulate cAMP was inhibited by a 10- to 100-fold molar excess of the specific inhibitors, PTH-(3-34) or PTHrP-(7-34). Inhibitors alone did not alter cAMP. Oxytocin also produced an increase in cAMP, but the effect was inconsistent and occurred only with high doses (0.1-1 microM). Using cells grown on coverslips and loaded with fura-2AM, intracellular Ca2+ was monitored in cells exposed to test peptides. Oxytocin (0.2-20 nM) produced rapid dose-related increases in intracellular Ca2+, with a peak and plateau characteristic of initial mobilization of intracellular Ca2+, followed by entry of extracellular Ca. The plateau was eliminated by the Ca channel antagonist La3+ or by perfusion of cells with Ca-free medium. PTHrP (10-100 nM) altered the intracellular Ca2+ response to oxytocin in 66% of 39 preparations tested. PTHrP inhibited the Ca2+ response when given before oxytocin or transiently decreased the plateau phase of the Ca2+ response when given after oxytocin. Analysis of cellular mRNA using reverse transcription polymerase chain reaction indicated that these cells express the gene for PTHrP, and immunohistochemistry using antiserum to PTHrP revealed positive staining of cells. Measurement of immunoreactive PTHrP in conditioned medium confirmed that these cells can synthesize and secrete the peptide. The finding of a response of this cell line to oxytocin provides functional evidence of their myoepithelial derivation.(ABSTRACT TRUNCATED AT 400 WORDS)