Phosphate transport in potato cuttings: Effect of gibberellic acid and abscisic acid

Apical cuttings of Solanum tuberosum L. cv. Sirtema were used al different stages of development to study long-distance transport of phosphate. The effects of two hormones, gibberellic acid (GA₃) and abscisic acid (ABA), on this process were also investigated. Before tuberization, phosphate (³²P) supplied to a single leaf was transported preferentially in the young and growing parts of the plant: apical bud, young leaves and roots. After tuberization, the tuber became the principal site of phosphate accumulation. GA³ treatment (10⁻⁴ M) of the tuber as well as of the leaves led to reduced transport of ³²P into the tuber. By contrast, treatment of the tuber with ABA (10⁻⁴M) did not change the ³²P distribution within the plant, while foliar spray with ABA greatly increased the transport into the tuber. The opposite effects of the two hormones on phosphate accumulation by tubers are discussed with regard to their opposite effects on the tuberization process.     

Identification of a calmodulin-stimulated (Ca2++ Mg2+)-ATPase in a plasma membrane fraction isolated from maize (Zea mays) leaves

Plasma membranes were isolated from light-grown, 14-day-old maize leaves ( Zea mays L . cv. Golden Cross Bantam) using aqueous two-phase partitioning. The plasma membrane (PM) fraction contained < 0.3% of the total chlorophyll, < 0.2% of the mitochondrial marker enzyme activity, minimal contamination by endomembranes and 34% of the total PM.
A calmodulin-stimulated (Ca2++ Mg2+)-ATPase was identified in the PM-enriched fraction. The Ca2++ calmodulin stimulation was dependent on Mg2+, saturated at ca 25 μM total Ca2+, had a pH maximum at 7.2 and was maximally stimulated by 600 n M bovine brain calmodulin. The stimulation was not greatly affected by the anion present and showed a divalent cation specificity of Ca2+ > Sr+2 ± Mn+2 > Co2+± Cu2+ > Ba2+. The napthalenesulfonamide W7, an antagonist of calmodulin action, completely inhibited the calmodulin stimulation at 175 μM , while the less active analogue W5 was ineffective at this concentration. La3+, an inhibitor of PM Ca2+ transport, showed a 50% inhibition of calmodulin-stimulated ATPase activity at ca 200 μM . Taken as a whole, these data demonstrate the presence of a calmodulinstimulated, (Ca2++ Mg2+)-ATPase on the cytoplasmic surface of the plasma membrane of maize leaf cells.     

Effect of salinity on phosphate accumulation and injury in soybean

Many soybean [Glycine max (L.) Merr.] genotypes that are grown in solution cultures are highly sensitive to the combination of both salinity and inorganic phosphate (Pi) in the substrate. This effect has been observed on numerous occasions on plants grown in a saline medium that contained a substantial amount of Ca (i.e., CaCl₂/NaCl=0.5 on a molar basis). Because Ca is important in regulating ion transport and membrane permeability, solution culture experiments were designed to examine the effects of various concentrations of Pi and ratios of CaCl₂/NaCl (0 to 0.5 on a molar basis) at a constant osmotic potential (−0.34 MPa) on this adverse interaction. Four soybean cultivars (‘Lee’, ‘Lee 74’ ‘Clark’ and ‘Clark 63’) were tested. No adverse salinity x Pi interaction was found on Lee at any ratio and leaf P and Cl were maintained below 300 and 200 mmol kg⁻¹ dry wt, respectively. Clark, Clark 63 and Lee 74 soybean plants, on the other hand, were severely injured by solution salinity (−0.34 MPa osmotic potential) when substrate Pi was ≥0.12 mM. Reduced substrate Ca did not intensify the salinity x Pi interaction. On the contrary, the onset of injury was hastened and more severe with increased CaCl₂/NaCl ratios in isotonic solutions. Shoot and root growth rates decreased as injury increased. Leaf P concentrations from these cultivars grown in saline solutions with 0.12 mM Pi were excessive (>600 mmol kg⁻¹ dry wt) compared with concentrations commonly found in soybean leaf tissue yet they were independent of the severity of injury. Since leaf Cl increased wiht increased CaCl₂/NaCl ratio, we suspect that the severity of foliar injury was related to the combined effects of excessive P and Cl within the tissue. Lee 74, the only injured cultivar examined that excluded Cl from its leaves, was less sensitive than either Clark cultivar and its injury was characteristically different. Other ion interactions were reported that may have played a role in injury susceptibility.     

Survival ofSclerotinia minor Jagger sclerotia in solarized soil

Calcium, magnesium and potassium dynamics in decomposing litter of three tree species were measured over a two-year period. The speices studied were flowering dogwood (Cornus florida), red maple (Acer rubrum) and chestnut oak (Quercus prinus). The order of decomposition was:C. florida>A. rubrum>Q. prinus.Calcium concentrations increased following any initial leaching losses. However, there were net releases of Ca from all three litter types since mass loss exceeded the increases in concentration. Net release of Ca by the end of two years from all three species combined was 42% of initial inputs in litterfall. Magnesium concentrations increased in the second year, following decreases due to leaching during the first year inC. florida andA. rubrum litter. Net release of Mg by the end of two years was 58% of initial inputs. Potassium concentrations decreased rapidly and continued to decline throughout the study. Net release of K by the end of two years was 91% of initial inputs.These data on cation dynamics, and similar data on N, S and P dynamics from a previous study, were combined with annual litterfall data to estimate the release of selected nutrients from foliar litter of these tree species at the end of one and two years of decomposition. The relative mobility of all six elements examined in relation to mass loss after two years was; K>Mg>mass>Ca>S>P>N.     

Characterization of a delayed rectifier K+ channel in NG108-15 neuroblastomaX glioma cells: Gating kinetics and the effects of enrichment of membrane phospholipids with arachidonic acid

Summary The calcium sensitivity of exocytosis from electroper-meabilized chromaffin cells is increased by activators of protein kinase C, such as TPA and certain phorbol esters, diacylglycerols, and mezerein. A range of putative inhibitors of protein kinase C block both the phorbol ester-sensitive component of secretion and also the underlying insensitive component. These inhibitors are also shown to inhibit medulla protein kinase C activity in vitro. The extent of secretion is reduced when electropermeabilized cells are exposed to Ca²⁺ levels much in excess of 50 m. The onset of inhibition is faster than the relatively slow rate of Ca-dependent exocytosis and is insensitive to inhibitors of proteolysis. Adrenal medulla protein kinase C activity is also irreversibly inhibited by high Ca²⁺ concentrations. Both the secretory response and the protein kinase C activity in vitro have similar nucleotide and cation specificities. Although these data do not definitely establish an involvement of protein kinase C in exocytosis, none argue against it.Deceased     

Phosphate sequestration by glycerol and its effects on photosynthetic carbon assimilation by leaves

Glycerol induced a limitation on photosynthetic carbon assimilation by phosphate when supplied to leaves of barley (Hordeum vulgare L.) and spinach (Spinacia oleracea L.). This limitation by phosphate was evidenced by (i) reversibility of the inhibition of photosynthesis by glycerol by feeding orthophosphate (ii) a decrease in light-saturated rates of photosynthesis and saturation at a lower irradiance, (iii) the promotion of oscillations in photosynthetic CO₂ assimilation and in chlorophyll fluorescence, (iv) decreases in the pools of hexose monophosphates and triose phosphates and increases in the ratio of glycerate-3-phosphate to triose phosphate, (v) decreased photochemical quenching of chlorophyll fluorescence, and increased non-photochemical quenching, specifically of the component which relaxed rapidly, indicating that thylakoid energisation had increased. In barley there was a massive accumulation of glycerol-3-phosphate and an increase in the period of the oscillations, but in spinach the accumulation of glycerol-3-phosphate was comparatively slight. The mechanism(s) by which glycerol feeding affects photosynthetic carbon assimilation are discussed in the light of these results.Abbreviations Chl chlorophyll - C _i intercellular concentration of CO₂ - P phosphate - PGA glycerate-3-phosphate - Pi orthophosphate - triose-P sum of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate     

Calmodulin plays a dominant role in determining neurotransmitter regulation of neuronal adenylate cyclase

Ca2+, through the mediation of calmodulin, stimulates the activity of brain adenylate cyclase. The growing awareness that fluctuating Ca2+ concentrations play a major role in intracellular signalling prompted the present study, which aimed to investigate the implications for neurotransmitter (receptor) regulation of enzymatic activity of this calmodulin regulation. The role of Ca2+/calmodulin in regulating neurotransmitter-mediated inhibition and stimulation was assessed in a number of rat brain areas. Ca2+/calmodulin stimulated adenylate cyclase activity in EGTA-washed plasma preparations from each region studied--from 1.3-fold (in striatum) to 3.4-fold (in cerebral cortex). The fold-stimulation produced by Ca2+/calmodulin was decreased in the presence of GTP, forskolin, or Mn2+. In EGTA-washed membranes, receptor-mediated inhibition of adenylate cyclase was strictly dependent upon Ca2+/calmodulin stimulation in all regions, except striatum. A requirement for Mg2+ in combination with Ca2+/calmodulin to observe neurotransmitter-mediated inhibition was also observed. In contrast, receptor-mediated stimulation of activity was much greater in the absence of Ca2+/calmodulin. The findings demonstrate that ambient Ca2+ concentrations, in concert with endogenous calmodulin, may play a central role in dictating whether inhibition or stimulation of adenylate cyclase by neurotransmitters may proceed.     

Low molecular weight factor in bovine caudal epididymal fluid that stimulates calcium uptake in caput spermatozoa

Secretions from the mammalian epididymis contain proteins that bind to developing sperm and are presumed to play a role in sperm maturation. The biochemical functions in sperm of most of these proteins are not known. In this report we describe the presence of a low molecular weight compound in bovine caudal epididymal luminal fluid (CF) that has a potent stimulatory effect on calcium (⁴⁵Ca²⁺) uptake in immature caput epididymal spermatozoa. The studies were initially undertaken to characterize the effect of the protein caltrin, present in bovine seminal plasma (BSP), on calcium uptake into caput spermatozoa. Caltrin is known to block calcium influx into mature bovine sperm. Unexpectedly, the kinetics of calcium uptake into caput sperm showed a biphasic response when treated with BSP, namely, a stimulation of uptake at 1 to 5 min and inhibition of uptake after this time. Since caudal sperm do not show this biphasic response, we reasoned that BSP contained a factor derived from CF that must interact with developing sperm before the binding of caltrin to sperm can prevent further calcium uptake. We first demonstrated that preincubation of caput sperm with CF eliminated the biphasic calcium uptake effect induced in caput sperm by BSP and that caudal fluid alone had a potent stimulatory effect on calcium uptake in caput sperm. Half-maximal stimulation (fivefold over control) occurred at a caudal fluid protein concentration of 0.27 mg/ml. Partial purification of the factor indicates that it is of low molecular weight (MW ～ 1,000), but further chemical characterization has not been carried out and its epididymal site of origin is not known. The results indicate that the regulation of intracellular calcium levels in sperm differs in immature and mature bovine sperm in that an epididymal factor promotes calcium uptake during epididymal maturation, and the seminal fluid protein caltrin prevents it at ejaculation.     

Initiation and characterization of primary mouse kidney epithelial cultures

Summary Primary cultures of murine renal epithelial cells were established from a preparation of proximal tubule fragments. Confluent cultures exhibited multiple dome formation, indicating the presence of tight junctions and an intact transcellular transport process. Ultrastructural analysis revealed a monolayer of polarized cells, with a sparse but clearly defined microvillar surface facing the growth medium and a basolateral surface attached to the substratum. Cultures grown on collagen gels did not show domes. The epithelial monolayer exhibited several differentiated functions of the proximal tubule: a) parathyroid hormone (PTH)-stimulated cAMP synthesis; b) production of 24,25-dihydroxyvitamin D₃ from 25-hydroxyvitamin D₃; c) high alkaline phosphatase activity; and d) Na⁺-dependent transport of phosphate (Pi) and α-methylglucoside (α-MG). The sugar uptake was selectively inhibited by phlorizin, a competitive inhibitor of glucose uptake at the luminal membrane. Kinetic analysis revealed independent transport systems for Pi and α-MG, with Km values corresponding to the high affinity systems identified in brush border membrane vesicles derived from the proximal tubule. Pi uptake by the epithelial monolayers was regulated by the concentration of Pi in the growth medium. Phorbol esters and PTH did not exert an effect on Pi and α-MG transport in mouse primary cultures. The present study demonstrates that primary cultures provide a useful in vitro preparation to investigate renal proximal tubular function. Cindy Bell was the recipient of an MRC Studentship Award. This work was supported by the MRC (Group in Medical Genetics). This is publication number 88011 of the McGill University-Montreal Children's Hospital Research Institute.     

Beta-adrenergic receptor characteristics of postnatal rat myocardial cell preparations

Summary Primary mycolardial cell cultures and freshly isolated cardiac cells in suspension resprensent two isolated, whole cell models for investigating cellular transsarcolemmal⁴⁵Ca⁺⁺ exchange in response to a receptor-coupled stimulus. Studies were performed to characterize beta-adrenergic receptor binding, beta-adrenergic receptor mediated cellular calcium (⁴⁵Ca⁺⁺) exchange, and viability in purified primary myocardial cell cultures and freshly isolated cardiac cells in suspension obtained from 3-to 3-d-old Sprague-Dawley rats. In addition, beta-adrenergic receptor binding was characterized in whole-heart crude membrane preparations. All three preparations had saturable beta-adrenergic binding sites with the antagonist [¹²⁵I]iodopindolol ([¹²⁵I]IPIN). The suspensions had a significantly lower B _max (42±6 fmol/mg protein) than the membranes and cultures (77±8 and 95±10 fmol/mg protein, respectively). The K _D of the cultures (218±2.0 pM) was significantly higher than that for the suspensions (107 ±1.3 pM) and membranes (93±1.3 pM). Viability was significantly lower in the suspensions (57%) when compared to 94% viability in myocardial cell cultures after 3 h of incubation in Kreb's Henseleit buffer. Incubation of the cultures with 5.0×10⁻⁷ M isoproterenol resulted in a significant increase in⁴⁵Ca⁺⁺ exchange as early as 15 s. In contrast,⁴⁵Ca⁺⁺ exchange into the suspensions was not increased. Although both primary cell cultures and cardiac cells in suspension possess saturable beta-adrenergic receptors, only the monolayer cultures exhibited functional beta-adrenergic receptor-mediated⁴⁵Ca⁺⁺ exchange. Of the two intact cell models investigated, these data suggest that primary myocardial cell cultures are more suitable than cell suspensions for investigating beta-adrenergic receptor binding and functions in the postnatal rat heart. This research was supported by The University of Texas Research Institute, a grant from the Texas Advanced Research Technology Program awarded to S. W. Leslie and R. E. Wilcox, and contract 223-86-2109 from the Food and Drug Administration.