Calcium regulation of flagellar curvature and swimming pattern in triton X-100--extracted rat sperm

Free Ca2+ changes the curvature of epididymal rat sperm flagella in demembranated sperm models. The radius of curvature of the flagellar midpiece region was measured and found to be a continuous function of the free Ca2+ concentration. Below 10(-7) M free Ca2+, the sperm flagella assumed a pronounced curvature in the same direction as the sperm head. The curvature reversed direction at 2.5 x 10(-6) M Ca2+ to assume a tight, hook-like bend at concentrations of 10(-5) to 10(-4) M free Ca2+. Sodium vanadate at 2 x 10(-6) M blocked flagellar motility, but did not inhibit the Ca2+-mediated change in curvature. Nickel ion at 0.2 mM and cadmium ion at 1 microM interfered with the transition and induced the low Ca2+ configuration of the flagellum. The forces that maintain the Ca2+-dependent curvature are locally produced, as dissection of the flagella into segments did not significantly alter the curvature of the excised portions. Irrespective of the induced pattern of curvature, the sperm exhibited coordinated, repetitive flagellar beating in the presence of ATP and cAMP. At 0.3 mM ATP the flagellar waves propagated along the principal piece while the level of free Ca2+ controlled the overall curvature. When Ca2+-treated sperm models with hooked midpieces were subjected to higher concentrations of ATP (1-5 mM), some cells exhibited a pattern of movement similar to hyperactivated motility in capacitated live sperm. This type of motility involved repetitive reversals of the Ca2+-induced bend in the midpiece, as well as waves propagated along the principal piece. The free Ca2+ available to the flagellum therefore appeared to modify both the pattern of motility and the flagellar curvature.     

Regulation of flagellar bending by cAMP and Ca2+ in hamster sperm

Hamster sperm were immotile in the medium at free Ca2+ concentrations ([Ca2+]) below 1 x 10(-4) M. The flagellum was acutely bent in the opposite direction to the curve of the hook-shaped heads. This phenomenon seemed to be caused by the decrease in the intracellular cAMP concentration, since the cAMP concentration was low at [Ca2+] below 1 x 10(-4) M and increased abruptly at 1 x 10(-3) M, at which sperm were swimming actively. In addition, sperm became motile due to treatment with 8-bromo-cAMP, a membrane permeable analogue of cAMP, in a medium without Ca2+. These results suggested that extracellular Ca2+ is involved in the regulation of flagellar movement via increasing intracellular cAMP concentration. By the treatment with W-13, a calmodulin inhibitor, sperm also became motile, although cAMP concentration remained at a low level. These results suggested that cAMP is not always required for the flagellar movement when the function of calmodulin is depressed.     

Decrease of internal free calcium and human sperm movement

In order to elucidate the effects of calcium on the movement of human spermatozoa, studies were conducted using motile cells selected by swim-up migration at 37 degrees C in 5% CO2 in air in a synthetic BWW medium containing 1.7 x 10(-3) M CaCl2 or BWW without added calcium (BWW-Ca). Preliminary experiments have confirmed that the addition of EGTA (5 x 10(-3); 10(-2) M) to BWW medium decreased the intracellular calcium concentration ((Ca++)i) of spermatozoa, as measured in cells loaded with a fluorescent Ca++ indicator, Quin-2. Concomitant measurements of (Ca++)i and sperm movement (analysed by videomicrography at 200 f/s at room temperature) were carried out on Quin-2 loaded cells incubated in BWW-Ca medium plus EGTA (10(-5) M; 10(-4) M; 10(-3) M). Under these conditions a decrease in (Ca++)i was observed and associated with a decrease in mean amplitude of lateral head displacement (ALH). Analysis using an automatic analyser (Hamilton Thorn at 37 degrees C) confirmed these results: the percentage of spermatozoa swimming with ALH greater than or equal to 6 microns is decreased when the external free calcium in BWW-Ca is decreased by the addition of 10(-5) M, 10(-4) M, or 10(-3) M EGTA. Flagellar analysis of the sperm population characterized by ALH greater than or equal to 6 microns showed a large proximal curvature of the tail associated with a low propagation wave velocity and a low beat frequency as compared to the spermatozoa with ALH less than 6 microns with similar progressive velocities. These characteristics result in a high flagellar beat efficiency (in terms of head displacement per beat). The disappearance of this pattern of movement when intracellular calcium is lowered indicates that calcium plays a complex role in the relationship between curvature and wave propagation. The ability of spermatozoa to modulate their movement in response to an alteration in the intracellular calcium level confirms the role of calcium in controlling flagellar movement in intact cells.     

Calcium sensors in sea urchin sperm flagella

The asymmetry of ATP-reactivated flagellar bending waves of Triton-demembrated sea urchin spermatozoa has been measured over a range of free Ca2+ ion concentrations from 10(-9) to 10(-4) M. Detailed examination of the gradual response of asymmetry to Ca2+ ion concentration over this wide range indicates the presence of two Ca2+ sensors. A high-affinity sensor operates at Ca2+ concentrations near 10(-7.5) M. A lower-affinity sensor operates at Ca2+ concentrations above 10(-6) M, in the typical range for calmodulin-mediated responses. Incubation of demembranated sperm flagella at high Ca2+ concentrations to release calmodulin is required to enable these Ca2+ responses to be observed. This treatment also causes a decrease in the apparent affinity of the flagella for calmodulin, as determined by measuring the increase in asymmetry in response to addition of exogenous calmodulin at low Ca2+ concentration.     

Regulation of activation state and flagellar wave form in epididymal rat sperm: evidence for the involvement of both Ca2+ and cAMP

Rat sperm from the cauda epididymis exhibit increased motility, longevity, and a distinct circular pattern of flagellar curvature in response to 5 mM procaine-HCl or 0.1 mM 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8), reagents that are thought to play a role in the immobilization of free cellular calcium. Triton X-100-extracted sperm models will exhibit the same pattern of motility and curvature as procaine- or TMB-8-activated cells, but only when calcium is removed by a strong chelating agent, and in the presence of cAMP (3 microM). Demembranated sperm models produced from epididymal rat sperm are quiescent unless cAMP is added. In these sperm models, the presence or absence of free calcium mediates a transition in flagellar curvature. The increased activity of the procaine-treated intact cells was not accompanied by a change in cellular ATP content, nor was ATP availability the limiting factor in the quiescent sperm. Therefore, the increased motility produced by procaine is probably mediated by a fall in free intracellular Ca2+ accompanied by a rise in cAMP. Our finding that calcium controls the curvature of sperm flagella may explain altered patterns of flagellar beating, such as the hyperactivated motility that sperm exhibit in the female reproductive tract.     

Basal body reorientation mediated by a Ca2+-modulated contractile protein

A rapid, Ca2+-dependent change in the angle between basal bodies (up to 180 degrees) is associated with light-induced reversal of swimming direction (the "photophobic" response) in a number of flagellated green algae. In isolated, detergent-extracted, reactivated flagellar apparatus complexes of Spermatozopsis similis, axonemal beat form conversion to the symmetrical/undulating flagellar pattern and basal body reorientation (from the antiparallel to the parallel configuration) are simultaneously induced at greater than or equal to 10(-7) M Ca2+. Basal body reorientation, however, is independent of flagellar beating since it is induced at greater than or equal to 10(-7) M Ca2+ when flagellar beating is inhibited (i.e., in the presence of 1 microM orthovanadate in reactivation solutions; in the absence of ATP or dithiothreitol in isolation and reactivation solutions), or when axonemes are mechanically removed from flagellar apparatuses. Although frequent axonemal beat form reversals were induced by varying the Ca2+ concentration, antiparallel basal body configuration could not be restored in isolated flagellar apparatuses. Observations of the photophobic response in vivo indicate that even though the flagella resume the asymmetric, breaststroke beat form 1-2 s after photostimulation, antiparallel basal body configuration is not restored until a few minutes later. Using an antibody generated against the 20-kD Ca2+-modulated contractile protein of striated flagellar roots of Tetraselmis striata (Salisbury, J. L., A. Baron, B. Surek, and M. Melkonian, 1984, J. Cell Biol., 99:962-970), we have found the distal connecting fiber of Spermatozopsis similis to be immunoreactive by indirect immunofluorescence and immunogold electron microscopy. Electrophoretic and immunoblot analysis indicates that the antigen of S. similis flagellar apparatuses consists, like the Tetraselmis protein, of two acidic isoforms of 20 kD. We conclude that the distal basal body connecting fiber is a contractile organelle and reorients basal bodies during the photophobic response in certain flagellated green algae.     

Vasopressin-induced increases in cellular free calcium concentration measured in single cells of rat renal papillary collecting tubule

We determined the cellular free calcium concentration [Ca2+]i in response to arginine vasopressin (AVP) using single cells of cultured rat renal papillary collecting tubule cells. AVP at a concentration of 1 x 10(-10) M or higher significantly increased [Ca2+]i in a dose-dependent manner. The prompt increase in [Ca2+]i induced by AVP was completely blocked by the V1V2 antagonist, but not by the V1 antagonist. Also, an antidiuretic agonist of 1-deamino-8-D-arginine vasopressin (dDAVP) increased [Ca2+]i, which was blocked by the pretreatment with the V1 V2 antagonist. An AVP-induced increase in [Ca2+]i was still demonstrable in cells pretreated with Ca2(+)-free medium containing 1 x 10(-3) M EGTA, or a blocker of cellular Ca2+ uptake, 5 x 10(-5) M verapamil. These results indicate that AVP increases [Ca2+]i through the V2 receptor in renal papillary collecting tubule cells where cAMP is a well-known second messenger for AVP, and that cellular free Ca2+ mobilization depends on both the intracellular and extracellular Ca2+.     

Evidence for an increased sensitivity to Ca2+ in the flagella of sperm from tw32/+mice

The majority of sperm from mice carrying the tw32 haplotype undergo hyperactivation sooner than sperm from +/+ mice of the same strains (Olds-Clarke, Dev Biol 131:475-482, 1989). To investigate the mechanism underlying this abnormal motility, the Ca2+ sensitivity of their flagellar apparatus was compared to that of age- and strain-matched controls using Triton X-100-extracted sperm. Under these conditions, the curvature of the sperm flagellum is controlled by the free calcium concentration. Sperm from mice carrying the tw32 haplotype consistently exhibited a change in flagellar curvature at lower free calcium concentrations than controls. In addition, intact sperm from tw32/+ mice were much more likely than congenic control sperm to have a hook-like bend in the midpiece, which persisted throughout most of the beat cycle. Sperm exhibiting the hooked middle piece could be converted to a more normal appearance by 2 mM procaine, which immobilizes cytoplasmic calcium. Thus an increased sensitivity of the sperm motor apparatus to calcium could be the cause of the precocious hyperactivation of sperm from mice carrying the tw32 haplotype.     

Direct measurement of the passive stiffness of rat sperm and implications to the mechanism of the calcium response

Glass microprobes were used to measure the stiffness of the flagella of Triton X-100-extracted rat sperm models. The sperm models were treated with 50 microM sodium vanadate and 0.1 mM Mg-ATP to evaluate the stiffness of the passive flagellar structure without the influence of the dynein motor proteins. The passive stiffness was determined to be 4.6 (+/- 1.1) x 10(-19) N x m(2). Rat sperm models exposed to greater than 10(-5) M calcium ions exhibit a strong bend in the basal 40 microm of the flagellum, resulting in a fishhook-like appearance. The torque required to bend a passive rat sperm flagellum into the fishhook-like configuration was determined. The result was compared to the previously published measurement of the torque required to straighten the flagella of rat sperm in the Ca(2+)-induced fishhook configuration [Moritz et al., 2001: Cell Motil. Cytoskeleton 49:33-40]. The torque required to induce a fishhook in a passive flagellum was 2.7 (+/- 0.7) x 10(-14) N x m and the torque to straighten an active Ca(2+)-induced fishhook was 2.6 (+/- 1.4) x 10(-14) N x m. These values are identical within the limit of error of the measurement technique. This finding suggests that the fishhook configuration observed in the Ca(2+) response of rat sperm is the result of a Newtonian equilibrium, where active torque produced by dynein is counterbalanced by an equal and opposite passive torque that results from bending the flagellum. Consistent with this mechanism, the Ca(2+)-induced fishhook configuration is progressively relaxed by incremental increases in sodium vanadate concentration. This supports an active role of the dynein motors in producing the torque for the response. When rat sperm respond to Ca(2+), the bend in the flagellum always forms in the direction opposite the curvature of the asymmetric sperm head. Based on this polarity, the bending torque for the Ca(2+) response must result from the action of the dyneins on outer doublets 1 through 4.     

Regulation by calcium and calmodulin of adenylate cyclase from rabbit intestinal epithelium

The effect of calcium on adenylate cyclase from rabbit small intestine has been studied using a particulate preparation obtained from isolated epithelial cells. Both basal and vasoactive intestinal peptide-stimulated activities were inhibited by calcium concentrations in the micromolar range. In the presence of calmodulin, a biphasic response was obtained. At low calcium concentration (4 X 10(-9)-6 X 10(-8) M) the enzyme was activated up to 50%. As the Ca2+ concentration was increased, the enzyme was concomitantly inhibited. Half-maximal inhibition of calmodulin-dependent activity was obtained at 1 microM free Ca2+. The activation of the enzyme was also dependent on the concentration of Mg2+. At less than 1 microM Ca2+, the enzyme exhibited a biphasic response, being activated at below 3 mM Mg2+ and inhibited at higher concentrations. At Ca2+ concentrations that were inhibitory, the enzyme did not show the biphasic response to Mg2+. At concentrations above 3 mM, the maximal rate (Vmax) remained constant. Vmax was inversely proportional to the concentration of Ca2+ present. Calmodulin altered Vmax when acting on vasoactive intestinal peptide-stimulated enzyme. Calmodulin had no effect on the Km for hormone activation. The calmodulin-dependent activity was inhibited by incubation with trifluoperazine.     

Hyperactivated motility of bull sperm is triggered at the axoneme by Ca2+ and not cAMP

Hyperactivated motility, a swimming pattern of mammalian sperm in the oviduct, is essential for fertilization in vivo. It is characterized by high-amplitude flagellar waves and, usually, highly asymmetrical flagellar beating. It had been suggested, but not tested, that Ca2+ and cAMP switch on hyperactivation by directly affecting the flagellar axoneme. In this study, the direct affects of these agents on the axoneme were tested by using detergent-demembranated bull sperm. As confirmed by TEM, treatment of sperm with 0.2% Triton X-100 disrupted the plasma, acrosomal, and inner mitochondrial membranes, leaving axonemes intact. In the presence of 2 mM ATP, the percentage of reactivated sperm that were hyperactivated increased to 80% when free Ca2+ was increased from 50 to 400 nM. The effect of the Ca2+ in this range was to increase beat asymmetry by increasing the curvature of the principal bend. No additional increases were observed above 400 nM free Ca2+, but motility was suppressed at 1 mM. The ability of Ca2+ to produce hyperactivation depended on ATP availability, such that more ATP was required to produce the high amplitude flagellar bends characteristic of hyperactivated motility than to produce activated motility. Cyclic AMP was not required for reactivation, nor for hyperactivation. Production of hyperactivated motility also required an alkaline environment (pH 7.9-8.5). These results suggest that, provided sufficient ATP is present and pH is sufficiently alkaline, Ca2+ switches on hyperactivation by enabling curvature of the principal bends to increase.     

The interaction of pH and cyclic adenosine 3',5'-monophosphate on activation of motility in Triton X-100 extracted bull sperm

The motility of demembranated bull sperm was found to be governed by the concentrations of cyclic adenosine 3', 5'-monophosphate (cAMP) and Ca2+ at low pH (6.6-7.1), and was less sensitive to these variables at higher pH (7.4-7.8). Although motility was generally found to increase with increasing pH in the range from 6.6 to 7.8, the addition of exogenous cAMP markedly and selectively improved the motility at the lower end of the range (pH 6.6-7.1). In the presence of 10 microM cAMP, low Ca2+ (8.0 X 10(-8) M), and a high concentration of Mg-adenosine 5'-triphosphate (ATP, 8 mM), demembranated sperm at pH 6.8 and 7.1 exhibited swimming similar to that of live ejaculated sperm. At a free Ca2+ concentration of 4.4 X 10(-5) M, the motility was rapidly inhibited at pH 6.8-7.1, whereas at pH 7.4-7.8, the activity was not greatly affected. Since calcium is known to antagonize the cAMP pathway by activating Ca2+-dependent phosphodiesterase and Ca2+-dependent phosphatase, this further supports the idea that cAMP-dependent activation is crucial for motility at low pH. Our results demonstrate that the flagellar axoneme can function normally at relatively acidic pH, and produce vigorous swimming at high levels of ATP. The ATP content of live sperm was measured and found to be high enough (approximately 8 mM) to support the vigorous motility seen at pH 6.6-7.1 in the models.(ABSTRACT TRUNCATED AT 250 WORDS)     

Existence of two calcium currents recorded at normal calcium concentrations in single frog atrial cells

A low threshold calcium current (ICALT) was found in Cs+-loaded frog atrial cells in addition to the classical (high threshold) calcium current (ICaHT), and was investigated at physiological Ca2+ concentrations using the whole-cell patch-clamp technique. The threshold potentials were approximately -60 mV for ICaLT and -40 mV for ICaHT. The amplitude and time course of ICaLT were almost unaffected by exchanging Ca2+ for Ba2+ or Sr2+, while those of ICaLT were modified. ICaLT was inhibited by Ni2+ (40 x 10(-6) M) but was not affected by Cd2+ (20 x 10(-6) M) while ICaHT was inhibited by Cd2+ and only slightly reduced by Ni2+ at the same concentrations. Co2+ (10(-3) M) inhibited both types of calcium currents while La3+ (5 x 10(-6) M) had a greater blocking effect on ICaHT. ICaLT was neither modified by dihydropyridines (nisoldipine, Bay K) nor by adrenergic agonists (adrenaline, noradrenaline, isoprenaline), in contrast with the effects of these agents on ICaHT. Angiotensin II (40 x 10(-9) M) increased and atrial natriuretic factor (0.1 x 10(-6) M) decreased ICaLT while ICaHT, was not modified by these two substances.     

The Adenosine Analogue N6-L-Phenylisopropyladenosine Inhibits Catecholamine Secretion from Bovine Adrenal Medulla Cells by Inhibiting Calcium Influx

We reported earlier that adenine nucleotides and adenosine inhibit acetylcholine-induced catecholamine secretion from bovine adrenal medulla chromaffin cells. In this article, we used an adenosine analogue, N6-L-phenylisopropyladenosine (PIA), to study the mechanism underlying inhibition of catecholamine secretion by adenosine. PIA inhibits secretion induced by a nicotinic agonist, 1,1-dimethyl-4-phenylpiperazinium, or by elevated external K+. The half-maximal effect on 1,1-dimethyl-4-phenylpiperazinium-induced secretion occurred at approximately 5 x 10(-5) M. The inhibition is immediate and reversible. Fura-2 measurements of cytosolic free Ca2+ indicate that PIA inhibits Ca2+ elevation caused by stimulation; measurements of 45Ca2+ influx show that PIA inhibits uptake of Ca2+. PIA does not inhibit calcium-evoked secretion from digitonin-permeabilized cells, nor does PIA cause any significant change in the dependence of catecholamine secretion on calcium concentration. These data suggest that inhibition by PIA occurs at the level of the voltage-sensitive calcium channel.     

Pharmacological actions of the calcium antagonist propyl-methylenedioxyindene in skinned vascular smooth muscle

Previous studies provided strong evidence that propyl-methylenedioxyindene (pr-MDI) interfered with calcium at an intracellular site. To further characterize the mechanism of action of pr-MDI, its pharmacological actions on chemically skinned vascular smooth muscle were examined. Rat caudal artery strips were chemically skinned with saponin (0.15 mg/mL for 1 h). The efficiency of the skinning was evidenced by a loss of contractile response to 74 mM K+. The intactness of the regulatory and contractile proteins was ascertained by the ability of the skinned tissue to contract in response to Ca2+ (free Ca2+ concentration of 10(-4) or 10(-6)M). Caffeine (25 mM) induced contraction was used as an index of the functional integrity of the sarcoplasmic reticulum in the skinned preparations. Contraction of the skinned artery with a free Ca2+ concentration of 10(-6)M was significantly obtunded by 1 X 10(-4)M trifluoperazine (a calmodulin antagonist) but not by 1 X 10(-4)M pr-MDI. Contraction of the skinned artery evoked by 25 mM caffeine in the absence of extracellular calcium was significantly obtunded by 1 X 10(-4)M pr-MDI but not by 1 X 10(-6)M nifedipine (a calcium channel blocker). The results indicate that pr-MDI acts intracellular to block calcium mobilization from the sarcoplasmic reticulum without directly interfering with the regulatory and contractile proteins.     

Regulation of abalone sperm cyclic AMP concentrations and the acrosome reaction by calcium and methylxanthines

When Ca2+ is added to abalone sperm (Haliotis rufescens) in Ca2+-free artificial seawater (CaFASW) to a final concentration of 9.6 mM a 4-fold elevation in sperm cAMP occurs within 15-30 sec. The methylxanthines, theophylline and 1-methyl-3-isobutylxanthine (MIX), also elevate sperm cAMP concentrations. In CaFASW, either compound causes up to a 3-fold increase in cAMP within 15-30 sec. MIX (150 microM), added to sperm in the presence of 9.6 mM Ca2+, elevates sperm cAMP 100-fold within 15-30 sec and also triggers the acrosome reaction (AR) in the same period. Under identical conditions theophylline (1.67 mM) is much less potent at elevating cAMP and inducing the AR. The effects of methylxanthines on cAMP of sperm incubated in the presence of Ca2+ appear to represent a potentiation by these compounds of the action of Ca2+. Neither compound induces the AR in the absence of Ca2+. All of the observed effects on sperm cAMP and the AR are dependent on Ca2+ and methylxanthine concentrations. Added cyclic nucleotides or their derivatives do not induce the AR in either the absence or presence of Ca2+. Experiments with isolated sperm heads and flagella indicate that the dramatic stimulatory response of sperm cAMP to Ca2+ plus MIX is present in the head region (acrosome, nucleus, midpiece) of the cell. The data suggest that the dramatic elevation of cAMP by MIX in the presence of Ca2+ may occur directly by an inhibition of phosphodiesterase activity and indirectly by an increase in cellular Ca2+. A strong temporal correlation between the cAMP elevation and the abalone AR exists, although cAMP elevation by itself does not act as the primary mediator of this exocytotic event.     

Noncyclooxygenase metabolites of arachidonic acid amplify the vasopressin-induced Ca2+ signal in glomerular mesangial cells by releasing Ca2+ from intracellular stores

Noncyclooxygenase metabolites of arachidonic acid may be potent modulators of the mitogenic response of renal mesangial cells to the mitogenic vasoactive peptide arginine vasopressin (AVP). Since Ca2+ is a critical second messenger in the response of mesangial cells to AVP, and Ca2+ has been implicated in the regulation of growth, we determined whether noncyclooxygenase metabolites altered the phospholipase C-Ca2+ signalling cascade which is activated by AVP. Pretreatment of mesangial cells for 10 min with lipoxygenase and cytochrome P450 monooxygenase inhibitors, nordihydroguaiaretic acid (NDGA, 10(-5) M) or SKF-525A (2.5 x 10(-5) M), but not the cyclooxygenase inhibitor indomethacin (2 x 10(-5) M), reduced the magnitude of the AVP (10(-8) and 10(-7) M)-induced increase in cytosolic free Ca2+ concentration ([Ca2+]i) without affecting inositol trisphosphate production. With 10(-8) M AVP, [Ca2+]i increased to 250 +/- 47 nM in NDGA-treated cells versus 401 +/- 59 nM in control cells (p less than 0.01). [Ca2+]i, measured 2 min after exposure to AVP, was also lower with NDGA (152 +/- 21 nM) when compared with AVP alone (220 +/- 22 nM, p less than 0.01). 14,15-epoxyeicosatrienoic acid (EET) (10(-8) M), which had no effect on inositol trisphosphate production, completely reversed the NDGA-induced inhibition of the [Ca2+]i transient, whereas 5-hydroperoxyeicosatetraenoic acid (HPETE) (5 x 10(-7) M) did not. Pretreatment with higher concentrations of 14,15-EET (10(-7)-10(-6) M) markedly potentiated the AVP-induced increase in [Ca2+]i. NDGA-induced inhibition of the AVP-generated [Ca2+]i transient was also observed when cells were incubated in low Ca2+ media ([Ca2+] less than 5 x 10(-8) M), suggesting that NDGA pretreatment impaired intracellular release of Ca2+. Since NDGA had no direct effect on inositol 1,4,5-trisphosphate-induced Ca2+ release, we postulated that NDGA blocked production of a metabolite that releases Ca2+ from intracellular stores. 14,15-EET and 15-HPETE, but not 15-hydroxyeicosatetraenoic acid (each at 3 x 10(-7) M), raised [Ca2+]i when added directly to cells in low Ca2+ media. In permeabilized cells 14,15-EET and 15-HPETE (10(-7) M) potently released Ca2+ from intracellular stores. In summary, noncyclooxygenase metabolites of arachidonic acid, and in particular P450 metabolites, are potent endogenous amplifiers of the AVP-induced [Ca2+]i signal by mechanisms not directly involving phospholipase C activation. This effect is mediated, at least in part, by enhanced release of Ca2+ from intracellular storage sites by an inositol 1,4,5-trisphosphate-independent mechanism.     

Calcium binding to mixed cardiolipin-phosphatidylcholine bilayers as studied by deuterium nuclear magnetic resonance

Calcium binding to bilayer membranes containing cardiolipin (CDL) mixed with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was investigated by using phosphorus-31 and deuterium nuclear magnetic resonance (NMR) spectroscopy. The destabilizing effect of Ca2+ on CDL bilayers, including the formation of hexagonal H11 and isotropic phases, was eliminated when CDL was mixed with sufficiently large proportion of POPC. Thus, for the mixture CDL-POPC (1:9 M/M), 31P NMR spectra retained a line shape typical of fluid bilayer lipids even in the presence of 1.0 M Ca2+. Specifically head-group-deuteriated CDL or POPC showed in this mixture 2H NMR spectra indicating that both lipids remained in a fluidlike bilayer at Ca2+ concentrations up to 1.0 M. Any phase separation of Ca2-CDL clusters could be excluded. The residence time of Ca2+ at an individual head group binding site was shorter than 10(-6) s. The deuterium quadrupole splitting, delta nu Q, of POPC deuteriated at the alpha-methylene segment of the choline head group was found to be linearly related to the number of bound calcium ions, X2, for the CDL-POPC (1:9 M/M) mixture. The effective surface charge density, sigma, could be determined from the measured amount of bound Ca2+. Subsequently, the surface potential, psi 0, and the concentration of free Ca2+ ions at the plane of ion binding were calculated by employing the Gouy-Chapman theory. Various possible models of the equilibrium binding of Ca2+ could then be tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microtubule sliding movement in tilapia sperm flagella axoneme is regulated by Ca2+/calmodulin-dependent protein phosphorylation

Demembranated euryhaline tilapia Oreochromis mossambicus sperm were reactivated in the presence of concentrations in excess of 10(-6) M Ca(2+). Motility features changed when Ca(2+) concentrations were increased from 10(-6) to 10(-5) M. Although the beat frequency did not increase, the shear angle and wave amplitude of flagellar beating increased, suggesting that the sliding velocity of microtubules in the axoneme, which represents dynein activity, rises with an increase in Ca(2+). Thus, it is possible that Ca(2+) binds to flagellar proteins to activate flagellar motility as a result of the enhanced dynein activity. One Ca(2+)-binding protein (18 kDa, pI 4.0), calmodulin (CaM), was detected by (45)Ca overlay assay and immunologically. A CaM antagonist, W-7, suppressed the reactivation ratio and swimming speed, suggesting that the 18 kDa Ca(2+)-binding protein is CaM and that CaM regulates flagellar motility. CaMKIV was detected immunologically as a single 48 kDa band in both the fraction of low ion extract of the axoneme and the remnant of the axoneme, suggesting that CaMKIV binds to distinct positions in the axoneme. It is possible that CaMKIV phosphorylates the axonemal proteins in a Ca(2+)/CaM-dependent manner for regulating the dynein activity. A (32)P-uptake in the axoneme showed that 48, 75, 120, 200, 250, 380, and 400 kDa proteins were phosphorylated in a Ca(2+)/CaM kinase-dependent manner. Proteins (380 kDa) were phosphorylated in the presence of 10(-5) M Ca(2+). It is possible that an increase in Ca(2+) induces Ca(2+)/CaM kinase-dependent regulation, including protein phosphorylation for activation/regulation of dynein activity in flagellar axoneme.     

Nicorandil is a potent dilator of endothelin-induced vascular contraction

Nicorandil, an antianginal drug, is known to open K+ channel and to increase cGMP production. The effects of nicorandil on vascular contraction induced by endothelin (ET), a potent newly discovered vasoconstrictor peptide, were investigated using helical strips from rat thoracic aorta. ET at a concentration of 5 x 10(-9) M induced strong and persistent contraction in the presence of extracellular Ca2+ and similar persistent but smaller contraction in the absence of extracellular Ca2+. Nicorandil at concentrations greater than 10(-7) M, strongly and dose-dependently inhibited ET-induced contraction in the presence of extracellular Ca2+. Nicorandil also suppressed ET-induced contraction in the presence of 10(-4) M methylene blue, an inhibitor of cGMP production, in the presence of extracellular Ca2+ but not in the absence of extracellular Ca2+. ET-induced contraction was also inhibited to lesser extents by the Ca2+ channel blockers nicardipine and verapamil. Nicorandil also strongly suppressed ET-induced increase in cytosolic free Ca2+ concentration in cultured vascular smooth muscle cells. These results suggest that nicorandil is a potent dilator of ET-induced vasoconstriction.