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.

Intracellular calcium: molecules and pools.

The complex nature of intracellular calcium storage pools has been examined at many levels in the past year. Additional molecules associated with calcium stores have been identified and their localization examined. The convergence of molecular biology, cell biology and biochemistry has now allowed the details of calcium signalling to be meaningfully explored.

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.

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.

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.

Rabbit cardiac and slow-twitch muscle express the same phospholamban gene.

The nucleotide sequences of cDNAs encoding phospholamban were found to be virtually identical when the cDNA clones were isolated from rabbit slow-twitch (soleus) and rabbit cardiac muscle libraries. These findings demonstrate that both types of muscle express the same phospholamban gene. The deduced amino acid sequences of rabbit and dog phospholamban were identical except for a change from Asp (dog) to Glu (rabbit) at position 2. The nucleotide sequences of the 5'- and the very long 3'-untranslated regions of rabbit and dog phospholamban cDNAs also exhibited a high percentage of identity.

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.

Differences in the subcellular localization of calreticulin and organellar Ca(2+)-ATPase in neurons.

It has become clear that calcium is an important mediator in the transduction of signals due to ligand binding to cell surface receptors. Cytosolic calcium is typically maintained at low levels in both muscle and non-muscle cells and intracellular sequestering of calcium appears to be important in this process. The identification of intracellular calcium pools has been the subject of much recent study, and it has been proposed that such pools would contain three components: a calcium-activated pump or Ca(2+)-ATPase, a calcium channel such as the inositol trisphosphate receptor or ryanodine receptor, and a high-capacity calcium-binding protein such as calsequestrin or calreticulin. We report here on the localization of two components, the organellar Ca(2+)-ATPase (SERCA) and calreticulin, in neuronal tissues. Using immunofluorescence and subcellular fractionation, we have found that for the most part, these two proteins do not co-localize in neuron cell bodies, dendrites, or axons; but may co-localize at the axon terminal.

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.

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.

The catalytic subunits of the (Na+,K+)-ATPase alpha and alpha(+) isozymes are the products of different genes.

The sequences of the first 14 amino acids of the (Na+,K+)-ATPase catalytic subunits from rat kidney (alpha) and rat brain axolemma (alpha(+)) have been determined. They are: (alpha), NH2-Gly-Arg-Asp-Lys-Tyr-Glu-Pro-Ala-Ala-Val-Ser-Glu-His-Gly; (alpha(+)), NH2-Gly-Arg-Glu-Tyr-Ser-Pro-Ala-Ala-Glu-Val-Ala-Glu-Val-Gly. Although they are highly homologous, it is clear these sequences are also sufficiently different to conclude they are the products of different genes, or at least different exons of the same, differentially spliced, gene. Among mammals, the amino terminal sequence of the kidney alpha chain is essentially invariant. Thus this section of the (Na+,K+)-ATPase molecule is more highly conserved in one tissue between several species than between different tissues in the same species. This may reflect upon the difference in function of the alpha and alpha(+) isozymes of (Na+,K+)-ATPase.

Insulin affects the sodium affinity of the rat adipocyte (Na+,K+)-ATPase.

The K0.5 for intracellular sodium of the two forms of (Na+,K+)-ATPase which exist in rat adipocytes (Lytton, J., Lin, J. C., and Guidotti, G. (1985) J. Biol. Chem. 260, 1177-1184) has been determined by incubating the cells in the absence of potassium in buffers of varying sodium concentration; these conditions shut off the Na+ pump and allow sodium to equilibrate into the cell. The activity of Na+,K+)-ATPase was then monitored with 86Rb+/K+ pumping which was initiated by adding isotope and KCl to 5 mM, followed by a 3-min uptake period. Atomic absorption and 22Na+ tracer equilibration were used to determine the actual intracellular [Na+] under the different conditions. The K0.5 values thus obtained were 17 mM for alpha and 52 mM for alpha(+). Insulin treatment of rat adipocytes had no effect on the intracellular [Na+] nor on the Vmax of 86Rb+/K+ pumping, but did produce a shift in the sodium ion K0.5 values to 14 mM for alpha (p less than 0.025 versus control) and 33 mM for alpha(+) (p less than 0.005 versus control). This change in affinity can explain the selective stimulation of alpha(+) by insulin under normal incubation conditions. Measurement of the K0.5 for sodium ion of (Na+,K+)-ATPase in membranes isolated from adipocytes revealed only a single component of activation with a low K0.5 of 3.5 or 12 mM in the presence of 10 or 100 mM KCl, respectively. Insulin treatment of the isolated membranes or of the cells prior to membrane separation had no effect on these values.

Molecular cloning of cDNAs from human kidney coding for two alternatively spliced products of the cardiac Ca2+-ATPase gene.

Ca2+-ATPase molecules present in the microsomal fraction from non-muscle cells were examined immunologically. Rabbit whole brain, cerebellum, liver, kidney, and COS-1 cell microsomes all displayed a polypeptide of about 110 kDa which was immunoreactive with a polyclonal antiserum against the cardiac muscle sarcoplasmic reticulum Ca2+-ATPase molecule, but was not immunoreactive with a monoclonal antibody specific for the fast-twitch muscle Ca2+-ATPase. cDNAs encoding the full length of two Ca2+-ATPase molecules were isolated from a human kidney library using a mixture of nucleotide probes derived from both rabbit fast-twitch and cardiac muscle Ca2+-ATPase cDNAs. The human kidney cDNAs, HK1 and HK2, are the products of alternative splicing. HK2 codes for a protein identical to rabbit cardiac muscle Ca2+-ATPase, with the exception of 6 scattered amino acid replacements, whereas HK1 codes for a protein identical to that encoded by HK2, but with the carboxyl-terminal 4 amino acids replaced by an extended sequence of 49 amino acids. cDNAs of the HK1 type are by far the most abundant in the library. The partial structure of a 40-kilobase genomic DNA encoding all but the 5' end of the human cardiac Ca2+-ATPase is described. The exons which give rise to the alternatively spliced products were located by Southern blotting and sequencing, and the alternative splicing patterns were determined.

Molecular cloning and quantification of sarcoplasmic reticulum Ca(2+)-ATPase isoforms in rat muscles.

A cDNA encoding the full-length adult rat fast-twitch muscle Ca(2+)-adenosinetriphosphatase (ATPase) was cloned. The deduced amino acid sequence of this molecule has 97 and 90% identity with those of rabbit fast-twitch muscle and chicken skeletal muscle Ca(2+)-ATPases, respectively. Specific probes from the 3'-untranslated region of each sarcoplasmic or endoplasmic reticulum Ca(2+)-ATPase (SERCA) gene product and full-length cRNA transcript standards were used to determine the quantity of mRNA encoding each isoform in various rat muscles. Quantitative immunoblotting was also used to determine the protein content of each SERCA isoform. Fast-twitch fibers expressed both SERCA1 mRNA and protein at a level two- to fivefold higher than SERCA2 was expressed in slow-twitch fibers. We observed a protein-to-mRNA ratio that varied from approximately 500,000 molecules per molecule in the fast-twitch muscles to approximately 200,000 in cardiac and smooth muscles. There was no difference, however, between the ratio for different isoforms in the same muscle. The content of Ca2+ pump in a given muscle therefore depends on at least three factors: 1) the efficiency of gene transcription and message stability (fiber type dependent), 2) the efficiency of translation and protein stability (muscle identity dependent), and 3) fiber composition of the muscle.

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.

Identification of two molecular forms of (Na+,K+)-ATPase in rat adipocytes. Relation to insulin stimulation of the enzyme.

Two molecular forms of the (Na+,K+)-ATPase catalytic subunit have been identified in rat adipocyte plasma membranes using immunological techniques. The similarity between these two forms and those in brain (Sweadner, K. J. (1979) J. Biol. Chem. 254, 6060-6067) led us to use the same nomenclature: alpha and alpha(+). The K0.5 values of each form for ouabain (determined by inhibition of phosphorylation of the enzyme from [gamma-32P]ATP) were 3 X 10(-7)M for alpha(+) and 1 X 10(-5)M for alpha. These numbers correlate well with the K0.5 values for the two ouabain-inhibitable components of 86Rb+/K+ pumping in intact cells (1 X 10(-7) M and 4 X 10(-5)M). Quantitation of the Na+ pumps in plasma membranes demonstrated a total of 11.5 +/- 0.2 pmol/mg of membrane protein, of which 8.5 +/- 0.3 pmol/mg, or 75%, was alpha(+). Insulin stimulation of 86Rb+/K+ uptake in rat adipocytes was abolished by ouabain at a concentration sufficient to inhibit only alpha(+)(2-5 X 10(-6)M). Immunological techniques and ouabain inhibition of catalytic labeling of the enzyme from [gamma-32P]ATP demonstrated that alpha(+) was present in skeletal muscle membranes as well as in adipocyte membranes, but was absent from liver membranes. Since insulin stimulates increased Na+ pump activity in adipose and muscle tissue but not in liver, there is a correlation between hormonal regulation of (Na+,K+)-ATPase and the presence of alpha(+). We propose that alpha(+) is the hormonally-sensitive version of the enzyme.

Inhibition of sarco-endoplasmic reticulum calcium ATPases (SERCA) by polycyclic aromatic hydrocarbons: lack of evidence for direct effects on cloned rat enzymes.

Previous studies have demonstrated that immunosuppressive polycyclic aromatic hydrocarbons (PAHs) disrupt Ca2+ homeostasis leading to inhibition of the Ca(2+)-dependent pathways of T cell and B cell activation. The sustained Ca(2+)-elevation produced by immunosuppressive PAHs may result from the inhibition of Ca(2+)-ATPases in the endoplasmic reticulum (SERCA). The purpose of the present study was to determine whether PAHs directly inhibit cloned SERCA enzymes, and whether there is any selectivity for certain isoforms. PAHs were examined for their effects on purified cloned rat SERCA enzymes, including SERCA1, SERCA2a and SERCA3, transiently expressed in human embryonic kidney (HEK) cells. Results showed that known SERCA inhibitors, thapsigargin (100 nM) and 2,5-di(t-butyl)-1,4-benzohydroquinone (10 mumol), completely inhibited all rat SERCA isoforms, whereas 7,12-dimethylbenz(a)anthracene, benzo(a)pyrene, benzo(e)pyrene, anthracene, 3-methyl-cholanthrene, 9,10-dimethylanthracene and benz(a)anthracene at concentrations as high as 10 mumol appeared to have little inhibitory effect on any of the SERCA. The results demonstrating that PAHs do not inhibit cloned SERCA enzymes suggest that metabolism may be required for PAH-induced inhibition, or that other cellular elements, not present in the HEK transfection model, may be required for activity.