The Carotid Body "Tripartite Synapse": Role of Gliotransmission.

In mammals, cardiorespiratory reflexes originating in the carotid body (CB) help maintain homeostasis by matching oxygen supply to oxygen demand. CB output to the brainstem is shaped by synaptic interactions at a "tripartite synapse" consisting of chemosensory (type I) cells, abutting glial-like (type II) cells, and sensory (petrosal) nerve terminals. Type I cells are stimulated by several blood-borne metabolic stimuli, including the novel chemoexcitant lactate. During chemotransduction, type I cells depolarize and release a multitude of excitatory and inhibitory neurotransmitters/neuromodulators including ATP, dopamine (DA), histamine, and angiotensin II (ANG II). However, there is a growing appreciation that the type II cells may not be silent partners. Thus, similar to astrocytes at "tripartite synapses" in the CNS, type II cells may contribute to the afferent output by releasing "gliotransmitters" such as ATP. Here, we first consider whether type II cells can also sense lactate. Next, we review and update the evidence supporting the roles of ATP, DA, histamine, and ANG II in cross talk among the three main CB cellular elements. Importantly, we consider how conventional excitatory and inhibitory pathways, together with gliotransmission, help to coordinate activity within this network and thereby modulate afferent firing frequency during chemotransduction.

The effect of marine dissolved organic carbon on nickel accumulation in early life-stages of the sea urchin, Strongylocentrotus purpuratus.

Dissolved organic carbon (DOC) is known to ameliorate the toxicity of the trace metal nickel (Ni) to aquatic animals. In theory, this effect is mediated by the capacity of DOC to bind Ni, rendering it less bioavailable, with the resulting reduction in accumulation limiting toxicological effects. However, there is a lack of experimental data examining Ni accumulation in marine settings with natural sources of DOC. In the current study, radiolabelled Ni was used to examine the time- and concentration-dependence of Ni accumulation, using naturally sourced DOC, on developing larvae of the sea urchin Strongylocentrotus purpuratus. Contrary to prediction, the two tested natural DOC samples (collected from the eastern United States, DOC 2 (Seaview park, Rhode Island (SVP)) and DOC 7 (Aubudon Coastal Center, Connecticut)) which had previously been shown to protect against Ni toxicity, did not limit accumulation. The control (artificial seawater with no added DOC), and the DOC 2 sample could mostly be described as having saturable Ni uptake, whereas Ni uptake in the presence of DOC 7 was mostly linear. These data provide evidence that DOC modifies the bioavailability of Ni, through either indirect effects (e.g. membrane permeability) or by the absorption of DOC-Ni complexes. There was some evidence for regulation of Ni accumulation in later-stage embryos (96-h) where the bioconcentration factor for Ni declined with increasing Ni exposure concentration. These data have implications for predictive modelling approaches that rely on known relationships between Ni speciation, bioavailability and bioreactivity, by suggesting that these relationships may not hold for natural marine DOC samples in the developing sea urchin model system.

Expanding Role of Dopaminergic Inhibition in Hypercapnic Responses of Cultured Rat Carotid Body Cells: Involvement of Type II Glial Cells.

Dopamine (DA) is a well-studied neurochemical in the mammalian carotid body (CB), a chemosensory organ involved in O2 and CO2/H+ homeostasis. DA released from receptor (type I) cells during chemostimulation is predominantly inhibitory, acting via pre- and post-synaptic dopamine D2 receptors (D2R) on type I cells and afferent (petrosal) terminals respectively. By contrast, co-released ATP is excitatory at postsynaptic P2X2/3R, though paracrine P2Y2R activation of neighboring glial-like type II cells may boost further ATP release. Here, we tested the hypothesis that DA may also inhibit type II cell function. When applied alone, DA (10 µM) had negligible effects on basal [Ca2+]i in isolated rat type II cells. However, DA strongly inhibited [Ca2+]i elevations (Δ[Ca2+]i) evoked by the P2Y2R agonist UTP (100 µM), an effect opposed by the D2/3R antagonist, sulpiride (1-10 µM). As expected, acute hypercapnia (10% CO2; pH 7.4), or high K+ (30 mM) caused Δ[Ca2+]i in type I cells. However, these stimuli sometimes triggered a secondary, delayed Δ[Ca2+]i in nearby type II cells, attributable to crosstalk involving ATP-P2Y2R interactions. Interestingly sulpiride, or DA store-depletion using reserpine, potentiated both the frequency and magnitude of the secondary Δ[Ca2+]i in type II cells. In functional CB-petrosal neuron cocultures, sulpiride potentiated hypercapnia-induced Δ[Ca2+]i in type I cells, type II cells, and petrosal neurons. Moreover, stimulation of type II cells with UTP could directly evoke Δ[Ca2+]i in nearby petrosal neurons. Thus, dopaminergic inhibition of purinergic signalling in type II cells may help control the integrated sensory output of the CB during hypercapnia.

Evidence for protein kinase involvement in the 5-HT-[Ca2+ ]i -pannexin-1 signalling pathway in type II glial cells of the rat carotid body.

NEW FINDINGS: What is the central question of this study? The mammalian carotid body (CB) is a peripheral chemoreceptor organ involved in O2 and CO2 /H+ homeostasis. Recent studies suggest that 5-HT, released from CB receptor cells, can stimulate adjacent glial-like type II cells, leading to an increase in intracellular Ca2+ (Δ[Ca2+ ]i ) and activation of ATP-permeable pannexin-1 (Panx-1) channels. The aim of this study was to elucidate the role of protein kinases in the 5-HT-[Ca2+ ]i -Panx-1 signalling pathway. What is the main finding and its importance? Src family kinase and protein kinase A, acting downstream from Δ[Ca2+ ]i , played central roles in 5-HT-mediated Panx-1 channel activation. This provides new insight into mechanisms regulating CB excitation, especially in pathophysiological conditions. ABSTRACT: Chemoreceptor (type I) cells of the rodent carotid body (CB) synthesize and release several neurotransmitters/neuromodulators, including 5-hydroxytryptamine (5-HT), implicated in enhanced CB excitation after exposure to chronic intermittent hypoxia, e.g. sleep apnoea. However, recent studies suggest that 5-HT can robustly stimulate adjacent glial-like type II cells via ketanserin-sensitive 5-HT2 receptors, leading to intracellular Ca2+ elevation (Δ[Ca2+ ]i ) and activation of ATP-permeable pannexin-1 (Panx-1) channels. Using dissociated rat CB cultures, we investigated the role of protein kinases in the intracellular signalling pathways in type II cells. In isolated type II cells, 5-HT activated a Panx-1-like inward current (I5-HT ) that was reversibly inhibited by the Src family kinase inhibitor PP2 (1 µm), but not by its inactive analogue, PP3 (1 µm). Moreover, I5-HT was reversibly inhibited (>90%) by H89 (1 µm), a protein kinase A blocker, whereas the protein kinase C blocker GF109203X (2 µm) was largely ineffective. In contrast, the P2Y2R agonist UTP (100 µm) activated Panx-1-like currents that were reversibly inhibited (∼60%) by either H89 or GF109203X. Using fura-2 spectrofluorimetry, the 5-HT-induced Δ[Ca2+ ]i was unaffected by PP2, H89 and GF109293X, suggesting that the kinases acted downstream of the Ca2+ rise. Given that intracellular Ca2+ chelation was previously shown to block receptor-mediated Panx-1 current activation in type II cells, these data suggest that CB neuromodulators use overlapping, but not necessarily identical, signalling pathways to activate Panx-1 channels and release ATP, a CB excitatory neurotransmitter. In conclusion, these studies provide new mechanistic insight into 5-HT signalling in the CB that has pathophysiological relevance.

Characterization of cadmium and calcium fluxes along the gut, malpighian tubules, and anal papillae of the dipteran Chironomus riparius.

Chironomids are often one of the dominant organisms in significantly polluted freshwater. Many invertebrate studies have characterized whole-organism mechanisms of toxicity, for example, assessing cadmium (Cd) uptake via calcium (Ca) channels. However, with the use of the scanning ion-selective electrode technique and an innovative Cd-selective microelectrode, we analyze this relationship at the organ level using a realistic concentration of Cd and Ca in the hemolymph (blood). Generally, Cd fluxes follow the same directional pattern as Ca, although Ca fluxes are approximately 5 times higher than those of Cd. These results correlate well with previous studies indicating that chironomids have a higher affinity for Ca over Cd, which affords them tolerance to Cd toxicity. When saline Ca concentration was increased to 10 times physiological levels, Cd fluxes from the gut lumen into the cells of the midgut regions were reduced by 50 to 80%. Transport of Cd from hemolymph to tissue for the posterior midgut, Malpighian tubule, and proximal ceca was also reduced by approximately 50%. The present results indicate that Cd fluxes into or across the gut and Malpighian tubules are reduced by high Ca, suggesting that Cd may be transported in some cells by similar mechanisms. However, Cd was actively excreted at the anal papillae after a 48-h waterborne exposure to Cd, but this process was independent of Ca and instead may involve a P-glycoprotein-related pump to detoxify Cd. Environ Toxicol Chem 2018;37:2542-2549. © 2018 SETAC.

Sensory Processing and Integration at the Carotid Body Tripartite Synapse: Neurotransmitter Functions and Effects of Chronic Hypoxia.

Maintenance of homeostasis in the respiratory and cardiovascular systems depends on reflexes that are initiated at specialized peripheral chemoreceptors that sense changes in the chemical composition of arterial blood. In mammals, the bilaterally-paired carotid bodies (CBs) are the main peripheral chemoreceptor organs that are richly vascularized and are strategically located at the carotid bifurcation. The CBs contribute to the maintenance of O2, CO2/H+, and glucose homeostasis and have attracted much clinical interest because hyperactivity in these organs is associated with several pathophysiological conditions including sleep apnea, obstructive lung disease, heart failure, hypertension, and diabetes. In response to a decrease in O2 availability (hypoxia) and elevated CO2/H+ (acid hypercapnia), CB receptor type I (glomus) cells depolarize and release neurotransmitters that stimulate apposed chemoafferent nerve fibers. The central projections of those fibers in turn activate cardiorespiratory centers in the brainstem, leading to an increase in ventilation and sympathetic drive that helps restore blood PO2 and protect vital organs, e.g., the brain. Significant progress has been made in understanding how neurochemicals released from type I cells such as ATP, adenosine, dopamine, 5-HT, ACh, and angiotensin II help shape the CB afferent discharge during both normal and pathophysiological conditions. However, type I cells typically occur in clusters and in addition to their sensory innervation are ensheathed by the processes of neighboring glial-like, sustentacular type II cells. This morphological arrangement is reminiscent of a "tripartite synapse" and emerging evidence suggests that paracrine stimulation of type II cells by a variety of CB neurochemicals may trigger the release of "gliotransmitters" such as ATP via pannexin-1 channels. Further, recent data suggest novel mechanisms by which dopamine, acting via D2 receptors (D2R), may inhibit action potential firing at petrosal nerve endings. This review will update current ideas concerning the presynaptic and postsynaptic mechanisms that underlie chemosensory processing in the CB. Paracrine signaling pathways will be highlighted, and particularly those that allow the glial-like type II cells to participate in the integrated sensory response during exposures to chemostimuli, including acute and chronic hypoxia.

Role of glial-like type II cells as paracrine modulators of carotid body chemoreception.

Mammalian carotid bodies (CB) are chemosensory organs that mediate compensatory cardiorespiratory reflexes in response to low blood PO2 (hypoxemia) and elevated CO2/H+ (acid hypercapnia). The chemoreceptors are glomus or type I cells that occur in clusters enveloped by neighboring glial-like type II cells. During chemoexcitation type I cells depolarize, leading to Ca2+-dependent release of several neurotransmitters, some excitatory and others inhibitory, that help shape the afferent carotid sinus nerve (CSN) discharge. Among the predominantly excitatory neurotransmitters are the purines ATP and adenosine, whereas dopamine (DA) is inhibitory in most species. There is a consensus that ATP and adenosine, acting via postsynaptic ionotropic P2X2/3 receptors and pre- and/or postsynaptic A2 receptors respectively, are major contributors to the increased CSN discharge during chemoexcitation. However, it has been proposed that the CB sensory output is also tuned by paracrine signaling pathways, involving glial-like type II cells. Indeed, type II cells express functional receptors for several excitatory neurochemicals released by type I cells including ATP, 5-HT, ACh, angiotensin II, and endothelin-1. Stimulation of the corresponding G protein-coupled receptors increases intracellular Ca2+, leading to the further release of ATP through pannexin-1 channels. Recent evidence suggests that other CB neurochemicals, e.g., histamine and DA, may actually inhibit Ca2+ signaling in subpopulations of type II cells. Here, we review evidence supporting neurotransmitter-mediated crosstalk between type I and type II cells of the rat CB. We also consider the potential contribution of paracrine signaling and purinergic catabolic pathways to the integrated sensory output of the CB during chemotransduction.

Mechanisms of nickel toxicity to fish and invertebrates in marine and estuarine waters.

In freshwater settings the toxicity of the trace metal nickel (Ni) is relatively well understood. However, until recently, there was little knowledge regarding Ni toxicity in waters of higher salinity, where factors such as water chemistry and the physiology of estuarine and marine biota would be expected to alter toxicological impact. This review summarizes recent literature investigating Ni toxicity in marine and estuarine invertebrates and fish. As in freshwater, three main mechanisms of Ni toxicity exist: ionoregulatory impairment, inhibition of respiration, and promotion of oxidative stress. However, unlike in freshwater biota, where mechanisms of toxicity are largely Class-specific, the delineation of toxic mechanisms between different species is less defined. In general, despite changes in Ni speciation in marine waters, organism physiology appears to be the main driver of toxic impact, a fact that will need to be accounted for when adapting regulatory tools (such as bioavailability normalization) from freshwater to estuarine and marine environments.

Chronic nickel bioaccumulation and sub-cellular fractionation in two freshwater teleosts, the round goby and the rainbow trout, exposed simultaneously to waterborne and dietborne nickel.

Rainbow trout and round goby were exposed for 30 days to waterborne and dietary Ni in combination at two waterborne concentration ranges (6.2-12 µmol/L, 68-86 µmol/L), the lower of which is typical of contaminated environments. The prey (black worms; Lumbriculus variegatus) were exposed for 48 h in the effluent of the fish exposure tanks before being fed to the fish (ration=2% body weight/day). Ni in gills, gut, and prey was fractionated into biologically inactive metal [BIM=metal-rich granules (MRG) and metallothionein-like proteins (MT)] and biologically active metal [BAM=organelles (ORG) and heat-denaturable proteins (HDP)]. Gobies were more sensitive than trout to chronic Ni exposure. Possibly, this greater sensitivity may have been due to the goby's pre-exposure to pollutants at their collection site, as evidenced by ∼2-fold greater initial Ni concentrations in both gills and gut relative to trout. However, this was followed by ∼2-16× larger bioaccumulation in both the gills and the gut during the experimental exposure. On a subcellular level, ∼3-40× more Ni was associated with the BAM fraction of goby in comparison to trout. Comparison of the fractional distribution of Ni in the prey versus the gut tissue of the predators suggested that round goby were more efficient than rainbow trout in detoxifying Ni taken up from the diet. Assessing sub-cellular distribution of Ni in the gills and gut of two fish of different habitat and lifestyles revealed two different strategies of Ni bioaccumulation and sub-cellular distribution. On the one hand, trout exhibited an ability to regulate gill Ni bioaccumulation and maintain the majority of the Ni in the MT fraction of the BIM. In contrast goby exhibited large Ni spillovers to both the HDP and ORG fractions of the BAM in the gill. However, the same trend was not observed in the gut, where the potential acclimation of goby to pollutants from their collection site may have aided their ability to regulate Ni spillover to the BAM more so than in trout. Overall, chronic mortality observed in goby may be associated more with Ni bioaccumulation in gills than in gut; the former at either 4-d or 30-d was predictive of chronic Ni toxicity. BIM and BAM fractions of the goby gills were equally predictive of chronic (30-d) mortality. However, critical body residue (CBR50) values of the BIM fraction were ∼2-4× greater than CBR50 values of the BAM fraction, suggesting that goby are more sensitive to Ni bioaccumulation in the BAM fraction. There was insufficient mortality in trout to assess whether Ni bioaccumulation was predictive of chronic mortality.

Acute toxicity, critical body residues, Michaelis-Menten analysis of bioaccumulation, and ionoregulatory disturbance in response to waterborne nickel in four invertebrates: Chironomus riparius, Lymnaea stagnalis, Lumbriculus variegatus and Daphnia pulex.

We investigated the bioaccumulation and acute toxicity (48 h or 96 h) of Ni in four freshwater invertebrate species in two waters with hardness of 40 (soft water) and 140 mg L(-1) as CaCO(3) (hard water). Sensitivity order (most to least) was Lymnaea stagnalis > Daphnia pulex > Lumbriculus variegatus > Chironomus riparius. In all cases water hardness was protective against acute Ni toxicity with LC(50) values 3-3.5× higher in the hard water vs. soft water. In addition, higher water hardness significantly reduced Ni bioaccumulation in these organisms suggesting that competition by Ca and Mg for uptake at the biotic ligand may contribute to higher metal resistance. CBR50 values (Critical Body Residues) were less dependent on water chemistry (i.e. more consistent) than LC(50) values within and across species by ~2 fold. These data support one of the main advantages of the Tissue Residue Approach (TRA) where tissue concentrations are generally less variable than exposure concentrations with respect to toxicity. Whole body Ni bioaccumulation followed Michaelis-Menten kinetics in all organisms, with greater hardness tending to decrease B(max) with no consistent effect on K(d). Across species, acute Ni LC(50) values tended to increase with both K(d) and B(max) values - i.e. more sensitive species exhibited higher binding affinity and lower binding capacity for Ni, but there was no correlation with body size. With respect to biotic ligand modeling, log K(NiBL) values derived from Ni bioaccumulation correlated well with log K(NiBL) values derived from toxicity testing. Both whole body Na and Mg levels were disturbed, suggesting that disruption of ionoregulatory homeostasis is a mechanism of acute Ni toxicity. In L. stagnalis, Na depletion was a more sensitive endpoint than mortality, however, the opposite was true for the other organisms. This is the first study to show the relationship between Na and Ni.

The effects of salinity on acute and chronic nickel toxicity and bioaccumulation in two euryhaline crustaceans: Litopenaeus vannamei and Excirolana armata.

We investigated the influence of salinity (5 ppt versus 25 ppt) on acute (96-h LC(50)) and chronic toxicity (15-30 day LC(50)) of Ni in two euryhaline crustaceans, the shrimp (Litopenaeus vannamei) and the isopod (Excirolana armata). 96-h LC50 values were 41 µmolL(-1) and 362 µmolL(-1) for L. vannamei and 278 µmolL(-1) and >1000 µmolL(-1) for E. armata at 5 ppt and 25 ppt, respectively. Speciation analysis demonstrated that complexation with anions such as SO(4)(2-), HCO(3)(-) and Cl(-) at 25 ppt had a negligible effect on reducing the free Ni(2+) ion component in comparison to 5 ppt. The salinity-dependent differences in acute Ni toxicity could not be explained by differences in Ni bioaccumulation. Therefore, differences in physiology of the organisms at the two salinities may be the most likely factor contributing to differences in acute Ni toxicity. Chronic LC(50) values (2.7-23.2 µmolL(-1)) were similar in the two species, but salinity had no significant effect, indicating that water chemistry and osmoregulatory strategy do not influence chronic toxicity. However chronic (15-day) mortality in both species could be predicted by acute (96-h) Ni bioaccumulation patterns.

Cadmium transport by the gut and Malpighian tubules of Chironomus riparius.

Many aquatic insects are very insensitive to cadmium in short-term laboratory studies. LC50 values for larvae of the midge Chironomus riparius are over 25,000 times the Criterion Maximum Concentration in the United States Environmental Protection Agency (U.S. EPA (2000)) species sensitivity distribution (SSD). Excretion or sequestration of cadmium may contribute to insensitivity and we have therefore examined cadmium transport by isolated guts and renal tissues of C. riparius larvae. Regional differences of Cd transport along the gut were identified using a Cd(2+)-selective microelectrode in conjunction with the Scanning Ion-Selective Electrode Technique (SIET). Cd is transported into the anterior midgut (AMG) cells from the lumen and out of the cells into the hemolymph. The transport of Cd from the gut lumen to the hemolymph exposes other tissues such as the nervous system and muscles to Cd. The gut segments which remove Cd from the hemolymph at the highest rate are the posterior midgut (PMG) and the ileum. In addition, assays using an isolated Malpighian (renal) tubule preparation have shown that the Malpighian tubules (MT) both sequester and secrete Cd. For larvae bathed in 10 micromol l(-1) Cd, the tubules can secrete the entire hemolymph burden of Cd in approximately 15 h.