Phosphoarginine Research Articles

Some Biochemical Properties of the Upregulation of the Squid Nerve Na+/Ca2+ Exchanger by MgATP and Phosphoarginine

In squid nerve MgATP upregulation of Na+/Ca2+ exchange requires a soluble cytosolic regulatory protein (SCRP) of about 13 kDa; phosphoarginine (PA) stimulation does not. MgATP-gamma-S mimics MgATP. When a 30-10-kDa cytosolic fraction is exposed to 0.5 mM [32P]ATP in the same solution used for transport assays, and in the presence of native membrane vesicles, a 13-kDa and a 25-kDa band become phosphorylated. Membrane vesicles alone do not show these phosphorylated bands and heat denaturation of the cytosolic fraction prevents phosphorylation. Moreover, staurosporine, a general inhibitor of kinases, does not affect MgATP + SCRP stimulation of the exchanger or the phosphorylation of the 13 kDa but prevents phosphorylation of the 25-kDa cytosolic band. The 30-10-kDa fraction phosphorylated in the presence of staurosporine stimulates Na+/Ca2+ exchange in vesicles in the absence of ATP but with Mg2+ in the medium. The 30-10-kDa fraction is not phosphorylated by PA. In membrane vesicles two protein bands, at about 60 kDa and 70 kDa identified as the low molecular weight neurofilament (NF), are phosphorylated by PA, but not by MgATP. This phosphorylation is specific for PA, insensitive to staurosporine (similar to the PA-stimulated fluxes), and labile. In addition, co-immunoprecipitation was observed between the NF and the exchanger protein. Under the conditions of these experiments no phosphorylation of the exchanger is detected, either with MgATP or PA.

The Squid Preparation as a General Model for Ionic and Metabolic Na+/Ca2+Exchange Interactions

We propose an integrated kinetic model for the squid nerve Na+/Ca2+ exchanger based on experimental evidences obtained in dialyzed axons. This model satisfactorily explains the interrelationship between ionic (Na+(i)-H+(i)-Ca2+(i)) and metabolic (ATP, phosphoarginine (PA)) regulation of the exchanger. Data in dialyzed axons show that the Ca(i)-regulatory site located in the large intracellular loop plays a central role in the modulation by ATP by antagonizing the inhibitory Na+(i)-H+(i) synergism. We have used the Na(o)/Na(i) exchange mode to unequivocally measure the affinity of the Ca(i)-regulatory site. This allowed us to separate Ca(i)-regulatory from Ca(i)-transport sites and to estimate their respective affinities. In this work we show for the first time that under conditions of saturation of the Ca(i)-regulatory site (10 microM Ca2+(i), pH(i) 8.0), ATP have no effect on the Ca(i)-transport site. In addition, we have expanded our equilibrium kinetic model of ionic and metabolic interactions to a complete exchange cycle (circular model). This model, in which the Ca(i)-regulatory site plays a central role, accounts for the decrease in Na(i) inactivation, at high pH(i), high Ca2+(i,) and MgATP. Furthermore, the model also predicts the net Ca2+ movements across the exchanger based on the exchanger complexes redistribution both during physiological and pathological conditions (ischemia).

Sublethal Effects of Pentachlorophenol in Abalone (Haliotis rufescens) Veliger Larvae as Measured by 31P-NMR

Toxicity tests with larval marine organisms are widely used as water quality-monitoring tools. Often employing a single morphological endpoint, they provide only limited insight into toxic action mechanisms. Since mechanistic observations can provide more sensitive endpoints of toxicity, the goal of this study was to modify existing in vivo31P-NMR techniques to evaluate the effects of pentachlorophenol (PCP) on high-energy phosphates in veliger larvae of the red abalone (Haliotis rufescens), a species and life stage commonly used in developmental toxicity tests. Fluxes of phosphoarginine (PA), nucleoside triphosphates (NTPs), sugar phosphates (SPs), and inorganic phosphate (Pi), as well as in intracellular pH (pHi), were monitored via NMR using a flow-through exposure system. The exposure protocol included 1 h of clean seawater, 2 h of PCP (1.2 mg L−1) exposure, and 2 h of recovery in clean seawater. During PCP exposure, veligers displayed maximum declines in PA and NTP resonance intensities to 38±10% (P<0.01) and 61±16% (P<0.05) of reference values (represented by the average resonance intensities taken from 1 h of preexposure spectral acquisition). Inorganic phosphate concentrations rose to 302±63% (P<0.01), and pHi declined from a reference period average of 7.12±0.03 to 7.04±0.04 (P<0.05). On exposure to clean seawater, veligers recovered within 2 h, returning to 196±53, 89±12, and 89±13% of the reference intensities for Pi, PA, and NTPs, respectively (P>0.05 for all values), while pHi increased to 7.08±0.11. When compared with adult abalone, veliger larvae responded similarly to PCP, differing mainly in their rapid recovery (2 h for larvae versus 6 h adults) and more severe acidification (a 0.08 pH unit decline versus a 0.03 pH unit decline). This report describes an application of NMR for small (≥200 μ) marine organisms using commonly available vertical-bore NMR magnets; it should prove adaptable for use with other similar larval forms.

Initial changes in ATP equivalents in the post-mortem muscles of three species of lobster

SUMMARY: Changes in the content of ATP, phosphoarginine (PA), and lactic acid (Lac) were determined in the muscles of three species of lobster during storage at 0, 5, and 10°C. The ATP equivalent (Eq) of each species was calculated to evaluate the initial degradation of ATP in the postmortem stage. Both ATP and PA decreased simultaneously with Lac accumulation in the muscle of every lobster. The contents of the three components in Japanese spiny lobster changed slowly at 0 and 5°C and rapidly at 10°C. In the case of rock lobster, the changes at 0°C were slowest, followed by 5°C, and those at 10°C were fastest. In contrast, the changes in the three components of American lobster muscle exhibited less dependency on the temperatures. The decreases in Eq at 0, 5, and 10°C for American lobster were largest of the three species. The change in Eq in the initial 24 h was larger than that in the second 24 h for every lobster. It is suggested that each species of lobster had its own relationship between the storage temperature and the rate of ATP production, and degradation.

Influence of salinity on the actions of pentachlorophenol in haliotis as measured by in vivo 31P NMR spectroscopy

The actions of pentachlorophenol (PCP) under various water salinities was investigated in live, intact red ( Haliotis rufescens) and black abalone ( H. cracherodii) by in vivo 31P nuclear magnetic resonance (NMR) spectroscopy. Based on PCP toxicity in red abalone (6 h LC 50 1.6 mg l −1; 6 h NOEL=0.8 mg l −1), animals were exposed in flowing seawater (14°C) to a sublethal PCP concentration of 1.2 mg l −1 and a water salinity of either 25, 35 or 45 ‰. Phosphagen changes in foot muscle were monitored by surface-probe localized 31P NMR. Upon PCP exposure, phosphoarginine (PA) levels declined and inorganic phosphate (Pi) levels concurrently increased. Dosing was terminated at a standard metabolic endpoint (MEP), defined as the time for the spectral peak height for Pi to reach one-half that of PA. This was on average 3.3±0.2 and 6.8±0.7 h at ambient salinity; 5.3±0.7 and 7.1±0.7 h for high salinity; and 4.0±0.5 and 6.5±1.0 h at low salinity, for red and black abalone, respectively. During recovery, all biochemical endpoints returned to normal; however, both onset and intensity of effects were influenced by changes in seawater salinity. Disposition of [U- 14C]PCP in both species was also examined to determine if the effects observed with NMR had a kinetic basis. Three sets of abalone ( n=3) from each species were exposed to each salinity (25, 35 and 45 ‰), plus 1.2 mg l −1 of [U- 14C]PCP. Red abalone were exposed for 3.5 h and black abalone for both 3.5 and 6.5 h (they represent the lowest average MEP for both species). Residues were quantified via tissue digestion and liquid scintillation counting. The 3.5 h total concentration factors (TCFs) for red abalone were not significantly different among the three salinities ( P>0.05) and ranged from 104 to 133. However, low salinity seemed to deter the uptake of [U- 14C]PCP in black abalone, as the TCF was over three times lower for abalone exposed to 25 ‰ at both 3.5 h (TCF=19±11) and 6.5 h (TCF=38±8) than at 35 ‰ (3.5 h TCF=62±6; 6.5 h TCF=119±11) and 45 ‰ (3.5 h TCF=67±14, 6.5 h TCF=115±8). Furthermore, the 3.5 h TCF for red abalone exposed to PCP at all salinities was similar to the 6.5 h TCF for black abalone exposed at 35 and 45 ‰. Thus, black abalone appear to have greater resistance to the combined effects of PCP and salinity variations, which appears to be due to slower uptake of the biocide. While PCP is a potent uncoupler of mitochondrial oxidative phosphorylation, use of varying water salinities simulated an additional stress imposed on intertidal animals by transient or seasonal environmental changes.

CHANGES IN CONTENT OF OCTOPINE, ACIDIC OPINES, RELATED AMINO ACIDS AND PHOSPHOARGININE IN THE ADDUCTOR MUSCLE OF THREE SPECIES OF SCALLOP DURING STORAGE

The postmortem changes of octopine (Oct), strombine (Stn), tauropine (Trn), alanopine (Aln), related amino acids and phosphoarginine (PA) in the edible adductor muscles of Chlamys nobilis, Pecten albicans and Patinopecten yessoensis during storage at 0 and 5C were examined. For this purpose, new alternative HPLC methods for the analysis of octopine and acidic opines were developed. Among opines, Oct was the major postmortem glycolytic end product of all three scallops, followed by Stn, Trn and Aln, respectively. The accumulation of Oct during storage at 0 and 5C was similar. On the contrary, almost twice as much Stn accumulated when stored at 5C compared to 0C. Trn and Aln increased to a lesser extent. Arginine and PA decreased, while glycine, taurine and alanine increased during storage.

Effects of hypoxia and toxicant exposure on phosphoarginine, intracellular pH, and free Mg 2+ in abalone as measured by 31P-NMR

The effects of hypoxia, sodium azide and pentachlorophenol (PCP) exposure on high-energy phosphorylated compounds, intracellular pH (pH i) and intracellular free Mg 2+ (Mg f) in intact red abalone ( Haliotis rufescens) were determined using 31P-NMR. Abalone made hypoxic by bubbling sea water with N 2 showed modest changes in phosphoarginine (PA) and inorganic phosphate (P i) concentrations, no significant changes in pH i and a moderate decrease in Mg f that was not statistically significant. Azide (50 mg/l) exposed animals displayed severe declines in PA dropping to 0.53 of reference values, coupled with large increases in P i to 10.66 times resting concentrations that occurred just after the 2-hr exposure period. pH i also showed significant declines from a resting value of 7.17-7.06 ( P < 0.05) but fully recovered by the end of the 6-hr clean seawater recovery period, whereas Mg f concentrations declined slightly during the exposure period but increased by 18% at the end of the recovery period relative to reference Mg f. PCP (1.2 mg/l) exposed animals displayed similar increases and declines in P i and PA, respectively, as did azide-exposed animals by the end of the exposure period, but recovery was much slower and occurred in a bimodal fashion with some animals completely recovering at the end of 6 hr and others essentially stabilized at the end of the exposure period and did not show any significant changes during the recovery period.

Phosphate metabolites of Tenebrio molitor (Coleoptera: tenebrionidae) infected with metacestodes of Hymenolepis diminuta.

In vivo phosphorus-31 nuclear magnetic resonance (31P NMR) spectra and those of extracts of Tenebrio molitor L. infected with metacestodes of Hymenolepis diminuta R. showed modifications in the amounts of phosphorous-containing metabolites when compared with those of uninfected beetles. Infected females were more affected than infected males, having significantly more glucose-6-phosphate (Glu-6-P), glycerol-3-phosphate (Gly-3-P), and phosphorylethanolamine (PE), but less inorganic orthophosphate (Pi), glycerophosphorylethanolamine (GPE), gylcerolphosphorylcholine (GPC), phosphoarginine (PAr), and adenosine diphosphate (ADP). Infected males had more Glu-6-P and PE, but less Pi, GPE, and GPC. These changes directly reflected the phosphorylation potential of infected hosts, which increased approximately 100% and 50% in infected females and males, respectively.

Biochemical and morphological pathology of the foot of the schistosome vector Biomphalaria glabrata infected with Schistosoma mansoni

Infection by Schistosoma mansoni resulted in morphological and biochemical changes to the foot of its intermediate host, Biomphalaria glabrata. Migration through, and emergence of cercariae from, the foot was observed and evidenced by lesions on the ciliated foot surface. This was accompanied by a significant decrease in the velocity of movement by infected individuals. In vivo 31P NMR spectral analyses demonstrated that the foot of infected snails had a lower phosphoarginine (PA)/adenosine 5' triphosphate (ATP) ratio than that of uninfected controls. Moreover, kinetic experiments, employing saturation transfer, demonstrated the pseudo-first-order rate constant for the arginine kinase-catalysed exchange reaction in the forward direction, that is, PA-->ATP was decreased by infection. The reverse reaction was not observed by the NMR methods used. PA was depleted upon exposure to hypoxic conditions suggesting its traditional role in preserving ATP level. Partly oxidized metabolic end-products were not observed in snails maintained under aerobic conditions, but succinate, propionate, acetate and lactate rapidly accumulated under hypoxic conditions.

Interactive effects of pentachlorophenol and temperature in the abalone ( Haliotis rufescens) as measured by in vivo 31P-NMR spectroscopy

The influence of temperature on the effects of pentachlorophenol (PCP) was described in red abalones ( Haliotis rufescens) using in vivo 31P nuclear magnetic resonance spectroscopy (NMR). Specifically, two effects were measured: the exposure times required for onset of PCP effects; and both intensity of, and recovery from, such effects. Following acclimation to clean seawater, abalones were exposed to a sublethal water concentration of PCP (1.2 mg l −1) at either 9 or 19°C until the spectral peak area for inorganic monophosphate (P i) was one-half that of phosphoarginine (PA); temperatures represented the seasonal range in California. The endpoint served to compare the times required to reach equivalent metabolic condition, and insured that all individuals were metabolically equivalent at the onset of exposure to clean seawater; abalones were maintained in clean seawater for up to 16 h to monitor recovery. In foot muscle, PCP caused a decrease in concentration of PA and ATP, an increase in that of P i, and acidification of intracellular pH; effects were produced more rapidly at 19°C, as the endpoint was attained in less than half the time (2.4 h) as was that at 9°C (5.4 h; P < 0.05). Both the average response to, and the average extent of recovery from, the biocide were greater in abalones exposed at 19°C. Upon exposure to clean seawater, all effects generally reversed within 10 to 16 h, and control abalones maintained at the two temperatures alone exhibited no adverse effects. Finally, abalones exposed to 120 μg l −1 of PCP for 18 h at both 9 and 19°C exhibited no adverse effects, indicating that PCP does not impact abalones at current environmental concentrations. In conclusion, high temperature potentiates the time both to onset of and for recovery from the sublethal effects of PCP in the red abalone. Use of in vivo NMR allows measurement of multiple-stress interactions in live, intact aquatic organisms, facilitating the detailed elucidation of individual responses and the laboratory assessment of the environmental effects of pollutants.

Interactive effects of pentachlorophenol and hypoxia in the abalone ( Haliotis rufescens) as measured by in vivo 31P NMR spectroscopy

The effects of hypoxia and pentachlorophenol (PCP) were investigated in red abalones ( Haliotis rufescens) using flow-through exposure and in vivo 31P nuclear magnetic resonance spectroscopy (NMR). Following acclimation to clean seawater, three abalones were separately exposed to a 6-h sublethal concentration of PCP (1.2 mg 1 −1) until the spectral resonance for inorganic monophosphate (P i) was half-height to that of phosphoarginine (PA); the endpoint assured that all individuals were metabolically equivalent at the start of air exposure. They were exposed to clean seawater for 2 h to allow the effects from PCP to stabilize, then air for 45 min, and finally clean seawater for 15 h to check recovery. The effects of PCP in foot muscle included a decrease in the concentrations of PA and adenosine triphosphate ([PA] and [ATP], respectively), an increase in P i, and a decrease in intracellular pH (pH i). Upon air exposure, [PA] and pH i rapidly declined further, [P i] increased, and [ATP] did not change; the magnitudes were similar to those from emergence alone. After resubmergence, both [PA] and [P i] recovered rapidly from the effects of hypoxia, but slowly from those of PCP. While pH i initially declined further, both [ATP] and pH i recovered to near-initial levels after 15 h. In general, the metabolic effects of hypoxia did not change upon pretreatment with PCP; the biochemical interaction of the two factors was additive. Another three abalones were separately exposed to PCP at an environmentally relevant concentration of 120 μg 1 −1 for 6 h, air for 1 h, then the same PCP concentration for 2.5 h to check recovery. At the lower concentration, PCP produced no discernable effects, and those of hypoxia were similar to those without PCP. Since PCP produced no additional effects with or without hypoxia, it does not significantly impact red abalones at currently measured environmental concentrations. Use of in vivo NMR allows measurement of the interactive effects of multiple stress factors in live aquatic organisms, allowing assessment of the actual environmental effects of pollutants in the laboratory.

Characterization of the 31P NMR spectrum of Manduca sexta and effects of antimetabolites

High resolution 31P nuclear magnetic resonance spectroscopy (NMR) was successfully applied to 5th instar larvae of Manduca sexta. Conditions for in vivo analysis under non-saturating conditions are described. The 31P NMR spectrum of intact larvae was composed of six peaks. Their resonance frequencies are reported relative to orthophosphoric acid. Analysis of tissue extracts demonstrated the in vivo peaks to be composed of the β phosphorus resonance of nucleotide triphosphates (NTP) at −19.36 ppm; α phosphorus of NTP and nucleotide diphosphates (NDP) at −10.51 ppm; β and γ phosphorus of NDP and NTP, respectively, at −5.42 ppm; phosphoarginine (PA) at −3.45 ppm; inorganic phosphate (Pi) at +2.76 ppm and sugar phosphates at +3.34 ppm. The major sugar phosphate present in fat body extracts was trehalose-6-phosphate and this was the major phosphorus component of the spectrum of hemolymph. The spin-lattice relaxation times for each in vivo peak were determined. Titration of aqueous fat body and hemolymph extracts was carried out and the relationship between the chemical shift of Pi and pH determined. On this basis the pH of the hemolymph was estimated at approx. 6.7. The metabolic inhibitors, iodoacetate and dinitrophenol, had significant effects on the 31P NMR spectrum of intact larvae. Administration of iodoacetate caused a rapid increase in the levels of sugar phosphates together with decreases in NTP and PA. Dinitrophenol also caused declines in the relative levels of NTP and PA but sugar phosphates decreased as well. The experiments demonstrated the potential of in vivo NMR analysis for metabolic studies on high energy phosphate metabolites in M. sexta.

Some factors influencing sodium extrusion by internally dialyzed squid axons.

Squid giant axons were internally dialyzed by a technique previously described. In an axon exposed to cyanide seawater for 1 hr and dialyzed with an ATP-free medium, the Na efflux had a mean value of 1.3 pmole/cm(2)sec when [Na](i) was 88 mM, in quantitative agreement with flux ratio calculations for a purely passive Na movement. When ATP at a concentration of 5-10 mM was supplied to the axoplasm by dialysis, Na efflux rose almost 30-fold, while if phosphoarginine, 10 mM, was supplied instead of ATP, the Na efflux rose only about 15-fold. The substitution of Li for Na in the seawater outside did not affect the Na efflux from an axon supplied with ATP, while a change to K-free Na seawater reduced the Na efflux to about one-half. When special means were used to free an axon of virtually all ADP, the response of the Na efflux to dialysis with phosphoarginine (PA) at 10 mM was very small (an increment of ca. 3 pmole/cm(2)sec) and it can be concluded that more than 96% of the Na efflux from an axon is fueled by ATP rather than PA. Measurements of [ATP] in the fluid flowing out of the dialysis tube when the [ATP] supplied was 5 mM made it possible to have a continuous measurement of ATP consumption by the axon. This averaged 43 pmole/cm(2)sec. The ATP content of axons was also measured and averaged 4.4 mM. Estimates were made of the activities of the following enzymes in axoplasm: ATPase, adenylate kinase, and arginine phosphokinase. Values are scaled to 13 degrees C.