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Control of reactivation and microtubule sliding by calcium, strontium, and barium in detergent-extracted macrocilia of Beroë.

Macrocilia of the ctenophore Beroë are activated to beat continuously in the normal direction by membrane-mediated Ca2+ influx (Tamm: Journal of Comparative Physiology [A] 163:23-31, 1988a). Using saponin or Brij-58 permeabilized models of macrocilia, we show that ATP-reactivation of beating requires microM levels of free Ca2+, Ba2+, or Sr2+. Isolated macrocilia beat initially in reactivation solution (RS) containing Ca2+, Ba2+, or Sr2+ and then undergo microtubule sliding disintegration without added proteases. Addition of protease inhibitors to RS + 10(-5) M Ca2+ prevents sliding disruption. Pretreatment in wash solution (containing 1 mM EGTA) without protease inhibitors, followed by RS + 10(-5) M Ca2+ with protease inhibitors results in extensive sliding disintegration. However, treatment in wash solution followed by RS + protease inhibitors does not induce sliding. Therefore, Ca2+ is not required for proteolysis by endogenous proteases, but is necessary for sliding disintegration. Local iontophoretic application of Ca2+, Ba2+, or Sr2+ to permeabilized macrocilia in RS lacking these cations triggers motility and/or sliding disintegration. Extrusion of microtubules occurs from the tip or the base, depending on whether or not the macrocilium remains attached to its large actin bundle. Thin sheets of microtubules telescope out initially, due to synchronized sliding of subsets of doublet microtubules from parallel rows of axonemes. Macrocilia are one of the first examples of ATP-induced microtubule sliding which retains Ca2+ sensitivity. In addition, the finding that Ba2+ and Sr2+ also trigger active sliding provides an additional method for investigating the control of dynein-powered microtubule movements.

Animals↗

Human large granular lymphocytes and natural killing ultrastructural studies of strontium-induced degranulation.

Large granular lymphocytes (LGL) are a subpopulation of human peripheral blood mononuclear cells believed to contain the mediators of spontaneous cytotoxicity or natural killing. In the present study, the mononuclear cells were enriched for LGL by differential density centrifugation on Percoll gradients and examined by transmission electron microscopy. The ultrastructure of LGL and of their binding interaction with natural killer-susceptible target cells (K562) is described in detail. Some morphological similarity between LGL and cells of a myelocytic origin was observed. Studies reported elsewhere have shown that Sr2+, an alkaline earth ion known to degranulate granulocytes, inhibits NK cell function. Comparison of the morphology of control and SR2+-treated LGL showed that Sr2+ caused several characteristic changes in LGL ultrastructure and, indeed, led to their degranulation. The recovery of natural killer function seen following in vitro culture of SR2+-treated effector cells was accompanied by the reappearance of typical intracytoplasmic granules. These data strongly suggest that the granules of LGL are required for and, perhaps, involved in natural killer-mediated cytolysis.

Cell Communication↗

Pharmacological dissection of calcium channel subtype-related components of strontium inflow in large mossy fiber boutons of mouse hippocampus.

Several subtypes of voltage-dependent calcium channels (VDCCs) are present in the presynaptic terminals. In the mammalian hippocampus, P/Q-, N-, and R- but not L-type VDCCs are involved in the fast transmitter release from large mossy fiber (MF) boutons, which are associated with CA3 pyramidal cell dendrites. We investigated whether L-type VDCCs are indeed absent in these large MF boutons. With the use of Sr2+ as the Ca2+ substitute, the stimulus-evoked Sr2+ increment (delta[Sr2+]pre) was evaluated fluorometrically. Delta[Sr2+]pre appeared to be proportional to Sr2+ inflow through VDCCs and was specifically attenuated by conventional VDCC subtype-selective antagonists. The P/Q-type selective omega-agatoxin IVA (AgTx(IVA)) blocked delta[Sr2+]pre with an IC50 of 28 nM and by 30-35% at its maximum effective concentration of 0.5 microM. The N-type selective omega-conotoxin GVIA (CgTx(GVIA)) blocked delta[Sr2+]pre with an IC50 of 15 nM and by 20-25% at its maximum effective concentration of 1 microM. The R-type selective SNX-482 blocked delta[Sr2+]pre with an IC50 of 79 nM and by 20-25% at its maximum effective concentration of 1 microM. The effects of these toxins did not overlap at their maximum effective concentrations and about 70-80% of delta[Sr +]pre was blocked by the simultaneous exposure to these toxins. delta[Sr2+]pre component that is resistant to AgTx(IVA), CgTx(IVA), and SNX-482 was significantly potentiated by an L-type agonist, (S)-(-)-Bay K8644, and attenuated by an L-type antagonist, nimodipine, suggesting that L-type VDCCs are present in large MF terminals. The L-type agonist, (+/-)-Bay K8644, also potentiated Sr2+ inflow into individual boutons identified as large MF boutons under confocal microscopy. Almost similar results were observed for Ca2+ inflow-dependent fluorescence increments. L-type VDCCs appear to be present in large MF boutons and mediate a substantial Ca2+ inflow into presynaptic terminals during action potentials.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

Effects of high extracellular calcium and strontium on inositol polyphosphates in bovine parathyroid cells.

The addition of Ca2+ or a variety of divalent cations increases intracellular Ca2+ in parathyroid cells and suppresses secretion. Since 1,4,5-inositol trisphosphate (IP3) and 1,3,4,5-inositol tetrakisphosphate (IP4) mediate Ca2+ mobilization in other systems, we examined high Ca(2+)- and Sr(2+)-induced accumulation of IP3 and IP4 isomers by anion-exchange HPLC and measured 1,4,5-IP3 mass in parathyroid cells. Raising extracellular [Ca2+] from 0.5 to 3.0 mM increased 3H-1,4,5-IP3 within 5 s, which was confirmed by mass measurements. 3H-1,3,4-IP3 rose gradually by 10 s and increased for 60 s after the addition of Ca2+. Although we detected no change in 3H-1,3,4,5-IP4, the increase in 3H-1,3,4-IP3 suggests that 3H-1,3,4,5-IP4 was being formed. The addition of 4 mM SrCl2 produced similar changes in 1,4,5-IP3, which were confirmed by mass assay. 3H-1,3,4,5-IP4 did not change. However, Sr2+ induced a gradual increase in 3H-1,3,4-IP3, which remained above control levels for 5 minutes. Isotopic labeling studies in this system may underestimate changes in 1,4,5-IP3 mass, but both mass and radioisotopic analyses indicate that high extracellular Ca2+ and Sr2+ stimulate substantial increases in 1,4,5-IP3 without significant accumulation of 1,3,4,5-IP4. These studies suggest a role for 1,4,5-IP3 in intracellular Ca2+ mobilization by divalent cations in parathyroid cells.

Animals↗

Sensitivity of cultured and skinned chick myotube to calcium, strontium, and barium ions examined by recording isometric contractions.

Sensitivity of cultured chick myotubes to alkaline earth metal ions was investigated by recording contractile isometric tension through a semiconductor transducer. The myotubes were obtained by culturing myoblasts of chick embryo breast muscles, and skinned chemically before physiological experiments. Contractions developed in response to Ca2+ in a bathing medium higher than 3 x 10(-7) M and reached maximum at 1 x 10(-5) M. Sr2+ was less effective than Ca2+; the threshold concentration was 1 x 10(-5) M and the tension reached maximum at 1 x 10(-3) M. Ba2+ was the least effective among the three alkaline earth metal ions; only one fifth of the Ca(2+)-induced maximum tension was attained at 1 x 10(-3) M. The sensitivity was similar to that of the mature pectoral muscle fiber, a fast twitch muscle fiber, rather than that of the anterior latissimus dorsi, a slow tonic muscle fiber. The sensitivity was shown to be dependent on its troponin C by replacing it with troponin C from the mature pectoral or cardiac muscle. This indicates that TnC of a fast-muscle type is expressed in the cultured chick myotube as in the mature pectoral muscle. The contractile apparatus was thus shown to be well developed in the cultured myotube with characteristics similar to the mature fast twitch muscle fiber.

Animals↗

Strontium induces murine keratinocyte differentiation in vitro in the presence of serum and calcium.

Primary mouse keratinocytes in culture are induced to terminally differentiate by increasing extracellular Ca2+ concentrations (Cao) from 0.05 mM to > or = 0.1 mM. The addition of Sr2+ (> or = 2.5 mM) to medium containing 0.05 mM Ca2+ induces focal stratification and terminal differentiation, which are similar to that found after increasing the Cao to 0.12 mM. Sr2+ in 0.05 mM Ca2+ medium induces the expression of the differentiation-specific keratins, keratin 1 (K1), keratin 10 (K10), and the granular cell marker, filaggrin, as determined by both immunoblotting and immunofluorescence. Sr2+ induces the expression of those differentiation markers in a dose dependent manner, with an optimal concentration of 5 mM. In the absence of Ca2+ in the medium, the Sr2+ effects are reduced, and Sr2+ is ineffective when both Ca2+ and serum are deleted from the medium. Sr2+ treatment increases the ratio of fluorescence intensity of the intracellular Ca2+ sensitive probe, fura-2, indicating an associated rise in the level of intracellular free Ca2+ and/or Sr2+. At doses sufficient to induce differentiation, Sr2+ also increases the level of inositol phosphates in primary keratinocytes within 30 min. The uptake curves of 85Sr2+ by primary keratinocytes are similar to those of 45Ca2+. At low concentrations, the initial uptake of both 45Ca2+ and 85Sr2+ reaches a plateau within 1 hr; at higher concentrations, the uptake of both 45Ca2+ and 85Sr2+ increases continuously for 12 hr. In keratinocytes pre-equilibrated with 45Ca2+ in 0.05 mM Ca2+ medium, Sr2+ causes an increase of 45Ca2+ uptake, which is dependent on the presence of serum. These results suggest that Sr2+ utilizes the same signalling pathway as Ca2+ to induce keratinocyte terminal differentiation and that Ca2+ may be required to exert these effects.

Animals↗

Comparison of the fertilizing ability of human spermatozoa preincubated in calcium- and strontium-containing media.

Human spermatozoa freed from seminal plasma using a discontinuous Percoll gradient procedure were incubated overnight in modified Tyrode's media containing either CaCl2 (CA medium) or with SrCl2 instead of CaCl2 (SR medium). The following morning these spermatozoa were washed by centrifugation, resuspended in fresh CA medium and incubated for either 2 or 3 h with zona-free hamster eggs to test their fertilizing ability. Penetration rates were higher after 3 h of sperm-egg contact than after 2 h, and the spermatozoa preincubated in SR medium penetrated significantly more hamster oocytes than those preincubated in CA medium.

Animals↗

Strontium supports capacitation and the acrosome reaction in mouse sperm and rapidly activates mouse eggs.

Extracellular Ca2+ is required for capacitation and fertilization in the mouse, but very little is known about the ability of other divalent cations to substitute for Ca2+. In this study, Sr2+, Ba2+, and Mg2+ were evaluated for their ability to support capacitation, the acrosome reaction, hyperactivated motility, and fertilization. Ba2+ proved to be ineffective, but Mg2+-containing medium was able to support capacitation to a greater extent than unsupplemented Ca2+-deficient media; despite this, Ca2+ was required for fertilization. In contrast, Sr2+ proved capable of substituting for Ca2+ in all events. Furthermore, Sr2+-induced responses were indistinguishable from the corresponding Ca2+-induced ones: Sperm capacitated at the same rate and underwent the acrosome reaction to the same extent. However, demonstration of sperm:egg fusion in Sr2+ required the use of zona-free eggs. This was due not to the inability of the sperm to penetrate the zona but to the very rapid activation and cortical granule release by eggs in response to Sr2+. When zona-intact eggs were used, the block to polyspermy had been mounted by the time sperm had penetrated the zona. A 15 min exposure to Sr2+ was sufficient to block sperm fusion, but a longer exposure was required to ensure the resumption of meiosis in eggs; such a response was surprising in that the eggs were freshly ovulated and not susceptible to activation by many different treatments. Thus Sr2+ can profoundly affect both gametes in the mouse: It substitutes completely for Ca2+ in sperm responses and rapidly activates eggs, possibly by displacing Ca2+ from intracellular stores into the cytoplasm, where the Ca2+ can then trigger the various events of activation.

Acrosome↗

The ultrastructure of skeletal haemangiosarcomas induced in mice by strontium-90.

The commonest skeletal tumour occurring in male CBA and C3H mice injected with 7--13 muCi 90Sr (per mouse) at Harwell was diagnosed with the light microscope as haemangiosarcoma. On the other hand, Nilsson, using male CBA mice injected with similar amounts of 90Sr, recorded the predominant tumour as fibroblastic osteosarcoma. To resolve the apparent discrepancy, samples of non-osteogenic tumours induced by 90Sr at Harwell were examined with the electron microscope and their ultrastructure compared with that described for fibroblastic osteosarcoma by Nilsson. The tumours diagnosed as haemangiosarcoma at Harwell showed ultrastructural features not observed in the fibroblastic osteosarcomas. Vasoformation was marked, the vascular channels varying in shape, size and in the character of their lining cells. Some vascular channels were lined by cuboidal cells, crowded together, and surrounded by a basement membrane. Others were lined by an attenuated endothelium and often formed networks. The tumour cells showed great variation in shape, size and ultrastructure and were sometimes enclosed by a basement membrane. Thus the ultrastructure of the tumours described in this report supports the diagnosis of haemangiosarcoma, rather than fibroblastic osteosarcoma. The question of whether these haemangiosarcomas truly originate from vascular endothelium, or are mimics, is discussed.

Animals↗

Some biochemical aspects of the protective effect of strontium chloride on gamma-irradiated rats.

The effect of treatment with SrCl2 (10 mg 100 g-1) on rats 15 min prior to whole body gamma-irradiation (7.5 Gy) was studied. The hazardous effects of irradiation were greatly corrected in the treated group. The hyperglycemic effect and liver glycogen accumulation in the untreated group decreased to normal level. The enzymatic activities of serum alkaline phosphatase, alanine and aspartate aminotransferases, and lactate dehydrogenase were greatly affected, showing insignificant changes in the treated group of animals. Life span calculated on 50% survival was also significantly elongated by 36.3%. These results show the potentiality of SrCl2 as a radioprotective agent. A proposed mechanism is discussed.

Animals↗