Studies on the size,location and turnover of calcium pools accessible to growing Tetrahymena cells

Tetrahymena pyriformis cells in the logarithmic phase of growth accumulate 2.5–3.75 times as much calcium per unit volume as is present in the growth medium. It appears that most of this calcium is stored in a non-ionic form, with approximately 30% existing in the cilia, near its site of action in effecting ciliary reversal. The exchange of extracellular ⁴⁵Ca²⁺ with the major internal pools is extremely rapid, exhibiting a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of less than 0.5 h. Sites located on the cilia are responsible for 35–50% of Ca²⁺ influx, with the remainder entering through other positions on the cell surface.     

Calcium pools,calcium entry,and cell growth

The Ca²⁺ pump and Ca²⁺ release functions of intracellular Ca²⁺ pools have been well characterized. However, the nature and identity of Ca²⁺ pools as well as the physiological implications of Ca²⁺levels within them, have remained elusive. Ca²⁺ pools appear to be contained within the endoplasmic reticulum (ER); however, ER is a heterogeneous and widely distributed organelle, with numerous other functions than Ca²⁺ regulation. Studies described here center on trying to determine more about subcellular distribution of Ca²⁺ pools, the levels of Ca²⁺ within Ca²⁺ pools, and how these intraluminal Ca²⁺ levels may be physiologically related to ER function. Experiments utilizingin situ high resolution subcellular morphological analysis of ER loaded with ratiometric fluroescent Ca²⁺ dyes, indicate a wide distribution of inositol 1,4,5-trisphosphate (InsP₃)-sensitive Ca²⁺ pools within cells, and large changes in the levels of Ca²⁺ within pools following InsP₃-mediated Ca²⁺ release. Such changes in Ca²⁺ may be of great significance to the translation, translocation, and folding of proteins in ER, in particular with respect to the function of the now numerously described luminal Ca²⁺-sensitive chaperonin proteins. Studies have also focussed on the physiological role of pool Ca²⁺ changes with respect to cell growth. Emptying of pools using Ca²⁺ pump blockers can result in cells entering a stable quiescent G₀-like growth state. After treatment with the irreversible pump blocker, thapsigargin, cells remain in this state until they are stimulated with essential fatty acids whereupon new pump protein is synthesized, functional Ca²⁺ pools return, and cells reenter the cell cycle. During the Ca²⁺ pool-depleted growth-arrested state, cells express a Ca²⁺ influx channel that is distinct from the store-operated Ca²⁺ influx channels activated after short-term depletion of Ca²⁺ pools. Overall, these studies indicate that significant changes in intraluminal ER Ca²⁺ do occur and that such changes appear linked to alteration of essential ER functions as well as to the cell cycle-state and the growth of cells.     

Depletion and refilling of intracellular calcium pools in bovine chromaffin cells

To investigate Ca²⁺ uptake by Ca²⁺-depleted bovine chromaffin cells we depleted these cells of Ca²⁺ by incubating them in Ca²⁺-free buffer, then measured changes in cytoplasmic Ca²⁺ concentration ([Ca²⁺ ₁)⁴⁵Ca²⁺ uptake, and Mn²⁺ uptake in response to added Ca²⁺ or MN²⁺. In depleted cells, the increase in [Ca²⁺]_i after Ca²⁺ addition, and the Mn²⁺ and⁴⁵Ca²⁺ uptakes were higher than in control cells, and were inhibited by verapamil. The size of the intracellular Ca²⁺ pools in depleted cells increased after Ca²⁺ addition. The times for [Ca²⁺]_i rise and Mn²⁺ entry to reach plateau levels were much shorter than the time for refilling of intracellular Ca²⁺ stores. In Ca²⁺-depleted cells and cells which had been loaded with BAPTA,⁴⁵Ca²⁺ uptake was much higher than in control cells. These results suggest that extracellular Ca²⁺ enters the cytoplasm first before refilling the intracellular stores. The rate of Mn²⁺ influx depended on the level of filling of the Ca²⁺ stores, suggesting that some signalling takes place between the intracellular stores and Ca²⁺ entry pathways through the plasma membrane.Abbreviations used BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid - BAPTA/AM acetoxymethyl ester of BAPTA - [Ca²⁺]_i cytosolic Ca²⁺ concentration - IP₃ inositol 1,4,5-trisphosphate - tBHQ 2,5-di-(t-butyl)-1,4-benzohydroquinone This work was included in a thesis submitted by A.-L. Sui to the Department of Biochemistry, National Yang-Ming Medical College, in partial fulfillment of the requirements for the degree of Doctor of Philosophy     

Actin filament turnover regulated by cross-linking accounts for the size, shape, location, and number of actin bundles in Drosophila bristles

Drosophila bristle cells are shaped during growth by longitudinal bundles of cross-linked actin filaments attached to the plasma membrane. We used confocal and electron microscopy to examine actin bundle structure and found that during bristle elongation, snarls of uncross-linked actin filaments and small internal bundles also form in the shaft cytoplasm only to disappear within 4 min. Thus, formation and later removal of actin filaments are prominent features of growing bristles. These transient snarls and internal bundles can be stabilized by culturing elongating bristles with jasplakinolide, a membrane-permeant inhibitor of actin filament depolymerization, resulting in enormous numbers of internal bundles and uncross-linked filaments. Examination of bundle disassembly in mutant bristles shows that plasma membrane association and cross-bridging adjacent actin filaments together inhibits depolymerization. Thus, highly cross-bridged and membrane-bound actin filaments turn over slowly and persist, whereas poorly cross-linked filaments turnover more rapidly. We argue that the selection of stable bundles relative to poorly cross-bridged filaments can account for the size, shape, number, and location of the longitudinal actin bundles in bristles. As a result, filament turnover plays an important role in regulating cytoskeleton assembly and consequently cell shape.     

Rapid turnover of calcium in the endoplasmic reticulum during signaling. Studies with cameleon calcium indicators

HEK293 cells expressing the thyrotropin-releasing hormone (TRH) receptor were transfected with cameleon Ca(2+) indicators designed to measure the free Ca(2+) concentration in the cytoplasm, [Ca(2+)](cyt), and the endoplasmic reticulum (ER), [Ca(2+)](er). Basal [Ca(2+)](cyt) was about 50 nm; thyrotropin-releasing hormone (TRH) or other agonists increased [Ca(2+)](cyt) to 1 micrometer or higher. Basal [Ca(2+)](er) averaged 500 micrometer and fell to 50-100 micrometer over 10 min in the presence of thapsigargin. TRH consistently decreased [Ca(2+)](er) to 100 micrometer, independent of extracellular Ca(2+), whereas agonists for endogenous receptors generally caused a smaller decline. When added with thapsigargin, all agonists rapidly decreased [Ca(2+)](er) to 5-10 micrometer, indicating that there is substantial store refilling during signaling. TRH increased [Ca(2+)](cyt) and decreased [Ca(2+)](er) if applied after other agonists, whereas other agonists did not alter [Ca(2+)](cyt) or [Ca(2+)](er) if added after TRH. When Ca(2+) was added back to cells that had been incubated with TRH in Ca(2+)-free medium, [Ca(2+)](cyt) and [Ca(2+)](er) increased rapidly. The increase in [Ca(2+)](er) was only partially blocked by thapsigargin but was completely blocked if cells were loaded with 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. In conclusion, these new Ca(2+) indicators showed that basal [Ca(2+)](er) is approximately 500 micrometer, that [Ca(2+)](er) has to be >100 micrometer to support an increase in [Ca(2+)](cyt) by agonists, and that during signaling, intracellular Ca(2+) stores are continuously refilled with cytoplasmic Ca(2+) by the sarcoendoplasmic reticulum Ca(2+)-ATPase pump.     

Studies on the turnover of endogenous choline-containing phospholipids of cultured neuroblastoma cells

The effects of two polypeptide antibiotics, polymixin B and gramicidin S, on the intracellular pool size and turnover of guanosine tetraphosphate (ppGpp) were analyzed in stringent (relA+) and relaxed (relA) strains of Escherichia coli. When either one of these two drugs was added to stringent bacteria cultures at a final concentration that blocked protein and RNA synthesis, ppGpp was found to accumulate. Under similar conditions of inhibition of macromolecular synthesis, ppGpp also appeared to accumulate in relaxed bacteria. Moreover, in either type of strain, no significant accumulation of guanosine pentaphosphate (pppGpp) could be detected upon drug treatment. It was, therefore, concluded that polymixin and gramicidin elicit ppGpp accumulation through a mechanism independent of the relA gene product and, consequently, quite distinct from the stringent control system triggered by amino acid starvation. Further experiments performed by using tetracycline as an inhibitor of ppGpp synthesis, showed that the increase in the level of this nucleotide induced by drug action was due, in fact, to a strong restriction of its degradation rate.     

Nucleotide pools of growing,synchronized and stressed cultures of Saccharomyces cerevisiae

High pressure liquid chromatography has been used to study the acid soluble nucleotide pool of Saccharomyces cerevisiae under different conditions of growth. ATP, ADP, AMP, NAD, GTP, UTP, UDP, CTP, CDP, and UDP-sugars plus UMP could be separated and were found in concentrations higher than 0.1 mumol per g yeast cell dry weight (= detection limit). During glucose-limited continuous culture the levels of individual nucleotides depended on the growth rate, which was most pronounced with pyrimidine (uridine, cytidine) nucleotides. The energy charge (E.C.) remained high (0.9) at all growth rates (0.07-0.3 h-1). During synchronized growth at a constant growth rate (0.11 h-1) almost all nucleotide levels and the E.C. remained at constant values with the only exception of UDP-sugars and UMP of which increased levels were found during the phase of budding. Under conditions of metabolic stress (addition of antimycin A, deoxyglucose plus iodoacetate) pronounced changes in the levels of purine (adenine and guanine) nucleotides and the E.C. were observed. All other nucleotides were less influenced by these conditions. Only under these conditions IMP accumulation was observed. The results strongly argue against the significance of purine nucleotide or E.C. measurements under viable conditions. In contrast, changes in the levels of pyrimidine nucleotides seem to be indicative of changes in the flux through the metabolic pathways where they act as coenzymes.     

Quantitative partition of protein,carbohydrate and fat pools in growing pigs

A model combining data from balance experiments with data from oxidation of nutrients demonstrating the pools of protein, carbohydrate and fat and their partition in the body was presented.

Data from more than 200 experiments with growing pigs were used to fill up the “black boxes”; in the model and to discuss the pattern between catabolic and anabolic processes of the different nutrients.

With a ME‐intake of 1.3 MJ/kg^0.75 the proportion of retained protein from the protein pool varied from 50 to 75% depending on the age of animals and the protein quality. At a low protein intake and ME of 0.6 MJ/kg^0.75 the utilization of protein was reduced to about 25% as a substantial part of the protein pool was oxidized.

The carbohydrate group from the protein sources constituted less than 5% of the carbohydrate pool. The oxidation of carbohydrates was between 50–75% of the pool in all experiments, while the rest was used for lipogenesis. Even on a low feeding level the pattern occurred indicating a requirement for specific substances formed in the body.

Lipogenesis constituted 46–96% of the fat pool depending of the amount on digested fat in the different experiments. No oxidation of fat was observed in experiments with pigs (30–90 kg LW) on a high feeding level in spite of the broad variation in the amount of digested fat, indicating a complete storage of the fat pool into body fat. In experiments with piglets (3–9 kg LW) on the same energy intake fat oxidation of about 30% occurred, probably caused by the high energy requirement of the piglets. With a ME‐intake of 0.6 MJ/kg^0.75 the oxidation of fat was higher than the fat pool for pigs between 30–60 kg LW, thereby causing oxidation of body fat to cover the energy requirements.     

Two distinct calcium pools in the endoplasmic reticulum of HEK-293T cells

Agonist-sensitive intracellular Ca2+ stores may be heterogeneous and exhibit distinct functional features. We have studied the properties of intracellular Ca2+ stores using targeted aequorins for selective measurements in different subcellular compartments. Both, HEK-293T [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] and HeLa cells accumulated Ca2+ into the ER (endoplasmic reticulum) to near millimolar concentrations and the IP3-generating agonists, carbachol and ATP, mobilized this Ca2+ pool. We find in HEK-293T, but not in HeLa cells, a distinct agonist-releasable Ca2+ pool insensitive to the SERCA (sarco/endoplasmic reticulum Ca2+ ATPase) inhibitor TBH [2,5-di-(t-butyl)-benzohydroquinone]. TG (thapsigargin) and CPA (cyclopiazonic acid) completely emptied this pool, whereas lysosomal disruption or manoeuvres collapsing endomembrane pH gradients did not. Our results indicate that SERCA3d is important for filling the TBH-resistant store as: (i) SERCA3d is more abundant in HEK-293T than in HeLa cells; (ii) the SERCA 3 ATPase activity of HEK-293T cells is not fully blocked by TBH; and (iii) the expression of SERCA3d in HeLa cells generated a TBH-resistant agonist-mobilizable compartment in the ER. Therefore the distribution of SERCA isoforms may originate the heterogeneity of the ER Ca2+ stores and this may be the basis for store specialization in diverse functions. This adds to recent evidence indicating that SERCA3 isoforms may subserve important physiological and pathophysiological mechanisms.     

Pseudopterosin A inhibits phagocytosis and alters intracellular calcium turnover in a pertussis toxin sensitive site in Tetrahymena thermophila

The free living ciliate Tetrahymena thermophila was chosen as a cellular model in order to investigate the mode of action of the anti-inflammatory marine natural product Pseudopterosin A (PsA). In this paper we present evidence that PsA inhibits phagosome formation (KD=10.5 microM) and triggers a discrete intracellular calcium release (depletion) from a site in T. thermophila cells (KD=6.4 microM). Pre-treatment with the Gi/o protein inhibitor, pertussis toxin (PTX), inhibits PsA activity of both responses providing pharmacological evidence that the site of action for PsA is at a PTX sensitive G protein or a G protein coupled receptor (GPCR). Addition of extracellular calcium induced a concentration dependent increase in the incidence of phagosome formation (KD=30.3 microM) and was blocked by PsA pre-treatment. This particular effect of PsA on extracellular calcium was not blocked by PTX pre-treatment.