Stimulation of Ca efflux from fura-2-loaded platelets activated by thrombin or phorbol myristate acetate

We investigated the restoration of [Ca²⁺]_i in fura-2-loaded human platelets following discharge of internal Ca²⁺ stores in the absence of external Ca²⁺. After stimulation by thrombin [Ca²⁺]_i returned from a peak level of 0.6 μM to resting levels within 4 min. When ionomycin discharged the internal stores the recovery was slower with [Ca²⁺]_i still elevated at around 0.5 μM after 5 min. Thrombin added shortly after ionomycin could accelerate the recovery of [Ca²⁺]_i and restore resting levels within 5 min, an effect that was mimicked by phorbol-12-myristate-13-acetate (PMA). Since the continued presence of ionomycin precluded reuptake into the internal stores we conclude that thrombin and PMA stimulate Ca²⁺ efflux, perhaps via protein kinase C actions on a plasma membrane Ca²⁺ pump.     

The effects of substance P and carbachol on inositol tris- and tetrakisphosphate formation and cytosolic free calcium in rat parotid acinar cells. A correlation between inositol phosphate levels and calcium entry

Both substance P and carbachol produced increases in inositol tris- and tetrakisphosphate and increased cytosolic free [Ca2+] in dispersed parotid acinar cells loaded with fura-2. The increase in [Ca2+]i in response to each agonist was due to a combination of mobilization of internal Ca2+ and entry of extracellular Ca2+. Kinetic studies of the initial response to substance P, and measurement of peak [Ca2+]i, demonstrated that the initial rapid rise in [Ca2+]i was due to both internal release and entry of Ca2+. Substance P could evoke a greater initial increase in [Ca2+]i and inositol trisphosphate than could carbachol. However, after 1 min in the presence of external Ca2+, the maintained [Ca2+]i level in response to substance P was considerably smaller than that seen with carbachol, an effect apparently due to homologous desensitization of the substance P receptor. The two agonists each produced a similar 4-5-fold increase in inositol tetrakisphosphate levels within 30 s; this level was maintained in the presence of carbachol, but decreased with substance P. Similarly, the level of inositol (1,4,5)-trisphosphate decreased after prolonged incubation with substance P. Thus, the maintained level of [Ca2+]i, and by deduction Ca2+ entry, correlated with the levels of inositol (1,4,5)-trisphosphate and inositol tetrakisphosphate; a result consistent with a possible role for these inositol phosphates in the control of receptor-mediated Ca2+ channels.     

Growth,ammonia accumulation and glutamine synthetase activity in alfalfa (Medicago sativa L.) shoots and cell cultures treated with phosphinothricin

Summary Phosphinothricin is a non-selective herbicide which inhibits glutamine synthetase (EC 6.3.1.2) activity causing an overaccumulation of ammonia in higher plants. Alfalfa (Medicago sativa L) shoot tissue and petiole-derived callus exposed to phosphinothricin show 50 and 70% reductions, respectively, in glutamine synthetase activity with a concomitant rise of 10 and 20 fold, respectively, in endogenous ammonia. The diffusibility of ammonia may limit the use of a detoxifying gene, phosphinothricin acetyltransferase, as a selectable marker for alfalfa transformation. However, the addition of up to 40 times the standard levels of ammonium nitrate to the culture media used in this study had no effect on callus growth, although glutamine synthetase activity was inhibited by 50% and endogenous ammonia increased 27 fold. Therefore, ammonia accumulation may not be the primary cause of cell death in alfalfa after exposure to phosphinothricin. It follows that diffusion of ammonia from cell to cell would not restrict the selection for phosphinothricin acetyltransferase transformed cells, thereby indicating that this enzyme could be used as a selectable marker in transformation experiments.Abbreviations PPT Phosphinothricin - PAT Phosphinothricin acetyltransferase     

Allometric shape change and heterochrony in the freeliving coral Trachyphyllia bilobata (Duncan)

Regression analysis has been used to study the relationship between age, size, shape, and surface area in two ancestral-descendant populations of the Neogene Caribbean coral Trachyphyllia bilobata. Analyses of the relationship between size and age show that the relationship is isometric and that little difference occurs between populations in mean corallite length or height and in their rates of growth. Onset of columella growth is significantly earlier, however, in the descendant population. Studies of the relationship between size and shape show that growth is allometric, with shape change occurring in both corallum elongation and pinching of the corallite wall during ontogeny. In the descendant population, pinching and elongation initiate earlier in the ontogeny of the coral. These results suggest that the evolutionary development of the meandroid form in freeliving corals has been accomplished by heterochrony, involving a complex set of disassociated peramorphic changes in ontogeny accompanied by paedomorphic changes in astogeny. Further analyses show that the observed heterochronic changes serve to decrease corallum surface area which may in turn enhance sediment removal and nutrition in unstable habitats.     

Thrombin and ionomycin can raise platelet cytosolic Ca2+ to micromolar levels by discharge of internal Ca2+ stores: studies using fura-2

In the presence of 1 mM EGTA, the addition of the calcium ionophore ionomycin to human platelets loaded with 30 microM fura-2 could elevate [Ca2+]i from less than 100 nM to a maximum of greater than 3 microM, presumably by discharge of Ca2+ from internal stores. Under the same conditions thrombin could maximally increase [Ca2+]i to a peak of greater than 1 microM which then declined to near resting levels within 3-4 minutes; by contrast in platelets loaded with 1 mM quin2 thrombin could raise [Ca2+]i to only about 200 nM. In the presence of 1 mM Ca2+ the peak response to thrombin in fura-2-loaded platelets was higher (1.4 microM) than that observed in the presence of EGTA (1.1 microM) and the elevation in [Ca2+] was prolonged, presumably by Ca2+ influx. These results with fura-2-loaded platelets indicate that mobilisation of internal Ca2+ can contribute a substantial proportion of the early peak [Ca2+]i evoked by thrombin directly confirming the deductions from previous work with different loadings of quin2. Under natural conditions the major role of Ca2+ influx may be to prolong the [Ca2+]i rise rather than to make it larger.     

Platelet activating factor raises intracellular calcium ion concentration in macrophages

Peritoneal cells from thioglycollate-stimulated mice were allowed to adhere to coverglasses for 2 h to give a dense monolayer of adherent cells greater than 95% of which were macrophages. After incubation with the tetra-acetoxymethyl ester of quin2, coverglasses were rinsed with Ca2+-free saline, oriented at a 45 degree angle in square cuvettes containing a magnetically driven stir bar, and analyzed for changes in quin2 fluorescence in a spectrofluorimeter. Such fluorescence, taken as an indication of intracellular calcium ion concentration ([Ca2+]i), increased as exogenous calcium ion concentration ([Ca2+]o) was raised to 1 mM. At [Ca2+]o approximately equal to 10 microM, [Ca2+]i = 72 +/- 14 nM (n = 26); at [Ca2+]o = 1 mM, [Ca2+]i = 140-220 nM, levels not increased by N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine, a membrane-permeant chelator of heavy metals than can quench quin2. Addition of mouse alpha + beta fibroblast interferon, lipopolysaccharide, thrombin, collagen, vasopressin, ADP, compound 48/80, or U46619 did not change [Ca2+]i. However, addition of platelet activating factor (PAF) (2-20 ng/ml) raised [Ca2+]i by 480 nM within 1 min if [Ca2+]o = 1 mM. In the presence of 5 mM EGTA, PAF raised [Ca2+]i by 25 nM. This suggests that PAF causes influx of exogenous Ca2+, as well as releasing some Ca2+ from intracellular stores. Consistent with these results, when PAF was added to 1 mM Ca2+ in the presence of 100 microM Cd2+ or Mn2+ to block Ca2+ influx, [Ca2+]i increased by only intermediate amounts; at the times of such dampened peak response, [Ca2+]i could be raised within 1 min to normal PAF-stimulated levels by chelation of the exogenous heavy metals with diethylenetriaminepentaacetic acid. Normal PAF responses were observed in the presence of indomethacin. The lowest dose of PAF observed to raise [Ca2+]i was 0.1 ng/ml. Response of [Ca2+]i to 2-20 ng/ml PAF was transient, and second applications had no effect. The PAF response also was seen in cell suspensions. These results suggest that an increase in [Ca2+]i may be an early event in PAF activation of macrophages.     

Inhibition by forskolin of cytosolic calcium rise, shape change and aggregation in quin2-loaded human platelets

The adenylate cyclase stimulator forskolin was used to study the inhibitory effects of elevated cAMP on the activation of washed human platelets loaded with the fluorescent Ca2+ indicator quin2. In the presence of 10 microM isobutylmethylxanthine forskolin inhibited rises in [Ca2+]i evoked by thrombin and platelet-activating factor (PAF) due to both Ca2+ influx and release from internal stores with similar potency. Aggregation evoked by thrombin and PAF was suppressed whilst partial shape-change persisted, even in the absence of a measurable rise in [Ca2+]i. Forskolin did not affect the rise in [Ca2+]i evoked by Ca2+ ionophore; aggregation was suppressed but shape-change persisted.     

Radiation induced formation of giant cells in Saccharomyces uvarum

Summary Thin sections of budding yeast cells and giant gells grown after X-irradiation have been examined by electron microscopy. The different steps of cross-wall formation during budding were documented with unirradiated cells. With X-ray induced giant cells cytokinesis was shown to be absent. Neither primary nor secondary septae appeared thus cell separation did not occur. Despite this fact both macromolecular synthesis and bud growth continued, giving rise to the formation of giant cells.