Depletion Of Extracellular Ca Research Articles

Interleukin-6 Synthesis Induced by Prostaglandin E 2: Cross-Talk Regulation by Protein Kinase C

We previously showed that prostaglandin E 2 (PGE 2) stimulates multiple intracellular signaling pathways as follows: by activation of adenylate cyclase; phosphoinositide (PI)-hydrolyzing phospholipase C and phosphatidylcholine (PC)-hydrolyzing phospholipase D; and by induction of Ca 2+ influx in osteoblast-like MC3T3-E1 cells. In this study, we investigated the effect of PGE 2 on the synthesis of interleukin-6 (IL-6) and its regulatory mechanism in MC3T3-E1 cells. PGE 2 significantly stimulated IL-6 secretion in a dose-dependent manner in the range between 1 nmol/L and 10 μmol/L. A23187, a calcium ionophore, or dibutyryl-cAMP significantly induced IL-6 secretion. The effect of a combination of A23187 and dibutyryl-cAMP on IL-6 secretion was additive. The depletion of extracellular Ca 2+ by EGTA reduced the PGE 2-induced IL-6 secretion. EP 1 receptor antagonist inhibited the PGE 2-induced IL-6 secretion. H-89, an inhibitor of cAMP-dependent protein kinase, decreased the PGE 2-induced IL-6 secretion. EP 2 receptor agonist alone stimulated IL-6 secretion. However, EP 4 receptor antagonist had little effect on IL-6 secretion. Calphostin C, a specific inhibitor of protein kinase C (PKC), enhanced the secretion of IL-6 induced by PGE 2. The stimulative effect of PGE 2 on IL-6 secretion was significantly enhanced in PKC downregulated MC3T3-E1 cells. Pertussis toxin enhanced PGE 2-induced IL-6 secretion. These results strongly suggest that PGE 2 stimulates IL-6 synthesis through both Ca 2+ mobilization from extracellular space via EP 1 receptor and cAMP production via EP 2 receptor in osteoblast-like cells, and that the PKC activation by PGE 2 itself regulates oversynthesis of IL-6.

Synapses in hippocampus occupy only 1–2% of cell membranes and are spaced less than half-micron apart: a quantitative ultrastructural analysis with discussion of physiological implications

Relatively little information exists regarding the spatial structure of synaptic neuropil in the brain. The present electron microscopic study employs unbiased stereological techniques and Monte Carlo simulations to characterise quantitatively the spatial organisation of synaptic circuitry in the dentate gyrus of the hippocampus, an area of particular importance in mechanisms of learning and the subject of a number of experimental neurobiological models of synaptic plasticity such as long-term potentiation. Firstly, tissue shrinkage/expansion resulting from embedding was assessed by imaging 300-μm thick hippocampal slices in the course of the entire embedding protocol, giving a value of 94.3±1.1% for distance measures and 84.3±2.8% for volumetric measures. Secondly, numeric synaptic density, N V, was estimated using the disector. Thirdly, accumulated area of post-synaptic densities (PSDs) per tissue volume, S V, and the overall cell membrane area per tissue volume, S* V, were assessed using unbiased stereological rules coupled with image analysis of single sections. Finally, the mean area of individual PSDs was derived as a ratio S V/ N V giving: 0.0394 μm 2 for axo-spinous PSDs (thus representing ∼1.3% of total cell membranes) and 0.0769 μm 2 for dendritic shaft PSDs (∼0.25% of total cell membranes). From these data, the mean nearest neighbour distance between synapses was estimated using Monte Carlo simulations of a random 3D arrangement of synapses constrained by PSD sizes (a truncated Poisson process), giving a value of 0.48–0.51 μm. The physiological importance of the morphometric data obtained is discussed in terms of assessing (i) the role of synaptic environment in modifying synaptic efficacy and (ii) the plausibility of cross talk between synapses in relation to extrasynaptic neurotransmitter diffusion and transient depletion of extracellular Ca 2+.

Mastoparan induces an increase in cytosolic calcium ion concentration and subsequent activation of protein kinases in tobacco suspension culture cells

Mastoparan induced a transient elevation of cytosolic free Ca 2+ concentration ([Ca 2+] cyt) in tobacco suspension culture cells. The mastoparan-induced [Ca 2+] cyt elevation was inhibited by 8-( N, N-diethylamino)-octyl 3,4,5-trimethoxybenzoate-HCl and neomycin but not by depletion of extracellular Ca 2+, suggesting that the elevation was the result of Ca 2+ release from the intracellular stores caused by stimulation of phosphoinositide turnover. Hydrogen peroxide which has been shown to induce an oxidative burst in soybean cells by mastoparan treatment [L. Legendre, P.F. Heinstein, P.S. Low, Evidence for participation of GTP-binding proteins in elicitation of the rapid oxidative burst in cultured soybean cells, J. Biol. Chem., 267 (1992) 20140–20147], also induced a transient [Ca 2+] cyt elevation in the tobacco cells. However, mastoparan did not induce an oxidative burst in the tobacco cells. Activation of a 50, a 75 and a 80 kDa protein kinases after the mastoparan-induced [Ca 2+] cyt elevation was shown by an in-gel protein kinase assay. This activation was inhibited by neomycin, suggesting that the [Ca 2+] cyt elevation is necessary for the mastoparan-induced activation of the protein kinases. The activation was inhibited also by pretreatment with staurosporine and was sustained by pretreatment with calyculin A, suggesting that the protein kinase activity is regulated by protein phosphorylation/dephosphorylation. The present report shows that mastoparan induces an increase in [Ca 2+] cyt without oxidative burst and subsequent activation of protein kinases in tobacco cells.

Warm cells revealed by microfluorimetry of Ca2+ in cultured dorsal root ganglion neurons

Abstract Warm cells were identified by Fura-PE3-based microfluorimetry of Ca 2+ in cultured dorsal root ganglion (DRG) neurons. In response to a physiologically relevant stimulus temperature (43°C), a subpopulation of small DRG neurons from new born rats increased the intracellular Ca 2+ concentration ([Ca 2+ ] i ). Seven percent of the cells responded to the warm stimulus. The stimulus evoked elevation in [Ca 2+ ] i from 52.5±9.5 nM (mean±S.D., n =18) to 171.0±15.6 nM in cells between 15 and 25 μ m in diameter. The depletion of extracellular Ca 2+ diminished the Ca 2+ elevation. The Na + -free condition also diminished the response. We concluded that the heat stimulation opens nonselective cation channels in putative warm cells from DRG neurons.

Basic Fibroblast Growth Factor Induces Interleukin-6 Synthesis in Osteoblasts: Autoregulation by Protein Kinase C

We previously reported that basic fibroblast growth factor (bFGF) stimulates both phospholipases C and D via independent pathways in osteoblastlike MC3T3-E1 cells. In this study, we investigated the effect of bFGF on interleukin-6 (IL-6) synthesis in these cells. bFGF stimulated the IL-6 synthesis dose-dependently in the range between 1 and 30 ng/ml. The depletion of extracellular Ca 2+ by EGTA suppressed the bFGF-induced IL-6 synthesis. TMB-8, an inhibitor of intracellular Ca 2+ mobilization, also inhibited the IL-6 synthesis by bFGF. bFGF stimulated the Ca 2+ influx from extracellular space. Genistein, a tyrosine kinase inhibitor, suppressed the bFGF-induced Ca 2+ influx. Staurosporine, an inhibitor for protein kinases, enhanced the bFGF-induced IL-6 synthesis. Calphostin C, a highly potent and specific inhibitor for protein kinase C (PKC), also enhanced the IL-6 synthesis by bFGF. The bFGF-induced IL-6 synthesis was amplified in PKC down-regulated cells. U-73122, a phospholipase C inhibitor, enhanced the bFGF-induced IL-6 synthesis. Propranolol, a phosphatidic acid phosphohydrolase inhibitor, also enhanced the IL-6 synthesis by bFGF. These results strongly suggest that bFGF stimulates IL-6 synthesis, which depends on intracellular Ca 2+ mobilization in osteoblastlike cells, and that the IL-6 synthesis by bFGF is autoregulated due to PKC activation.

Substance P increases intracellular Ca 2+ in individual rat pituitary lactotrophs, somatotrophs, and gonadotrophs

The present study has investigated transients in the intracellular calcium concentration [Ca 2+] i in response to substance P (SP) in single pituitary cells. SP raised [Ca 2+] i in three subtypes of pituitary cells: lactotrophs, somatotrophs, and gonadotrophs. In all three cell subtypes the [Ca 2+] i response to SP was amplitude-modulated and a concentration of 100 nM was necessary to elicit well pronounced two phased [Ca 2+] i transients. The first phase was associated with increased generation of inositol 1,4,5-trisphosphate (Ins(1,4,5) P 3) in all three cell types. In lactotrophs, the second phase, but not the first, was blunted by depletion of extracellular Ca 2+ (Ca 2+ free EGTA incubation buffer) and by addition of dopamine (1 μM). In somatotrophs, the second phase of the SP-induced [Ca 2+] i response was inhibited by depletion of extracellular Ca 2+ and by addition of somatostatin (100 nM), while the first phase was unaffected by this treatment. In gonadotrophs, the second phase, but not the first, was inhibited by the Ca 2+ channel blocker methoxyverapamil and depletion of extracellular Ca 2+. SP was compared with other agonists having an action on lactotrophs, somatotrophs or gonadotrophs. These experiments demonstrated that SP was a weaker agonist in terms of maximal [Ca 2+] i response than thyrotropin-releasing hormone (TRH) (in lactotrophs), growth hormone-releasing hexapeptide (in somatotrophs) and GnRH (in gonadotrophs). On the basis of these results it is concluded that SP exerts direct Ca 2+ mobilizing effects in single lactotrophs, somatotrophs, and gonadotrophs derived from male peripubertal rats. The first phase in SP-induced [Ca 2+] i transients is likely to be brought about by inositol 1,4,5-trisphosphate-mediated Ca 2+ release from internal stores while the second phase reflects an influx of calcium through voltage-gated calcium channels.

Function of Ca 2+ in phosphatidylcholine-hydrolyzing phospholipase d activation in osteoblast-like cells

We investigated the function of Ca 2+ in the activation of phosphatidylcholine (PC)-hydrolyzing phospholipase D (PLD) in osteoblast-like MC3T3-E1 cells. Fetal calf serum (FCS) stimulated the formation of choline in a dose-dependent manner in the range between 0.6% and 10%. The effect of a combination of FCS and 12- O-tetradecanoylphor-bol-13-acetate, a protein kinase C (PKC) activator, on the formation of choline was additive. Staurosporine, an inhibitor of protein kinases, enhanced the formation of choline induced by FCS. BAPTA/AM, a chelator of intracellular Ca 2+, inhibited the formation of choline induced by FCS. The depletion of extracellular Ca 2+ by EGTA markedly reduced the FCS-induced formation of choline. SK&F 96365, an inhibitor of receptor-operated Cat' Ca 2+y, significantly inhibited the choline formation induced by FCS. On the other hand, nifedipine, an inhibitor of L-type voltage-dependent Ca 2+ channels, had little effect on the choline formation. TMB-8, an inhibitor of Ca 2+, mobilization from intracellular Ca 2+ store, significantly inhibited FCS-induced choline formation. These results strongly suggest that Ca 2+ mobilization, through both the influx via receptor-operated Ca 2+ channel and the release from intracellular Ca 2+ store, plays an important role in the activation of PLD in osteoblast-like cells.

Membrane-associated phospholipase D activity in neural cell line PC12

Stimulation of PC12 cells with carbachol (Cch) or phorbol 12-myristate 13-acetate (PMA) induced [ 3H]phosphatidylbutanol (PBut) production. Depletion of extracellular Ca 2+ abolished the Cch-mediated phospholipase D (PLD) activation, indicating the requirement of Ca 2+ influx. Two different types of PLD activity, oleate-dependent and GTPγS-dependent, were examined by using exogenous [ 3H]phosphatidylcholine substrate. PLD activity of the membrane fraction of PC12 cells was highly dependent on oleate and independent of GTPγS. This profile is in sharp contrast to that observed in HL60 cells showing the profound GTPγS-induced activation of PLD. The oleate-dependent PLD activity of PC12 membrane was inhibited by high concentrations of Ca 2+ and Mg 2+. These results indicate that Ca 2+ may not directly activate PLD but through some Ca 2+-dependent mechanism(s) in Cch-stimulated cells.

PLD activation in Chinese hamster ovary (CHO) cells transfected with PGF2α receptor cDNA

Pertussis toxin-insensitive GTP-binding protein was observed to be involved in prostaglandin F2 α(PGF2 α)-induced phosphoinositide metabolism in Chinese hamster ovary (CHO) cells transfected with PGF2 α receptor cDNA (CHO-PGF2 α·R cells) (Ito, S. et al. Biochem. Biophys. Res. Commun. 200: 756, 1994). In the present study, we investigated PGF2 α-induced PLD activation in CHO-PGF2 α·R cells. PLD activation was examined by measuring the production of [ 3H]phosphatidylbutanol ([ 3H]PBut), a specific product of the PLD-catalyzed transphosphatidylation reaction. PGF2 α-induced [ 3H]PBut formation was concentration-dependent with the maximal level obtained at 1 μM PGF2 α. The maximal [ 3H]PBut formation was observed at 2 min after addition of PGF2 α. Depletion of extracellular Ca 2+ with EGTA suppressed PGF2 α-induced PLD activation by 50%. PKC inhibitors Ro31–8425 and calphostin C inhibited PGF2 α-induced [ 3H]PBut formation by 50%. PTK inhibitors genistein and herbimycin A failed to inhibit PGF2 α-induced PLD activation. A combination of maximal effective concentrations of PGF2 α (1 μM) and PMA (100 nM) enhanced PLD activation in an additive manner. Pretreatment of the cells with PMA for 2 h down-regulated PKC α and decreased PGF2 α-induced PLD activation. These results suggest that PLD activation by PGF2 α is mediated by both PKC-dependent and -independent pathways and that PKC α is involved in the former pathway.

Tyrosine kinase is involved in angiotensin II-stimulated phospholipase D activation in aortic smooth muscle cells: Function of Ca 2+ influx

In the present study, we examined the effect of angiotensin II (Ang II) on phosphatidylcholine-hydrolyzing phospholipase D activity in subcultured rat aortic smooth muscle cells (SMC). Ang II dose-dependently stimulated the formation of choline and inositol phosphates. The effect of Ang II on the formation of inositol phosphates (EC 50 was 0.249 ± 0.091 nM) was more potent than that on the formation of choline (EC 50 was 2.39 ± 1.29 nM). A combination of Ang II and 12- O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, additively stimulated the formation of choline. Staurosporine, an inhibitor of protein kinases, inhibited the TPA-induced formation of choline, but had little effect on the Ang II-induced choline formation. Ang II stimulated Ca 2+ influx from extracellular space time- and dose-dependently. The depletion of extracellular Ca 2+ by (ethylenebis(oxyethylenenitrilo)) tetraacetic acid (EGTA) significantly reduced the Ang II-induced formation of choline. Genistein and tyrphostin, protein tyrosine kinase inhibitors, significantly suppressed the Ang II-induced Ca 2+ influx. Genistein and tyrphostin also suppressed the Ang II-induced formation of choline. These results suggest that Ang II stimulates phosphatidylcholine-hydrolyzing phospholipase D due to Ca 2+ influx from the extracellular space in rat aortic SMC, and that protein tyrosine kinase is involved in the Ang II-induced Ca 2+ influx, resulting in the promotion of phosphatidylcholine hydrolysis.

Function of Ca 2+ influx in phospholipase D activation induced by prostaglandin F 2α in osteoblast-like cells: Involvement of tyrosine kinase

We previously reported that prostaglandin F 2α (PGF 2α) induces Ca 2+ influx from the extracellular space via protein tyrosine kinase in osteoblast-like MC3T3-E1 cells and that PGF 2α stimulates phosphatidylcholine-hydrolyzing phospholipase D in these cells (6, 12). In this study, we examined the relationship between the tyrosine kinase-regulated Ca 2+ influx by PGF 2α and the activation of phospholipase D in MC3T3-E1 cells. The depletion of extracellular Ca 2+ by [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) markedly reduced the PGF 2α-induced formation of choline. Genistein, an inhibitor of protein tyrosine kinases, which by itself had little effect on choline formation, significantly suppressed the formation of choline induced by PGF 2α in a dose-dependent manner. Tyrphostin, an inhibitor of protein tyrosine kinases chemically distinct from genistein, also suppressed the PGF 2α-induced formation of choline. Sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, significantly enhanced the PGF 2α-induced formation of choline. These results strongly suggest that the phospholipase D activation by PGF 2α is dependent on extracellular Ca 2+ in osteoblast-like cells and that protein tyrosine kinase is involved in the activation of phospholipase D.

Protein tyrosine phosphorylation in human platelets induced by interaction between glycoprotein Ib and von Willebrand factor

The interaction between von Willebrand factor (vWF) and glycoprotein Ib (GPIb) induced by ristocetin or botrocetin resulted in associated platelet aggregation, protein tyrosine phosphorylation (PTP) of a 64 kDa protein, as detected by a monoclonal antibody against phosphotyrosine (PY-20), and intracellular Ca 2+ elevation that is largely dependent upon Ca 2+ influx in human platelets. It is of interest that 75–80, 97 and 125 kDa proteins which are strongly tyrosine-phosphorylated in platelet activation induced by thrombin and other agonists were not detected. Neither vWF nor a coaggregating agent (ristocetin or botrocetin) alone induced aggregation, [Ca 2+] i elevation or the 64 kDa PTP. NMC-4, an antibody which inhibits both ristocetin- or botrocetin-induced vWF binding to GPIb, abolished the appearance of the 64 kDa PTP as well as other responses, suggesting that it is specifically induced by the GPIb-vWF interaction. Aspirin, or ONO-3708, a competitive inhibitor of thromboxane A 2, did not modify the 64 kDa PTP, while [Ca 2+] i elevation was moderately suppressed. Depletion of extracellular Ca 2+ or RGD peptides suppressed neither the 64 kDa PTP nor aggregation. H-7, a protein kinase C inhibitor, did not inhibit the 64 kDa PTP, while staurosporine, a potent protein kinase inhibitor, inhibited the 64 kDa PTP and Ca 2+ influx, but not aggregation, in a dose-dependent manner. It is suggested that the 64 kDa PTP is associated with platelet aggregation induced by the interaction between GPIb and vWF.

Mechanism of phospholipase D activation induced by prostaglandin D 2 in osteoblast-like cells: Function of Ca 2+/calmodulin

Prostaglandin D 2 (PGD 2) stimulated the formation of choline in a dose-dependent manner in the range between 10 nM and 10 μM. The effect of PGD 2 on the formation of inositol phosphates (EC 50 was 20 nM) was more potent than that on the formation of choline (EC 50 was 0.5 μM). The formation of choline stimulated by a combination of PGD 2 and 12- O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, was additive. Staurosporine, an inhibitor for protein kinases, enhanced the PGD 2-induced formation of choline, but H-7, another inhibitor for protein kinases, had little effect. PGD 2 stimulated Ca 2+ influx from extracellular space dose-dependently. The depletion of extracellular Ca 2+ by EGTA reduced the PGD 2-induced formation of choline. W-7 and trifluoperazine dihydrochloride, antagonists of calmodulin, dose-dependently inhibited the PGD 2-induced choline formation. These results strongly suggest that PGD 2 activates phospholipase D in a Ca 2+/calmodulin dependent manner in osteoblast-like cells, and that protein kinase C is not essential for the PGD 2-induced activation of phospholipase D.

Lipopolysaccharide-mediated signal transduction through phospholipase D activation in monocytic cell lines

Lipopolysaccharide (LPS)-induced phospholipase D (PLD) activation was investigated in undifferentiated monocytic leukemic cell lines THP-1 and U-937. Treatment of THP-1 or U-937 cells labelled with [ 32P]orthophosphate, [ 32P]acyl GPC or [ 3H]alkyl GPC with LPS, in the presence of 0.5% ethanol, resulted in the accumulation of labelled phosphatidylethanol (PEt) through PLD activation. LPS-mediated PLD activation of THP-1 or U-937 was inhibited by staurosporine (2 μM) and by protein kinase C (PKC) down-regulation with 12- O-tetradecanoylphorbol 13-acetate (TPA) suggesting a role for PKC. In addition to LPS, TPA, ionomycin and cell-permeant analogs of diacylglycerol also stimulated [ 3H]PEt accumulation. The TPA-induced PEt accumulation was also completely abolished by staurosporine or down-regulation of PKC (> 95% inhibition). Furthermore, the LPS-mediated [ 32P]PEt formation was attenuated by either depletion of extracellular Ca 2+ with EGTA (5 mM) or chelation of intracellular Ca 2+ by BAPTA (30 μM). These results indicate that an increase in intracellular Ca 2+ is necessary for LPS-mediated PLD activation. Further support for PKC activation by LPS was obtained by determining PKC activity in an in vitro assay of histone H1 phosphorylation using [γ- 32P]ATP. In untreated THP-1 cells, approximately 64% of the PKC activity was localized in the cytosol and 36% in the membrane fraction. Treatment of the cells with LPS (10 μg/ml, for 2 h) resulted in an increase of 10% of the membrane-associated PKC activity and a corresponding decrease in the cytosol fraction. These data provide evidence that one of the mechanisms of LPS-mediated signal transduction in human monocytic cell lines involves activation of PLD.

Mechanism of arachidonic acid liberation in ethanol-treated mouse peritoneal macrophages

Ethanol has previously been found to induce the release of unmetabolized free arachidonic acid from resident peritoneal macrophages (Diez, E., Balsinde, J., Aracil, M. and Schüller, A. (1987) Biochim. Biophys. Acta 921, 82–89). The molecular mechanism by which ethanol promoted this effect has been investigated in this report. The results reported herein suggest that ethanol stimulated [ 3H]arachidonate liberation from prelabeled macrophages by inhibiting AA reesterification into phospholipids rather than by enhancing phospholipase A 2 activity. Evidence supporting this view was 3-fold. First, ethanol-induced [ 3H]arachidonic acid release was neither affected by depletion of extracellular Ca 2+ nor by cell treatment with the protein kinase C inhibitor staurosporine, two conditions which are known to prevent agonist-induced phospholipase A 2 activation in macrophages. Second, ethanol appreciably inhibited the uptake and esterification of [ 3H]arachidonate into phospholipids of unlabeled cells. Third, combined treatment with ethanol and albumin resulted in an additive release of radiolabel. Altogether, the results of this study underscore the importance of the reacylation pathway in controlling free arachidonic acid levels in macrophages.

Pulsed electric current enhances the phorbol ester induced oxidative burst in human neutrophils

Oxidative burst (OB) response in human neutrophils, measured with chemiluminescence (CL), has been used to determine whether pulsed electric current (PEC) might induce a functional response in these electrically nonexcitable cells, and also whether it might modify cellular response to tumor-promoting phorbol ester (PMA). Five minutes of PEC treatment caused no significant changes in neutrophil CL levels in HBSS (1.2 mM Ca 2+ concentration) as well as in HBSS-EGTA, where the extracellular Ca 2+ concentration was reduced to less than 30 nM. The CL level of PMA-activated neutrophils in HBSS was 52% higher than in HBSS-EGTA. In HBSS the CL level, after the combined PMA and PEC treatment, was 53% higher than in PMA-alone-treated neutrophils. Activation of the OB in HBSS-EGTA with PMA and PEC was 13% higher than in solely PMA treated neutrophils. The results suggest that in neutrophil OB response, the PEC effect is closely related with cellular calcium mobilization, since depletion of extracellular Ca 2+ decreased the PEC effect.

Circadian Rhythm in Amino Acid Uptake by Synechococcus RF-1

In the prokaryote Synechococcus RF-1, circadian changes in the uptake of l-leucine and 2-amino isobutyric acid were observed. Uptake rates in the light period were higher than in the dark period for cultures entrained by 12/12 hour light/dark cycles. The periodic changes in l-leucine uptake persisted for at least 72 hours into continuous light (L/L). The rhythm had a free-running period of about 24 hours in L/L at 29 degrees C. A single dark treatment of 12 hours could initiate rhythmic leucine uptake in an L/L culture. The phase of rhythm could be shifted by a pulse of low temperature (0 degrees C). The free-running periodicity was "temperature-compensated" from 21 to 37 degrees C. A 24 hour depletion of extracellular Ca(2+) before the free-running L/L condition reduced the variation in uptake rate but had little effect on the periodicity of the rhythm. The periodicity was also not affected by the introduction of 25 mm NaNO(3). The uptake rates for 20 natural amino acids were studied at 12 hour intervals in cultures exposed to 12/12 hour light/dark cycles. For eight of these amino acids (l-Val, l-Leu, l-Ile, l-Pro, l-Phe, l-Trp, l-Met, and l-Tyr), the light/dark uptake rate ratios had values greater than 3 and the rhythm persisted in L/L.

Diffusion around a cardiac calcium channel and the role of surface bound calcium

The diffusion of Ca as it converges to the external mouth of a Ca channel is examined. Diffusional limitation on Ca ions entering Ca channels during current flow, cause local extracellular Ca depletions. Such extracellular Ca depletions have been reported in cardiac muscle. The cardiac sarcolemma has a large number of low-affinity Ca binding sites that can buffer these local Ca depletions. For a hemisphere of extracellular space (of radius less than 0.33 microns) centered on the external mouth of a Ca channel the amount of Ca bound at the membrane surface exceeds that which is free within the associated hemisphere. The ratio of bound Ca/free Ca increases as r decreases, such that the [Ca] nearest the Ca channel is the most strongly buffered by sarcolemmal bound Ca. It is demonstrated that Ca ions coming from these sarcolemmal Ca binding sites contribute quantitatively to the integrated Ca current. The electric field generated by the local depletion of Ca near the channel mouth has little impact on the extent of Ca depletion, but if an additional electric field exists at the mouth of the channel, Ca depletion can be significantly altered. Other low-affinity Ca binding sites in the interstitium may also contribute to the buffering of extracellular Ca. The complex geometry of the extracellular space in cardiac muscle (e.g., transverse tubules and restrictions of extracellular space between cells) increases both the predicted Ca depletions (in the absence of binding) and the bound/free ratio. Thus, the impact of this surface Ca binding is greatly increased. By considering arrays of Ca channels in transverse tubules or in parallel planes (e.g., membranes of neighboring cells), extracellular Ca depletions are predicted which agree with those measured experimentally. Membrane Ca binding may also be expected to buffer increases in [Ca] around the inner mouth of Ca channels. It is demonstrated that in the absence of other intracellular systems most of the Ca entering the cell via Ca channels might be expected to be bound to the inner sarcolemmal surface. It is concluded that surface Ca binding may have a substantial impact on the processes of extracellular Ca depletion (and intracellular Ca accumulation).

A role for Ca 2+ in the cellular differentiation of root cap cells: A re-examination of root growth control mechanisms

Ultrastructural investigations of root caps ( Zea mays) have shown a correlation between the depletion of extracellular Ca 2+ resources by treatment of intact roots with 50 mM EGTA and changes in the activity of peripheral cap cells, involving reductions in the quantity and changes in the appearance of Golgi apparatus-derived material. In EGTA-treated roots the development of the Golgi apparatus of peripheral cap cells was severely altered and there was no evidence of a granular secretory product, which was encountered in the vesicles of dictyosomes of control roots. Vesicles of dictyosomes of EGTA-treated roots were small and the development of dictyosomes was reminiscent of that encountered in central cap cells of control roots. A decrease in amyloplast numbers brought about by EGTA was concomitant with reduced secretory activity. EGTA treatment was also associated with a redistribution of amyloplasts located in central cap cells. Decreased cap volume arising from EGTA treatment was considered indicative of a decline in overall cap activity. Proposals are made with regard to the integration of intercellular activities in the response of plant roots to stimulus-modulated signals. It is suggested that alterations in peripheral cap cell secretory activity arising from the redistribution of Ca 2+ may account for the anisotropic growth response of gravireactive roots.

Evidence for tight coupling of phospholipase activation and Ca 2+ influx during acrosome reaction of golden hamster spermatozoa

1. 1. Phospholipases have been proposed to play a key role in sperm acrosome reaction. To examine the activation mechanism of phospholipases and subsequently sperm fertilizing capacity, Ca 2+ fluxes and phospholipid turnover (breakdown and synthesis) were investigated in golden hamster spermatozoa during acrosome reaction. 2. 2. Upon exposure of the spermatozoa to 1.7 mM Ca 2+, a net uptake by the cells occurred in two distinguishable phases. 3. 3. Depletion of extracellular Ca 2+ by ethylene glycol bis(β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid (EGTA) at a time that an initial Ca 2+ uptake was observed to reach almost steady-state, prevented the secondary Ca 2+ uptake and acrosome reaction. 4. 4. The time course of an initial Ca 2+ uptake seemed to precede that of the acrosome reaction. 5. 5. Incubation of the spermatozoa with Ca 2+ in the presence of [ 3H]glycerol induced a rapid increase in labeling of phosphatidic acid, a key intermediate of phosphinositide turnover initiated by the action of phospholipase C, which appeared to parallel the time course of a first phase of Ca 2+. 6. 6. Phospholipase A 2 activation, detected by lysophospholipid formation, slightly delayed the initial events of first Ca 2+ uptake and phosphatidic acid production. 7. 7. It is concluded that first Ca 2+ entry into the cells, associated with phosphatidic acid production, activates a phospholipase A 2, leading to the production of substances, like lysophospholipids and fatty acids, which may contribute to acrosome reaction.