P2X7 receptors on osteoblasts couple to production of lysophosphatidic acid: a signaling axis promoting osteogenesis.

Nucleotides are released from cells in response to mechanical stimuli and signal in an autocrine/paracrine manner through cell surface P2 receptors. P2rx7-/- mice exhibit diminished appositional growth of long bones and impaired responses to mechanical loading. We find that calvarial sutures are wider in P2rx7-/- mice. Functional P2X7 receptors are expressed on osteoblasts in situ and in vitro. Activation of P2X7 receptors by exogenous nucleotides stimulates expression of osteoblast markers and enhances mineralization in cultures of rat calvarial cells. Moreover, osteogenesis is suppressed in calvarial cell cultures from P2rx7-/- mice compared with the wild type. P2X7 receptors couple to production of the potent lipid mediators lysophosphatidic acid (LPA) and prostaglandin E2. Either an LPA receptor antagonist or cyclooxygenase (COX) inhibitors abolish the stimulatory effects of P2X7 receptor activation on osteogenesis. We conclude that P2X7 receptors enhance osteoblast function through a cell-autonomous mechanism. Furthermore, a novel signaling axis links P2X7 receptors to production of LPA and COX metabolites, which in turn stimulate osteogenesis.

Renal responses to acute lead waterborne exposure in the freshwater rainbow trout (Oncorhynchus mykiss).

The possible nephrotoxic effects of waterborne lead exposure (as Pb(NO3)2) were investigated in the freshwater rainbow trout (Oncorhynchus mykiss). Kidney lead accumulation was time-dependent, increasing upon exposure to 0.57+/-0.01 mg dissolved Pb L(-1) for up to 96 h with a significantly higher burden occurring in the posterior kidney compared to the anterior segment. Urine analyses in trout exposed to 1.20+/-0.09 mg dissolved Pb L(-1) revealed a significant increase in urinary lead excretion rate throughout 96 h of exposure. Urine flow rate and glomerular filtration rate (GFR) were not impacted with the exception of a significant decrease in GFR from 84 to 96 h in lead-exposed trout. Urine pH decreased significantly over time in lead-exposed fish. Correspondingly, urine ammonia excretion rate showed a marked increase from 48 h onwards. In experimental fish, urine glucose excretion was significantly greater by 96 h while urine lactate, urea and protein excretion were not significantly altered by lead exposure. The urine excretion rate of Ca2+ increased significantly by approximately 43% after only 24 h of lead exposure, and was maintained at a higher rate than controls for up to 96 h. Magnesium excretion increased in a time-dependent fashion, reaching a two- to three-fold rise by 96 h. In contrast, rates of Na+ and Cl- excretion were decreased in experimental fish by approximately 30% by 48 h, this trend continuing for the duration of lead-exposure. There were no changes in any of these parameters in similarly treated control fish. Clearance ratio analyses indicated progressive decreases in the net reabsorption efficiencies of the renal system for Ca2+, Mg2+, Pb, and glucose, suggesting that the active tubular transport mechanisms for these substances were inhibited by lead exposure, while Na+, K+, Cl-, lactate, and protein reabsorptions were unaffected. Net ammonia secretion increased. We conclude that changes in renal function both reflect and help to minimize some of the associated disturbances in systemic physiology. Lead-induced ionoregulatory toxicity in rainbow trout, particularly the disturbance of Ca2+ homeostasis, is not exclusively a branchial phenomenon, but is in part a result of disruption of ionoregulatory mechanisms at the kidney. This action of lead outside the gills is critical to consider when developing guidelines for water quality.

Mechanisms behind Pb-induced disruption of Na+ and Cl- balance in rainbow trout (Oncorhynchus mykiss).

The mechanism of Pb-induced disruption of Na(+) and Cl(-) balance was investigated in the freshwater rainbow trout (Oncorhynchus mykiss). Na(+) and Cl(-) influx rates were reduced immediately in the presence of 2.40 +/- 0.24 and 1.25 +/- 0.14 muM Pb, with a small increase in efflux rates occurring after 24-h exposure. Waterborne Pb caused a significant decrease in the maximal rate of Na(+) influx without a change in transporter affinity, suggesting a noncompetitive disruption of Na(+) uptake by Pb. Phenamil and bafilomycin markedly reduced Na(+) influx rate but did not affect Pb accumulation at the gill. Time-course analysis in rainbow trout exposed to 0, 0.48, 2.4, and 4.8 microM Pb revealed time- and concentration-dependent branchial Pb accumulation. Na(+)-K(+)-ATPase activity was significantly reduced, with 4.8 microM exposure resulting in immediate enzyme inhibition and 0.48 and 2.4 microM exposures inhibiting activity by 24 h. Reduced activity was weakly correlated with gill Pb accumulation after 3- and 8-h exposures; this relationship strengthened by 24 h. Reduced Na(+) uptake was correlated with gill Pb burden after exposures of 3, 8, and 24 h. Immediate inhibition of branchial carbonic anhydrase activity occurred after 3-h exposure to 0.82 +/- 0.05 or 4.30 +/- 0.05 microM Pb and continued for up to 24 h. We conclude that Pb-induced disruption of Na(+) and Cl(-) homeostasis is in part a result of rapid inhibition of carbonic anhydrase activity and of binding of Pb with Na(+)-K(+)-ATPase, causing noncompetitive inhibition of Na(+) and Cl(-) influx.

Socially-mediated differences in brain monoamines in rainbow trout: effects of trace metal contaminants.

Monoaminergic systems play a crucial role in linking behaviour and physiology. Here the physiological and behavioural effects of metal exposure in relation to monoaminergic systems were considered by exposing rainbow trout dyads, demonstrating stable dominance relationships, to cadmium or lead. Fish exposed to 4 microg l(-1) cadmium accumulated more cadmium at the gill than fish held in control water. Fish exposed to 7 microg l(-1) cadmium had higher gill, liver and kidney cadmium concentrations. No significant lead accumulation was seen after exposure to 46 microg l(-1) for 48 h but exposure to 325 microg l(-1) lead caused an increase in gill, liver and kidney lead concentrations. Brain accumulation of both cadmium and lead was only seen after exposure to the highest concentrations. Exposure to 4 or 7 microg l(-1) cadmium, or 46 or 325 microg l(-1) lead for 48 h did not disrupt established dominance hierarchies. As expected with this stable behavioural situation, in control pairs, animals of different social status displayed different physiological profiles. Subordinate fish had higher concentrations of circulating plasma cortisol and telencephalic 5-hydroxyindoleacetic acid/5-hydroxytryptamine (serotonin) (5-HIAA/5-HT) ratios. However, these physiological profiles were affected by metal exposure, with a trend towards higher serotonergic activity in dominant fish. Dominants exposed to 325 microg l(-1) lead had significantly higher hypothalamic 5-HIAA/5-HT ratios when compared with subordinates. The results demonstrate that if stable social hierarchies are established in control water they may not be affected by exposure to cadmium and lead although physiological changes may be evident.

Intraspecific divergence of ionoregulatory physiology in the euryhaline teleost Fundulus heteroclitus: possible mechanisms of freshwater adaptation.

We examined intraspecific variation in ionoregulatory physiology within euryhaline killifish, Fundulus heteroclitus, to understand possible mechanisms of freshwater adaptation in fish. Pronounced differences in freshwater tolerance existed between northern (2% mortality) and southern (19% mortality) killifish populations after transfer from brackish water (10 g l(-1)) to freshwater. Differences in Na(+) regulation between each population might partially account for this difference in tolerance, because plasma Na(+) was decreased for a longer period in southern survivors than in northerns. Furthermore, northern fish increased Na(+)/K(+)-ATPase mRNA expression and activity in their gills to a greater extent 1-14 days after transfer than did southerns, which preceded higher whole-body net flux and unidirectional influx of Na(+) at 14 days. All observed differences in Na(+) regulation were small, however, and probably cannot account for the large differences in mortality. Differences in Cl(-) regulation also existed between populations. Plasma Cl(-) was maintained in northern fish, but in southerns, plasma Cl(-) decreased rapidly and remained low for the duration of the experiment. Correspondingly, net Cl(-) loss from southern fish remained high after transfer, while northerns eliminated Cl(-) loss altogether. Elevated Cl(-) loss from southern fish in freshwater was possibly due to a persistence of seawater gill morphology, as paracellular permeability (indicated by extrarenal clearance rate of PEG-4000) and apical crypt density in the gills (detected using scanning electron microscopy) were both higher than in northern fish. These large differences in the regulation of Cl(-) balance probably contributed to the marked differences in mortality after freshwater transfer. Glomerular filtration rate and urination frequency were also lower in southerns. Taken together, these data suggest that northern killifish are better adapted to freshwater environments and that minimizing Cl(-) imbalance appears to be the key physiological difference accounting for their greater freshwater tolerance.

Characterization of branchial lead-calcium interaction in the freshwater rainbow trout Oncorhynchus mykiss.

The mechanism of branchial lead uptake and interplay with Ca(2+) transport was investigated in the freshwater rainbow trout Oncorhynchus mykiss. Lead significantly reduced Ca(2+) influx by approximately 40% and 30% after exposure to 2.3+/-0.1 and 1.4+/-0.2 micromol l(-1) dissolved lead, respectively, for 0-48 h. Acute inhibition of Ca(2+) influx by lead exhibited typical Michaelis-Menten kinetics with an approximate 16-fold increase in K(m), whereas J(max) values did not significantly change, yielding an inhibitor constant (K(i,Pb)) of 0.48 micromol l(-1). Alternative analyses suggest the possibility of a mixed competitive/non-competitive interaction at the highest lead concentration tested (4.8 micromol l(-1)). Branchial lead accumulation was reduced with increasing waterborne Ca(2+) concentrations, suggesting a protective effect of Ca(2+) against lead uptake at the gill. The apical entries of Ca(2+) and lead were both inhibited (55% and 77%, respectively) by the addition of lanthanum (1 micromol l(-1)) to the exposure water. The use of cadmium (1 micromol l(-1)) and zinc (100 micromol l(-1)) as voltage-independent calcium channel competitors also reduced branchial lead uptake by approximately 56% and 47%, respectively. Nifedipine and verapamil (up to 100 micromol l(-1)), both voltage-dependent calcium channel blockers, had no effect on gill lead accumulation. CaCl(2) injection reduced both Ca(2+) and lead uptake by the gills. This suggests transport of lead through apical voltage-independent calcium channels, similar to the entry of Ca(2+). High-affinity Ca(2+)-ATPase activity was not acutely affected by lead, but a significant 80% reduction in activity occurred during exposure for 96 h to 5.5+/-0.4 micromol l(-1) dissolved lead, indicating a possible non-competitive component to lead-induced Ca(2+) disruption. The effect of lead on Ca(2+) efflux was investigated and found to be insignificant. We conclude that uptake of lead occurs, at least in part, by the same mechanism as Ca(2+), which results in disruption of Ca(2+) influx and ultimately Ca(2+) homeostasis.