Arachidonic acid potentiates acid-sensing ion channels in rat sensory neurons by a direct action.

Acid-sensing ion channels (ASICs) are activated by a decrease in extracellular pH. ASICs are expressed in nociceptive sensory neurons, and several lines of evidence suggest that they are responsible for signaling the pain caused by extracellular acidification, but little is understood of the modulation of ASICs by pro-inflammatory factors. Using whole-cell patch clamp we demonstrate that low pH evokes three distinct inward currents in rat dorsal root ganglion neurons: a slowly inactivating transient current, a rapidly inactivating transient current, and a sustained current. All three currents were potentiated by arachidonic acid (AA), to 123%, 171%, and 264% of peak current, respectively. Membrane stretch had no effect on proton-gated currents, implying that AA is unlikely to act via local membrane deformation. The current carried by heterologously expressed ASIC1a and ASIC3 was also potentiated by AA. AA potentiates ASIC activation by a direct mechanism, because inhibition of AA metabolism had no effect on potentiation, and potentiation of single ASIC2a channels could be observed in cell-free patches. Potentiation by lipids with the same chain length as AA increased as the number of double bonds was increased. AA is known to be released in inflammation and the results suggest that AA may be an important pro-inflammatory agent responsible for enhancing acid-mediated pain.

Characterization of the primary spinal afferent innervation of the mouse colon using retrograde labelling.

Visceral pain is the most common form of pain produced by disease and is thus of interest in the study of gastrointestinal (GI) complaints such as irritable bowel syndrome, in which sensory signals perceived as GI pain travel in extrinsic afferent neurones with cell bodies in the dorsal root ganglia (DRG). The DRG from which the primary spinal afferent innervation of the mouse descending colon arises are not well defined. This study has combined retrograde labelling and immunohistochemistry to identify and characterize these neurones. Small to medium-sized retrogradely labelled cell bodies were found in the DRG at levels T8-L1 and L6-S1. Calcitonin gene-related peptide (CGRP)- and P2X3-like immunoreactivity (LI) was seen in 81 and 32%, respectively, of retrogradely labelled cells, and 20% bound the Griffonia simplicifolia-derived isolectin IB4. CGRP-LI and IB4 were co-localized in 22% of retrogradely labelled cells, whilst P2X3-LI and IB4 were co-localized in 7% (vs 34% seen in the whole DRG population). Eighty-two per cent of retrogradely labelled cells exhibited vanilloid receptor 1-like immunoreactivity (VR1-LI). These data suggest that mouse colonic spinal primary afferent neurones are mostly peptidergic CGRP-containing, VR1-LI, C fibre afferents. In contrast to the general DRG population, a subset of neurones exist that are P2X3 receptor-LI but do not bind IB4.

Protein kinase C activation potentiates gating of the vanilloid receptor VR1 by capsaicin, protons, heat and anandamide.

1. The effects of activation of protein kinase C (PKC) on membrane currents gated by capsaicin, protons, heat and anandamide were investigated in primary sensory neurones from neonatal rat dorsal root ganglia (DRG) and in HEK293 cells (human embryonic kidney cell line) transiently or stably expressing the human vanilloid receptor hVR1. 2. Maximal activation of PKC by a brief application of phorbol 12-myristate 13-acetate (PMA) increased the mean membrane current activated by a low concentration of capsaicin by 1.65-fold in DRG neurones and 2.18-fold in stably transfected HEK293 cells. Bradykinin, which activates PKC, also enhanced the response to capsaicin in DRG neurones. The specific PKC inhibitor RO31-8220 prevented the enhancement caused by PMA. 3. Activation of PKC did not enhance the membrane current at high concentrations of capsaicin, showing that PKC activation increases the probability of channel opening rather than unmasking channels. 4. Application of PMA alone activated an inward current in HEK293 cells transiently transfected with VR1. The current was suppressed by the VR1 antagonist capsazepine. PMA did not, however, activate a current in the large majority of DRG neurones nor in HEK293 cells stably transfected with VR1. 5. Removing external Ca(2+) enhanced the response to a low concentration of capsaicin 2.40-fold in DRG neurones and 3.42-fold in HEK293 cells. Activation of PKC in zero Ca(2+) produced no further enhancement of the response to capsaicin in either DRG neurones or HEK293 cells stably transfected with VR1. 6. The effects of PKC activation on the membrane current gated by heat, anandamide and low pH were qualitatively similar to those on the capsaicin-gated current. 7. The absence of a current activated by PMA in most DRG neurones or in stably transfected HEK293 cells suggests that activation of PKC does not directly open VR1 channels, but instead increases the probability that they will be activated by capsaicin, heat, low pH or anandamide. Removal of calcium also potentiates activation, and PKC activation then has no further effect. The results are consistent with a model in which phosphorylation of VR1 by PKC increases the probability of channel gating by agonists, and in which dephosphorylation occurs by a calcium-dependent process.

Multidrug transporters in prokaryotic and eukaryotic cells: physiological functions and transport mechanisms.

Multidrug transporters mediate the extrusion of structurally unrelated drugs from prokaryotic and eukaryotic cells. As a result of this efflux activity, the cytoplasmic drug concentration in the cell is lowered to subtoxic levels and, hence, cells become multidrug resistant. The activity of multidrug transporters interferes with the drug-based control of tumours and infectious pathogenic microorganisms. There is an urgent need to understand the structure-function relationships in multidrug transporters that underlie their drug specificity and transport mechanism. Knowledge about the architecture of drug and modulator binding sites and the link between energy-generating and drug translocating functions of multidrug transporters may allow one to rationally design new drugs that can poison or circumvent the activity of these transport proteins. Furthermore, if one is to inhibit multidrug transporters in human cells, one should know more about their physiological substrates and functions. This review will summarize important new insights into the role that multidrug transporters in general, and P-glycoprotein and its bacterial homologue LmrA in particular, play in the physiology of the cell. In addition, the molecular basis of drug transport by these proteins will be discussed.

Modulation of the synaptic Ca2+ current in salamander photoreceptors by polyunsaturated fatty acids and retinoids.

Synaptic transmission between retinal photoreceptors and second-order neurones is controlled by an L-type Ca2+ conductance (gCa) in the photoreceptor inner segment. Modulation of this conductance therefore influences the flow of visual information to higher centres. Possible modulation of gCa by retinal factors was investigated using patch clamp and Ca2+ imaging. No significant modulation of gCa by retinal neurotransmitters nor by intracellular signalling pathways was found. gCa was inhibited by retinoids (all-trans retinal) and by polyunsaturated fatty acids (PUFAs) such as arachidonic acid and docosahexaenoic acid, which are known to be released in the retina by exposure to light. Some PUFAs tested are physiological substrates for the cyclo-oxygenase, lipoxygenase and epoxygenase pathways, but specific inhibitors of these pathways had no effect on the inhibition of gCa. Treatments designed to activate or inhibit G-protein-coupled pathways or protein kinases A and C similarly had no effect on the inhibition by PUFAs nor on gCa itself. Inhibitors of phosphatases 1 and 2A were also largely ineffective. The inhibition by PUFAs is, however, dependent on membrane potential, suggesting that it arises from a direct interaction of fatty acids with the Ca2+ channel. The effect was not use or frequency dependent, suggesting that the effect does not depend on channel gating state. Control by retinoids and by PUFAs may be an important mechanism by which the Ca2+ conductance, and consequently the transmission of the visual signal, is modulated at the first retinal synapse.

Lysophospholipids trigger calcium signals but not DNA synthesis in cortical astrocytes.

Astrocytes generate calcium signals and proliferate in response to a growth factor-like lipid bound to plasma and serum albumin, in a process likely to be important in the formation of glial scars. A number of potential candidates for the physiologically active lipid were investigated. Lysophosphatidic acid, lysophosphatidylcholine, sphingomyelin, and platelet-activating factor all elicited calcium signals of varying magnitudes in cortical astrocytes, although only lysophosphatidic acid elicited calcium signals comparable in amplitude to those induced by the active physiological lipid. None of these lipids, however, caused cell division in astrocytes. There is therefore no invariable relationship between the ability of lipids to induce calcium signals and mitogenic activity. None of the lipids investigated demonstrate the activity of the natural lipid factor in generating both calcium signals and mitotic activity in astrocytes.

Specific involvement of PKC-epsilon in sensitization of the neuronal response to painful heat.

Pain is unique among sensations in that the perceived intensity increases, or sensitizes, during exposure to a strong stimulus. One important mediator of sensitization is bradykinin (BK), a peptide released as a consequence of tissue damage. BK enhances the membrane ionic current activated by heat in nociceptive neurons, using a pathway that involves activation of protein kinase C (PKC). We find that five PKC isoforms are present in sensory neurons but that only PKC-epsilon is translocated to the cell membrane by BK. The heat response is sensitized when constitutively active PKC-epsilon is incorporated into nociceptive neurons. Conversely, BK-induced sensitization is suppressed by a specific peptide inhibitor of PKC-epsilon. We conclude that PKC-epsilon is principally responsible for sensitization of the heat response in nociceptors by bradykinin.

Ion channels gated by heat.

All animals need to sense temperature to avoid hostile environments and to regulate their internal homeostasis. A particularly obvious example is that animals need to avoid damagingly hot stimuli. The mechanisms by which temperature is sensed have until recently been mysterious, but in the last couple of years, we have begun to understand how noxious thermal stimuli are detected by sensory neurons. Heat has been found to open a nonselective cation channel in primary sensory neurons, probably by a direct action. In a separate study, an ion channel gated by capsaicin, the active ingredient of chili peppers, was cloned from sensory neurons. This channel (vanilloid receptor subtype 1, VR1) is gated by heat in a manner similar to the native heat-activated channel, and our current best guess is that this channel is the molecular substrate for the detection of painful heat. Both the heat channel and VR1 are modulated in interesting ways. The response of the heat channel is potentiated by phosphorylation by protein kinase C, whereas VR1 is potentiated by externally applied protons. Protein kinase C is known to be activated by a variety of inflammatory mediators, including bradykinin, whereas extracellular acidification is characteristically produced by anoxia and inflammation. Both modulatory pathways are likely, therefore, to have important physiological correlates in terms of the enhanced pain (hyperalgesia) produced by tissue damage and inflammation. Future work should focus on establishing, in molecular terms, how a single ion channel can detect heat and how the detection threshold can be modulated by hyperalgesic stimuli.

Albumin elicits calcium signals from astrocytes in brain slices from neonatal rat cortex.

1. Albumin causes calcium signals and mitosis in cultured astrocytes, but it has not been established whether astrocytes in intact brain also respond to albumin. The effect of albumin on intracellular calcium concentration ([Ca2+]i) in single cells was therefore studied in acutely isolated cortical brain slices from the neonatal rat. 2. Physiological concentrations of albumin from plasma and from serum produced an increase in [Ca2+]i in a subpopulation of cortical cells. Trains of transient elevations in [Ca2+]i (Ca2+ spikes) were seen in 41 % of these cells. 3. The cells responding to albumin are identified as astrocytes because the neurone-specific agonist NMDA caused much smaller and slower responses in these cells. On the other hand NMDA-responsive cells, which are probably neurones, exhibited only small and slow responses to albumin. The residual responses of astrocytes to NMDA and neurones to albumin are likely to be due to crosstalk with adjacent neurones and astrocytes, respectively. 4. Methanol extraction of albumin removes a polar lipid and abolishes the ability of albumin to increase intracellular calcium. 5. Astrocyte calcium signalling caused by albumin may have important physiological consequences when the blood-brain barrier breaks down and allows albumin to enter the CNS.

Plasma albumin induces calcium waves in rat cortical astrocytes.

Changes in intracellular calcium were monitored in cultured cortical astrocytes stimulated with albumin. Albumin elicited intracellular calcium mobilisation from intracellular stores, inducing repetitive intracellular calcium oscillations. The oscillations were not blocked by ryanodine, a blocker of the Ca-induced Ca release mechanism, and the release occurred from the same store as is accessed by glutamate and bradykinin, both of which release calcium by an IP3-dependent mechanism. Calcium signals induced by albumin appear therefore to occur via a pure IP3-dependent mechanism. When albumin was applied to confluent monolayers of astrocytes, the oscillations in individual cells were initially unsynchronised, but after several minutes of application, the Ca2 oscillations were observed to synchronise and spread through the astrocyte network as a wave. These intercellular calcium waves were inhibited by the gap junction blocker halothane. Using the fluorescence recovery after photobleaching (FRAP) technique, we demonstrate that the development of propagated waves with prolonged exposure to albumin does not result from an increase in cell coupling. The development of calcium waves on exposure to albumin may be important in the formation of glial scars in the CNS after breakdown of the blood-brain barrier.

Protein kinase C-mediated phosphorylation does not regulate drug transport by the human multidrug resistance P-glycoprotein.

P-glycoprotein (P-gp) is an active transporter that can confer multidrug resistance by pumping cytotoxic drugs out of cells and tumors. P-gp is phosphorylated at several sites in the "linker" region, which separates the two halves of the molecule. To examine the role of phosphorylation in drug transport, we mutated P-gp such that it could no longer be phosphorylated by protein kinase C (PKC). When expressed in yeast, the ability of the mutant proteins to confer drug resistance, or to mediate [3H]vinblastine accumulation in secretory vesicles, was indistinguishable from that of wild type P-gp. A matched pair of mammalian cell lines were generated expressing wild type P-gp and a non-phosphorylatable mutant protein. Mutation of the phosphorylation sites did not alter P-gp expression or its subcellular localization. The transport properties of the mutant and wild type proteins were indistinguishable. Thus, phosphorylation of the linker of P-gp by PKC does not affect the rate of drug transport. In light of these data, the use of agents that alter PKC activity to reverse multidrug resistance in the clinic should be considered with caution.

Albumin stimulates uptake of calcium into subcellular stores in rat cortical astrocytes.

1. When albumin from either plasma or serum is applied at low concentrations to cortical astrocytes a decrease in the level of [Ca2+]i is observed. At higher concentrations trains of calcium spikes are seen. 2. Removal of the polar lipids which are normally bound to native albumin abolishes the ability to induce spikes, but the decrease in [Ca2+]i is unaffected. The decrease is abolished by the denaturation of albumin and is not reproduced by a number of other proteins, and is therefore a specific action of albumin. We conclude that native albumin has a dual agonist action: the decrease in [Ca2+]i is induced by the albumin protein molecule, while the spikes are induced by a lipid normally bound to it. 3. The decrease is rapid (fastest tau = 12 s) and the rate is dependent on the concentration of albumin. [Ca2+]i falls from 77 nM to around 34 nM in the presence of saturating levels of albumin, and this level appears to be maintained indefinitely. 4. The decrease is due to an uptake of calcium into subcellular stores, as it is not abolished by removal of external Ca2+ or Na+ but is abolished by thapsigargin and cyclopiazonic acid, which are specific inhibitors of the endoplasmic reticulum Ca(2+)-ATPase. 5. When the state of store filling after albumin application is probed with a pulse of glutamate it can be seen that stores fill with the same time course as the decrease in [Ca2+]i. The low level of [Ca2+]i in albumin must therefore be maintained by a suppression of calcium influx rather than by a continued uptake into stores. 6. The calcium uptake potentiates the efficacy of low concentrations of calcium-releasing agonists such as glutamate and bradykinin by almost an order of magnitude. 7. A possible function for the calcium uptake caused by albumin is to potentiate the production of calcium spike trains by promoting refilling of calcium stores in the intervals between spikes. The uptake may play a role in the response of astrocytes to damage in the CNS.

Rods, cones and calcium.

A transduction cascade in the outer segments of vertebrate photoreceptors amplifies the visual signal, resulting in the metabolism of cGMP and the closure of ionic channels. The intracellular calcium concentration declines after a light response, and this decline is the key regulator responsible for controlling the gain of the transduction cascade. Calcium turnover in the outer segment is determined by three processes: influx through light-sensitive channels; buffering within the outer segment; and extrusion by a Na/Ca,K exchange mechanism.

Plasma albumin is a potent trigger of calcium signals and DNA synthesis in astrocytes.

Cells in the central nervous system are normally prevented from coming into contact with albumin and other protein components of blood by the existence of a tight blood-brain barrier. Astrocytes and other glial cells proliferate to form glial scars when the blood-brain barrier is breached. In this report we show that albumin is an important blood component responsible for inducing astrocyte proliferation. Albumin also generates maintained trains of calcium spikes in astrocytes. Neither activity depends on blood coagulation, as albumins from both serum and plasma are approximately equally effective. Methanol extraction of albumin abolishes both actions, and recombination of the methanol-extracted factor with extracted albumin restores full activity indistinguishable from that of native albumin. The factor is sensitive to lipase, and the solvent extraction profile is that of a polar lipid.

Drug efflux mediated by the human multidrug resistance P-glycoprotein is inhibited by cell swelling.

P-glycoprotein (P-gp), the product of the human multidrug resistance (MDR1) gene, confers multidrug resistance on cells by acting as an ATP-dependent drug transporter. A method using confocal microscopy was developed to measure the transport activity of P-gp from the rate of movement of doxorubicin, a fluorescent substrate of P-gp, across the membrane of a single cell. Recent work has shown that expression of P-gp enhances the activation of chloride channels in response to cell swelling, suggesting that membrane stretch might switch P-gp from a drug-transporting mode to a mode in which it activates chloride channels. In agreement with this idea, we find that cell swelling inhibits drug efflux in cells expressing P-gp but is without effect on the slower background efflux in cells not expressing P-gp and in cells transiently transfected with a mutated MDR1 in which the ATP hydrolysis sites had been inactivated. The identification of a novel means for inhibiting P-gp-mediated drug transport may have implications for the reversal of multidrug resistance during chemotherapy.

The mechanism of ion transport by the Na(+)-Ca2+,K+ exchange in rods isolated from the salamander retina.

1. Membrane currents caused by the operation of electrogenic Na(+)-Ca2+,K+ exchange were recorded from isolated rod outer segments under voltage-clamp using a whole-cell electrode. 2. Reversed mode exchange currents (Na+i-Ca2+o,K+o) were recorded with a high internal [Na+] and when both Ca2+ and K+ were present in the external solution. Omission of either Ca2+ or K+ completely suppressed both the reversed exchange current and the entry of Ca2+. 3. The charge transferred by the exchange per Ca2+ ion transported was identical in both forward and reversed modes. 4. The reversed exchange current declined as Ca2+ accumulated inside the outer segment, and the form of this decline was consistent with a first-order inhibition by internal Ca2+. 5. The reversed exchange current was increased e-fold by a 230 mV depolarization over the range -51 to +29 mV. 6. The activation of reversed exchange by external Ca2+ was well described by first-order kinetics with a Michaelis constant, KappCao, of 34 microM in the presence of 20 mM external K+. KappCao was reduced by lowering external [K+], was increased by adding external Na+ and was unaffected by membrane potential. 7. External K+ also activated the exchange in a first-order manner with a Michaelis constant, KappKo, of 151 microM in the presence of 0.5 mM external Ca2+. KappKo was reduced by lowering external [Ca2+], increased by adding external Na+ and was unaffected by membrane potential. 8. When the level of internal Ca2+ was increased via reversed exchange, KappCao diminished in proportion to the reduction in the maximum current, but KappKo remained approximately constant. 9. These observations cannot be reconciled with simple models of the exchange in which ions bind simultaneously at opposite faces of the membrane before transport occurs. The results are broadly consistent with a consecutive model of the exchange in which unbinding of Na+ at either the external or the internal membrane surface is followed by binding of Ca2+ and then K+, and are fully reproduced by a model in which Ca2+ binds before all of the Na+ has dissociated from the exchange molecule.

Temperature dependence of the light response in rat rods.

1. The effects of temperature on the light responses of rat rods have been investigated over the range 17-40 degrees C. 2. The amplitude of the light-sensitive current increased with temperature with a mean temperature coefficient (Q10) of 2.47. 3. The amplitude of the Na(+)-Ca2+, K+ exchange current decreased with temperature when expressed as a fraction of the light-sensitive current, showing that the light-sensitive channel becomes less permeable to calcium as the temperature is raised. The time constant of relaxation of the exchange current was little affected by temperature. 4. The flash intensity required to give a half-saturating response increased with temperature with a mean Q10 of 1.68. 5. The responses to single photoisomerizations were determined from amplitude histograms of the responses to dim-flash trains. The amplitude of the response to a single photoisomerization decreased with temperature when expressed as a fraction of the light-sensitive current, but the change was not sufficient to account for the overall decrease in sensitivity. 6. The fraction of dim flashes that produced a photoisomerization decreased with temperature. This decrease in photon capture efficiency together with the decrease in the relative size of the single photon event fully accounts for the observed change in sensitivity. 7. The speed of the falling phase of the dim-flash response was accelerated more by warming than the rising phase, and it was therefore not possible to superimpose light responses at different temperatures by a simple change in time scale.

Calcium homeostasis in the outer segments of retinal rods from the tiger salamander.

1. The processes regulating intracellular calcium in the outer segments of salamander rods have been investigated. The main preparation used was the isolated rod loaded with the Ca(2+)-sensitive photoprotein aequorin, from which outer segment membrane current and free [Ca2+]i could be recorded simultaneously. Two other preparations were also used: outer segment membrane current was recorded from intact, isolated rods using a suction pipette, and from detached outer segments using a whole-cell pipette. 2. Measurements of free intracellular [Ca2+] in Ringer solution were obtained from two aequorin-loaded rods. Mean [Ca2+]i in darkness was 0.41 microM, and after a bright flash [Ca2+]i fell to below detectable levels ( < 0.3 microM). No release of intracellular Ca2+ by a bright flash of light could be detected ( < 0.2 microM). 3. Application of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) caused an increase in the size of the light-sensitive current and a rise in [Ca2+]i, but application of IBMX either when the light-sensitive channels had been closed by a bright light or in the absence of external Ca2+ caused no detectable rise in [Ca2+]i. It is concluded that IBMX increases [Ca2+]i by opening light-sensitive channels, and does not release Ca2+ from stores within the outer segment. 4. Removal of external Na+ caused a rise in [Ca2+]i to around 2 microM and completely suppressed the light-sensitive current. 5. The Na(+)-Ca2+, K+ exchange current in aequorin-loaded rods was activated in first-order manner by internal free calcium, with a mean Michaelis constant, KCa, of 1.6 microM. 6. The KCa of the Na(+)-Ca2+, K+ exchange was increased by elevating internal [Na+]. 7. The Michaelis relation between [Ca2+]i and the activity of the Na(+)-Ca2+, K+ exchange was used to calculate the change in [Ca2+]i occurring during the response to a bright light. In aequorin-loaded rods in Ringer solution the mean change in free [Ca2+]i after a bright flash was 0.34 microM. In these rods 10% of the dark current was carried by Ca2+. 8. Most of the calcium entering the outer segment was taken up rapidly and reversibly by buffer systems. The time constant of equilibration between free and rapidly bound Ca2+ was less than 20 ms. No slow component of calcium uptake was detected. 9. Two components of calcium buffering could be distinguished in the outer segments of aequorin-loaded rods.(ABSTRACT TRUNCATED AT 400 WORDS)

Net charge transport during sodium-dependent calcium extrusion in isolated salamander rod outer segments.

The light-sensitive current and the current associated with the extrusion of internal Ca2+ in exchange for external Na+ have been recorded from detached rod outer segments from the salamander retina by the use of the whole-cell voltage clamp technique. No significant current-carrying mechanisms are present in the outer segment membrane apart from the light-sensitive conductance and the Na:Ca,K exchange, and exchange currents can therefore be recorded directly without the use of subtraction procedures or pharmacological blockers. The charge moved by the exchange was studied by loading outer segments with a known amount of calcium and then recording the exchange current on return to a Na(+)-containing solution. Calcium is not sequestered to any significant extent in a slowly exchanging internal store, as the charge recovered is unaffected if admission of the Na(+)-containing solution is delayed for 40 s. The number of charges flowing into the cell in exchange for each Ca2+ ion extruded was found not to deviate significantly from one over a wide range of ionic conditions and membrane potentials. These results show that the stoichiometry of the exchange is fixed over a wide range of conditions, and that the size of the inward exchange current is therefore directly proportional to the rate of Ca2+ efflux through the carrier.