Low-copy nuclear markers in Isoëtes (Isoëtaceae) identified with transcriptomes.

PREMISE OF THE STUDY: Few genetic markers provide phylogenetic information in closely related species of Isoëtes (Isoëtaceae). We describe the development of primers for several putative low-copy nuclear markers to resolve the phylogeny of Isoëtes, particularly in the southeastern United States. METHODS AND RESULTS: We identified regions of interest in Isoëtes transcriptomes based on low-copy genes in other plants. Primers were designed for these regions and tested with 16 taxa of Isoëtes and one species of Lycopodium. Parts of the pgiC, gapC, and IBR3 gene regions show phylogenetic signal within the North American and Mediterranean clades of Isoëtes. CONCLUSIONS: Transcriptome data prove useful for identification and primer design of low-copy genes. Three new markers show potential for inferring phylogenies in regional clades of Isoëtes, and possibly across the entire genus.

Reduced Protein Expression of the Na+/Ca2++K+-Exchanger (SLC24A4) in Apical Plasma Membranes of Maturation Ameloblasts of Fluorotic Mice.

Exposure of forming enamel to fluoride results into formation of hypomineralized enamel. We tested whether enamel hypomineralization was caused by lower expression of the NCKX4/SLC24A4 Ca2+-transporter by ameloblasts. Three commercial antibodies against NCKX4 were tested on enamel organs of wild-type and Nckx4-null mice, one of which (a mouse monoclonal) was specific. This antibody gave a prominent staining of the apical plasma membranes of maturation ameloblasts, starting at early maturation. The layer of immuno-positive ameloblasts contained narrow gaps without immunostaining or with reduced staining. In fluorotic mouse incisors, the quantity of NCKX4 protein in ameloblasts as assessed by western blotting was not different from that in non-fluorotic ameloblasts. However, immunostaining of the apical plasma membranes of fluorotic ameloblasts was strongly reduced or absent suggesting that trafficking of NCKX4 to the apical membrane was strongly reduced. Exposure to fluoride may reduce NCKX4-mediated transport of Ca2+ by maturation stage ameloblasts which delays ameloblast modulation and reduces enamel mineralization.

Isoetes mississippiensis: A new quillwort from Mississippi, USA.

Isoetes mississippiensis S.W. Leonard, W.C. Taylor, L.J. Musselman and R.D. Bray (Isoetaceae, Lycopodiophyta) is a new species known from two sites along tributaries of the Pearl River in southern Mississippi. This species is distinguished from other species in the southeastern United States by a combination of character states including a basic diploid (2n=22) chromosome count, laevigate megaspores, and a narrow velum covering less than one-third of the adaxial sporangium wall.

Anatomical evidence for a non-synaptic influence of the K+ -dependent Na+/Ca2+ -exchanger, NCKX2, on hippocampal plasticity.

The K(+)-dependent Na(+)/Ca(2)-exchanger (NCKX) family is encoded by five related genes, of which NCKX2 (solute carrier family 24, member 2) is the most abundant member present in the brain. Nckx2 knockout mice display profound loss of hippocampal long-term potentiation, and selective deficits in motor learning and spatial working memory. However, the molecular mechanisms underlying these changes have not been established. Thus, the overall goal of this project was to identify the exact subcellular localization of NCKX2 in the hippocampus, as an important step toward understanding the physiological role for NCKX2 in neuronal plasticity. To achieve this goal, we used dual immunofluorescent confocal microscopy and immunoelectron microscopy. Our data demonstrate that the majority of NCKX2 is co-localized with the dendritic marker, microtubule associated protein 2. A smaller fraction is co-localized with the presynaptic marker, synapsin 1, and the smallest amount is co-localized with the glutamatergic spine marker, N-methyl-d-aspartate receptor 1. The data from immunoelectron microscopy are consistent with the observations from dual immunofluorescence, and show that the highest fraction of NCKX2 is located on the plasma membrane of small oblique dendrites, particularly in CA1 neurons of the stratum radiatum. In the molecular layer, a greater fraction of NCKX2 is associated with axon terminals and, in addition, a fraction of NCKX2 is found not associated with the plasma membrane but located in the cytoplasm. These studies describe for the first time the exact location of NCKX2 in the hippocampus of adult mice and suggest that the function of NCKX2 in neuronal plasticity in hippocampal CA1 neurons may be mediated by its kinetic effect on the local Ca(2+) concentration that influences dendritic integration. At other hippocampal locations NCKX2 has a somewhat different spatial distribution, consistent with published reports of NCKX2 expression in other brain regions, suggesting that NCKX2 contributes to Ca(2+) homeostasis in distinct ways in different brain neurons.

Structural attributes of the hypogeous holoparasite Hydnora triceps Drege & Meyer (Hydnoraceae).

The morphology of the hypogeous root holoparasite Hydnora triceps is highly reduced, and as with many holoparasites, the vegetative body is difficult to interpret. The vegetative body of H. triceps has been historically considered a "pilot root" studded with lateral appendages known as "haustorial roots." We found the vegetative body of H. triceps to consist of a rhizome with a thickened root-cap-like structure that covered a vegetative shoot apical meristem. From the apical meristem, procambial strands originated and developed into endarch collateral vascular bundles arranged radially around a pith without an interfascicular cambium. Xylem vessels had scalariform pitting and simple perforation plates. A continuous periderm without root hairs was observed. Increase in girth was attributed to cork and fascicular cambia. "Haustorial roots" or bumps on the surface of the vegetative body were exogenous, contained meristems and were the origins of vegetative branching, budding, and haustoria. The haustoria of H. triceps were cylindrical and penetrated the host root stele. Phloem and xylem elements were observed within the endophyte, and direct xylem to host-xylem contacts were observed. The arrangement of vascular tissues and xylem anatomy of H. triceps are likely plesiomorphic features in light of Hydnoraceae's placement in the Piperales.

Overexpression of Na+/Ca2+ exchanger alters contractility and SR Ca2+ content in adult rat myocytes.

The functional consequences of overexpression of rat heart Na+/Ca2+ exchanger (NCX1) were investigated in adult rat myocytes in primary culture. When maintained under continued electrical field stimulation conditions, cultured adult rat myocytes retained normal contractile function compared with freshly isolated myocytes for at least 48 h. Infection of myocytes by adenovirus expressing green fluorescent protein (GFP) resulted in >95% infection as ascertained by GFP fluorescence, but contraction amplitude at 6-, 24-, and 48-h postinfection was not affected. When they were examined 48 h after infection, myocytes infected by adenovirus expressing both GFP and NCX1 had similar cell sizes but exhibited significantly altered contraction amplitudes and intracellular Ca2+ concentration ([Ca2+]i) transients, and lower resting and diastolic [Ca2+]i when compared with myocytes infected by the adenovirus expressing GFP alone. The effects of NCX1 overexpression on sarcoplasmic reticulum (SR) Ca2+ content depended on extracellular Ca2+ concentration ([Ca2+]o), with a decrease at low [Ca2+]o and an increase at high [Ca2+]o. The half-times for [Ca2+]i transient decline were similar, suggesting little to no changes in SR Ca2+-ATPase activity. Western blots demonstrated a significant (P < or = 0.02) threefold increase in NCX1 but no changes in SR Ca2+-ATPase and calsequestrin abundance in myocytes 48 h after infection by adenovirus expressing both GFP and NCX1 compared with those infected by adenovirus expressing GFP alone. We conclude that overexpression of NCX1 in adult rat myocytes incubated at high [Ca2+]o resulted in enhanced Ca2+ influx via reverse NCX1 function, as evidenced by greater SR Ca2+ content, larger twitch, and [Ca2+]i transient amplitudes. Forward NCX1 function was also increased, as indicated by lower resting and diastolic [Ca2+]i.

Electrophysiological characterization and ionic stoichiometry of the rat brain K(+)-dependent NA(+)/CA(2+) exchanger, NCKX2.

We have recently described a novel K(+)-dependent Na(+)/Ca(2+) exchanger, NCKX2, that is abundantly expressed in brain neurons (Tsoi, M., Rhee, K.-H., Bungard, D., Li, X.-F., Lee, S.-L., Auer, R. N., and Lytton, J. (1998) J. Biol. Chem. 273, 4115--4162). The precise role for NCKX2 in neuronal Ca(2+) homeostasis is not yet clearly understood but will depend upon the functional properties of the molecule. Here, we have performed whole-cell patch clamp analysis to characterize cation dependences and ion stoichiometry for rat brain NCKX2, heterologously expressed in HEK293 cells. Outward currents generated by reverse NCKX2 exchange depended on external Ca(2+) with a K(12) of 1.4 or 101 microm without or with 1 mm Mg(2+), and on external K(+) with a K(1/2) of about 12 or 36 mm with choline or Li(+) as counter ion, respectively. Na(+) inhibited outward currents with a K(1/2) of about 60 mm. Inward currents generated by forward NCKX2 exchange depended upon external Na(+) with a K(1/2) of 30 mm and a Hill coefficient of 2.8. K(+) inhibited the inward currents by a maximum of 40%, with a K(1/2) of 2 mm or less, depending upon the conditions. The transport stoichiometry of NCKX2 was determined by observing the change in reversal potential as individual ion gradients were altered. Our data support a stoichiometry for rat brain NCKX2 of 4 Na(+):(1 Ca(2+) + 1 K(+)). These findings provide the first electrophysiological characterization of rat brain NCKX2, and the first evidence that a single recombinantly expressed NCKX polypeptide encodes a K(+)-transporting Na(+)/Ca(2+) exchanger with a transport stoichiometry of 4 Na(+):(1 Ca(2+) + 1 K(+)).

Molecular cloning of a third member of the potassium-dependent sodium-calcium exchanger gene family, NCKX3.

We describe here the identification and characterization of a novel member of the family of K(+)-dependent Na(+)/Ca(2+) exchangers, NCKX3 (gene SLC24A3). Human NCKX3 encodes a protein of 644 amino acids that displayed a high level of sequence identity to the other family members, rod NCKX1 and cone/neuronal NCKX2, in the hydrophobic regions surrounding the "alpha -repeat" sequences thought to form the ion-binding pocket for transport. Outside of these regions NCKX3 showed no significant identity to other known proteins. As anticipated from this sequence similarity, NCKX3 displayed K(+)-dependent Na(+)/Ca(2+) exchanger activity when assayed in heterologous expression systems, using digital imaging of fura-2 fluorescence, electrophysiology, or radioactive (45)Ca(2+) uptake. The N-terminal region of NCKX3, although not essential for expression, increased functional activity at least 10-fold and may represent a cleavable signal sequence. NCKX3 transcripts were most abundant in brain, with highest levels found in selected thalamic nuclei, in hippocampal CA1 neurons, and in layer IV of the cerebral cortex. Many other tissues also expressed NCKX3 at lower levels, especially aorta, uterus, and intestine, which are rich in smooth muscle. The discovery of NCKX3 thus expands the K(+)-dependent Na(+)/Ca(2+) exchanger family and suggests this class of transporter has a more widespread role in cellular Ca(2+) handling than previously appreciated.

Regulation of SERCA Ca2+ pump expression by cytoplasmic Ca2+ in vascular smooth muscle cells.

Vascular smooth muscle cells (VSMC) express three isoforms of the sarcoplasmic or endoplasmic reticulum Ca2+-ATPase (SERCA) pump; SERCA2b predominates (91%), whereas SERCA2a (6%) and SERCA3 (3%) are present in much smaller amounts. Treatment with thapsigargin (Tg) or A-23187 increased the level of mRNA encoding SERCA2b four- to fivefold; SERCA3 increased about 10-fold, whereas SERCA2a was unchanged. Ca2+ chelation prevented the Tg-induced SERCA2b increase, whereas Ca2+ elevation itself increased SERCA2b expression. These responses were discordant with those of 78-kDa glucose-regulated protein/immunoglobulin-binding protein (grp78/BiP), an endoplasmic reticulum stress-response protein. SERCA2b mRNA elevation was much larger than could be accounted for by the observed increase in message stability. The induction of SERCA2b by Tg did not require protein synthesis, nor was it affected by inhibitors of calcineurin, protein kinase C, Ca2+/calmodulin-dependent protein kinase, or tyrosine protein kinases. Treatment with the nonselective protein kinase inhibitor H-7 prevented Tg-induced SERCA2b expression from occurring, whereas another nonselective inhibitor, staurosporine, was without effect. We conclude that changes in cytosolic Ca2+ control the expression of SERCA2b in VSMC via a mechanism involving a currently uncharacterized, H-7-sensitive but staurosporine-insensitive, protein kinase.

Alternatively spliced isoforms of the rat eye sodium/calcium+potassium exchanger NCKX1.

We have investigated the structure, function, and expression of the rat eye sodium/calcium+potassium exchanger NCKX1. The sequence of independent rat NCKX1 clones and the analysis of rat eye mRNA by RT-PCR revealed a region of alternative splicing that comprised four exons and encoded a stretch of 113 amino acids near the beginning of the large cytosolic loop. In comparison with other NCKX1 molecules and the rat NCKX2 protein, rat NCKX1 was highly conserved within the hydrophobic regions but was quite divergent in the two large hydrophilic loops. The only exception was the region of the cytosolic loop encoded by the second alternatively spliced exon, which was approximately 60% identical. Similar to bovine, but different from human, rat NCKX1 possessed an acidic motif that was repeated 14 times in the cytoplasmic loop. Analysis of NCKX1 expression in different rat tissues by Northern blot revealed a very high level of expression of a 7-kb transcript in the eye but also lower levels of transcripts of various lengths in other tissues. The recombinant rat NCKX1 protein was tagged in the extracellular loop with the FLAG epitope and expressed in HEK-293 cells. Surface delivery and potassium-dependent sodium/calcium exchange activity were observed for each spliced variant.

Physiological and molecular characterization of the Na+/Ca2+ exchanger in human platelets.

In this report, we have demonstrated that Na+/Ca2+ exchanger activity in a human megakaryocytic cell line (CHRF-288 cells) is K+ dependent, similar to the properties previously described for Na+/Ca2+ exchange activity in human platelets. With the use of RT-PCR techniques and mRNA, the exchanger expressed in CHRF-288 cells was found to be identical to that expressed in human retinal rods. Northern blot analysis of the mRNA for the human retinal rod exchanger in CHRF-288 cells revealed a major transcript at 5.8 kb with two minor bands at 4.9 and 6.8 kb. mRNA for the retinal rod exchanger was also identified in human platelets. Using Ba2+ influx as a measure of Na+/Ca2+ exchange activity in human platelets, we have demonstrated that exchange activity is driven by the transmembrane gradient for K+ as well as that for Na+. We propose that the K+ dependence of the platelet Na+/Ca2+ exchanger could make platelets especially sensitive to daily fluctuations in salt intake.

Functional characterization and m-RNA expression of 5-HT receptors mediating contraction in human umbilical artery.

5-HT1-like and 5-HT2 receptors have both been described to mediate contractions to 5-HT in the human umbilical artery (HUA). However, the nature of the 5-HT receptor subtypes is unknown. 2 In isometric force studies with ring preparations of HUA alpha-methyl-5-hydroxytryptamine (alpha-Me-5-HT) and 5-hydroxytryptamine (5-HT) contracted HUA with pED50 values of 8.04 and 7.74, respectively. In the presence of a subthreshold concentration of another vasoconstrictor sumatriptan and 5-nonyloxytryptamine elicited concentration-dependent contractions with pEC50 values of 7.21 and 7.67, respectively. In the presence of the selective 5-HT1B/D receptor antagonist GR127935, contractile responses elicited by sumatriptan and 5-nonyloxytryptamine were competitively antagonized (pKB 9.01 and 9.02, respectively). In the experiments with 5-HT, GR127935 appeared to be non-competitive with shallow Schild plot slopes. The data were fitted with two linear regression lines and the calculated pKB of the high affinity component (8.90) was comparable to that expected for GR127935 at the 5-HT1B/1D receptor. Several 5-HT2 selective receptor antagonists (spiperone, cyproheptadine, pirenperone) competitively inhibited responses to 5-HT. The selective 5-HT2A antagonist ketanserin against sumatriptan and 5-nonyloxytryptamine behaved as a weak antagonist while against 5-HT demonstrated a competitive antagonism (pKB 8.56). Using specific primers for human 5-HT1B, 5-HT1D and 5-HT2A receptor genes, the reverse transcriptase-polymerase chain reaction revealed mRNA expression of 5-HT1B and 5-HT2A receptors in the HUA. The results suggest that the HUA has a functional population of 5-HT1B and 5-HT2A receptor subtypes which are involved in the contractile response to 5-HT. Contractions mediated by 5-HT1B receptors can be 'uncovered' by exposure to other vasoactive agents.

The relationship between Ca2+-ATPase and freely exchangeable Ca2+ in the dense tubules: a study in platelets from women.

The main aims of this work were to examine in women: the relationship between the freely exchangeable Ca2+ (FECa2+) in the dense tubules and the activity of the sarco(endo)plasmic reticulum (SER) Ca2+-ATPase (SERCA) in platelets, and the relationship of these parameters with blood pressure and serum lipoproteins. Platelets from 14 white and 13 black women in good health were studied. The FECa2+ was measured as the ionomycin-evoked Ca2+ release (in the presence of thapsigargin) in Ca2+-free medium. SERCA activity was measured as the thapsigargin sensitive, Ca2+ dependent and ouabain resistant, ATP hydrolyses in platelet membranes. Relative expressions of SERCA 2 and 3 isoforms and Ras-related protein (Rap) 1 in platelet membranes were determined by Western immunoblots. Highly significant correlations were observed for FECa2+ in the dense tubules with: 1) the maximal reaction velocity (Vmax) of the SERCA (r = 0.592, P = .0014), and 2) Rapl (r = 0.551, P = .0035). In addition, negative correlations were observed between FECa2+ in the dense tubules and age. No correlations were observed for these variables with blood pressure or serum lipoproteins. We conclude the FECa2+ and the Vmax of the SERCA are reliable indicators of Ca2+ load in platelets from women. However, in women, unlike previous observations in men, these platelet parameters are not correlated with blood pressure and serum lipoproteins.

A circularized sodium-calcium exchanger exon 2 transcript.

Previous reports of Na/Ca exchanger gene 1 (NCX1) expression have revealed a major RNA transcript of 7 kilobase pairs (kb), minor transcripts of approximately 13 and approximately 4 kb, and a relatively abundant 1.8-kb RNA band. In the present report we demonstrate that the 1.8-kb message, which has a tissue and subcellular distribution matching that of full-length NCX1 but is not polyadenylated, corresponds to a perfectly circularized exon 2 species. The circular transcript contained the normal NCX1 start codon, a new stop codon introduced as a consequence of circularization, and encoded a protein corresponding to the NH2-terminal portion of NCX1, terminating just after amino acid 600 in the cytoplasmic loop. A linear version of the circular transcript was prepared and transfected into HEK-293 cells. A protein, matching the predicted size of approximately 70 kDa, was expressed, and the transfected cells possessed Na/Ca exchange activity. Although in native tissue we could not detect a protein corresponding exactly to that predicted from the circular transcript, a prominent band of slightly shorter size, possibly representing further proteolytic processing of circular transcript protein, was observed in membranes from LLC-MK2 cells and rat kidney.

Functional characterization of alternatively spliced human SERCA3 transcripts.

The sarcoplasmic (or endoplasmic) reticulum Ca2+-ATPase (SERCA)-3 has been implicated in the possible dysregulation of Ca2+ homeostasis that accompanies the pathology of hypertension and diabetes. We report the molecular cloning of two alternatively spliced transcripts from the human SERCA3 gene, ATP2A3, that encode proteins that differ at their carboxy termini by 36 amino acids. SERCA3 transcripts were most abundantly expressed in lymphoid tissues, intestine, pancreas, and prostate. The two human SERCA3 proteins encoded by alternatively spliced transcripts were recognized by the monoclonal antibody PL/IM430 and demonstrated Ca2+ uptake and ATPase activity with an apparent Ca2+ affinity 0.5 pCa unit lower than that of other SERCA gene products. The subcellular distribution of SERCA3 protein was indistinguishable from that of SERCA2b, with expression in the nuclear envelope and in the endoplasmic reticulum throughout the cell. Two variant SERCA3 constructs, huS3-I and huS3-II, were isolated that encode proteins with three amino acid differences: Ala-673 (in huS3-I) substituted for Thr (in huS3-II), Ile-817 substituted for Met, and an insertion of Glu-994. huS3-I displayed a 10-fold lower capacity to transport Ca2+ than huS3-II.

Enhanced myocardial contractility and increased Ca2+ transport function in transgenic hearts expressing the fast-twitch skeletal muscle sarcoplasmic reticulum Ca2+-ATPase.

In this study, we investigated whether the fast-twitch skeletal muscle sarco(endo)plasmic reticulum Ca2+ transport pump (SERCA1a) can functionally substitute the cardiac SERCA2a isoform and how its overexpression affects cardiac contractility. For this purpose, we generated transgenic (TG) mice that specifically overexpress SERCA1a in the heart, using the cardiac-specific alpha-myosin heavy chain promoter. Ectopic expression of SERCA1a resulted in a 2.5-fold increase in the amount of total SERCA protein. At the same time, the level of the endogenous SERCA2a protein was decreased by 50%, whereas the level of other muscle proteins, including calsequestrin, phospholamban, actin, and tropomyosin, remained unchanged. The steady-state level of SERCA phosphoenzyme intermediate was increased 2.5-fold, and the maximal velocity of Ca2+ uptake was increased 1.7-fold in TG hearts, demonstrating that the overexpressed protein is functional. Although the basal cytosolic calcium signal was decreased by 38% in TG cardiomyocytes, the amplitude of cytosolic calcium signal was increased by 71.8%. The rate of calcium resequestration was also increased in TG myocytes, which was reflected by a 51.6% decrease in the normalized time to 80% decay of calcium signal. This resulted in considerably increased peak rates of myocyte shortening and relengthening (50.0% and 66.6%, respectively). Cardiac functional analysis using isolated work-performing heart preparations revealed significantly faster rates of contraction and relaxation in TG hearts (41.9% and 39.5%, respectively). The time to peak pressure and the time to half-relaxation were shorter (29.1% and 32.7%, respectively). In conclusion, our study demonstrates that the SERCA1a pump can functionally substitute endogenous SERCA2a, and its overexpression significantly enhances Ca2+ transport and contractile function of the myocardium. These results also demonstrate that the SERCA pump level is a critical determinant of cardiac contractility.

Upregulation of atrial natriuretic peptide gene expression in remnant kidney of rats with reduced renal mass.

Atrial natriuretic peptide (ANP) is synthesized in the kidney but its physiologic significance there is unclear. To determine whether renal expression of the ANP gene is regulated, renal ANP mRNA expression was assessed in remnant kidneys after 5/6 nephrectomy in Munich-Wistar rats. In normal sodium intake groups, ANP mRNA expression in the remnant kidney was significantly increased by 5.0 +/- 0.8-fold (n = 7, mean +/- SEM) at 4 d when compared with sham-operated controls (n = 6, all sham-operated groups) (*P < 0.001 by Scheffe's test) and by 28.3 +/- 5.1-fold at 14 d. This latter response was markedly diminished to 7.6 +/- 2.1-fold (n = 7, versus sham) in rats maintained on a low sodium diet. At 4 d, on the other hand, no significant downregulation was observed with dietary sodium restriction. Because natriuretic peptides have previously been shown by us to play a major role in the adaptive responses of remnant nephrons to renal mass ablation, these data suggest that ANP of renal origin may contribute to the overall mechanism for enhancing sodium excretion in the face of declining nephron number.

Molecular cloning of a novel potassium-dependent sodium-calcium exchanger from rat brain.

We have isolated a novel cDNA clone from rat cerebral cortex encoding a protein of 670 amino acids (NCKX2) that has significant similarity to the 1199-amino acid-long Na/Ca-K exchanger of bovine rod outer segment (NCKX1). NCKX2 transcripts are 10.5 kilobase pairs in length and are expressed abundantly in neurons throughout the brain and with much lower abundance in selected other tissues. The predicted topology of the rat NCKX2 protein is very similar to that of bovine NCKX1, beginning with a solitary transmembrane segment (M0), which is removed as a "signal peptide" in bovine NCKX1, an extracellular loop, a cluster of five transmembrane spanning segments (M1 to M5), a long cytoplasmic loop, and a final hydrophobic cluster (M6 to M11). Within the hydrophobic clusters, rat NCKX2 shares 80% identity and 91% similarity with bovine NCKX1. The two larger hydrophilic loops are much shorter in NCKX2 than in NCKX1, accounting largely for the difference in length between the two proteins, and are dissimilar in sequence except for a 32-amino acid stretch with 69% identity in the cytosolic loop. NCKX2 was epitope-tagged in the extracellular domain and was shown to be expressed at the surface of transfected HEK cells. Analysis of NCKX2 function by fluorescent imaging of fura-2-loaded transfected cells demonstrated that NCKX2 is a potassium-dependent sodium/calcium exchanger.

Ca2+ in the dense tubules: a model of platelet Ca2+ load.

In this work, we explored the relationship between the freely exchangeable Ca2+ (FECa2+) in the dense tubules (DT) and the sarco(endo)plasmic reticulum (SER) Ca2+-ATPase (SERCA) in circulating human platelets and examined the relationship between blood pressure (BP) and these platelet parameters. Studying platelets from 32 healthy men, we showed that the maximal reaction velocity (Vmax) of the SERCA significantly correlated with FECa2+ in the DT and with the protein expressions of SERCA 2 and 3. BP positively correlated with both the Vmax of the SERCA (r=.462, P=.010) and the FECa2+ sequestered in the DT (r=.492, P=.005). The relationships between these platelet Ca2+ parameters and BP were in part confounded by increased levels of serum triglycerides and diminished HDL cholesterol with a higher BP. No correlation was observed between the resting cytosolic Ca2+ and BP. Collectively, these findings indicate that (1) an increase in the cellular Ca2+ load in platelets is expressed by a higher activity of the SERCA and an increase in the expressions of SERCA 2 and 3 proteins, coupled with an increase in the FECa2+ in the DT, and (2) a higher BP is associated with an increase in platelet Ca2+ load in human beings, expressed by a rise in the FECa2+ in the DT and the upregulation of SERCA activity.

Alternative promoters and cardiac muscle cell-specific expression of the Na+/Ca2+ exchanger gene.

Many studies have investigated the regulation of the Na+/ Ca2+ exchanger, NCX1, but limited data exist on transcriptional regulation of the NCX1 gene. We have identified the transcription start sites of three tissue-specific alternative promoters of NCX1 transcripts from rat heart, kidney, and brain. We have characterized the cardiac NCX1 promoter, from which the most abundant quantities of NCX1 transcripts are expressed. Transfection of primary cardiac myocytes, CHO cells, and COS-7 cells with overlapping genomic DNA fragments spanning the NCX1 cardiac transcription start site has uncovered a cardiac cell-specific minimum promoter from -137 to +85. The cardiac NCX1 promoter is TATA-less but has putative binding sites for cardiac-specific GATA factors, an E box, and an Inr as well as multiple active enhancers. The kidney NCX1 promoter has a typical TATA box and binding sites for several tissue-specific factors. The brain NCX1 promoter is very GC-rich and possesses several Sp-1 binding sites consistent with its ubiquitous expression.