Real-time analysis of the role of Ca(2+) in flagellar movement and motility in single sea urchin sperm

Eggs of many marine and mammalian species attract sperm by releasing chemoattractants that modify the bending properties of flagella to redirect sperm paths toward the egg. This process, called chemotaxis, is dependent on extracellular Ca(2+). We used stroboscopic fluorescence imaging to measure intracellular Ca(2+) concentration ([Ca(2+)]i) in the flagella of swimming sea urchin sperm. Uncaging of cyclic GMP induced Ca(2+) entry via at least two distinct pathways, and we identified a nimodipine-sensitive pathway, compartmentalized in the flagella, as a key regulator of flagellar bending and directed motility changes. We found that, contrary to current models, the degree of flagellar bending does not vary in proportion to the overall [Ca(2+)]i. Instead we propose a new model whereby flagella bending is increased by Ca(2+) flux through the nimodipine-sensitive pathway, and is unaffected by [Ca(2+)]i increases through alternative pathways.     

Increase of cGMP, cADP-ribose and inositol 1,4,5-trisphosphate preceding Ca(2+) transients in fertilization of sea urchin eggs.

Transient increases, or oscillations, of cytoplasmic free Ca(2+) concentration, [Ca(2+)](i), occur during fertilization of animal egg cells. In sea urchin eggs, the increased Ca(2+) is derived from intracellular stores, but the principal signaling and release system involved has not yet been agreed upon. Possible candidates are the inositol 1,4,5-trisphosphate receptor/channel (IP(3)R) and the ryanodine receptor/channel (RyR) which is activated by cGMP or cyclic ADP-ribose (cADPR). Thus, it seemed that direct measurements of the likely second messenger candidates during sea urchin fertilization would be essential to an understanding of the Ca(2+) signaling pathway. We therefore measured the cGMP, cADPR and inositol 1,4,5-trisphosphate (IP(3)) contents of sea urchin eggs during the early stages of fertilization and compared these with the [Ca(2+)](i) rise in the presence or absence of an inhibitor against soluble guanylate cyclase. We obtained three major experimental results: (1) cytosolic cGMP levels began to rise first, followed by cADPR and IP(3) levels, all almost doubling before the explosive increase of [Ca(2+)](i); (2) most of the rise in IP(3) occurred after the Ca(2+) peak; IP(3) production could also be induced by the artificial elevation of [Ca(2+)](i), suggesting the large increase in IP(3) is a consequence, rather than a cause, of the Ca(2+) transient; (3) the measured increase in cGMP was produced by the soluble guanylate cyclase of eggs, and inhibition of soluble guanylate cyclase of eggs diminished the production of both cADPR and IP(3) and the [Ca(2+)](i) increase without the delay of Ca(2+) transients. Taken together, these results suggest that the RyR pathway involving cGMP and cADPR is not solely responsible for the initiating event, but contributes to the Ca(2+) transients by stimulating IP(3) production during fertilization of sea urchin eggs.     

Na+ /Ca2+ exchanger contributes to asterosap-induced elevation of intracellular Ca2+ concentration in starfish spermatozoa

Asterosap, a group of equally active isoforms of sperm-activating peptides from the egg jelly of the starfish Asterias amurensis, functions as a chemotactic factor for sperm. It transiently increases the intracellular cGMP level of sperm, which in turn induces a transient elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)). Using a fluorescent Ca(2+)-sensitive dye, Fluo-4 AM, we measured the changes in sperm [Ca(2+)](i) in response to asterosap. KB-R7943 (KB), a selective inhibitor of Na(+)/Ca(2+) exchanger (NCX), significantly inhibited the asterosap-induced transient elevation of [Ca(2+)](i), suggesting that asterosap influences [Ca(2+)](i) through activation of a K+-dependent NCX (NCKX). An NCKX activity of starfish sperm also shows K(+) dependency like other NCKXs. Therefore, we cloned an NCKX from the starfish testes and predicted that it codes for a 616 amino acid protein that is a member of the NCKX family. Pharmacological evidence suggests that this exchanger participates in the asterosap-induced Ca(2+) entry into sperm.     

Potentiation by stannous chloride of calcium entry into osteoblastic MC3T3-E1 cells through voltage-dependent L-type calcium channels

The present study was undertaken to confirm that L-type Ca(2+) channels are involved in Ca(2+) entry into osteoblastic MC3T3-E1 cells and to examine the effect of SnCl2, a Ca(2+)]-channel activator, on the intracellular Ca(2+)concentration ([Ca(2+)]i). High K(+)concentration-dependently raised the [Ca(2+)]i. All of the L-type Ca(2+)channel blockers used here, such as nifedipine, nicardipine, verapamil, and diltiazem, and CdCl2 (a non-selective blocker) inhibited the high K(+)-induced [Ca(2+)]i rise, but v-conotoxin GVIA (an N-type blocker) and NiCl2(a T-type blocker) had no effect. Application of SnCl2 alone did not change the [Ca(2+)]i. However, in the presence of high K(+), SnCl2 enhanced the high K(+)-induced [Ca(2+)]i rise, which was inhibited by Ca(2+)]-free medium or nifedipine. In the case where high K(+)was applied prior to SnCl2, SnCl2 alone raised the [Ca(2+)]i by itself. In conclusion, MC3T3-E1 cells possess the voltage-dependent L-type Ca(2+)] channels and SnCl2 facilitates the Ca(2+) entry through the L-type ones under the condition of the membrane depolarization. There is the possibility that Ca(2+) release from intracellular Ca(2+) stores is involved in the action of SnCl2.     

Sea urchin sperm head plasma membranes: characteristics and egg jelly induced Ca2+ and Na+ uptake

Sea urchin sperm respond to egg factors with changes in the ionic permeability of their plasma membrane. It has been previously shown that plasma membranes isolated preferentially from sea urchin sperm flagella respond to egg jelly increasing their Ca2+ and Na+ uptake (Darszon et al. (1984) Eur. J. Biochem. 144, 515-522). However, the egg jelly induced acrosome reaction occurs in the sperm head, and there is evidence for an heterogeneous distribution of plasma membrane components within the various regions of this cell. We here report a method for purifying sperm head membranes using positively charged beads according to Jacobson (1977) Biochim. Biophys. Acta 471, 331-335). Under the transmission electron microscope these membranes appeared homogeneous and apparently free of internal membranes. The yield of the preparation was 0.9% of the total protein in the sperm homogenate. The preparation contained less than 5% of the mitochondrial marker cytochrome oxidase, and 10% of the total DNA/mg protein. Surface labeling with 125I indicated a 2.5-3-fold enrichment in specific activity of the head membranes with respect to whole sperm. The SDS band pattern and the lipid composition of this preparation were different from those of isolated flagellar membranes. Phosphatidylcholine was higher in the head membranes, while phosphatidylserine and phosphatidylethanolamine were lower. The head membranes displayed a 1.7-2.3-fold higher Ca2+-ATPase activity and a 2.5-fold lower Na+/K+-ATPase activity, than the flagellar membranes. These results are consistent with a heterogeneous distribution of membrane components along the sea urchin sperm plasma membranes. Isolated head membranes sonicated in the presence of soybean phospholipid liposomes responded to egg jelly with a species-specific increase in Ca2+ and Na+ uptake. As in whole sperm, Ca2+ uptake was inhibited by the Ca2+ channel blocker nisoldipine. A close analog of this compound, [3H]nitrendipine, binds with high affinity to head membranes in a saturable, reversible manner, showing a Kd and Bmax of 31 nM and 5.3 pmol/mg protein, respectively.     

A sustained increase in intracellular Ca(2+) is required for the acrosome reaction in sea urchin sperm.

The acrosome reaction (AR), necessary for fertilization in many species, requires an increase in intracellular Ca(2+) ([Ca(2+)](i)). In sea urchin sperm, the AR is triggered by an egg-jelly factor: the associated [Ca(2+)](i) elevation lasts minutes and involves two Ca(2+) permeable channels. Both the opening of the second channel and the onset of the AR occur approximately 5 s after treatment with egg factor, suggesting that these events are linked. In agreement, removal of Ca(2+) from sea water or addition of Ca(2+) channel blockers at the time when opening of the second channel is first detected inhibits AR and causes a "rapid" (t(1/2) = 3--15 s) decrease in [Ca(2+)](i) and partial inhibition of the intracellular pH change associated with the AR. Simultaneous addition of NH(4)Cl and either EGTA, Co(2+), or Ni(2+) 5 s after egg factor prevents the partial inhibition of the evoked pH(i) change observed but does not reverse AR inhibition. Therefore, the sustained increase in [Ca(2+)](i) caused by the second Ca(2+) channel is needed for the sperm AR. Experiments with agents that induce capacitative Ca(2+) uptake (thapsigargin and cyclopiazonic acid) suggest that the second channel opened during the AR could be a store-operated Ca(2+) channel.     

Proteins associated with soluble adenylyl cyclase in sea urchin sperm flagella

Adenylyl cyclases (ACs) synthesize cAMP and are present in cells as transmembrane AC and soluble AC (sAC). In sperm, the cAMP produced regulates ion channels and it also activates protein kinase-A that in turn phosphorylates specific axonemal proteins to activate flagellar motility. In mammalian sperm, sAC localizes to the midpiece of flagella, whereas in sea urchin sperm sAC is along the entire flagellum. Here we show that in sea urchin sperm, sAC is complexed with proteins of the plasma membrane and axoneme. Immunoprecipitation shows that a minimum of 10 proteins is tightly associated with sAC. Mass spectrometry of peptides derived from these proteins shows them to be: axonemal dynein heavy chains 7 and 9, sperm specific Na+/H+ exchanger, cyclic nucleotide-gated ion channel, sperm specific creatine kinase, membrane bound guanylyl cyclase, cyclic GMP specific phosphodiesterase 5A, the receptor for the egg peptide speract, and alpha- and beta-tubulins. The sAC-associated proteins could be important in linking membrane signal transduction to energy utilisation in the regulation of flagellar motility.     

Mechanism regulating Ca2+-dependent mechanosensory behaviour in sea urchin spermatozoa

Flagellar movement of the sea urchin sperm is regulated by intracellular Ca(2+). Flagellasialin, a polysialic acid-containing glycoprotein, as well as other membrane proteins seems responsible for the Ca(2+) control. To elucidate the mechanism of Ca(2+) dynamics underlying flagellar movement, we analysed the sperm's mechanosensory behavioural responses by using microtechniques. In sea water containing 10 mM Ca(2+), the sperm swim in circular paths. When a mechanical stimulus was applied to the sperm head with a glass microstylus, the sperm showed a series of flagellar responses, consisting of a stoppage of beating (quiescence) and a recovery of swimming in a straight path, followed by swimming in a circular path again; as the result the sperm avoided the obstacle. Ca(2+)-imaging with Fluo-4 showed that the intracellular Ca(2+) was high in the quiescence and gradually decreased after that. The effects of blockers and antibodies against candidate components revealed that the Ca(2+) influx was induced by Ca(2+) channels and the Ca(2+) efflux was induced by a flagellasialin-related Ca(2+)-efflux system, plasma membrane Ca(2+)-ATPases and the K(+)-dependent Na(+)/Ca(2+) exchanger. The results show that the Ca(2+)-dependent mechanosensory behaviour of the sea urchin sperm is regulated by organized functioning of the membrane environment including the plasma membrane proteins and flagellasialin.     

Development of calcium releasing activity induced by inositol trisphosphate and cyclic ADP-ribose during in vitro maturation of sea urchin oocytes

During fertilization of sea urchin eggs, the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) transiently increases (Ca(2+) transient). Increased [Ca(2+)](i) results from a rapid release from intracellular stores, mediated by one or both of two signaling pathways; inositol 1,4,5-trisphosphate (IP(3)) and IP(3) receptor (IP(3)R) or cyclic GMP (cGMP), cyclic ADP-ribose (cADPR) and ryanodine receptor (RyR). During fertilization, cGMP and cADPR increase preceding the Ca(2+) transient, suggesting their contribution to this. If the RyR pathway contributed to the Ca(2+) transient, its Ca(2+) releasing activity would develop in parallel with that of the IP(3) system during maturation of oocytes. Sea urchin oocytes were cultivated in vitro and Ca(2+) transients induced by photolysis of caged IP(3) or caged cADPR were measured during maturation. Oocytes spontaneously began to maturate in seawater. More than 50% of oocytes underwent germinal vesicle breakdown within 25 h and the second meiosis within 35 h, but it took more than 24 h until they became functionally identical to in vivo-matured eggs. Both IP(3) and cADPR induced Ca(2+) transients comparable to those of in vivo-matured eggs later than 24 h from the second meiosis. However, cADPR induced a small Ca(2+) transient even before meiosis, whereas IP(3) and sperm almost did not.     

The rate of change in Ca(2+) concentration controls sperm chemotaxis

During chemotaxis and phototaxis, sperm, algae, marine zooplankton, and other microswimmers move on helical paths or drifting circles by rhythmically bending cell protrusions called motile cilia or flagella. Sperm of marine invertebrates navigate in a chemoattractant gradient by adjusting the flagellar waveform and, thereby, the swimming path. The waveform is periodically modulated by Ca(2+) oscillations. How Ca(2+) signals elicit steering responses and shape the path is unknown. We unveil the signal transfer between the changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) and path curvature (κ). We show that κ is modulated by the time derivative d[Ca(2+)](i)/dt rather than the absolute [Ca(2+)](i). Furthermore, simulation of swimming paths using various Ca(2+) waveforms reproduces the wealth of swimming paths observed for sperm of marine invertebrates. We propose a cellular mechanism for a chemical differentiator that computes a time derivative. The cytoskeleton of cilia, the axoneme, is highly conserved. Thus, motile ciliated cells in general might use a similar cellular computation to translate changes of [Ca(2+)](i) into motion.     

What Is the Core Oscillator in the Speract-Activated Pathway of the Strongylocentrotus purpuratus Sperm Flagellum?

Sperm chemotaxis has an important role in fertilization. Most of our knowledge regarding this phenomenon comes from studies in organisms whose fertilization occurs externally, like sea urchins. Sea urchin spermatozoa respond to sperm-activating peptides, which diffuse from the egg jelly coat and interact with their receptor in the flagellum, triggering several physiological responses: changes in membrane potential, intracellular pH, cyclic nucleotide levels, and intracellular Ca2+ concentration ([Ca2+]). In particular, flagellar [Ca2+] has been shown to oscillate. These [Ca2+] oscillations are correlated with changes in the flagellar shape and so with the regulation of the sperm swimming paths. In this study, we demonstrate, from a mathematical modeling perspective, that the reported speract-activated signaling pathway in Strongylocentrotus purpuratus (speract being a sperm-activating peptide specific to this species) has the necessary elements to replicate the reported [Ca2+] oscillations. We further investigate which elements of this signaling pathway constitute the core oscillator.     

Altering the speract-induced ion permeability changes that generate flagellar Ca2+ spikes regulates their kinetics and sea urchin sperm motility

Speract, an egg-derived sperm-activating peptide, induces changes in intracellular Ca2+, Na+, pH, cAMP, cGMP, and membrane potential in sperm of the sea urchin Strongylocentrotus purpuratus. Ca2+ is a key regulator of motility in all sperm and, in many marine species, is required for generating turns interspersed with straighter swimming paths that are essential for chemotaxis towards the egg. We show that speract triggers a train of increases in flagellar Ca2+, and that each individual Ca2+ fluctuation induces a transient increase in flagellar asymmetry that leads to a turn. We also find that modifying the amplitude, duration and interval between individual Ca2+ fluctuations by treating sperm with niflumic acid, an inhibitor of Ca2+-activated Cl(-) channels, correspondingly alters the properties of the sperm turns. We conclude that Ca2+ entry through a fast flagellar pathway not only induces sperm turns, but the kinetics of Ca2+ entry may shape the nature of these turns, and that these kinetics are tuned by other channels, possibly including Cl(-) channels. In addition, the speract-induced changes in sperm motility closely resemble those seen during chemotaxis in other marine organisms, yet speract is not a chemoattractant. This implies the Ca2+-induced motility changes are necessary but not sufficient for chemotaxis.     

A bipartite Ca2+-regulated nucleoside-diphosphate kinase system within the Chlamydomonas flagellum. The regulatory subunit p72

Regulation of flagellar activity in Chlamydomonas involves both Ca(2+) and cAMP-mediated signaling pathways. However, Chlamydomonas and sea urchin sperm flagella also exhibit nucleoside-diphosphate kinase (NDK) activity, suggesting a requirement for GTP within this highly conserved organelle. In sea urchin sperm, the NDK catalytic subunit is an integral component of the outer dynein arm. Here we describe a modular protein (p72) from the Chlamydomonas flagellum that consists of three domains closely related to the presumptive regulatory segment of rat NDK-7 followed by two EF-hands that are predicted to bind Ca(2+). There are close homologues of p72 in both mammalian and insect genomes. The p72 protein is tightly associated with the flagellar axoneme and is located along the entire length except at the transition zone. Cross-linking experiments suggest that p72 interacts with two or three additional axonemal polypeptides. The sensitivity of p72 to tryptic digestion differed considerably in the presence and the absence of Ca(2+), suggesting that it indeed binds this ligand. These studies indicate that the flagellar NDK system is bipartite with the regulatory and catalytic components residing on different polypeptides. We propose that Ca(2+) regulation of flagellar motility in Chlamydomonas may be achieved in part through a downstream GTP-mediated signaling pathway.     

Speract induces calcium oscillations in the sperm tail

Sea urchin sperm motility is modulated by sperm-activating peptides. One such peptide, speract, induces changes in intracellular free calcium concentration ([Ca2+]i). High resolution imaging of single sperm reveals that speract-induced changes in [Ca2+]i have a complex spatiotemporal structure. [Ca2+]i increases arise in the tail as periodic oscillations; [Ca2+]i increases in the sperm head lag those in the tail and appear to result from the summation of the tail signal transduction events. The period depends on speract concentration. Infrequent spontaneous [Ca2+]i transients were also seen in the tail of unstimulated sperm, again with the head lagging the tail. Speract-induced fluctuations were sensitive to membrane potential and calcium channel blockers, and were potentiated by niflumic acid, an anion channel blocker. 3-isobutyl-1-methylxanthine, which potentiates the cGMP/cAMP-signaling pathways, abolished the [Ca2+]i fluctuations in the tail, leading to a very delayed and sustained [Ca2+]i increase in the head. These data point to a model in which a messenger generated periodically in the tail diffuses to the head. Sperm are highly polarized cells. Our results indicate that a clear understanding of the link between [Ca2+]i and sperm motility will only be gained by analysis of [Ca2+]i signals at the level of the single sperm.     

Roles of osmolality, calcium - Potassium antagonist and calcium in activation and flagellar beating pattern of sturgeon sperm

The present study shows the roles of osmolality, calcium (Ca(2+))-potassium (K(+)) antagonist and Ca(2+) in sperm activation and flagellar beating of a sturgeon species, sterlet (Acipenser ruthenus). Sperm motility was activated at hypoosmolality relative to seminal plasma and suppressed at 175 mOsmol kg(-1). Sperm activation was totally suppressed by 0.35mM K(+), but Ca(2+) could fully reverse K(+) inhibitory effect at Ca(2+): K(+) ratio of 0.25. Neither EGTA (a chelator of Ca(2+) ions) nor nifedipine (a Ca(2+) channel blocker) prevented sperm activation. But, sperm motility and velocity were significantly decreased by EGTA, nifedipine and an inhibitor for Ca(2+)/calmodulin activated phosphodiesterase (w-7) that suggest role of Ca(2+) signaling after triggering sperm activation through hypoosmolality. Symmetric flagellar beating was also turned to asymmetric after activation in w-7, which is an evidence for modulation of Ca(2+)-binding proteins activity. Sturgeon sperm, similar to salmonids, is immotile in seminal plasma due to high K(+) concentrations, but the mechanism of sperm activation seems to be closer to other fish species where osmolality prohibits sperm activation in seminal plasma. In these species, hypoosmolality is the primary signal for sperm Ca(2+)-dependent signaling of axonemal beating.     

Na(+)] in the subsarcolemmal 'fuzzy' space and modulation of [Ca(2+)](i) and contraction in cardiac myocytes

The strength of the heart beat depends on the amplitude and time course of the transient increase in [Ca(2+)] in the myocytes with each cycle. [Na(+)](i) modulates cardiac contraction through its effect on the Ca(2+) flux through the Na/Ca exchanger. Cardiac excitation-contraction coupling has been postulated to occur in a microdomain or 'fuzzy' space at the junction of the T-tubules and the sarcoplasmic reticulum. This 'fuzzy' space is well described for the Ca(2+) fluxes and the interaction between the L-type Ca(2+) channel, the Ca(2+) release channel of the sarcoplasmic reticulum and the Na/Ca exchanger. Co-localization of the Na(+) transporters, in particular the Na/K pump and the Na(+) channel, within this 'fuzzy' space is not as well established. The functional and morphological characteristics of the 'fuzzy' space for Na(+) and its interaction with the Ca(2+) handling suggest that this space is not strictly co-inciding with the Ca(2+) microdomain. In this space [Na(+)] can be several-fold higher or lower than [Na(+)] in the bulk cytosol. This has implications for modulation of [Ca(2+)](i) during a single beat as well as during alterations in Na(+) fluxes seen in pathological conditions.     

Characteristics of a store-operated calcium-permeable channel: sarcoendoplasmic reticulum calcium pump function controls channel gating

We examined the single channel properties and regulation of store-operated calcium channels (SOCC). In human submandibular gland cells, carbachol (CCh) induced flickery channel activity while thapsigargin (Tg) induced burst-like activity, with relatively lower open probability (NP(o)) and longer mean open time. Tg- and CCh-activated channels were permeable to Na(+) and Ba(2+), but not to NMDG, in the absence of Ca(2+). The channels exhibited similar Ca(2+), Na(+), and Ba(2+) conductances and were inhibited by 2-aminoethoxydiphenylborate, xestospongin C, Gd(3+), and La(3+). CCh stimulated flickery activity changed to burst-like activity by (i) addition of Tg, (ii) using Na(+) instead of Ca(2+), (iii) using Ca(2+)-free bath solution, or (iv) buffering [Ca(2+)](i) with BAPTA-AM. Buffering [Ca(2+)](i) induced a 2-fold increase in NP(o) of Tg-stimulated SOCC. Reducing free [Ca(2+)] in the endoplasmic reticulum with the divalent cation chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), induced burst-like channel activity similar to that seen with CCh + Tg. Thus, SOCC is activated by stimulation of muscarinic receptors, inhibition of the sarcoendoplasmic Ca(2+) pump, and lowering [Ca(2+)] in the internal store. Importantly, SOCC activity depends on [Ca(2+)](i) and the free [Ca(2+)] in the internal store. These novel findings reveal that SERCA plays a major role in the gating of SOCC by (i) refilling the internal Ca(2+) store(s) and (ii) decreasing the [Ca(2+)](i)-dependent inhibition.     

The NO pathway acts late during the fertilization response in sea urchin eggs

Both the inositol 1,4,5-trisphosphate (InsP(3)) and ryanodine receptor pathways contribute to the Ca(2+) transient at fertilization in sea urchin eggs. To date, the precise contribution of each pathway has been difficult to ascertain. Evidence has accumulated to suggest that the InsP(3) receptor pathway has a primary role in causing Ca(2+) release and egg activation. However, this was recently called into question by a report implicating NO as the primary egg activator. In the present study we pursue the hypothesis that NO is a primary egg activator in sea urchin eggs and build on previous findings that an NO/cGMP/cyclic ADP-ribose (cADPR) pathway is active at fertilization in sea urchin eggs to define its role. Using a fluorescence indicator of NO levels, we have measured both NO and Ca(2+) at fertilization and establish that NO levels rise after, not before, the Ca(2+) wave is initiated and that this rise is Ca(2+)-dependent. By inhibiting the increase in NO at fertilization, we find not that the Ca(2+) transient is abolished but that the duration of the transient is significantly reduced. The latency and rise time of the transient are unaffected. This effect is mirrored by the inhibition of cGMP and cADPR signaling in sea urchin eggs at fertilization. We establish that cADPR is generated at fertilization, at a time comparable to the time of the rise in NO levels. We conclude that NO is unlikely to be a primary egg activator but, rather, acts after the initiation of the Ca(2+) wave to regulate the duration of the fertilization Ca(2+) transient.     

Ca2+ signaling and exocytosis in pituitary corticotropes

The secretion of adrenocorticotrophin (ACTH) from corticotropes is a key component in the endocrine response to stress. The resting potential of corticotropes is set by the basal activities of TWIK-related K(+) (TREK)-1 channel. Corticotrophin-releasing hormone (CRH), the major ACTH secretagogue, closes the background TREK-1 channels via the cAMP-dependent pathway, resulting in depolarization and a sustained rise in cytosolic [Ca(2+)] ([Ca(2+)](i)). By contrast, arginine vasopressin and norepinephrine evoke Ca(2+) release from the inositol trisphosphate (IP(3))-sensitive store, resulting in the activation of small conductance Ca(2+)-activated K(+) channels and hyperpolarization. Following [Ca(2+)](i) rise, cytosolic Ca(2+) is taken into the mitochondria via the uniporter. Mitochondrial inhibition slows the decay of the Ca(2+) signal and enhances the depolarization-triggered exocytotic response. Both voltage-gated Ca(2+) channel activation and intracellular Ca(2+) release generate spatial Ca(2+) gradients near the exocytic sites such that the local [Ca(2+)] is ~3-fold higher than the average [Ca(2+)](i). The stimulation of mitochondrial metabolism during the agonist-induced Ca(2+) signal and the robust endocytosis following stimulated exocytosis enable corticotropes to maintain sustained secretion during the diurnal ACTH surge. Arachidonic acid (AA) which is generated during CRH stimulation activates TREK-1 channels and causes hyperpolarization. Thus, corticotropes may regulate ACTH release via an autocrine feedback mechanism.     

A Ca(2+)- and voltage-modulated flagellar ion channel is a component of the mechanoshock response in the unicellular green alga Spermatozopsis similis

In flagellate green algae, behavioral responses to photo- and mechanoshock are induced by different external stimuli within 10-15 ms. In the accompanying changes in flagella beat, Ca(2+) has important regulatory roles. Although the axonemal Ca(2+) responsive elements are well characterized, analyses of flagellar channels involved in Ca(2+) signalling as well as other ion channels at the single-channel level were not yet conducted in green algae. To gain a further understanding of these important signaling elements in movement responses, intact flagella of Spermatozopsis similis were isolated and characterized and the solubilized flagellar membrane proteins were reconstituted into liposomes. We observed three types of channel activity, two of which were weakly anion and cation-selective and in the high-conductance regime typical for porin-like solute channels. The dominating channel activity was a voltage dependent, rectifying, low conductance (Lambda=80 pS in 50 mM KCl) cation-selective channel modulated by, and highly permeable to, Ca(2+) ions (SFC1: Spermatozopsis flagellar cation channel 1). Depolarizations necessary to activate SFC1 probably only occur in vivo during avoidance reactions of this alga. Ca(2+)-activation of SFC1 points to a direct link to Ca(2+)-mediated signaling pathway(s) in the flagella. Both the response to mechanoshock and SFC1 activity were inhibited by Gd(3+) and Ba(2+), thus supporting our assumption that SFC1 represents a major flagellar ion channel involved in this green algal avoidance reaction.