On the mechanism of stimulation of H+ transport in gastric mucosa by Ca++ ionophore A23187. II. Ca++-cyclic AMP interactions

The possibility of interactions between calcium and cyclic AMP (cAMP) in the mechanism of stimulation of H+ transport by A23187 was studied in the isolated gastric mucosa of the toad Bufo marinus. A23187 stimulated H+ secretion and histamine release. The amount of histamine released by A23187 did not explain the degree of stimulation. Metiamide partially inhibited the response to A23187. Ca++ ionophore produced an overstimulation of secretion after H+ transport had been induced by supramaximal effective concentrations of histamine (10-4 M). In the presence of metiamide, IMX potentiated the response to A23187. Also, in the same condition (metiamide treated) the effects of db-cAMP and A23187 were additive. The results are consistent with an interaction between Ca++ and ionophore-released histamine at the oxyntic cell in the stimulation by A23187. The stimulatory response may be the result of a potentiation between calcium and cAMP at the intracellular level.     

On the mechanism of stimulation of H+ transport in gastric mucosa by Ca++ ionophore A23187: I. Pathways for cytosolic calcium increase

The pathways for cytosolic Ca⁺⁺ increase under A23187 stimulation of H⁺ secretion were studied in the isolated gastric mucosa of the toad $\text{Bufo marinus}$. A23187 produced a more potent stimulation of secretion when added to the mucosal side which did not contain calcium. Measurements of ionophore incorporation by fluorometric methods indicated that A23187 incorporates into oxyntic cells intracellularly. The presence of divalent cations inhibited incorporation. This may be the reason for a more potent action when A23187 was added from the mucosal side. With-drawal of calcium from serosal solution largely inhibited the secretory response to A23187 added to the mucosal side. Reintroduction of calcium into the serosal side in the presence of ionophore elicited H⁺ secretion. The results are consistent with an uptake of A23187 from the mucosal side into cellular organelles and basolateral membranes. Calcium entry through the serosal side may be responsible for triggering secretion. Although A23187 likely releases calcium from intracellular stores, its rate of release may not be sufficient to bring about a full stimulation of secretion in serosal-Ca⁺⁺-free conditions.     

Role of calcium in the regulation of sugar transport in the avian erythrocyte: Effects of the calcium ionophore,A23187

The tissue/medium distribution of the nonmetabolized glucose analog [¹⁴C]-3-0-methyl-D-glucose was measured in pigeon erythrocytes and related to changes in ⁴⁵Ca uptake and efflux, total calcium content and ATP levels. Sugar transport was not affected by changes in external Ca²⁺. However, both sugar and ⁴⁵Ca influx were increased by the Ca-ionophore A23187. In the absence of external Ca²⁺, the ionophore caused a delayed increase in sugar transport and net loss of calcium, probably through releasing Ca²⁺ from internal storage sites into the cytoplasm. Increasing internal Na⁺ through Na⁺ pump inhibition or using the sodium ionophore monensin did not alter influx of sugar or ⁴⁵Ca, indicating Na⁺-Ca²⁺ exchange was absent in these cells. The results are consistent with A23187 causing increased Ca²⁺ influx or release from mitochondrial storage and the resulting rise in cytoplasmic Ca²⁺ stimulating hexose transport. Experiments with low Mg⁺⁺ and high K⁺ media and measurements of ATP levels exclude alternative explanations for the action of A23187. We conclude that sugar transport regulation in avian erythrocytes is Ca²⁺-dependent and resembles that in muscle in its basic mechanism. It differs in the response to some modulating agents, largely because of a different pattern of Ca²⁺ fluxes in these cells.     

Effect of dopa-decarboxylase inhibition on Aedes aegypti eggs: evidence for sclerotization

Newly deposited fertilized and unfertilized Aedes aegypti eggs are soft and white. Within a short time they darken and harden. Injection of a potent dopa decarboxylase inhibitor (dl)-3-(3,4-dihydroxyphenyl)-2-hydrazino-2-methylpropionic acid (α-MDH) into females, subsequent to a blood meal, results in oviposited eggs which are pale in colour. Moreover, such fertilized eggs do not hatch. The severity of both effects seems to be positively correlated and is dependent upon the time of α-MDH injection.Extracts of mature ovaries are capable of converting dopa to dopamine in the absence of a pretreatment with α-MDH. Mature ovaries obtained from females who had been previously injected with α-MDH could not accomplish this conversion. The inhibitor does not seem to have any effect on dopa oxidase activity and subsequent melanization. We conclude that dopamine is synthesized by blood-fed females via decarboxylation of dopa by dopa decarboxylase and propose that the normal darkening and hardening of A. aegypti eggs is a result of sclerotization.     

Effects of the calcium ionophore A-23187 on the regulation of sugar transport in muscle

The distribution of (¹⁴C)-3-0-methyl-D-glucose and of (⁴⁵Ca) was followed in perifused left atria and intact hemidiaphragms of the rat. The carboxylic calcium ionophore A-23187 affected sugar and Ca²⁺ influx in parallel, with low concentrations inhibiting and higher ones stimulating influx under basal conditions. The stimulation of sugar transport by insulin, high concentrations of adrenaline or ouabain, or by K⁺-free medium was antagonized by the calcium ionophore. Likewise, A-23187 counteracted the depression of sugar transport caused by low concentrations of ouabain or adrenaline. These results support a role of Ca²⁺ in the regulation of sugar transport in muscle. However, increased influx of Ca²⁺ cannot explain all the effects of A-23187. It is suggested that the ionophore may also act by releasing Ca²⁺ from intracellular storage and binding sites.     

Ionomycin-mediated calcium transport in rigid and fluid liposomes

The antibiotic ionophore ionomycin translocates Ca from an aqueous medium into or across an organic immiscible phase. At pH 8.0, ionomycin translocates less Ca than A23187, the effects of these ionophores being additive to one another. The capacity of ionomycin to translocate Ca across the organic phase is dramatically decreased when the pH of the aqueous media is reduced from 8.0 to 7.5 or lower values. Ionomycin also mediates Ca exchange-diffusion in liposomes, the magnitude of such a process being greater in fluid than in rigid liposomes. At a physiological pH (7.4), ionomycin is unexpectedly as potent as A23187 in mediating Ca transport in fluid liposomes. These findings suggest that the capacity of ionophores to translocate Ca across model membranes depends on both the transverse and lateral mobility of the ionophoretic molecules. The relative importance of the latter phenomenon itself largely depends on the stoichiometry of the Ca-ionophore complex.     

Interaction of covalently closed circular PM-2 DNA and hedamycin.

The interaction of hedamycin with covalently closed circular PM-2 DNA was examined. Hedamycin produced strand breakage detectable in alkaline sucrose gradients. Under neutral conditions hedamycin inhibited ethidium bromide binding and induced conformational changes in PM-2 DNA.     

Protein substrate specificity of a calmodulin-dependent protein kinase isolated from bovine heart

The protein substrate specificity of a calmodulin-dependent protein kinase activity from the cytosolic fraction of bovine heart was examined. Prior to the experiments, the kinase activity was purified more than 50-fold with a recovery of greater than 10% of the homogenate activity. Two endogenous protein substrates of molecular weight 57,000 and 73,000 were phosphorylated in these kinase preparations. The kinase preparation was also able to phosphorylate exogenous synapsin, phospholamban, glycogen synthase, MAP-2, myelin basic proteins and κ-casein, but not tubulin, pyruvate kinase, the regulatory subunit of cAMP protein kinase II, myosin light chain or phosphorylase b. High levels of calmodulin were required for activation of the kinase activity toward the 57,000 and 73,000 molecular weight endogenous substrates (K_0.5 = 93 +/- 5 nM), glycogen synthase (K_0.5 = 127 +/- 10 nM), and κ-casein (K_0.5 = 321 +/- 107 nM). The kinase possessed a high affinity for glycogen synthase (half maximal activity at 0.9 +/- 0.4 μM) but a low affinity for κ-casein (21 +/- 2 μM). Sucrose density gradient centrifugation separated the calmodulin-dependent protein kinase activity into two fractions with apparent molecular weights of approximately 900,000 and 100,000. Both fractions phosphorylated the endogenous 57,000 molecular weight substrate and glycogen synthase similarly. These results indicate that cardiac calmodulin-dependent protein kinase previously observed to phosphorylate endogenous protein substrate possesses a wide range of substrate specificity.     

The effects of a macrophage-derived cytotoxin on the growth and metabolism of target cells.

Peritoneal macrophages obtained from Sarcoma I (SaI)-immune CS7BL/6 mice release a heat-labile cytotoxin, specific macrophage cytotoxin (SMC), following a two hour interaction with appropriate target cells. Specific macrophage cytotoxin specifically inhibits A/Jax spleen cells from mitogenically responding to concanavalin A, whereas syngeneic CS7BL/6 spleen cells are unaffected. Treatment of target cells with SMC results in early alterations in RNA and DNA metabolism. The uptake and incorporation of ³H-uridine was found to be initially elevated while intracellular levels of ³H-thymidine became markedly reduced. Furthermore, the cytotoxic action of SMC was found to be rapidly accelerated and amplified by low levels of actinomycin-D.     

Ca entry at rest and during prolonged depolarization in dialyzed squid axons

Ca influx has been studied in squid axons under internal dialysis control. In axons dialyzed with "normal" physiological conditions (Nai = 40-50 mM, Cai2+ = 0.06-0.1 microM, ATP = 2 mM, Ki = 310 mM), 70% of the resting Ca influx is sensitive to external TTX (K0.5 congruent to 5 nM), 20% of it can be accounted by the reversal of the Na-Ca exchange, and the remaining fraction (10%) is insensitive to TTX, D-600, and Nai. The Ca antagonic drug D-600 (50-100 microM) has an inhibitory effect on the resting Ca influx. This compound was found to affect both the TTX sensitive and the Nai-dependent Ca influx components. In the presence of Nai and ATP, Cai2+ activates the carrier mediated Ca entry (Nai-dependent Ca influx). Most of the activation occurs in the submicromolar range of Cai2+ concentrations (K0.5 congruent to 0.6 microM). In the absence of Nai and/or ATP, no activation of Ca influx by Cai2+ was found up to about 5 microM Cai2+. Prolonged depolarization with high Ko causes an increase in Ca influx sustained for long time (minutes). Depolarizing the axons by removing Ki causes the same effect. This depolarization-induced Ca entry was only observed in axons containing Nai. In the absence of Nai, Ca influx decreases with increasing Ko. The activation of the carrier mediated Ca entry (electrogenic Na/Ca exchange) by membrane depolarization was found to be markedly dependent on the magnitude of Ca2+ i. Increasing the magnitude of Ca2+ i from 0.1 to 0.6 microM causes a ten fold increase in the extra Ca influx induced by a K-depolarization.     

The effect of calcium oxalate crystallization kinetics on the kinetics of calcium uptake and calcium ATPase activity of sarcoplasmic reticulum vesicles

The ionophore A23187 is a potent inhibitor of oxalate supported calcium uptake if added before uptake is initiated by ATP and is a much weaker inhibitor of uptake once uptake has been initiated. This observation is shown to be due to a failure of oxalate to capture the transported calcium at the beginning of uptake because the rate of calcium oxalate crystallization is initially slow, thereby allowing the ionophore to release the accumulated calcium. This hypothesis is supported by the observation that calcium oxalate crystallization shows a lag phase which is absent when calcium oxalate seeds are in the reaction system. Once calcium uptake has progressed, calcium oxalate seeds are present in the sarcoplasmic reticulum and calcium oxalate crystallization proceeds sufficiently rapidly that the ionophore cannot compete successfully for calcium. That A23187 and oxalate compete for intravesicular ionic calcium is shown by the stimulation which each produces in ATPase activity and by the dependence of ionophore activity on oxalate concentration.The failure of calcium oxalate crystallization to reach equilibrium during the early phase of calcium uptake caused us to examine whether at any time during calcium uptake, crystallization reaches equilibrium. Skeletal sarcoplasmic reticulum accumulated calcium at such a high rate that oxalate, in concentrations up to 20mM, was unable to clamp intravesicular calcium at equilibrium values. The lower rate of calcium accumulation by cardiac sarcoplasmic reticulum and/or perhaps its greater permeability to oxalate apparently allows intravesicular calcium to be clamped by oxalate.     

The sequestration of Ca2+ by mitochondria in rat heart cells

Rat heart ventricular cells, purified by Percoll density gradient centrifugation, were incubated in the presence of 1.3 mM CaCl2. After 20 min incubation, samples of the cells were lysed in medium containing 0.3 mM digitonin, ruthenium red and EGTA, and a mitochondrial fraction was isolated at intervals thereafter. Extrapolation of the mitochondrial 45Ca2+ contents to zero time enabled the endogenous 45Ca2+ to be estimated at the time of cell lysis. The lysis conditions yielded essentially complete release of lactate dehydrogenase from the cells, but caused negligible damage to the mitochondria as judged by their retention of glutamate dehydrogenase, and their ability to accumulate and retain Ca2+ in the absence of ruthenium red and EGTA. The data indicate that about 13% of total cell Ca2+ only may be mitochondrial in vivo.     

Calcium influx is not required for thyrotropin-releasing hormone stimulation of prolactin release from GH3 cells

TRH stimulation of prolactin release from GH3 cells is dependent on Ca2+; however, whether TRH-induced influx of extracellular Ca2+ is required for stimulated secretion remains controversial. We studied prolactin release from cells incubated in medium containing 110 mM K+ and 2 mM EGTA which abolished the electrical and Ca2+ concentration gradients that usually promote Ca2+ influx. TRH caused prolactin release and 45Ca2+ efflux from cells incubated under these conditions. In static incubations, TRH stimulated prolactin secretion from 11.4 +/- 1.2 to 19 +/- 1.8 ng/ml in control incubations and from 3.2 +/- 0.6 to 6.2 +/- 0.8 ng/ml from cells incubated in medium with 120 mM K+ and 2 mM EGTA. We conclude that Ca2+ influx is not required for TRH stimulation of prolactin release from GH3 cells.     

Transport and control of Ca2+ by pigeon erythrocytes. I. Survey of some cell responses to a range of A23187 doses in the presence of Ca2+

Cells were subjected to a range of 45Ca2+ influx loads with A23187. We measured cell 45Ca2+ with time and A23187 dose, and the apparent 45Ca2+ influxes (identical to "J(in,app)") at Ca2+ steady state. We also measured endogeneous exchangeable and total cell Ca2+, which were 50 and 17-220 microM respectively. The effects of A23187 and Ca2+ on cell ATP, swelling, net Cl- permeability, and cell morphology were measured. These were modest and do not affect our conclusions. J(in,app) congruent to 3 X 10(-4) [A23187]2.9 X [Ca2+(o)]mumoles/l X min with 92-552 microM [Ca2+(o)] (identical to external Ca2+ concentration) and 0-7 microM A23187. J(in,app) was increased an order of magnitude by vanadate and is probably much less than the true influx. The least unlikely explanation found for the high [A23187] exponent, 2.9, was that most of the Ca2+ crossing the membrane is expelled by the pump before it can move deeper into the cell. Calcium pumping increased rapidly in response to increased influx, but the steady state cell 45Ca2+ was approximately proportional to J(in,app) rather than approximately constant between 10 and 120 mumoles/l X min with 184 microM [Ca2+(o)]. This is not the result expected from a simple feedback mechanism. At high A23187 doses the pump appears fully activated resulting in a linear relation between cell/medium 45Ca2+ and [A23187]-2. From the plot we calculated alpha identical to free/total exchangeable Ca2+ = 0.38 +/- 0.08 (n = 3) and a maximum pump rate, "Pmax" = 78 mumole/l X min. Pmax is underestimated insofar as J(in,app) is less than the true influx.     

Column chromatography and electrophoresis of bovine uterine lumenal proteins collected during the estrous cycle

Uteri of 29 normally cycling Holstein and Jersey cows were non-surgically flushed with 50 ml sterile 1.5% saline, and fluids were recovered to evaluate biochemical methods for determination of qualitative changes in uterine lumenal protein at known stages of the estrous cycle. Total protein (mg) and number of red blood cells (million/ml) were 17.7 and 9.8; 7.6 and 7.1; 9.1 and 6.0; and 26.2 and 2.8 at day 0, 5, 10 and 15 of the bovine estrous cycle. Column chromatography (Sephacryl S-200) of uterine secretions revealed seven uterine specific peaks at ambient temperatures. One peak may be a hemoglobin contaminant. Five uterine specific protein peaks representing proteins greater than 160 000, ～ 25 000 and less than 13 700 mol. wt. (3) were eluted with high performance liquid chromatography. Native polyacrylamide disc gel electrophoresis fractionated uterine fluid into as many as 13 bands. There were differences in six protein bands between uterine fluid and plasma. The consistency between Sephacryl S-200 and high performance liquid chromatography is the presence of three to four low molecular weight (< 13 700) uterine specific proteins. Sephacryl S-200 chromatography resulted in elucidation of a uterine specific protein approximately 60 000 mol. wt. not found with high performance liquid chromatography. However, proteins with mol. wt. > 160 000 and approximately 25 000 were found with high performance liquid chromatography. Results indicate no differences in protein class during the estrous cycle and that red blood cell contamination must be monitored during qualitative evaluation of uterine proteins.     

The mechanism of regulation of fibroblastic cell replication. III. A central role for nutrient limitation: a conditioning effect due to serum imprinting of the cell response

Haploid Saccharomyces cerevisiae cells of mating type a, but not α, produce and secrete a diffusible substance, designated a factor. The a factor transiently arrests cells of mating type α, but not a, at a very early stage of the cell cycle, prior to budding and to the initiation of DNA synthesis. While the cells are arrested at this stage, few, if any, of the functions required for the ensuing cell cycle are carried out. This stage of the cell cycle coincides with the stage at which α factor, produced by cells of mating type a, specifically arrests cells of mating type a [2]. It seems probable that the reciprocally acting a and α factors together provide the mechanism by which haploid cells are synchronized to the appropriate stage of the cell cycle as a prelude to conjugation.     

Suppressor cell influence in selected strains of inbred rats: I. Strain-dependent mitogen- and antigen-related low responsiveness

Lymphocytes derived from Lewis (LE), Brown-Norway (BN), or the F₁ hybrid (LBNF₁) rats respond in vitro to the mitogens phytohemagglutinin, concanavalin A, and pokeweed mitogen. The magnitude of the response, as determined by incorporation of ³H-thymidine, ¹⁴C-adenine, and ³H-leucine, was highest for LE and lowest for BN animals. These proliferation response differences were observed for lymph node lymphocytes and peripheral blood lymphocytes. The response to antigen, as measured by lymphocyte transformation, reflected the mitogen responsiveness of the strains tested, i.e., LE animals responded to a higher level than did BN animals. Equivalent levels of antibody were found in all animals immunized with antigen. In addition, BN rats are suppressed to a greater magnitude than are LE rats when both strains are primed, rechallenged, and assayed via lymphocyte transformation to the test antigen.     

Stimulation of arachidonic acid metabolism in primary cultures of osteoblast-like cells by hormones and drugs

The effects of parathyroid hormone (PTH), dihydroxycholecalciferol (1,25-(OH)₂ D₃), thrombin, epidermal growth factor (EGF) and 12-o-tetradecanoylphorbol-13-acetate (PMA) on the biosynthesis and release of arachidonic acid metabolites were studied in primary cultures of osteoblast-like cells isolated from 18-day-old chick embryo calvaria. Cells were labelled with (¹⁴C)-arachidonic acid for 30 h. The radioactive eicosanoids were extracted from the cell culture media after a further 30 h stimulation period and analysed on a PRP-1 column by HPLC. The radioactive products were characterized by co-elution of (³H) standard prostanoids. Osteoblasts showed a basal release of the prostanoids 6-keto-PGF_1α, TXB₂, PGF_2α, PGE₂, PGD₂ and PGB₂, the latter being the most abundant one. Indomethacin (10⁻⁵ M) effectively inhibited the basal release, but not that of an as yet unidentified compound. The release of prostanoids was stimulated by PTH (2 U/ml), thrombin (0.4 NIH/ml), EGF (50 ng/ml) and PMA (25 ng/ml), the latter being by far the most potent one. 1,25-(OH)₂D₃ was found to slightly inhibit the prostanoid release. These results indicate: (1) primary cultures of osteoblasts synthesize several prostaglandins, thromboxane B₂ and one unidentified product. (2) the action on bone of PTH and the various drugs tested may be, at least partly, mediated by an increased prostaglandin production by osteoblasts. Clearly this does not apply to 1,25-(OH)₂D₃.