Calcium transients in single adrenal chromaffin cells detected with aequorin

The effect of 55 mM K+ and nicotine on intracellular free calcium was monitored in bovine adrenal chromaffin cells microinjected with aequorin. In contrast to results with quin 2, which suggested that stimulation of chromaffin cells resulted in sustained rises in free calcium, aequorin measurements showed that 55 mM K+ and nicotine resulted in a transient (60-90 s) elevation of free calcium. The peak free calcium and duration of the transient elicited by nicotine were dose-dependent. The concentration of nicotine (10 microM) giving a maximal secretory response gave a peak rise in free calcium of up to 1 microM. 55 mM K+ which only releases 30% of the catecholamine released by 10 microM nicotine generated a calcium transient indistinguishable from that due to 10 microM nicotine. These results support the idea that nicotine agonists generate an alternative second messenger in addition to the rise in free calcium.     

Complete overlap of caffeine- and K+ depolarization-sensitive intracellular calcium storage site in cultured rat arterial smooth muscle cells

Changes in the concentration of cytosolic free calcium were recorded microfluorometrically in rat vascular smooth muscle cells in primary culture and loaded with quin-2. The effects of caffeine and high extracellular K+ on the release of calcium from the intracellular storage sites were determined. In the absence of extracellular calcium, both the depolarization of plasma membrane with excess extracellular K+ and the application of caffeine induced a transient and dose-dependent elevation of the cytosolic free calcium concentration, with durations of 4 and 2 min, respectively. Transient elevations of calcium repeatedly appeared in response to both repetitive depolarization (100 mM K+) and caffeine (10 mM) applications with progressive reductions in peak levels. In either case, the fifth or later treatments induced little or no rise in levels of the cytosolic calcium. The amount of released calcium induced by high K+ depolarization after (n-1) time applications (1 less than or equal to n less than or equal to 5) of caffeine was equal to that induced by the n-th application of caffeine. The amount of released calcium induced by caffeine after (n-1) time exposures (1 less than or equal to n less than or equal to 5) to K+ depolarization was equal to that observed during the n-th exposure to K+ depolarization. These results indicate that caffeine- and depolarization-sensitive intracellular calcium storage sites may be identical and that caffeine and K+, in optimal concentrations, will release an equal amount of calcium from the same storage site in cultured arterial smooth muscle cells, irrespective of the amount of stored calcium.     

Effect of orthophosphate and oxalate on the cold-induced release of calcium from sarcoplasmic reticulum preparations from rabbit skeletal muscle.

The cold-induced release of calcium from sarcoplasmic reticulum preparations from both white and red muscles of the rabbit was studied. Part of the release was due to the increase in pH of the reaction mixture with cooling. Calcium release was greatly reduced or completely prevented by the inclusion of oxalate or inorganic orthophosphate in the medium. No release occurred in 5 mM oxalate. With phosphate, the proportion of the calcium previously taken up at 23 degrees C that was released at 0 degrees C became progressively smaller as the phosphate concentration was increased. When the pH was adjusted to be the same at 0 degrees C as at 23 degrees C there was little release from white muscle preparations in 10 mM phosphate and no release when the phosphate concentration was 20 mM or more. With red muscle preparations calcium was released at higher phosphate concentrations, 8% of the amount previously taken up still being released at 50 mM phosphate and a smaller amount at 100 mM phosphate. The effects of oxalate and phosphate can be explained in terms of the reduction in free calcium concentration inside the vesicles by calcium precipitants, and a difference in the temperature coefficients of calcium inflow and outflow.     

Regulation by calcium and calmodulin of adenylate cyclase from rabbit intestinal epithelium

The effect of calcium on adenylate cyclase from rabbit small intestine has been studied using a particulate preparation obtained from isolated epithelial cells. Both basal and vasoactive intestinal peptide-stimulated activities were inhibited by calcium concentrations in the micromolar range. In the presence of calmodulin, a biphasic response was obtained. At low calcium concentration (4 X 10(-9)-6 X 10(-8) M) the enzyme was activated up to 50%. As the Ca2+ concentration was increased, the enzyme was concomitantly inhibited. Half-maximal inhibition of calmodulin-dependent activity was obtained at 1 microM free Ca2+. The activation of the enzyme was also dependent on the concentration of Mg2+. At less than 1 microM Ca2+, the enzyme exhibited a biphasic response, being activated at below 3 mM Mg2+ and inhibited at higher concentrations. At Ca2+ concentrations that were inhibitory, the enzyme did not show the biphasic response to Mg2+. At concentrations above 3 mM, the maximal rate (Vmax) remained constant. Vmax was inversely proportional to the concentration of Ca2+ present. Calmodulin altered Vmax when acting on vasoactive intestinal peptide-stimulated enzyme. Calmodulin had no effect on the Km for hormone activation. The calmodulin-dependent activity was inhibited by incubation with trifluoperazine.     

P2-purinoceptor-stimulated phosphoinositide turnover in chick myotubes. Calcium mobilization and the role of guanyl nucleotide-binding proteins

ATP, a trigger of P2-purinoceptor-mediated polyphosphoinositide (PI) turnover in cultured myotubes, increased cytosolic calcium levels in a time- and dose-dependent manner (quin2 fluorescence). The calcium was released from intracellular stores, as acute addition of 5 mM EGTA was without significant effect. Adenosine 5'-(3-thiotriphosphate) and 5'-adenylyl imidodiphosphate also increased intracellular levels of inositol phosphates (InsP) and cytosolic calcium levels. Treatment with cholera or pertussis toxin of myotube cultures did not affect the P2-purinoceptor-mediated InsP increase although PI turnover in permeabilized myotubes was stimulated by guanosine 5'-(3-thiotriphosphate). The results suggest that myotube P2-purinoceptors trigger PI turnover and increase intracellular free calcium levels, via a mechanism insensitive to ADP-ribosylation, by cholera or pertussis toxin of guanyl nucleotide-binding (G) proteins. However, the presence of a phospholipase C-coupled G-protein was otherwise demonstrated.     

Influence of Prolactin and Calcium Gluconate Concentration on Permeation and Intestinal Absorption of Ca(II) Ions

The in vitro permeation and absorption of calcium ions across the small intestine were measured at different concentrations of calcium gluconate solutions (1.0, 10.0 and 20.0 mM) with or without prolactin. The calcium ions permeated through the small intestine from a donor environment to an acceptor environment that mimicked the conditions in the stomach to ileum segment of the digestive tract. The permeation and absorption of calcium were directly dependent on the calcium concentration of the solutions. At 10 and 20 mM permeation was significantly higher than that at 1.0 mM (p < 0.05). In the presence of prolactin both permeation and absorption increase considerably. At the lowest concentration (1.0 mM) simulating calcium deficiency, there was compensation by the small intestine, suggesting that such deficiency stimulates its mobilization from intestinal tissue. Prolactin enhances the calcium mobilization process even at sufficient calcium intakes. It is suggested that prolactin takes part in regulation of calcium homeostasis in the organism.     

The role of calcium in the regulation of glucose uptake in isolated working rat heart

Catecholamines or ischemia may increase myocardial glucose uptake by an increase in intracellular calcium. We tested the hypothesis that increasing or decreasing extracellular calcium supply would change glucose uptake. Hearts were perfused for 60 min at a physiological workload with Krebs-Henseleit buffer containing glucose (5 mM) and oleate (0.4 mM; bound to 1% BSA). Calcium concentration was 2.5 mM. In group A (control; n = 12), insulin (1 mU/ml) was added at 30 min. In Group B (n = 7), the calcium concentration was increased to 5.0 and 7.5 mM at 20 min and 40 min, respectively. In Group C (n = 7), verapamil was added at 20 min (0.25 M) and 40 min (1.0 M) to decrease calcium influx. In group D (n = 7), EDTA was added at 20 min (0.5 mM) and at 40 min (1.5 mM) to decrease the free extracellular calcium. Glucose uptake was measured by ³H₂O production from [2-³H]glucose and cardiac work was measured simultaneously. Cardiac power in group B was 8.24 ± 0.60 mW at 2.5 mM calcium, 9.45 ± 0.50 mW at 5 mM calcium and 7.99 ± 0.99 mW at 7.5 mM calcium (n.s.). The addition of verapamil decreased contractile function in a dose-dependent manner (8.50 ± 0.74 vs. 3.11 ± 0.84 vs. 1.48 ± 0.39 mW, p < 0.01) suggesting that verapamil decreased cytosolic calcium concentration. A similar dose-dependent reduction in contractile performance was observed in the EDTA group (8.44 ± 0.81 vs. 7.42 ± 0.96 vs. 4.03 ± 1.32 mW, p < 0.01). Glucose uptake was 1.35 ± 0.11 mol/min/g dry weight under control conditions. Glucose uptake increased threefold with the addition of insulin. Increasing extracellular [Ca²⁺] did not affect glucose uptake. Decreasing Ca²⁺ availability showed a trend towards a decrease in glucose uptake (n.s.), which was minor compared to the decrease in contractile function. We conclude that extracellular calcium does not regulate glucose uptake in the isolated working rat heart in the presence of glucose and fatty acids as substrates. The trend of decreased glucose uptake when calcium supply was limited may be due to dramatically reduced energy demand and not directly due to changes in calcium.     

Intestinal zinc uptake in freshwater rainbow trout: evidence for apical pathways associated with potassium efflux and modified by calcium

Understanding the mechanisms of intestinal zinc uptake in fish is of considerable interest from both nutritional and toxicological perspectives. In this study, properties of zinc transport across the apical membrane of freshwater rainbow trout intestinal epithelia were examined using right-side-out brush border membrane vesicles (BBMV's). Extravesicular calcium was found to have complex actions on zinc uptake. At a low zinc concentration of 1 microM, calcium (0.1-2 mM) significantly stimulated zinc uptake. In contrast, calcium inhibited zinc uptake at higher zinc levels (100 microM). Lanthanum and cadmium in the external medium did not block zinc uptake, suggesting that interactions between zinc and calcium were not exerted at a calcium channel. Copper also failed to exercise any inhibitory action. Zinc association with the BBMV's was enhanced by an outward potassium gradient. This stimulatory effect was only present at a zinc concentration of 100 microM. The potassium channel blocker, tetraethylammonium chloride inhibited zinc uptake at this relatively high zinc concentration, suggesting the presence of a low affinity zinc uptake pathway linked to potassium efflux. The present study provides evidence that the mechanism of intestinal zinc uptake in rainbow trout is pharmacologically very different from that of the piscine gill and the mammalian intestine.     

Evidence on the role of three calcium pools in Ca-ionophore A23187-stimulated rat blood platelet aggregation.

The effect of Ca-ionophore A23187 on activation of rat blood platelets was investigated to elucidate the involvement of extracellular and intracellular Ca2+ ions. Platelet aggregation induced by 10 concentrations of the stimulus was studied in Ca-free medium as well as in the presence of EGTA and/or calcium. In Ca-free medium, A23187 induced platelet aggregation in a dose-dependent way; the mean effective concentration was 1.43 +/- 0.08 mumol/l. The stimulatory effect of ionophore was potentiated by addition of 0.01 and 0.1 mM calcium and inhibited when the calcium concentration was increased to 1 mmol/l. In the presence of EGTA, A23187-stimulated aggregation of isolated rat platelets was recorded only at a 10-times higher ionophore concentration and was then reduced to 30% in comparison with aggregation in Ca-free medium. The inhibitory effect of 1 mM EGTA was abolished by addition of 2 mM calcium. We suggest the participation of at least three calcium pools in the stimulation of rat platelets by A23187, i.e. the extracellular pool, the membrane-associated pool and the pool displacing calcium intracellularly.     

Role of calcium in the release of EC 3.4.24.15 and EC 3.4.24.16 from endothelial cells

Summary The two closely related soluble zinc metalloendopeptidases EC 3.4.24.15 (EP24.15) and EC 3.4.24.16 (EP24.16) readily hydrolyze the vasocative peptide bradykinin in vitro, and therefore may play a role in cardiovascular regulation. Although primarily soluble cytosolic enzymes, both secreted and membrane-associated forms of both peptidases have been reported. However, these enzymes have neither a transmembrane domain nor a signal sequence; thus, the mechanisms of membrane anchoring and secretion are unknown. In the present study, secreted/released EP24.15 and EP24.16 activity from aortic endothelial cells in culture was assessed by the cleavage of a specific quenched fluorescent substrate. An increase in enzyme activity released from endothelial cells, which express both peptidases, was seen following incubation with calcium-free media. In the AtT-20 endocrine cell (mouse pituitary corticotrope), which predominantly expresses EP24.15, the release of activity into media was unaffected by calcium removal. The release of enzyme activity from endothelial cells was inversely proportional to calcium concentrations ranging between 0.01 mM (activity equivalent to calcium-free media) and 0.5 mM (activity equivalent to normal media). Cleavage of the EP24.16-specific substrate AcNT_8–13 indicated that the increase in enzyme activity released upon incubation with calcium-free medium was due at least in part to the release of EP24.16. These results suggest that EP24.15 and EP24.16 are secreted from endothelial cells, and that removal of calcium selectively enhances the release of EP24.16 by an as yet unknown mechanism.     

Signalling pathways involved in the detection of peptones by murine small intestinal enteroendocrine L-cells

Glucagon like peptide-1 is an insulinotropic hormone released from intestinal L-cells in response to food ingestion. Here, we investigated mechanisms underlying the sensing of peptones by primary small intestinal L-cells. Meat, casein and vegetable-derived peptones (5 mg/ml), the L-amino acids Phe, Trp, Gln and Ala (20 mM each), and the dipeptide glycine-sarcosine (20 mM) stimulated GLP-1 secretion from primary cultures prepared from the small intestine. Further mechanistic studies were performed with meat peptone, and revealed the elevation of intracellular calcium in L-cells. Inhibition of the calcium sensing receptor (CaSR), transient receptor potential (TRP) channels and Q-type voltage gated calcium channels (VGCC) significantly attenuated peptone-stimulated GLP-1 release and reduced intracellular Ca²⁺ responses. CaSR inhibition also attenuated the GLP-1 secretory response to Gln. Targeting these pathways in L-cells could be used to increase endogenous production of GLP-1 and offer exploitable avenues for the development of therapeutics to treat diabetes and obesity.     

Comparison of gastric and intestinal antisecretory and protective effects of prostacyclin and its stable thia-imino-analogue (Hoe 892) in conscious rats

The effects of prostacyclin (PGI2) and its stable thia-thimo-analogue (Hoe 892) on gastric and intestinal secretions and gastric mucosal lesions have been determined in conscious rats. Both PGI2 and Hoe 892 given subcutaneously (s.c.) reduced dose-dependent gastric acid secretion, the ID50 (dose producing 50% inhibition) being about 48.6 and 11.8 micrograms/kg, respectively. In contrast, intragastric (i.g.) PGI2 and Hoe 892 did not cause any change in gastric acid secretion at doses ranging from 1 to 100 micrograms/kg. Both PGI2 and Hoe 892 reduced significantly intestinal fluid secretion (antienteropooling activity). PGI2 and Hoe 892 given i.g. or s.c. reduced dose-dependent gastric ulcer formation induced by acidified aspirin (ASA), Hoe 892 being somewhat less potent than PGI2. Both PGI2 and Hoe 892 were equally effective against gastric mucosal necrosis induced by absolute ethanol and this effect was observed both after i.g. and s.c. administration of these agents. We conclude that stable thia-imino-PGI2 analogue, Hoe 892, has similar gastric and intestinal antisecretory and protective activity as PGI2 and may be useful in the prevention of gastric damage by various noxious agents.     

Role of calcium in the release of EC 3.4.24.15 and EC 3.4.24.16 from endothelial cells

The two closely related soluble zinc metalloendopeptidases EC 3.4.24.15 (EP24.15) and EC 3.4.24.16 (EP24.16) readily hydrolyze the vasoactive peptide bradykinin in vitro, and therefore may play a role in cardiovascular regulation. Although primarily soluble cytosolic enzymes, both secreted and membrane-associated forms of both peptidases have been reported. However, these enzymes have neither a transmembrane domain nor a signal sequence; thus, the mechanisms of membrane anchoring and secretion are unknown. In the present study, secreted/released EP24.15 and EP24.16 activity from aortic endothelial cells in culture was assessed by the cleavage of a specific quenched fluorescent substrate. An increase in enzyme activity released from endothelial cells, which express both peptidases, was seen following incubation with calcium-free media. In the AtT-20 endocrine cell (mouse pituitary corticotrope), which predominantly expresses EP24.15, the release of activity into media was unaffected by calcium removal. The release of enzyme activity from endothelial cells was inversely proportional to calcium concentrations ranging between 0.01 mM (activity equivalent to calcium-free media) and 0.5 mM (activity equivalent to normal media). Cleavage of the EP24.16-specific substrate AcNT_8-13 indicated that the increase in enzyme activity released upon incubation with calcium-free medium was due at least in part to the release of EP24.16. These results suggest that EP24.15 and EP24.16 are secreted from endothelial cells, and that removal of calcium selectively enhances the release of EP24.16 by an as yet unknown mechanism.     

Characterization of a phospholipase A2 in hamster lung and in vitro and in vivo effects of bleomycin on this enzyme

Phospholipase A2 (PLA2) activity from adult hamster lung was characterized using L-alpha-1-palmitoyl-2-arachidonyl-[arachidonyl-1-14C]-phosphatidylcholine as the substrate. The released [14C]-arachidonic acid was separated by TLC. The enzyme activity increased with increasing incubation time (0-120 minutes), calcium ion concentration (0-25.0 mM) and protein (0-2.0 mg). The optimum pH was 8.0. Deoxycholate had a concentration dependent (0.1 to 0.5 mM) inhibitory effect on the activity. PLA2 specific activity was the highest in mitochondrial fraction. PLA2 activity following incubation with bleomycin was increased in a dose related fashion. In vivo study showed that both PLA2 activity and collagen content in hamster lung were significantly elevated at 14 days followed intratracheal instillation of bleomycin. The activation of PLA2 may play an important role in bleomycin-induced pulmonary toxicity.     

Excitation-contraction coupling model to estimate the recirculating fraction of activator calcium in intact cardiac muscle.

Potentiated contractions were evoked with rapid pace pause maneuver in 14 length-clamped ferret papillary muscles paced 12 times/min at 25 degrees C. At 1.25 mM [Ca2+]o the average steady-state force was 2.94 +/- 1.08 g/mm2 and the potentiated contraction averaged 10.96 +/- 1.61 g/mm2. At 5.0 mM [Ca2+]o the steady-state force increased to 6.18 +/- 1.23 g/mm2 and the potentiated contraction averaged 12.08 +/- 1.15 g/mm2. Under the conditions of these experiments the potentiated contraction obtained at 5.0 mM [Ca2+]o is equal to the maximum twitch tension (Fmax) these muscles can generate. We have previously shown that Fmax is an equivalent of maximal calcium activated force. Since there is a beat to beat nearly exponential decay of the evoked potentiation, the fraction (= fraction x) of the potentiation that is not dissipated with each beat is nearly constant. Using an excitation-contraction coupling model we have previously found that x reflects a measure of the recirculating fraction of activator calcium. Because the tension-calcium relationship is better characterized by a sigmoidal curve, we have now incorporated the Hill equation in the model. To account for the inverse relationship between [Ca2+]i and the magnitude of the slow inward current, a term for negative feedback (h) was also included. We have determined the quantity (x-h) because x and h could not be determined separately. The quantity (x-h) was denoted as x'. The average values of x' at 1.25 and 5.0 mM [Ca2+]o were significantly different (p less than 0.0001), approximately 20% at the lower [Ca2+]o and about 50% at the higher [Ca2+]o.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of the calcium permeability of erythrocyte membranes by perfringolysin O

Calcium ions inhibited perfringolysin O-induced hemolysis at a concentration lower than 1 mM, but not the hemolysis by digitonin at 10 mM. The introduction of calcium ions into ghosts inhibited the lysis more strongly than the addition of calcium ions outside ghosts. When erythrocytes were treated with perfringolysin O in the presence of 1 mM CaCl2 containing 45CaCl2, the radioactivities inside cells rapidly increased during incubation. On the other hand, when perfringolysin O-treated erythrocytes were incubated in a calcium-free medium, the erythrocytes released calcium ions at a 3.3-fold higher rate than untreated cells. These results suggested that perfringolysin O accelerated both the calcium influx into and efflux from erythrocytes.     

Spontaneous and propagated contractions in rat cardiac trabeculae

Sarcomere length measurement by microscopic and laser diffraction techniques in trabeculae of rat heart, superfused with Krebs-Henseleit solution at 21 degrees C, showed spontaneous local sarcomere shortening after electrically stimulated twitches. The contractions originated in a region of several hundred micrometers throughout the width of the muscle close to the end of the preparation that was damaged by dissection. The contractions propagated at a constant velocity along the trabeculae. The velocity of propagation increased from 0 to 10 mm/s in proportion to the number of stimuli (3-30) in a train of electrically evoked twitches at 2 Hz and at an external calcium ion concentration ([Ca++]o) of 1.5 mM. At a constant number of stimuli (n), the velocity of propagation increased from 0 to 15 mm/s with [Ca++]o increasing from 1 to 7 mM. In addition, increase of n and [Ca++]o led to an increase of the extent of local sarcomere shortening during the spontaneous contractions, and the occurrence of multiple contractions. Spontaneous contractions with much internal shortening and a high velocity of propagation frequently induced spontaneous synchronized contractions and eventually arrhythmias. Propagation of spontaneous contractions at low and variable velocity is consistent with the hypothesis that calcium leakage into damaged cells causes spontaneous calcium release from the overloaded sarcoplasmic reticulum in the damaged cells. This process propagates as a result of diffusion of calcium into adjacent cells, which triggers calcium release from their sarcoplasmic reticulum. We postulate that the propagation velocity depends on the intracellular calcium ion concentration, with increases with n and [Ca++]o.     

Optimization of calcium pectinate gel beads for sustained-release of catechin using response surface methodology

Response surface methodology was used to optimize bead preparation conditions, including CaCl₂ concentration (X₁), hydroxypropylmethylcellulose concentration (X₂), and bead-hardening time (X₃), for the sustained-release of catechin from the calcium pectinate gel beads reinforced with liposomes and hydroxypropylmethylcellulose into simulated gastric fluid (SGF) and intestinal fluid (SIF). The optimized values of X₁, X₂, and X₃ were found to be 5.82%, 0.08%, and 10.29 min, respectively. The beads prepared according to the optimized conditions released only about half of the entrapped catechin into SGF while most of the entrapped catechin was released into SIF after 24 h incubation.     

Voltage-gated and calcium-gated calcium release during depolarization of skeletal muscle fibers.

The role of elevated intracellular calcium concentration ([Ca2+]) in activating calcium release from the sarcoplasmic reticulum (SR) was studied in skeletal muscle fibers microinjected with strong calcium buffers. After the injection of 3.8 +/- 0.5 mM (mean +/- S.E. of mean, n = 16) BAPTA (1,2-bis[o-aminophenoxy]ethane- N,N,N',N'-tetraacetic acid) or 2.2-2.8 mM fura-2 the normal increase in [Ca2+] during a depolarizing pulse was virtually eliminated. Even though calcium was released from the SR the kinetics of this release were markedly altered: the extensive buffering selectively eliminated the early peak component of SR calcium release with no effect on the maintained steady level. Microinjections of similar volumes but with low concentrations of fura-2 had no significant effect on the release waveform. The calcium released by voltage-dependent activation during depolarization may thus be involved in activating further calcium release, that is, in a calcium-induced calcium release mechanism.