Mechanisms of phosphate uptake into brush-border membrane vesicles from goat jejunum

This study concerns the uptake of inorganic phosphate into brush-border membrane vesicles prepared from jejunal tissues of either control or Ca-and/or P-depleted goats. The brush-border membrane vesicles showed a time-dependent accumulation of inorganic phosphate with a typical overshoot phenomenon in the presence of an inwardly directed Na⁺ gradient. The Na⁺-dependent inorganic phosphate uptake was completely inhibited by application of 5 mmol·l^-1 sodium arsenate. Half-maximal stimulation of inorganic phosphate uptake into brush-border membrane vesicles was found with Na⁺ concentrations in the order of 5 mmol·l^-1. Inorganic phosphate accumulation was not affected by a K⁺ diffusion potential (inside negative), suggesting an electroneutral transport process. Stoichiometry suggested an interaction of two or more Na ions with one inorganic phosphate ion at pH 7.4. Na⁺-dependent inorganic phosphate uptake into jejunal brush-border membrane vesicles from normal goats as a function of inorganic phosphate concentration showed typical Michaelis-Menten kinetic with V _max=0.42±0.08 nmol·mg^-1 protein per 15 s^-1 and K _m=0.03±0.01 mmol·l^-1 (n=4, x ±SEM). Long-term P depletion had no effect on these kinetic parameters. Increased plasma calcitriol concentrations in Ca-depleted goats, however, were associated with significant increases of V _max by 35–80%, irrespective of the level of P intake. In the presence of an inwardly directed Na⁺ gradient inorganic phosphate uptake was significantly stimulated by almost 60% when the external pH was decreased to 5.4 (pH_out/pH_in=5.4/7.4). The proton gradient had no effect on inorganic phosphate uptake in absence of Na⁺. In summary, in goats Na⁺ and calcitriol-dependent mechanisms are involved in inorganic phosphate transport into jejunal brush-border membrane vesicles which can be stimulated by protons.Abbreviations AP activity of alkaline phosphatase - BBMV brush-border membrane vesicles - EGTA ethyleneglycol-triacetic acid - n _app apparent Hill coefficient - P _i inorganic phosphate - PTH parathyroid hormone     

Characterization of phosphorus uptake in slow- and fast-growing southern pine seedlings grown in solution culture

The effects of low-P growth conditions on growth and net P acquisition were examined in two species of pine that are indigenous to P-deficient soils of the Atlantic Coastal Plain: pond pine (Pinus serotina Michx.), a moderately-fast growing pine, and a slow-growing seed source of loblolly pine P. taeda L.) from Texas. Short-term ³²P uptake experiments were conducted using intact nonmycorrhizal seedlings that had been grown for 7 weeks in continuously-flowing solution culture at 5 or 100 µM P. Growth and P uptake of pond pine were more responsive to a higher P supply than the slow-growing loblolly pine. Pond pine seedlings in the 100 µM P treatment were twice the size of those grown in 5 µM P and accumulated almost five times as much seedling P. In contrast, seedling biomass of loblolly pine increased by only 8% under high-P growth conditions, and seedlings accumulated twice as much P, reflecting the higher P concentrations in shoot and root tissues. Although rates of unidirectional influx of ³²P were 22 and 61% higher under low-P growth conditions in pond and loblolly pine, respectively, net uptake rates in seedlings from the 5 µM P treatment were over three times those of seedlings grown in 100 µM P. These results suggest that unidirectional efflux out of the root was controlling net uptake of P as much, if not more, than unidirectional influx. Efflux of³² P out of root tissue, particularly older tissue, decreased in seedlings grown under low-P conditions, possibly due to a reduction in the size of the phosphorus pool available for efflux, i.e. the soluble P_i pool. Over 75% of the total root P in both loblolly and pond pine seedlings grown in 100 µM P treatment was present as organic P, suggesting that organic P, particularly phytate, may represent important storage pools in roots of woody species. Within each species, higher rates of influx and net uptake in seedlings from the low-P treatment were associated with lower P concentrations in shoot and root tissue, and shoot FW:root FW ratios. Efflux may represent a short-term means of regulating net P uptake, while the demand for P created by growth and storage may represent a long-term regulation.     

Up-Regulation of Functional Voltage-Dependent Sodium Channels by Insulin in Cultured Bovine Adrenal Chromaffin Cells

Abstract: Treatment of cultured bovine adrenal chromaffin cells with 100 nM insulin raised [³H]saxitoxin ([³H]STX) binding in a time-dependent manner (t_1/2 = 26 h). Insulin (100 nM for 4 days) increased the B_max of [³H]STX binding by 49% without changing the K_D value and also augmented the maximal influx of ²²Na⁺ due to 560 µM veratridine by 39% without altering the EC₅₀ value of veratridine. The stimulatory effect of insulin on ²²Na⁺ influx was concentration-dependent with an EC₅₀ of 3 nM, whereas insulin-like growth factor (IGF)-I had little effect at 1 nM. Ptychodiscus brevis toxin-3 allosterically potentiated veratridine (100 µM)-induced ²²Na⁺ influx by approximately twofold in both insulin-treated cells and untreated cells. Veratridine-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels and catecholamine secretion were also enhanced by insulin treatment, whereas insulin did not alter nicotine-induced ²²Na⁺ influx via the nicotinic receptor-ion channel complex and high-K⁺ (direct activation of voltage-dependent Ca²⁺ channels)-induced ⁴⁵Ca²⁺ influx. Stimulatory effects of insulin on [³H]STX binding and veratridine-induced ²²Na⁺ influx were nullified by simultaneous treatment with either 5,6-dichlorobenzimidazole riboside, an inhibitor of RNA synthesis, or cycloheximide, an inhibitor of protein synthesis, whereas insulin treatment did not appreciably increase the level of mRNA encoding the Na⁺ channel α-subunit. These results suggest that the binding of insulin to insulin (but not IGF-I) receptors mediates the up-regulation of functional Na⁺ channel expression at plasma membranes; this up-regulation may be due, at least in part, to the de novo synthesis of an as yet unidentified protein(s).     

Effects of Protein Kinase Inhibitors on Morphology and Function of Cultured Bovine Adrenal Chromaffin Cells: KN-62 Inhibits Secretory Function by Blocking Stimulated Ca2+ Entry

Abstract: In cultured bovine adrenal chromaffin cells, a nonselective protein kinase inhibitor, staurosporine, inhibits secretory function and induces neurite outgrowth. In the present study, effects of other nonselective protein kinase inhibitors (K-252a, H-7, and H-8) and reportedly selective protein kinase inhibitors (KN-62 and chelerythrine chloride) were examined on bovine adrenal chromaffin cell morphology, secretory function, and ⁴⁵Ca²⁺ uptake. Treatment of chromaffin cells with 10 µ M K-252a, 50 µ M H-7, or 50 µ M H-8 induced changes in cell morphology within 3 h; these compounds also induced a time-dependent inhibition of stimulated catecholamine release. Chelerythrine chloride, a selective inhibitor of Ca²⁺/phospholipid-dependent protein kinase, did not induce outgrowth or inhibit secretory function under our treatment conditions. KN-62, a selective inhibitor of Ca²⁺/calmodulin-dependent protein kinase II (CaMK II), significantly inhibited stimulated catecholamine release (IC₅₀ value of 0.32 µ M ), but had no effect on cell morphology. The reduction of secretory function induced by 1 µ M KN-62 was significant within 5 min and rapidly reversible. Unlike H-7, H-8, and staurosporine, KN-62 significantly inhibited stimulated ⁴⁵Ca²⁺ uptake. KN-04, a structural analogue of KN-62 that does not inhibit CaMK II, inhibited stimulated ⁴⁵Ca²⁺ uptake and catecholamine release like KN-62. These studies indicate that KN-62 inhibits secretory function via the direct blockade of activated Ca²⁺ influx. The nonselective inhibitors, K-252a, H-7, H-8, and staurosporine, inhibit secretory function by another mechanism, perhaps one involving alterations in the cytoskeleton.     

Protein Kinase C-Mediated Down-Regulation of Voltage-Dependent Sodium Channels in Adrenal Chromaffin Cells

Abstract: Treatment of cultured bovine adrenal chromaffin cells with 12- O -tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C (PKC), decreased [³H]saxitoxin ([³H]STX) binding in a concentration (IC₅₀ = 19 n M )- and time ( t _1/2 = 4.5 h)-dependent manner. TPA (100 n M for 15 h) lowered the B _max of [³H]STX binding by 53% without altering the K _D value. Phorbol 12,13-dibutyrate (PDBu) also reduced [³H]STX binding, whereas 4α-TPA, an inactive analogue, had no effect. The inhibitory effect of TPA was abolished when H-7 (an inhibitor of PKC), but not H-89 (an inhibitor of cyclic AMP-dependent protein kinase), was included in the culture medium for 1 h before and during TPA treatment. Simultaneous treatment with TPA in combination with either actinomycin D or cycloheximide, an inhibitor of protein synthesis, nullified the effect of TPA. TPA treatment also attenuated veratridine-induced ²²Na⁺ influx but did not alter the affinity of veratridine for Na channels as well as an allosteric potentiation of veratridine-induced ²²Na⁺ influx by brevetoxin. These results suggest that an activation of PKC down-regulates the density of Na channels without altering their pharmacological features; this down-regulation is mediated via the de novo synthesis of an as yet unidentified protein(s), rather than an immediate effect of Na channel phosphorylation.     

Molecular players of auxin transport systems: advances in genomic and molecular events*

Phytohormone auxin plays an indispensable role in the plethora of plant developmental process starting from the cell division, and cell elongation to morphogenesis. Auxins are transported to different parts of the plant by different sophisticated transporter molecules known as ‘auxin transporters’.There are four auxin transporter families that have been reported so far in the plant kingdom which includes AUX/LAX (AUXIN-RESISTANT1–LIKES), PIN (PIN-FORMED, auxin efflux carriers), ABCB ((ATP-binding cassette-B (ABCB)/P-glycoprotein (PGP)) and PILS (PIN-Likes). Auxin influx and efflux carriers are distributed in a polar fashion in the plasma membrane whereas ABCB and PILS are present in a non-polar fashion. Other than AUX/LAX, other auxin transporters harbor N-and C-terminal conserved domains along with a variable hydrophilic loop in the transmembrane domain. The AUX/LAX, ABCB and PIN transporters mediate long distance auxin transport whereas PILS and PIN5 protein involved in intracellular auxin homeostasis.     

Exopolyphosphatases of the yeast Saccharomyces cerevisiae

Separate compartments of the yeast cell possess their own exopolyphosphatases differing from each other in their properties and dependence on culture conditions. The low-molecular-mass exopolyphosphatases of the cytosol, cell envelope, and mitochondrial matrix are encoded by the PPX1 gene, while the high-molecular-mass exopolyphosphatase of the cytosol and those of the vacuoles, mitochondrial membranes, and nuclei are presumably encoded by their own genes. Based on recent works, a preliminary classification of the yeast exopolyphosphatases is proposed.     

Effect of nickel on nutrient removal by selected indigenous protozoan species in wastewater systems

Nutrient and heavy metal pollutions are major concern worldwide. This study aimed at comparing the effect of Ni²⁺ on nutrient removal efficiency of four indigenous wastewater protozoan species (Aspidisca sp., Paramecium sp., Peranema sp., Trachelophyllum sp.). Specific physicochemical parameters and microbial growth/die-off were measured using standard methods. The results revealed that protozoan species were able to simultaneously remove phosphate, nitrate and Ni²⁺ at concentrations ranging between 66.4–99.36%, 56.19–99.88% and 45.98–85.69%, respectively. Peranema sp. appeared to be the isolates with the highest removal of nutrients (Phosphate-99.36% and Nitrate-99.88%) while Paramecium sp. showed higher removal of Ni²⁺ at 85.69% and low removal of nutrients. Aspidisca sp. was the most sensitive isolate to Ni²⁺ but with significant nutrient removal (Phosphate-66.4% and Nitrate-56.19%) at 10 mg-N²⁺/L followed by an inhibition of nutrient removal at Ni²⁺ concentration greater than 10 mg/L. Significant correlation between the growth rate and nutrient removal (r = 0.806/0.799, p < 0.05 for phosphate and nitrate, respectively) was noted. Except for Peranema sp. which revealed better nutrient removal ability at 10 mg-Ni²⁺/L, an increase in Ni²⁺ concentration had a significant effect on nutrient removal efficiency of these indigenous protozoan species. This study suggests that although Ni²⁺ appeared to be toxic to microbial isolates, its effect at a low concentration (10 mg-Ni²⁺/L) towards these isolates can be used to enhance the wastewater treatment process for the removal of nutrients. Peranema sp., which was able to remove both Ni²⁺ and nutrients from wastewater mixed-liquor, can also be used for bioremediation of wastewater systems.