A comparison of the metal content of some benthic species from coastal waters of the Florida panhandle using high-resolution inductively coupled plasma mass spectrometry (ICP-MS) analysis.

Benthic marine invertebrates, sediment, and water from several locations along the Florida panhandle coast from St. Joseph Bay in the west to the mouth of the Wakulla River in the east, including from several river estuaries, were analyzed by double focusing ICP-MS (Finnigan MAT ELEMENT) for Cd, Hg, Pb, Cu, Zn, and As. All were detected in all samples. Sponges generally contained higher levels of Cd than other species. Microciona prolifera sponges from St. Joseph Bay had higher As levels (8.1-13.6 microg/g dry weight) than sponges collected from Dickerson Bay or Appalachee Bay (2.20-9.7) and higher Cd levels (0.43-0.73) than that of a single Microciona specimen collected from Dickerson Bay (0.29). Water content of As was about 20-30x higher in St. Joseph Bay than in any other location, and sediment levels of Cd were about 9x higher. Cu and Zn were higher in organic sediment from St. Joseph Bay than they were in other areas. The Pb content of several sponge species and two of tunicates was considerably higher than in other species. The uptake of most metals in this study (except As) appeared to be affected by the metal, genus, species, and location as much as by levels in either water or sediment. In general, sponges and tunicates seemed to accumulate higher levels than most other species, possibly a function of high filtration rates. The ICP-MS method is useful for environmental studies, but the instrument requires considerable maintenance.

Effects of experimental manipulation of pH and salinity on Cd(2+) uptake by the sponge Microciona prolifera and on sponge cell aggregation induced by Ca(2+) and Cd(2+).

Marine sponges (Microciona prolifera) collected in St. Joseph Bay, Florida panhandle, were exposed for 2 h to pH/salinity unit combinations of 7.4/30, 6.3/30, 7.4/11, and 6.3/11. Cell suspensions from these were aggregated with 24 microM of either CaCl(2) or CdCl(2). Cells exposed to the low/low (11/6.3) combination aggregated spontaneously (no added stimulus) in 8/11 experiments, suggesting a significant disturbance of normal function, possibly involving disrupted ion uptake. In all other combinations aggregation proceeded normally and there were no statistically significant differences among the groups. CdCl(2) was as effective an aggregation stimulus as CaCl(2). The calcium channel blocker verapamil (100 microM) reduced calcium-induced aggregation by 15% but had no effect on cadmium (Cd)-induced aggregation, indicating that L-type calcium channels do not play a major role in aggregation induced by these divalent cations. Sponge tissue was exposed for 48 h to the same pH/salinity combinations but containing Cd (15 or 150 microg/ml) and then dried and analyzed for Cd. All sponges concentrated Cd but those exposed to low salinity concentrated it most (in one case x13). Low pH alone had no appreciable effect but appeared to increase the effect of low salinity. One sponge with a native Cd content of 47.2 microg/g dry weight had the highest acquired Cd content. The results of this study indicate that low levels of salinity and pH, similar to those we recorded in the study area, facilitate the accumulation of Cd, but not via L-type calcium channels, and disrupt normal aggregation responses of the cell. These results may help explain a previous observation that cells from M. prolifera from this area, with high native levels of Cd, failed to aggregate in response to CaCl(2)[Philp RB (1999) Comp Biochem Physiol 124C:41-49] and also the frequent die-offs of Microciona that have virtually eliminated this sponge from its local habitat.

Cadmium content of the marine sponge Microciona prolifera, other sponges, water and sediment from the eastern Florida panhandle: possible effects on Microciona cell aggregation and potential roles of low pH and low salinity.

Marine sponges from shallow bays and estuaries along the Florida panhandle contained cadmium (Cd), 1.78-27.9 microg g(-1) (dry weight). Levels > 7 microg g(-1) were never found in a variety of other benthic invertebrates. Water levels of Cd were of the order 0.22-0.43 microg ml(-1) and did not differ significantly between the two study areas (Panacea and St. Joseph Bay). Cd in organic sediment however, was significantly (P = 0.033) higher around Panacea (0.343 +/- 0.063 S.E.M.) than in St. Joseph Bay (0.18 +/-0.223 S.E.M.). Salinity of 10 and pH 6.5 were recorded following heavy rains, and can constitute a hostile environment for these organisms. Temporal variations of Cd and Cu but not Zn were observed in Microciona prolifera over a period of 2 years in St. Joseph Bay, and a negative correlation was observed between Cd content and the Ca(2+)-induced aggregation of Microciona cells suspended in Ca2+/Mg(2+)-free artificial seawater. Cells unresponsive to Ca2+ aggregated in response to releasers of Ca2+ from internal stores (thapsigargin, A23187 and tetredecylamine), suggesting that they were viable but dormant. In vitro studies showed that Cd2+ is about equipotent with Ca2+ in inducing aggregation of Microciona cells, whereas lower concentrations (1-2 x 10(-3) M), added 10 min before inducing aggregation with CaCl2, inhibited aggregation, possibly by blocking Ca2+ uptake. Microciona contained a metallothionein (MT)-like protein. Taken together these results suggest that naturally-acquired Cd may interfere with cell regulatory processes in Microciona. Possible effects of low pH and salinity on Cd uptake and detrimental effects on cell function are discussed.

Nitrous oxide enhances Na+/Ca++ exchange in the neuroblastoma cell line SK-N-SH.

Changes in the concentration of cytosolic free calcium ([Ca++]i) play fundamental roles in the initiation and regulation of many neuronal processes. Altered regulation of [Ca++]i has been implicated in the action of some anesthetics. We investigated the effects of nitrous oxide (N2O) on Ca++ mobilization and membrane potential in the human neuroblastoma cell line SK-N-SH. [Ca++]i was monitored by fluorescence spectrophotometry of cells loaded with fura-2 or fluo-3. N2O reversibly suppressed carbachol-stimulated increases in [Ca++]i. N2O also inhibited increases in [Ca++]i induced by calcium ionophore or depolarization suggesting a mechanism involving enhanced efflux or sequestration of cytosolic Ca++. The inhibitory effect of N2O was attenuated when the transmembrane Na+ gradient was altered either by suspending cells in nominally Na(+)-free buffer or by pretreating cells with ouabain. The inhibitory effect of N2O was also attenuated by the Na+/Ca++ exchange inhibitor 3,4-dichlorobenzamil. The effects of N2O on membrane potential were measured fluorimetrically using bis(1,3-dibutylthiobarbituric acid)-trimethine oxonol. In the presence of N2O, resting membrane potential was hyperpolarized, a condition that would favor Ca++ efflux mediated by the electrogenic Na+/Ca++ exchanger. Taken together, these findings indicate that N2O suppresses carbachol-stimulated increases in [Ca++]i by enhancing Na+/Ca++ exchange activity. Enhancement of neuronal Na+/Ca++ exchange may contribute to the anesthetic action of N2O.

Pressure antagonism of nitrous oxide depression of intracellular calcium in a neuroblastoma cell line.

While the ability of increased pressure to reverse anaesthesia has been well documented, the cellular or molecular mechanism(s) responsible for their mutual antagonism have remained elusive. Previous work in our laboratory, using diverse cell types, has indicated several processes requiring Ca2+ are affected in opposite directions by hydrostatic pressure [as represented by helium (He)], narcotic gases, and some anesthetics. Here we report on the effects of elevated pressures of He, and of 1 atm abs of the anesthetic gas nitrous oxide (N2O), when present alone and in combination, on calcium mobilization in the human neuroblastoma cell line SK-N-SH. Cytosolic-free Ca2+ ([Ca2+]i) was monitored by fluorescence spectrophotometry in cell suspensions loaded with the intracellular Ca2+ indicator fura-2. N2O reversibly depressed the carbachol-stimulated increase in [Ca2+]i (P < 0.01). The application of both 18 and 35 atm abs He attenuated this N2O-induced depression of carbachol-stimulated increase in [Ca2+]i. These findings support the hypothesis that pressure/anesthetic antagonism may be due in part to effects on neuronal [Ca2+]i and its regulation.

Effects of high hydrostatic pressures on secondary structure of acetylcholinesterase with and without carbachol.

Ultra-high hydrostatic pressures (to 13 kbar) were applied to acetylcholinesterase (AChE) in the presence and absence of 1 mM carbachol (a muscarinic agonist) by means of a piston-and-cylinder system designed for use with Fourier transform infrared spectroscopy. At normal atmospheric pressure, carbachol decreased the number of intramolecular hydrogen bonds and the anti-parallel beta-sheet structure. In the absence of carbachol, pressure dramatically increased the number of intermolecular hydrogen bonds but decreased the alpha-helical, beta-sheet, and anti-parallel beta-sheet segments. In the presence of carbachol, pressure had the opposite effects, decreasing the number of intermolecular hydrogen bonds and increasing the alpha-helix: beta-sheet ratio. Thus in the absence of an attached ligand, the enzyme molecule was vulnerable to pressure-induced distortions that would most likely impair its function. These effects were observed in the absence of a lipid component, indicating that pure proteins are vulnerable to pressure-induced changes in configuration that could affect function.

Effects of elevated pressures of inert gases on cytosolic free Ca2+ of cultured human neuroblastoma cells stimulated with carbachol: relevance to high pressure neurological syndrome.

Suspended cells of the human neuroblastoma line SK-N-SH were exposed to elevated pressures of non-narcotic helium (He) and the narcotic gases nitrogen (N2), and argon (Ar) and stimulated with carbachol. He, 18 and 36 atmospheres absolute (ATA), equivalent to 544 and 1120 feet of seawater, potentiated the increase in [Ca2+]i induced by carbachol, as measured by Fura-2. Carbachol-stimulated increases in [Ca2+]i were not significantly altered from values in 1 ATA air by either N2 or Ar at the same pressures. The response to carbachol of cells exposed to 36 ATA of He and slowly decompressed to 1 ATA was indistinguishable from that of cells never exposed to pressure. Thus this pressure-potentiated increase in [Ca2+]i is compatible with excitation, is reversible and is not elicited by narcotic gases. It was observed, moreover, at pressures encountered by commercial deep-sea divers. The High Pressure Neurological Syndrome (HPNS) encountered by divers breathing He/O2 mixtures at high pressures, and its known antagonism by N2, may be due in part to effects on neuronal [Ca2+]i levels since an increase in these would most likely result in an excitatory response.

Effects of elevated pressures of inert gases on cytosolic free Ca2+ of human platelets stimulated with ADP.

Platelets were exposed to elevated pressures of helium (He), nitrogen (N2), and He/xenon (He/Xe, 85/15), and stimulated with ADP (5 microM). He to 36 atmospheres absolute (ATA) inhibited the ADP-stimulated increase in [Ca2+]i, measured by Fura-2/AM, with most of the effect occurring by 9 ATA. N2 caused a similar but initially greater effect and it was bimodal, with inhibition being less at 36 ATA than at 18 ATA. N2 also significantly depressed basal levels at 18 and 36 ATA. In the He/Xe mixture, the effect on ADP-stimulated [Ca2+]i was no different from He alone at the same pressures, but basal levels were significantly depressed. In a calcium-free medium both He and N2 moderately depressed the already-reduced response to ADP stimulation but only N2 significantly depressed basal levels at all pressures. These results indicate that raised pressures of inert narcotic gases, as well as pressure per se (represented by He), can affect cell [Ca2+]i sufficiently to have physiological consequences. This, together with previous findings in this laboratory, may have implications for some of the neurological problems associated with deep diving.

Effects of gaseous anesthetics and ultrashort and short-acting barbiturates on human blood platelet free cytosolic calcium: relevance to their effects on platelet aggregation.

The effects of elevated pressures (to 6 atmospheres absolute (ATA)) of nitrous oxide (N2O) and of xenon (Xe), and barbiturates on platelet free cytosolic calcium ([Ca2+]i) and platelet aggregation were studied. N2O inhibited the ADP-induced rise in [Ca2+]i whereas Xe had no effect. Neither affected basal levels. Pentobarbital and methohexital had little effect on basal or stimulated levels in the presence or "absence" of extracellular Ca2+; but both, at concentrations > 10(-4) M, inhibited platelet aggregation induced by adenosine diphosphate. Thiopental increased basal and stimulated [Ca2+]i when extracellular Ca2+ was present, but not when it was absent, and displayed a bimodal effect with low and high doses being more active than intermediate ones. It also potentiated aggregation. Methitural displayed similar, but nonsignificant, effects. These patterns held for all agents whether or not acetylsalicylic acid was present. Pentobarbital and methohexital inhibited phorbol myristate acetate aggregation in low extracellular calcium and no potentiation was seen with thiopental. In the absence of extracellular Ca2+, no potentiation was observed in stimulated platelets. Potentiation of aggregation previously reported for Xe does not involve increased Ca2+ uptake and did not occur in the absence of extracellular Ca2+. A common mechanism of action for these agents cannot be inferred from their effects on platelet aggregation or [Ca2+]i, as their pharmacological profiles differ markedly. It is evident that their inhibitory properties in this cell are not dependent on extracellular Ca2+, whereas the potentiation observed with pentobarbital, and formerly with Xe, is so dependent.

Effects of pressure and gaseous anesthetics on the aggregation of human blood platelets and marine sponge cells: similarities in responses.

1. Aggregation of blood platelets and marine sponge cells was inhibited by nitrous oxide (N2O) and helium (He) at elevated pressure but potentiated by xenon (Xe) despite the fact that Xe and N2O are equipotent gaseous anesthetics. 2. The above aggregations are dependent on an extracellular source of calcium. 3. Platelet aggregation induced by phorbol myristate is independent of extracellular calcium and was inhibited by both N2O and Xe and by high pressures of He. 4. The results argue against a common mechanism for Xe and N2O and suggest that pressure may affect calcium interactions with binding site.

Effect of pressure on the release of endogenous dopamine from rat striatum and the role of sodium-calcium exchange.

Exposure to environmental pressures in excess of 20 atm abs can precipitate a hyperexcitability state known as high pressure neurologic syndrome (HPNS). Little is known about the underlying neurochemical basis of this syndrome. An in vitro model of the synthesis and release of endogenous dopamine (DA) from rat striatal slices has been used to examine the mechanism underlying the effects of high pressures of He. He at 100 atm abs produced changes in DA release which were strikingly similar to those of the cardiac glycoside, ouabain. Neither pressure nor ouabain (1-10 microM) had any significant effects on the spontaneous (nonevoked) release of DA or its metabolite 3,4-dihydroxyphenylacetic acid, but both pressure and ouabain significantly enhanced the stimulated release of DA which was evoked by a 6-min exposure to 35 mM KCl (P less than 0.05 and P less than 0.001). In both cases, this effect was dependent on the presence of extracellular Ca2+. Augmentation of evoked DA release by both ouabain and He pressure was reversed (P less than 0.05) by 3,4-dichlorobenzamil, a selective antagonist of the membrane Na+/Ca2+ exchange mechanism. The results suggest that pressure exerts its effects on DA release by increasing intracellular-free Ca2+ exchange after pressure-inhibition of the activity of the membrane Na,K-ATPase.

Pharmacological studies on the mechanism of pressure inhibition of human platelet aggregation.

Platelet shape change and aggregation in response to adenosine diphosphate (ADP), epinephrine, collagen, thrombin, ristocetin, and phorbol myristate acetate (PMA) were studied photometrically at 1 ATA air and 35 ATA helium (He). Pressure inhibited aggregation in response to all agents except PMA. Dose response curves were constructed for ADP and epinephrine at 1 ATA and 35 ATA in the presence of acetylsalicyclic acid (ASA) 2.5 . 10(-4) M, which prevents aggregation due to released ADP and other granular constituents thus allowing study of the effect of the added stimulus alone. Pressure inhibited aggregation and yielded a shallower dose response curve. Pharmacologically, this would be interpreted as non-competitive blockade or reduced availability of receptors. All of the agonist agents inhibited by pressure are dependent upon extracellular Ca2+ for their function. All unmask other receptors (integrins) for adhesive proteins, principally fibrinogen. These integrins incorporate Ca2+ to become active. In contrast, PMA-induced aggregation, and shape change, are independent of extracellular Ca2+ and were unaffected by pressure. It is proposed that pressure may distort Ca2(+)-dependent surface glycoprotein receptors in a manner that reduces ligand affinity and hence inhibits platelet aggregation. The possibility that other calcium-mediated cell processes are affected by pressure cannot be discounted.

Isolated human blood platelets discriminate between anaesthetic and non-anaesthetic gases at high pressures.

We have compared the effects of the anaesthetic gases nitrogen and argon on adenosine diphosphate (ADP)-induced human blood platelet aggregation with the effects of the non-anaesthetic gas helium. All three gases showed dose-dependent inhibition of platelet aggregation. For nitrogen and argon there was a linear relationship between gas pressure and inhibition of aggregation over the range 15-68 atmospheres absolute (atm abs), whereas helium had a threshold for inhibition of approximately 34 atm abs. The inhibition by all gases was reversible after slow decompression. At pressures greater than 55 atm abs, nitrogen produced less inhibition than helium, indicating anaesthetic-pressure antagonism. Whereas pressure alone and the anaesthetic gases inhibited aggregation, the platelet shape change elicited by ADP was resistant to both nitrogen and helium, indicating that ADP binding and the early events in platelet activation were relatively unaffected by these conditions.

Hyperbaric He but not N2 augments Ca2+-dependent dopamine release from rat striatum.

Endogenous dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were measured by high performance liquid chromatography with electrochemical detection in perfusate from continuously superfused rat brain striatal slices, and the effects of various pressures of He and N2 were determined. He at 24 and 100 atmospheres absolute (ATA) significantly (P less than 0.01 and less than 0.05) increased the release of DA evoked by a 6-min exposure to 35 mM K+, whereas He at 48 ATA did not. Experiments conducted in a Ca2+-free medium showed that only the extracellular Ca2+-dependent component of release was affected by pressure. Similar increases in DA release were observed when DA reuptake and metabolism were blocked with cocaine and pargyline, although statistical significance was not achieved. N2 did not significantly affect DA release at 12, 24, 48, or 100 ATA. The results indicate that He (= hydrostatic pressure) augments Ca2+-dependent DA release and that substitution of N2 negates this effect. The relevance of these observations to the phenomena of high pressure neurologic syndrome in divers and the anesthetic reversal of pressure effects is discussed.

Memory deficit caused by compressed air equivalent to 36 meters of seawater.

Twenty-four students from a diving school undertook a hyperbaric chamber dive to a pressure equal to 36 m of seawater. Tests of cognitive function and manual dexterity, performed in the chamber during the 35-min bottom time and before, or after, the dive included immediate and delayed free recall of words presented as 7 lists of 15 each, recognition of previously presented words, number identification, and a forceps pickup of ball bearings. Delayed free recall and immediate free recall (primacy region) were significantly impaired, whereas manual dexterity and recognition memory were not. These are in keeping with previously reported findings but indicate that significant impairment of memory may occur in experienced divers at operational depths for air diving. Lack of effect on recognition memory suggests that cueing strategies might be useful for debriefing divers.

Failure of platelets to enhance aggregation of temperature-stabilized neutrophils: effects of warming and of drugs.

Polymorphonuclear leukocytes (PMNs) were isolated on a Ficoll-Hypaque gradient, suspended in modified Hank's buffer, and aggregated alone or in the presence of washed platelets (4 or 8/PMN). Platelets had no effect on formyl-methionyl-leucyl-phenylalanine (FMLP)-induced aggregation of PMNs that had been allowed to equilibrate at 37 degrees C for 5 min after storage at room temperature. Pretreatment of platelets with an inhibitor of cyclooxygenase (ASA) or lipoxygenase (nordihydroguaiaretic acid, NDGA) produced no significant effect whereas pretreatment with an inhibitor of both enzymes (eicosatetraynoic acid) or of phospholipase (methylprednisolone sodium succinate) caused a modest but statistically-significant inhibition of PMN aggregation which appeared to be a direct effect on PMNs rather than through platelets. The warming of PMNs from 0 degrees C or 22 degrees C to 37 degrees C produced a spontaneous, reversible aggregation within 2 or 3 min, the extent of which was dependent on the degree of temperature change. This aggregation was enhanced by the presence of platelets in a 'dose' (count) dependent fashion. This enhancement was not decreased by any of the aforementioned drugs, in fact, the aggregation was augmented by all drugs, the difference being statistically significant for NDGA. Thus different mechanisms appear to be involved in spontaneous vs FMLP-induced aggregation. The role of platelets in PMN aggregation remains to be elucidated but the importance of controlling for the effects of temperature changes in such studies is self-evident.

Effects of nitrogen and helium at pressure on drug-induced stereotypy and locomotion in mice.

Stereotyped behavior was induced in mice by apomorphine (5-50 mg X kg-1 s.c.) and d-amphetamine (3-5 mg X kg-1 i.p.), and increased locomotor activity was induced by d-amphetamine (8 and 10 mg X kg-1 i.p.) and by morphine sulfate (15 and 30 mg X kg-1 i.p.). Experiments were conducted at 1, 4, and 7 ATA. All mice were myringotomized under ether anesthesia 3 d before behavioral studies to minimize disturbances due to pressure differences across the tympanic membrane during compression. Compressed air significantly increased locomotor activity induced by d-amphetamine and morphine sulfate whereas He/O2 had no effect, suggesting that the change was due to the narcotic effect of N2. Drug-induced stereotyped behavior was affected variably (usually depressed) by compressed air and not by He/O2. Since both stereotypy and locomotor activity induced by these drugs involve dopamine receptor systems, the results suggest that compressed air does not influence all such membrane receptors in like manner. Evidence for receptor plasticity is discussed.