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Molecular and functional characterization of the human platelet Na(+) /Ca(2+) exchangers.

ABSTRACT: BACKGROUND AND PURPOSE The Na(+) /Ca(2+) exchanger is a bi-directional transporter that plays an important role in maintaining the concentration of cytosolic Ca(2+) ([Ca(2+) ](i) ) of quiescent platelets and increasing it during activation with some, but not all, agonists. There are two classes of Na(+) /Ca(2+) exchangers: K(+) -independent Na(+) /Ca(2+) exchanger (NCX) and K(+) -dependent Na(+) /Ca(2+) exchanger (NCKX). Platelets have previously been shown to express NCKX1. However, initial studies from our laboratory suggest that NCX may also play a role in platelet activation. The objective of this study was to determine if the human platelet expresses functional NCXs. EXPERIMENTAL APPROACH RT-PCR, DNA sequencing and Western blot analysis were utilized to characterize the human platelet Na(+) /Ca(2+) exchangers. Their function during quiescence and collagen-induced activation was determined by measuring [Ca(2+) ](i) with calcium-green/fura-red in response to: changes in the Na(+) and K(+) gradient, NCX pharmacological inhibitors (CBDMB, KB-R7943 and SEA0400) and antibodies specific to extracellular epitopes of the exchangers. KEY RESULTS Human platelets express NCX1.3, NCX3.2 and NCX3.4. The NCXs operate in the Ca(2+) efflux mode in resting platelets and also during their activation with thrombin but not collagen. Collagen-induced increase in [Ca(2+) ](i) was reduced with the pharmacological inhibitors of NCX (CBDMB, KB-R7943 or SEA0400), anti-NCX1 and anti-NCX3. In contrast, anti-NCKX1 enhanced the collagen-induced increase in [Ca(2+) ](i) . CONCLUSIONS AND IMPLICATIONS Human platelets express K(+) -independent Na(+) /Ca(2+) exchangers NCX1.3, NCX3.2 and NCX3.4. During collagen activation, NCX1 and NCX3 transiently reverse to promote Ca(2+) influx, whereas NCKX1 continues to operate in the Ca(2+) efflux mode to reduce [Ca(2+) ](i) .

SUBMITTER: Roberts DE

PROVIDER: S-EPMC3312489 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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Molecular and functional characterization of the human platelet Na(+) /Ca(2+) exchangers.

Roberts Diane E DE Matsuda Toshio T Bose Ratna R

British journal of pharmacology 20120201 4

BACKGROUND AND PURPOSE The Na(+) /Ca(2+) exchanger is a bi-directional transporter that plays an important role in maintaining the concentration of cytosolic Ca(2+) ([Ca(2+) ](i) ) of quiescent platelets and increasing it during activation with some, but not all, agonists. There are two classes of Na(+) /Ca(2+) exchangers: K(+) -independent Na(+) /Ca(2+) exchanger (NCX) and K(+) -dependent Na(+) /Ca(2+) exchanger (NCKX). Platelets have previously been shown to express NCKX1. However, initia ...[more]

PMID: 21790537

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Project description:BackgroundWhile calcium is known to play a crucial role in mammalian sperm physiology, how it flows in and out of the male gamete is not completely understood. Herein, we investigated the involvement of Na+/Ca2+ exchangers (NCX) in mammalian sperm capacitation. Using the pig as an animal model, we first confirmed the presence of NCX1 and NCX2 isoforms in the sperm midpiece. Next, we partially or totally blocked Ca2+ outflux (forward transport) via NCX1/NCX2 with different concentrations of SEA0400 (2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline; 0, 0.5, 5 and 50 µM) and Ca2+ influx (reverse transport) with SN6 (ethyl 2-[[4-[(4-nitrophenyl)methoxy]phenyl]methyl]-1,3-thiazolidine-4-carboxylate; 0, 0.3, 3 or 30 µM). Sperm were incubated under capacitating conditions for 180 min; after 120 min, progesterone was added to induce the acrosome reaction. At 0, 60, 120, 130, and 180 min, sperm motility, membrane lipid disorder, acrosome integrity, mitochondrial membrane potential (MMP), tyrosine phosphorylation of sperm proteins, and intracellular levels of Ca2+, reactive oxygen species (ROS) and superoxides were evaluated.ResultsPartial and complete blockage of Ca2+ outflux and influx via NCX induced a significant reduction of sperm motility after progesterone addition. Early alterations on sperm kinematics were also observed, the effects being more obvious in totally blocked than in partially blocked samples. Decreased sperm motility and kinematics were related to both defective tyrosine phosphorylation and mitochondrial activity, the latter being associated to diminished MMP and ROS levels. As NCX blockage did not affect the lipid disorder of plasma membrane, the impaired acrosome integrity could result from reduced tyrosine phosphorylation.ConclusionsInhibition of outflux and influx of Ca2+ triggered similar effects, thus indicating that both forward and reverse Ca2+ transport through NCX exchangers are essential for sperm capacitation.

Dataset Information

Molecular and functional characterization of the human platelet Na(+) /Ca(2+) exchangers.

Publications

Molecular and functional characterization of the human platelet Na(+) /Ca(2+) exchangers.

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