Two distinct Ca(2+) signaling pathways modulate sperm flagellar beating patterns in mice

Hyperactivation, a swimming pattern of mammalian sperm in the oviduct, is essential for fertilization. It is characterized by asymmetrical flagellar beating and an increase of cytoplasmic Ca(2+). We observed that some mouse sperm swimming in the oviduct produce high-amplitude pro-hook bends (bends in the direction of the hook on the head), whereas other sperm produce high-amplitude anti-hook bends. Switching direction of the major bends could serve to redirect sperm toward oocytes. We hypothesized that different Ca(2+) signaling pathways produce high-amplitude pro-hook and anti-hook bends. In vitro, sperm that hyperactivated during capacitation (because of activation of CATSPER plasma membrane Ca(2+) channels) developed high-amplitude pro-hook bends. The CATSPER activators procaine and 4-aminopyridine (4-AP) also induced high-amplitude pro-hook bends. Thimerosal, which triggers a Ca(2+) release from internal stores, induced high-amplitude anti-hook bends. Activation of CATSPER channels is facilitated by a pH rise, so both Ca(2+) and pH responses to treatments with 4-AP and thimerosal were monitored. Thimerosal triggered a Ca(2+) increase that initiated at the base of the flagellum, whereas 4-AP initiated a rise in the proximal principal piece. Only 4-AP triggered a flagellar pH rise. Proteins were extracted from sperm for examination of phosphorylation patterns induced by Ca(2+) signaling. Procaine and 4-AP induced phosphorylation of proteins on threonine and serine, whereas thimerosal primarily induced dephosphorylation of proteins. Tyrosine phosphorylation was unaffected. We concluded that hyperactivation, which is associated with capacitation, can be modulated by release of Ca(2+) from intracellular stores to reverse the direction of the dominant flagellar bend and, thus, redirect sperm.     

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.     

Distinct Ca2+ channels maintain a high motility state of the sperm that may be needed for penetration of egg jelly of the newt,Cynops pyrrhogaster

Activation state of sperm motility named “hyperactivation” enables mammalian sperm to progress through the oviductal matrix, although a similar state of sperm motility is unknown in non‐mammalian vertebrates at fertilization. Here, we found a high motility state of the sperm in the newt Cynops pyrrhogaster. It was predominantly caused in egg jelly extract (JE) and characterized by a high wave velocity of the undulating membrane (UM) that was significantly higher at the posterior midpiece. An insemination assay suggested that the high motility state might be needed for sperm to penetrate the egg jelly, which is the accumulated oviductal matrix. Specific characteristics of the high motility state were completely abrogated by a high concentration of verapamil, which blocks the L‐type and T‐type voltage‐dependent Ca²⁺ channels (VDCCs). Mibefradil, a dominant blocker of T‐type VDCCs, suppressed the wave of the UM at the posterior midpiece with separate wave propagation from both the anterior midpiece and the posterior principal piece. In addition, nitrendipine, a dominant L‐type VDCC blocker, weakened the wave of the UM, especially in the anterior midpiece. Live Ca²⁺ imaging showed that, compared with the intact sperm in the JE, the relative intracellular Ca²⁺ level changed especially in the anterior and posterior ends of the midpiece of the blocker‐treated sperm. These suggest that different types of Ca²⁺ channels mediate the intracellular Ca²⁺ level predominantly in the anterior and posterior ends of the midpiece to maintain the high motility state of the newt sperm.     

Characterization of the intracellular calcium store at the base of the sperm flagellum that regulates hyperactivated motility

Hyperactivated sperm motility is usually characterized by high-amplitude flagellar bends and asymmetrical flagellar beating. There is evidence that an inositol 1,4,5-trisphosphate (IP3) receptor-gated Ca2+ store in the base of the flagellum provides Ca2+ to initiate hyperactivation; however, the identity of the store was not known. Ca2+ stores are membrane-bounded organelles, and the only two membrane-bounded organelles found in this region of sperm are the redundant nuclear envelope (RNE) and mitochondria. Transmission electron micrographs revealed two different compartments of RNE, one enriched with nuclear pores and the other containing few pores but extensive membranous structures with enlarged cisternae. Immunolabeling showed that IP3 receptors and calreticulin are located in the region containing enlarged cisternae. In other cell types, mitochondria adjacent to Ca2+ stores are actively involved in modulating Ca2+ signals by taking up Ca2+ released from stores and also may respond by increasing production of NADH and ATP to support increased energy demand. Nevertheless, bull sperm did not show an increase in NADH when Ca2+ was released from intracellular stores by thapsigargin to induce hyperactivation. Consistently, no net increase in ATP production was detected when sperm were hyperactivated, although ATP was hydrolyzed at a greater rate. Furthermore, blocking Ca2+ efflux from mitochondria by CGP-37157, a specific inhibitor of the mitochondrial Na+/Ca2+ exchanger, did not inhibit the development of hyperactivated motility. We concluded that the intracellular Ca2+ store is the part of RNE that contains enlarged cisternae and that Ca2+ is released directly to the axoneme to trigger hyperactivated motility without the active participation of mitochondria.     

Catsper3 and Catsper4 are essential for sperm hyperactivated motility and male fertility in the mouse

Catsper3 and Catsper4 are two recently identified testis-specific genes homologous to Catsper1 and Catsper2 that have been shown to play an essential role in sperm hyperactivated motility and male fertility in mice. Here we report that Catsper3 and Catsper4 knockout male mice are completely infertile due to a quick loss of motility and a lack of hyperactivated motility under capacitating conditions. Our data demonstrate that both CATSPER3 and CATSPER4 are required for hyperactivated sperm motility during capacitation and for male fertility. The present study also demands a revisit to the idiopathic male infertility patients who show normal sperm counts and normal initial motility for defects in sperm hyperactivated motility and for potential CATSPER gene mutations. The CATSPER channel also may be an excellent drug target for male contraceptives.     

Bovine sperm hyperactivation is promoted by alkaline-stimulated Ca2+ influx

Sperm hyperactivated motility is characterized by high flagellar bend amplitude and asymmetrical beating, which are detected by computer-assisted sperm motility analysis as increased curvilinear velocity and lateral head movement. It is required for sperm penetration of the oocyte zona pellucida during fertilization and is induced by an increase in flagellar Ca(2+). Our objective was to determine whether pH plays a role in promoting Ca signaling of hyperactivated motility. The cell-permeant weak base NH(4)Cl increased curvilinear velocity and amplitude of lateral head movement of bovine sperm, indicative of hyperactivation. Fluorometric recordings of sperm loaded with BCECF-AM or fluo3-AM, revealed that NH(4)Cl evoked elevations of intracellular pH and Ca(2+), respectively, with the rise in pH occurring more rapidly than that of Ca(2+). Single-cell image analysis showed increased Ca(2+) levels in the flagellum in response to NH(4)Cl. When extracellular Ca(2+) was lowered with BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) prior to treatment with NH(4)Cl, intracellular pH was increased, but elevation of Ca(2+) and hyperactivation were diminished. This suggests that the rise in intracellular pH precedes an influx of Ca(2+). The Ca(2+) channel blocker Ni(2+) also diminished NH(4)Cl stimulation of hyperactivation, demonstrating that Ca(2+) entry is required for maximal expression of hyperactivation. Ca(2+) ionophore produced an increase in Ca(2+) that was 3-fold greater than that produced by NH(4)Cl; however, it produced a weaker hyperactivation response. These results indicate that a rise in pH increases intracellular Ca(2+)and promotes hyperactivation primarily by stimulating Ca(2+) influx, but also by other mechanisms.     

Contributions of extracellular and intracellular Ca2+ to regulation of sperm motility: Release of intracellular stores can hyperactivate CatSper1 and CatSper2 null sperm

In order to fertilize, mammalian sperm must hyperactivate. Hyperactivation is triggered by increased flagellar Ca(2+), which switches flagellar beating from a symmetrical to an asymmetrical pattern by increasing bending to one side. Thimerosal, which releases Ca(2+) from internal stores, induced hyperactivation in mouse sperm within seconds, even when extracellular Ca(2+) was buffered with BAPTA to approximately 30 nM. In sperm from CatSper1 or CatSper2 null mice, which lack functional flagellar alkaline-activated calcium currents, 50 microM thimerosal raised the flagellar bend amplitudes from abnormally low levels to normal pre-hyperactivated levels and, in 20-40% of sperm, induced hyperactivation. Addition of 1 mM Ni(2+) diminished the response. This suggests that intracellular Ca(2+) is abnormally low in the null sperm flagella. When intracellular Ca(2+) was reduced by BAPTA-AM in wild-type sperm, they exhibited flagellar beat patterns more closely resembling those of null sperm. Altogether, these results indicate that extracellular Ca(2+) is required to supplement store-released Ca(2+) to produce maximal and sustained hyperactivation and that CatSper1 and CatSper2 are key elements of the major Ca(2+) entry pathways that support not only hyperactivated motility but possibly also normal pre-hyperactivated motility.     

SLO3 K+ Channels Control Calcium Entry through CATSPER Channels in Sperm

Here we show how a sperm-specific potassium channel (SLO3) controls Ca²⁺ entry into sperm through a sperm-specific Ca²⁺ channel, CATSPER, in a totally unanticipated manner. The genetic deletion of either of those channels confers male infertility in mice. During sperm capacitation SLO3 hyperpolarizes the sperm, whereas CATSPER allows Ca²⁺ entry. These two channels may be functionally connected, but it had not been demonstrated that SLO3-dependent hyperpolarization is required for Ca²⁺ entry through CATSPER channels, nor has a functional mechanism linking the two channels been shown. In this study we show that Ca²⁺ entry through CATSPER channels is deficient in Slo3 mutant sperm lacking hyperpolarization; we also present evidence supporting the hypothesis that SLO3 channels activate CATSPER channels indirectly by promoting a rise in intracellular pH through a voltage-dependent mechanism. This mechanism may work through a Na⁺/H⁺ exchanger (sNHE) and/or a bicarbonate transporter, which utilizes the inward driving force of the Na⁺ gradient, rendering it intrinsically voltage-dependent. In addition, the sperm-specific Na⁺/H⁺ exchanger (sNHE) possess a putative voltage sensor that might be activated by membrane hyperpolarization, thus increasing the voltage sensitivity of internal alkalization.     

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.     

An inositol 1,4,5-trisphosphate receptor-gated intracellular Ca(2+) store is involved in regulating sperm hyperactivated motility.

Hyperactivated motility, a swimming pattern displayed by mammalian sperm in the oviduct around the time of ovulation, is essential to fertilization. Ca(2+) has been shown to be crucial for the initiation and maintenance of hyperactivated motility. Nevertheless, how Ca(2+) reaches the axoneme in the core of the flagellum to switch on hyperactivation is unknown. Ca(2+)-releasing agents were used to determine whether an intracellular store provides Ca(2+) to the axoneme. Hyperactivation was induced immediately in bull sperm by thapsigargin, caffeine, and thimerosal. The responses were dose-dependent and were induced in both capacitated and uncapacitated sperm. When external Ca(2+) was buffered below 50 nM with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, the response to caffeine was significantly reduced; however, the responses to thapsigargin and thimerosal were not affected. This indicates caffeine-induced hyperactivation depends on external Ca(2+) influx, whereas hyperactivation by thapsigargin and thimerosal do not. Acrosome reactions were not induced by these treatments; therefore, an acrosomal store was probably not involved. Indirect immunofluorescence labeling showed type I inositol 1,4,5-trisphosphate receptors (IP(3)R) in the acrosome and neck region, but no ryanodine receptors (RyR) were found using anti-RyR antibodies or BODIPY FL-X ryanodine. These data indicate that there is an IP(3)R-gated Ca(2+) store in the neck region of sperm that regulates hyperactivated motility.     

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.     

Hyperactivation of monkey spermatozoa is triggered by Ca2+ and completed by cAMP

Digital image analysis of the flagellar movements of cynomolgus macaque spermatozoa hyperactivated by caffeine and cAMP was carried out to understand the change in flagellar movements during hyperactivation. The degree of flagellar bending increased remarkably after hyperactivation, especially at the base of the midpiece. Mainly two beating patterns were seen in the hyperactivated monkey sperm flagella: remarkably asymmetrical flagellar bends of large amplitude and relatively symmetrical flagellar bends of large amplitude. The asymmetrical bends were often seen in the early stage of hyperactivation, whereas the symmetrical bends executed nonprogressive, figure-of-eight movement. Beat frequency of the hyperactivated spermatozoa significantly decreased while wavelength of flagellar waves roughly doubled. To determine the conditions under which the axonemes of hyperactivated sperm flagella have asymmetrical or symmetrical bends, the plasma membranes of monkey spermatozoa were extracted with Triton X-100 and motility was reactivated with MgATP(2-) under various conditions. The asymmetrical flagellar bends were brought about by Ca(2+), whereas the symmetrical flagellar bends resulted from low levels of Ca(2+) and high levels of cAMP. Under these conditions, beat frequency and wavelength of flagellar waves of demembranated, reactivated spermatozoa were similar to those of the hyperactivated spermatozoa. These results suggest that during hyperactivation of monkey spermatozoa intracellular Ca(2+) concentrations first rise, and then decrease while cAMP concentrations increase simultaneously.     

Simultaneous knockout of Slo3 and CatSper1 abolishes all alkalization- and voltage-activated current in mouse spermatozoa

During passage through the female reproductive tract, mammalian sperm undergo a maturation process termed capacitation that renders sperm competent to produce fertilization. Capacitation involves a sequence of changes in biochemical and electrical properties, the onset of a hyperactivated swimming behavior, and development of the ability to undergo successful fusion and penetration with an egg. In mouse sperm, the development of hyperactivated motility is dependent on cytosolic alkalization that then results in an increase in cytosolic Ca²⁺. The elevation of Ca²⁺ is thought to be primarily driven by the concerted interplay of two alkalization-activated currents, a K⁺ current (KSPER) composed of pore-forming subunits encoded by the Kcnu1 gene (also termed Slo3) and a Ca²⁺ current arising from a family of CATSPER subunits. After deletion of any of four CATSPER subunit genes (CATSPER1–4), the major remaining current in mouse sperm is alkalization-activated KSPER current. After genetic deletion of the Slo3 gene, KSPER current is abolished, but there remains a small voltage-activated K⁺ current hypothesized to reflect monovalent flux through CATSPER. Here, we address two questions. First, does the residual outward K⁺ current present in the Slo3 ^−/− sperm arise from CATSPER? Second, can any additional membrane K⁺ currents be detected in mouse sperm by patch-clamp methods other than CATSPER and KSPER? Here, using mice bred to lack both SLO3 and CATSPER1 subunits, we show conclusively that the voltage-activated outward current present in Slo3 ^−/− sperm is abolished when CATSPER is also deleted. Any leak currents that may play a role in setting the resting membrane potential in noncapacitated sperm are likely smaller than the pipette leak current and thus cannot be resolved within the limitation of the patch-clamp technique. Together, KSPER and CATSPER appear to be the sole ion channels present in mouse sperm that regulate membrane potential and Ca²⁺ influx in response to alkalization.     

Intracellular Ca(2+)-Mg(2+)-ATPase regulates calcium influx and acrosomal exocytosis in bull and ram spermatozoa.

Calcium influx is required for the mammalian sperm acrosome reaction (AR), an exocytotic event occurring in the sperm head prior to fertilization. We show here that thapsigargin, a highly specific inhibitor of the microsomal Ca(2+)-Mg(2+)-ATPase (Ca(2+) pump), can initiate acrosomal exocytosis in capacitated bovine and ram spermatozoa. Initiation of acrosomal exocytosis by thapsigargin requires an influx of Ca(2+), since incubation of cells in the absence of added Ca(2+) or in the presence of the calcium channel blocker, La(3+), completely inhibited thapsigargin-induced acrosomal exocytosis. ATP-Dependent calcium accumulation into nonmitochondrial stores was detected in permeabilized sperm in the presence of ATP and mitochondrial uncoupler. This activity was inhibited by thapsigargin. Thapsigargin elevated the intracellular Ca(2+) concentration ([Ca(2+)](i)), and this increase was inhibited when extracellular Ca(2+) was chelated by EGTA, indicating that this rise in Ca(2+) is derived from the external medium. This rise of [Ca(2+)](i) took place first in the head and later in the midpiece of the spermatozoon. However, immunostaining using a polyclonal antibody directed against the purified inositol 1,4,5-tris-phosphate receptor (IP(3)-R) identified specific staining in the acrosome region, in the postacrosome, and along the tail, but not in the midpiece region. No staining in the acrosome region was observed in sperm without acrosome, indicating that the acrosome cap was stained in intact sperm. The presence of IP(3)-R in the anterior acrosomal region as well as the induction, by thapsigargin, of intracellular Ca(2+) elevation in the acrosomal region and acrosomal exocytosis, implicates the acrosome as a potential cellular Ca(2+) store. We suggest here that the cytosolic Ca(2+) is actively transported into the acrosome by an ATP-dependent, thapsigargin-sensitive Ca(2+) pump and that the accumulated Ca(2+) is released from the acrosome via an IP(3)-gated calcium channel. The ability of thapsigargin to increase [Ca(2+)](i) could be due to depletion of Ca(2+) in the acrosome, resulting in the opening of a capacitative calcium entry channel in the plasma membrane. The effect of thapsigargin on elevated [Ca(2+)](i) in capacitated cells was 2-fold higher than that in noncapacitated sperm, suggesting that the intracellular Ca pump is active during capacitation and that this pump may have a role in regulating [Ca(2+)](i) during capacitation and the AR.     

Lipid rafts function in Ca2+ signaling responsible for activation of sperm motility and chemotaxis in the ascidian Ciona intestinalis

Lipid rafts are specialized membrane microdomains that function as signaling platforms across plasma membranes of many animal and plant cells. Although there are several studies implicating the role of lipid rafts in capacitation of mammalian sperm, the function of these structures in sperm motility activation and chemotaxis remains unknown. In the ascidian Ciona intestinalis, egg-derived sperm activating- and attracting-factor (SAAF) induces both activation of sperm motility and sperm chemotaxis to the egg. Here we found that a lipid raft disrupter, methyl-β-cyclodextrin (MCD), inhibited both SAAF-induced sperm motility activation and chemotaxis. MCD inhibited both SAAF-promoted synthesis of intracellular cyclic AMP and sperm motility induced by ionophore-mediated Ca(2+) entry, but not that induced by valinomycin-mediated hyperpolarization. Ca(2+)-imaging revealed that lipid raft disruption inhibited Ca(2+) influx upon activation of sperm motility. The Ca(2+)-activated adenylyl cyclase was clearly inhibited by MCD in isolated lipid rafts. The results suggest that sperm lipid rafts function in signaling upstream of cAMP synthesis, most likely in SAAF-induced Ca(2+) influx, and are required for Ca(2+)-dependent pathways underlying activation and chemotaxis in Ciona sperm.     

Store-operated calcium channels trigger exocytosis of the sea urchin sperm acrosomal vesicle

The acrosome reaction (AR) of sperm is a prerequisite for fusion with the egg. In sea urchins, the complete AR (CAR) consists of exocytosis of the acrosomal vesicle (AV) and polymerization of acrosomal actin to form the approximately 1 micro m long acrosomal process. The fucose sulfate polymer (FSP) of egg jelly stimulates Ca(2+) entry through two distinct Ca(2+) channels and induces the CAR. Here we report that the second channel is blocked by SKF96365 (SKF), an inhibitor of store-operated channels. SKF also blocks the thapsigargin (TG), trifluoperazine (TFP), and calmidizolium (CMZ) stimulated Ca(2+) entry into sperm. These data indicate that the second Ca(2+) channel is a store-operated channel (SOC) that may be regulated by calmodulin. The TG, TFP, and CMZ-induced intracellular Ca(2+) elevations are similar to those induced by FSP, but the sperm acrosomal process does not polymerize. An antibody to bindin, the major protein of the AV, showed that in a significant percentage of these drug-treated sperm, the AV had undergone exocytosis. When NH(4)Cl was added to increase intracellular pH, the TG-treated sperm polymerized actin to form the acrosomal process. We conclude that the second Ca(2+) channel of sea urchin sperm is a SOC that triggers AV exocytosis.     

Spatial microenvironment defines Ca2+ entry and Ca2+ release in salivary gland cells

The difference of Ca(2+) mobilization induced by muscarinic receptor activation between parotid acinar and duct cells was examined. Oxotremorine, a muscarinic-cholinergic agonist, induced intracellular Ca(2+) release and extracellular Ca(2+) entry through store-operated Ca(2+) entry (SOC) and non-SOC channels in acinar cells, but it activated only Ca(2+) entry from non-SOC channels in duct cells. RT-PCR experiments showed that both types of cells expressed the same muscarinic receptor, M3. Given that ATP activated the intracellular Ca(2+) stores, the machinery for intracellular Ca(2+) release was intact in the duct cells. By immunocytochemical experiments, IP(3)R2 colocalized with M3 receptors in the plasma membrane area of acinar cells; in duct cells, IP(3)R2 resided in the region on the opposite side of the M3 receptors. On the other hand, purinergic P2Y2 receptors were found in the apical area of duct cells where they colocalized with IP(3)R2. These results suggest that the expression of the IP(3)Rs near G-protein-coupled receptors is necessary for the activation of intracellular Ca(2+) stores. Therefore, the microenvironment probably affects intracellular Ca(2+) release and Ca(2+) entry.     

Regulation of store-operated calcium channels by the intermediary metabolite pyruvic acid

A rise in cytosolic Ca(2+) concentration is used as a key activation signal in virtually all animal cells, where it triggers a range of responses, including neurotransmitter release, muscle contraction, and cell growth and proliferation. A major route for Ca(2+) influx is through store-operated Ca(2+) channels. One important intracellular target for Ca(2+) entry through store-operated channels is the mitochondrion, which increases aerobic metabolism and ATP production after Ca(2+) uptake. Here, we reveal a novel feedback pathway whereby pyruvic acid, a critical rate-limiting substrate for mitochondrial respiration, increases store-operated entry by reducing inactivation of the channels. Importantly, the effects of pyruvic acid are manifest at physiologically relevant concentrations and membrane potentials. The reduction in the inactivation of calcium release-activated calcium (CRAC) channels by pyruvate is highly specific in that it is not mimicked by other intermediary metabolic acids, does not require its metabolism, is independent of its Ca(2+) buffering action, and does not involve mitochondrial Ca(2+) uptake or ATP production. These results reveal a new and direct link between intermediary metabolism and ion-channel gating and identify pyruvate as a potential signaling messenger linking energy demand to calcium-channel function.     

Mitochondrial Ca2+ homeostasis in human NADH:ubiquinone oxidoreductase deficiency

NADH:ubiquinone oxidoreductase or complex I is a large multisubunit assembly of the mitochondrial inner membrane that channels high-energy electrons from metabolic NADH into the electron transport chain (ETC). Its dysfunction is associated with a range of progressive neurological disorders, often characterized by a very early onset and short devastating course. To better understand the cytopathological mechanisms of these disorders, we use live cell luminometry and imaging microscopy of patient skin fibroblasts with mutations in nuclear-encoded subunits of the complex. Here, we present an overview of our recent work, showing that mitochondrial membrane potential, Ca(2+) handling and ATP production are to a variable extent impaired among a large cohort of patient fibroblast lines. From the results obtained, the picture emerges that a reduction in cellular complex I activity leads to a depolarization of the mitochondrial membrane potential, resulting in a decreased supply of mitochondrial ATP to the Ca(2+)-ATPases of the intracellular stores and thus to a reduced Ca(2+) content of these stores. As a consequence, the increase in cytosolic Ca(2+) concentration evoked by a Ca(2+) mobilizing stimulus is decreased, leading to a reduction in mitochondrial Ca(2+) accumulation and ensuing ATP production and thus to a hampered energization of stimulus-induced cytosolic processes.