Thyroxine and vitamin D in the gorgonian Leptogorgia virgulata.

The gorgonian coral Leptogorgia virgulata contains thyroxine, or a thyroxine-like substance, referred to here as G-T(4). The G-T(4) levels were significantly higher in colonies collected in the summer vs. winter months. Using immunocytochemical techniques, G-T(4) was localized in the axis, polyp epithelium, and within the electron dense bodies of scleroblasts (spicule-forming cells), as well as on the periphery of spicules. G-T(4) was also localized in the mesoglea between closely adjacent scleroblasts. The effects of exogenous T(4) on the uptake of Ca(45) was determined in spicule, tissue and axis fractions of L. virgulata. The uptake of Ca(45) increased in T(4) treated spicules but decreased in the tissue fraction for all time periods tested. The uptake of Ca(45) into axes was not affected by exogenous T(4) until day 10 of the study. These data suggest that G-T(4) may function in the process of spicule formation. 1,25-dihydroxyvitamin D apparently is synthesized via ultraviolet radiation. Colonies deprived of ultraviolet radiation had significantly more 'irregular' spicules than colonies maintained in ultraviolet radiation. Exposure to sunlight therefore may be associated with the process of normal spicule formation.     

Scleroblast cultures from the gorgonianLeptogorgia virgulata (Lamarck) (Coelenterata: Gorgonacea)

Summary Scleroblasts were separated from fragmented tissue of growing tips ofLeptogorgia virgulata and cultured using a modification of the technique of Rannou. Replacement of fetal bovine serum with horse serum seemed to increase scleroblast viability. Cell adhesion occurred from 14 to 43 d. Cultured scleroblasts demonstrated cell aggregation, spicule formation, and extrusion of spicules into the external medium. Cells showing spicules in the process of being extruded appeared on the average after 24 d of culture. Variability among cultures was marked with respect to both division and spicule formation. Healthy cultures were maintained for more than 4 mo. This work was supported by National Science Foundation grants PCM8201389 and DCB8502698. This is contribution No. 674 of Belle W. Baruch Institute for Marine Biology and Coastal Research, University of South Carolina.     

Ultrastructural investigation of spicule formation in the gorgonianLeptogorgia virgulata (Lamarck) (Coelenterata: Gorgonacea)

Summary Ultrastructural examination of original and regenerated branch tips of the gorgonianLeptogorgia virgulata reveals that spicule formation begins with the aggregation of scleroblasts in the mesoglea. Calcite crystal deposition occurs within a Golgi vacuole containing organic matrix. Vacuole size increases while matrix incorporation and subsequent crystal growth continue, filling the vacuole. At approximately this time, the scleroblasts dissociate and wart formation begins. Further spicule growth stretches the cell into a thin envelope. Fusion of vacuole and plasma membrane followed by breach formation during spicule growth, as well as scleroblast atrophy or migration from mature spicules, result in the transition of the spicule from the intracellular to the extracellular environment. The results also reveal aborted spicules and digestive bodies, implying possible relationships among calcification, detoxification, and waste management.Contribution No 436, Belle W. Baruch Institute for Marine Biology and Coastal Research, University of South Carolina, Columbia, South Carolina, 29208, USA     

An autoradiographic study of calcium transport in spicule formation in the gorgonian Leptogorgia virgulata (Lamarck) (Coelenterata: Gorgonacea)

Summary The route of calcium transport to the sites of spicule formation in the gorgonian Leptogorgia virgulata has been examined by the use of ⁴⁵Ca as a tracer in light- and electron-microscopic autoradiography. From 1 to 15 min after the 15-min incubation the tracer accumulates in the axis. After 15 min there is a movement of label out of the axis largely to the peripheral region of the axis, the axial epithelium, and the mesoglea. By 60 min much of the label is in the spicules reaching its maximum level at 120 min. When calcium enters the scleroblast from the mesoglea, it appears to be transported to the spicule by electron-dense bodies. There does not appear to be a simultaneous release of all ionic calcium from the axis, but rather a continuously increasing efflux which levels off at 60–120 min. Not all of the calcium reaching the axis will traverse it en route to spicules; instead, a portion of it apparently precipitates as an amorphous compound.Contribution No. 550; Belle W. Baruch Institute for Marine Biology and Coastal Research, University of South Carolina, Columbia, South Carolina 29208 USA     

Spicule Formation in the Invertebrates with Special Reference to the Gorgonian Leptogorgia virgulata

Many of the invertebrates possess calcium carbonate spicules.This paper is a review of the formation of these structuresin the Porifera, Coelenterata, Platyhelminthes, Mollusca, Echinodermataand Ascidiacea. Mature spicules appear to be extracellular structures.Sponge spicules initiate intercellularly then become extracellular.Alcyonarian, turbellarian, echinoid and ascidian spicule depositionbegins intracellularly and then becomes extracellular. The continuationof growth in the extracellular environment has not been documentedexcept for the echinoids. Placophoran spicules initiate andremain as extracellular structures. Early spicule growth seemsto occur from or within a single cell. However, cell aggregationand/or neighboring cells appear to be important to the processof spicule formation. The spicule forming cells, in general,are found in a collagenous medium which may be associated withspicule growth. The organic matrix from the spicules of the gorgonian Leptogorgiavirgulata is a glycoprotein. Autoradiography reveals that thismatrix is apparently synthesized in the rough endoplasmic reticulumand Golgi complexes and then transported to the spicule formingvacuole via Golgi vesicles. To gain information about the entryand transport of calcium ions, the effects of ouabain and vanadateon calcium uptake were examined. Ouabain had no effect on calciumuptake. Vanadate treatment increased the uptake of calcium inscleroblasts and epithelial tissue and decreased its uptakein spicules. This may suggest that vanadate sensitive ATPasesare involved in the pumping of calcium out of scleroblasts,out of epithelial cells into the mesoglea, and into scleroblastorganelles. Autoradiography using ⁴⁵Ca indicates that the majorityof these ions initially accumulate in the branch axis. The labelmoves through the axial epithelium to the mesoglea and reachesthe spiculeforming vacuoles in the scleroblasts via dense bodies     

Seasonal localization of a collagenous protein in the organic matrix of spicules from the gorgonian Leptogorgia virgulata (Cnidaria: Gorgonacea)

Spicules of the gorgonian Leptogorgia virgulata possess an insoluble matrix fraction that is predominantly collagenous in summer months. This collagenous component is largely absent in winter months. Using an antibody directed against the 140 kD collagenous protein (CP) of the insoluble matrix, immuno-gold labelling was employed to localized this protein at the transmission electron-microscopy level throughout the year, and in different areas of the gorgonian colonies. Within the tip regions, the 140 kD CP varied throughout the year in the spicules, electron-dense bodies (EDBs) of scleroblasts, polyp vesicles, desmocytes and axes. In the mid and base regions, the 140 kD CP varied throughout the year in the spicules, EDBs and lysosomes of scleroblasts, desmocytes and axes. This variation in the location and density of the label suggests a dynamic annual cycling of the collagenous component of the insoluble matrix. EDBs may transport a collagenous component of the matrix to the spicule-forming vacuole. A component of the 140 kD CP may be transported and/or degraded by polyp vesicles and lysosomes, respectively. The pattern of labelling of the axial region suggests that translocation and storage of a component of the collagenous protein may occur. Environmental factors may be responsible for the triggering of matrix cycling.     

Asymmetric inhibition of spicule formation in sea urchin embryos with low concentrations of gadolinium ion

As gastrulation proceeds during sea urchin embryogenesis, primary mesenchyme cells (PMCs) fuse to form syncytial cables, within which calcium is deposited as CaCO₃, and a pair of spicules is formed. Earlier studies suggested that calcium, previously sequestered by primary mesenchyme cells, is secreted and incorporated into growing spicules. We examined the effects of gadolinium ion (Gd³⁺), a Ca²⁺ channel blocker, on spicule formation. Gd³⁺ did not lead to a retardation of embryogenesis prior to the initiation of gastrulation and did not inhibit the ingression of PMCs from the blastula wall or their migration along the inner blastocoel surface. However, when embryos were raised in seawater containing submicromolar to a few micromolar Gd³⁺, of which levels are considered to be insufficient to block Ca²⁺ channels, a pair of triradiate spicules was formed asymmetrically. At 1–3 μmol/L Gd³⁺, many embryos formed only one spicule on either the left or right side, or embryos formed a very small second spicule. Induction of the spicule abnormality required the presence of Gd³⁺ specifically during late blastula stage prior to spicule formation. An accumulation or adsorption of Gd³⁺ was not detected anywhere in the embryos by X‐ray microanalysis, which suggests that Ca²⁺ channels were not inhibited. These results suggest that Gd³⁺ exerts an inhibitory effect on spicule formation through a mechanism that does not involve inhibition of Ca²⁺ channels.     

Rapid stimulatory effect of thyroxine on plasma membrane transport systems: Calcium uptake and neutral amino acid accumulation in immature rat testis

Although the biological effects of thyroid hormones are mediated by nuclear receptors (genomic mechanisms), interactions with receptors associated with the plasma membrane (non-genomic mechanisms) of target cells are not clear. In this study we investigated the rapid stimulatory effect of thyroxine (T₄) on ⁴⁵Ca²⁺ uptake as well as ionic currents and intracellular messengers involved in the stimulatory action of T₄ in amino acid accumulation in immature rat testes. Results indicated that 10^?9 M or 10^?6 M T₄ was able to increase immediately ⁴⁵Ca²⁺ uptake after 60 s of hormone exposure. These results indicate for the first time that voltage-dependent Ca²⁺ channels and ATP-dependent K⁺ channels can be seen as a set-point in the stimulatory effect of T₄ on amino acid accumulation. Apamin-sensitive small-conductance Ca²⁺-activated K⁺ channels (SK_Ca) and chloride channels were shown to be partially involved in this mechanism. The amino acid accumulation triggered by the PKC pathway suggests a functional link between different ion channel activities and the stimulatory effect of T₄ on amino acid accumulation. In conclusion, we show in this study a rapid and stimulatory effect of T₄ on calcium uptake and on amino acid accumulation, both events initiated at the plasma membrane, which strongly characterizes a non-genomic effect of T₄ in immature rat testes.     

Synthesis and transport of the organic matrix of the spicules in the gorgonian Leptogorgia virgulata (Lamarck) (Coelenterata: Gorgonacea)

Summary The sequence of the synthesis and transport of the organic matrix of spicules has been elucidated in the gorgonian Leptogorgia virgulata by use of ³H-aspartic acid as the tracer in electron-microscopic autoradiography. The entire process of matrix synthesis and transport takes approximately 2 h. It seems that the protein moiety of the organic matrix is synthesized in the RER prior to 5 min following the initial 10 min incubation in the tracer. At the 5 min chase the label is moving from the RER to the Golgi complexes where the carbohydrate moiety of the matrix is presumed to be synthesized. At the 5 to 15 min chases the label is transported out of the Golgi complexes via Golgi vesicles. This phase continues for 30 min. From 60 to 120 min the ³H-aspartic acid moves to the spicules. After 120 min the majority of the label has moved into the spicules. Silver grain counts over both multivesicular and electron-dense bodies remain at relatively low and constant levels over 4 h indicating that neither organelle is involved in the synthesis and transport of the organic matrix.Contribution No 512; Belle W. Baruch Institute for Marine Biology and Coastal Research, University of South Carolina, Columbia, South Carolina 29208, USA     

A tip-high,Ca<Superscript>2+</Superscript>-interdependent,reactive oxygen species gradient is associated with polarized growth in <Emphasis Type="Italic">Fucus serratus</Emphasis> zygotes

We report the existence of a tip-high reactive oxygen species (ROS) gradient in growing Fucus serratus zygotes, using both 5-(and 6-) chloromethyl-2′,7′-dichlorodihydrofluorescein and nitroblue tetrazolium staining to report ROS generation. Suppression of the ROS gradient inhibits polarized zygotic growth; conversely, exogenous ROS generation can redirect zygotic polarization following inhibition of endogenous ROS. Confocal imaging of fluo-4 dextran distributions suggests that the ROS gradient is interdependent on the tip-high [Ca²⁺]_cyt gradient which is known to be associated with polarized growth. Our data support a model in which localized production of ROS at the rhizoid tip stimulates formation of a localized tip-high [Ca²⁺]_cyt gradient. Such modulation of intracellular [Ca²⁺]_cyt signals by ROS is a common motif in many plant and algal systems and this study extends this mechanism to embryogenesis.     

Developmental changes in the45Ca2+ uptake byTrichoderma viride mycelium

The rate of the⁴⁵Ca²⁺ uptake by the submergedTrichoderma viride mycelium increased with the age of the culture from 6 h until a maximum which was reached at about 30 h, and then decreased until the uptake was virtually zero. The decrease in the rate of the⁴⁵Ca²⁺ uptake was accompanied by an increase of mycelial mass. The uptake rate could not be reactivated upon substituting the medium for a fresh one, without or with dilution of the mycelium. The results suggest that the rate of⁴⁵Ca²⁺ uptake reflects the biological age of the submerged culture. The surface-cultivated mycelium took up⁴⁵Ca²⁺ proportionally with time. The autoradiography of colonies showed that⁴⁵Ca²⁺ was distributed homogeneously throughout the mycelium during vegetative growth while conidiation was accompanied by a massive accumulation of⁴⁵Ca²⁺ in conidia. This work was supported by theSwiss National Science Foundation (joint Swiss-Slovak project 75LPJ041485) andSlovak Grant Agency (grant no. 1/1158/93).     

CRYSTAL PROPERTY OF THE LARVAL SEA URCHIN SPICULE*

A pair of pluteus skeletal spicules arises from a pair of calcareous granules via the triradiate form. In polarized light, each spicule behaves as though carved out of a single crystal of magnesian calcite. The optic axis lies perpendicular to the plane of the triradiate and parallel to the body rod of the pluteus. However, in the scanning electron microscope, the spicule surface appeared smooth or somewhat spongy and manifested no crystal faces. Neither etching nor fracturing revealed underlying crystalline texture. Nevertheless, rhombohedral calcite crystals could be grown epitaxially onto isolated spicules immersed in a medium containing CaCl₂ and NaHCO₃. The optic axes of all crystals coincided with the optic axis of the spicule on which they were grown. Corresponding faces of the crystals were all aligned parallel to each other despite the complex shape of each spicule. Where the left and right spicules joined, two mutually tilted sets of crystals were observed but not crystals of intermediate orientation. Thus, the sea urchin larval spicule is built from a stack of molecularly contiguous microcrystals but its overall shape is generated by the mesenchyme cells independent of the magnesian calcite crystal habit.     

Influence of endogenous and exogenous RNases on the variation of pollen cytosolic-free Ca2+ in Pyrus serotina Rehd

The objective of this study was to examine whether S-RNase plays a specific role in the pre-germinated Pyrus pollen. Effects of exogenous RNase and endogenous S-RNase on concentration of cytosolic-free calcium ([Ca²⁺]_i) variation of pre-germinated Pyrus pollen were studied. [Ca²⁺]_i variation caused by different RNases were complex. In 1 h after being cultured, exogenous RNase, RNase T₁ and RNase A, and endogenous incompatible ‘Hohsui’ RNase promoted the [Ca²⁺]_i of ‘Hohsui’ pollen. Acid proteins of ‘Hohsui’ had no remarkable influence on the [Ca²⁺]_i of self-pollen. Endogenous compatible ‘Kohsui’ RNase reduced the [Ca²⁺]_i of ‘Hohsui’ pollen, but compatible ‘Hohsui’ RNase can stimulate the [Ca²⁺]_i of ‘Kohsui’ pollen. RNase T₁, RNase A and incompatible ‘Kohsui’ S-RNase can also make ‘Kohsui’ pollen [Ca²⁺]_i increase. Different from ‘Hohsui’ pollen, acid proteins of ‘Hohsui’ pull down the ‘Kohsui’ pollen [Ca²⁺]_i remarkably. Conclusion can be made that during the prophase of pollen germination, endogenous S-RNase has no specific effect on pollen [Ca²⁺]_i changes.     

Integrin participates in the effect of thyroxine on plasma membrane in immature rat testis

Background

The secretory activity of Sertoli cells (SC) is dependent on ion channel functions and protein synthesis and is critical to ongoing spermatogenesis. The aim of this study was to investigate the mechanism of action associated with a non-metabolizable amino acid [¹⁴C]-MeAIB (α-(methyl-amino)isobutyric acid) accumulation stimulated by T₄ and the role of the integrin receptor in this event, and also to clarify whether the T₄ effect on MeAIB accumulation and on Ca²⁺ influx culminates in cell secretion.

Methods

We have studied the rapid and plasma membrane initiated effects of T₄ by using ⁴⁵Ca²⁺ uptake and [⁴⁵C]-MeAIB accumulation assays, respectively. Thymidine incorporation into DNA was used to monitor nuclear activity and quinacrine to analyze the secretory activity on SC.

Results

The stimulation of MeAIB accumulation by T₄ appears to be mediated by the integrin receptor in the plasma membrane since tetrac and RGD peptide were able to nullify the effect of this hormone. In addition, T₄ increases extracellular Ca²⁺ uptake and Ca²⁺ from intracellular stocks to enhance nuclear activity, but this genomic action seems not to influence SC secretion mediated by T₄. Also, the cytoskeleton and ClC-3 chloride channel contribute to the membrane-associated responses of SC.

Conclusions

T₄ integrin receptor activation ultimately determines the plasma membrane responses on amino acid transport in SC, but it is not involved in calcium influx, cell secretion or the nuclear effect of the hormone.

General significance

The integrin receptor activation by T₄ may take a role in plasma membrane processes involved in the male reproductive system.     

The effect of exogenous Ca2+ ions on pollen grain germination and pollen tube growth

Summary The involvement of exogenous calcium ions in the regulation of pollen tube formation has been investigated in Haemanthus albiflos L. and Oenothera biennis L. by following the changes that occur in pollen germination, tube growth, and ⁴⁵⁺Ca²⁺ uptake and distribution upon application of Verapamil (an inhibitor of calcium channels), lanthanum (a Ca²⁺ substitute), and ruthenium red (believed to raise the intracellular calcium level). It was found that exogenous Ca²⁺ takes part in the formation of the calcium gradient present in germinating pollen grains and growing pollen tubes. Ca²⁺ ions enter the cells through calcium channels. Raising or reducing ⁴⁵Ca²⁺ uptake causes disturbances in the germination of the pollen grains and in the growth of the pollen tubes.     

Phosphate and calcium uptake in beech (Fagus sylvatica) in the presence of aluminium and natural fulvic acids

In the present study we examine the effects of Al on the uptake of Ca²⁺ and H₂PO^-₄ in beech (Fagus sylvatica L.) grown in inorganic nutrient solutions and nutrient solutions supplied with natural fulvic acids (FA). All the solutions used were chemically well characterized. The uptake of Al by roots of intact plants exposed to solutions containing 0, 0.15 or 0.3 mM AlCl₃ for 24 h, was significantly less if FA (300 mg l⁻¹) were also present in the solutions. The Ca²⁺(⁴⁵Ca²⁺) uptake was less affected by Al in solutions supplied with FA than in solutions without FA. There was a strong negative correlation between the Al and Ca²⁺ uptake (r²=0.98). When the Al and Ca²⁺ (⁴⁵Ca²⁺) uptake were plotted as a function of the Al³⁺ activity (or concentration of inorganic mononuclear Al), almost the same response curves were obtained for the -FA and +FA treatments. We conclude that FA-complexed Al was not available for root uptake and therefore could not affect the Ca²⁺ uptake. The competitive effect of Al on the Ca²⁺ uptake was also shown in a 5-week cultivation experiment, where the Ca concentration in shoots decreased at an AlCl₃ concentration of 0.3 mM. The effect of Al on H₂PO⁻₄ uptake was more complex. The P content in roots and shoots was not significantly affected, compared with the control, by cultivation for 5 weeks in a solution supplied with 0.3 mM AlCl₃, despite a reduction of the H₂PO⁻₄ concentration in the nutrient solution to about one-tenth. At this concentration Al obviously had a positive effect on H₂PO⁻₄ uptake. The presence of FA decreased ³²P-phosphate uptake by more than 60% during 24 h, and the addition of 0.15 or 0.3 mM AlCl₃ to these solutions did not alter the uptake of ³²P-phosphate.     

Calmodulin stimulation of unidirectional calcium uptake by the endoplasmic reticulum of barley aleurone

The role of calmodulin (CaM) in gibberellic acid (GA₃)-stimulated Ca²⁺ uptake was investigated in endomembranes isolated from aleurone cells of barley (Hordeum vulgare L.). Unidirectional Ca²⁺ -uptake activity of endoplasmic reticulum (ER) was higher in membranes isolated from aleurone layers treated for 16 h with GA₃ and Ca²⁺ compared with those isolated from layers incubated in Ca²⁺ alone. However, the level of uptake from Ca²⁺-treated tissue could be stimulated to that of the GA₃-treated cells by applying exogenous CaM which increased the V _max of the Ca²⁺ transporter approximately threefold. Calcium uptake in ER from GA₃-treated tissue was inhibited by the CaM antagonist W7 in 50% of experiments, whereas the activity in membranes from non-GA₃-treated tissue was unaffected. Treatment with GA₃ also led to a twofold increase in CaM levels in aleurone layers within 4–6 h, paralleling the time course of the stimulation of Ca²⁺ uptake and preceding the stimulation of α-amylase secretion. We propose that the elevation of Ca²⁺ uptake into the ER induced by GA₃ may be coordinated and regulated by elevated levels of membrane-associated CaM and this may regulate Ca²⁺-dependent α-amylase synthesis in the lumen of the ER.     

An ultrastructural study on spicule formation in the pennatulid colony Renilla reniformis

Examination of the coenenchyme tissues of Renilla reniformis revealed two regions of crystal formation: the endoderm containing small oval deposits of unknown composition and the mesoglea containing larger elongated spicules composed of calcite. Spicule formation takes place intracellularly in scleroblasts and may be explained by the following sequential processes: an organic matrix consisting of a homogeneous ground substance and fibers is formed in a large vacuole. Calcite needles 0.4 μ in diameter develop in close association with matrix fibers and vesicles, and grow to form the central core of the spicule. Large electron dense bodies dominate the scleroblast cytoplasm during these early stages of growth. In a later stage, smaller needles 0.2 μ in diameter develop surrounding the core to form the distal lobes of the spicule. ‘Lollipop’-shaped vesicles containing fibers appear in the scleroblast cytoplasm at the onset of this stage. This material is released at the calcification front and presumably is incorporated into the spicule as an organic matrix of crystals in the distal lobes.     

Tentacle contraction inDiscophrya collini: The effects of ionophore A23187 and ruthenium red on Ca2+-induced contraction and uptake of extracellular calcium

Summary The tentacles of the suctorian protozoonDiscophrya collini are stimulated to contract by externally applied Ca²⁺. The role of extracellular Ca²⁺ in tentacle contraction was studied by monitoring⁴⁵Ca²⁺ uptake, using ionophore A23187 to facilitate membrane transport of calcium and ruthenium red (RR) as an inhibitor of transport. The degree of tentacle retraction was dependent upon external Ca²⁺ concentration and studies with⁴⁵Ca²⁺ using scintillation counting indicated a linear relationship between external Ca²⁺ concentration and Ca²⁺ uptake. Uptake of Ca²⁺ was enhanced in the presence of the ionophore while RR caused little inhibition.⁴⁵Ca²⁺ uptake was only partially inhibited by RR when cells were subjected to a Ca²⁺, ionophore and RR mixture. Grain counts from light microscope autoradiographs after treatment of cells with⁴⁵Ca²⁺/ionophore,⁴⁵Ca²⁺/RR or⁴⁵Ca²⁺ alone showed heavy, light and intermediate labelling respectively. In all instances the grains were evenly distributed within the cell.These observations are interpreted as supporting the suggestion that the ionophore enhances both the uptake of extracellular Ca²⁺ and release of Ca²⁺from an internal source, while the RR could only partially prevent movement of Ca²⁺ through the plasma mebrane. A model is presented suggesting that tentacle retraction is mediated by cytosolic Ca²⁺ levels which are determined by the fluxing of Ca²⁺ across the plasma membrane and the membrane of elongate dense bodies which act as internal Ca²⁺ reservoirs.     

Protective effects of antioxidative serotonin derivatives isolated from safflower against postischemic myocardial dysfunction

N-(p-Coumaroyl)serotonin (C) and N-feruroylserotonin (F) with antioxidative activity are present in safflower oil. The protective effects of C and F were investigated in perfused guinea-pig Langendorff hearts subjected to ischemia and reperfusion. Changes in cellular levels of high phosphorous energy, NO and Ca²⁺ in the heart together with simultaneous recordings of left ventricular developed pressure (LVDP) were monitored by an nitric oxide (NO) electrode, fluorometry and ³¹P-NMR. The rate of recovery of LVDP from ischemia by reperfusion was 30.8% in the control, while in the presence of C or F a gradual increase to 63.2 or 61.0% was observed. Changes of transient NO signals (T_NO) released from heart tissue in one contraction (LVDP) were observed to be upside-down with respect to transient fura-2-Ca²⁺ signals (T_Ca) and transient O₂ signals detected with a pO₂ electrode. At the final stage of ischemia, the intracellular concentration of Ca²⁺ ([Ca²⁺]_i) and the release of NO increased with no twitching and remained at a high steady level. The addition of C increased the NO level at the end of ischemia compared with the control, but [Ca²⁺]_i during ischemia decreased. On reperfusion, the increased diastolic level of T_Ca and T_NO returned rapidly to the control level with the recovery of LVDP. By in vitro EPR, C and F were found to directly quench the activity of active radicals. Therefore, it is concluded that the antioxidant effects of two derivatives isolated from safflower play an important role in ischemia-reperfusion hearts in close relation with NO.