Feedback mechanisms in the regulation of intracellular calcium ([Ca2+]i) in the peripheral nociceptive system: role of TRPV-1 and pain related receptors.

Multimodal stimuli like heat, cold, bacterial or mechanical events are able to elicit pain, which is necessary to guarantee survival. However, the control of pain is of major clinical importance. The perception and transduction of pain is differentially modulated in the peripheral and central nervous system (CNS): while peripheral structures modulate these signals, the perception of pain occurs in the CNS. In recent years major advances have been made in the understanding of the processes which are involved in pain sensation. For the peripheral pain reception, the importance of specific pain receptors of the transition receptor pore (TRP)-family (e.g. the TRPV-1 receptor) has been analyzed. These receptors/channels are localized at the cell membrane of nociceptive neurones as well as in membranes of intracellular calcium stores like the endoplasmic reticulum. While the associated channel conducts different ions, a major proportion is calcium. Therefore, this review focuses on (1) the modulations of intracellular calcium ([Ca2+]i) initiated by the activation of pain receptors and (2) the consequences of [Ca2+]i changes for the processing of pain signals at the peripheral side. The possible interference of TRPV-1 induced [Ca2+]i modulations to the function of other membrane receptors and channels, like voltage gated calcium, sodium or potassium channels, or co-expressed CB1-receptors will be discussed. The latter interactions are of specific interest since the analgetic properties of endo- and exo-cannabinoids are mediated by CB1 receptors and their activation significantly modulates the calcium induced release of pain related transmitters. Furthermore, multiple cross links between different pain modulating intracellular pathways and their dependence on [Ca2+]i modulations will be illuminated. Overall, this review will summarize new insights resulting in the understanding of the prominent influence of [Ca2+]i for processes which are involved in pain sensation.

IP(3) receptor antagonist, 2-APB, attenuates cisplatin induced Ca2+-influx in HeLa-S3 cells and prevents activation of calpain and induction of apoptosis.

BACKGROUND AND PURPOSE: Cisplatin drives specific types of tumour cells to apoptosis. In this study we investigate the involvement of intracellular calcium ([Ca(2+)](i)) in triggering apoptosis in two different cell lines. As cisplatin is used for the treatment of several forms of cancer we choose HeLa-S3 and U2-OS as two examples of tumour cell lines. EXPERIMENTAL APPROACH: Cisplatin (1 nM-10 microM) was applied to HeLa-S3 and U2-OS cells and [Ca(2+)](i) measured with fluo-4, using laser scanning microscopy. Inositol-1,4,5-trisphosphate (IP(3)) receptors were visualized with immunostaining. Membrane conductances were measured with patch-clamp techniques. Levels of calpain and caspases were assessed by western blots and apoptotic cells were stained with Hoechst 33342 and counted. KEY RESULTS: Cisplatin increases [Ca(2+)](i) concentration-dependently in HeLa-S3 but not in U2-OS cells. This elevation of [Ca(2+)](i) depended on extracellular Ca(2+) but was reduced by the IP(3) receptor blocker, 2-APB. This effect was not due to a Ca(2+) release triggered by Ca(2+) entry. Immunostaining showed IP(3)-receptors (type 1-3) at the cellular membrane of HeLa-S3 cells, but not in U2-OS cells. Electrophysiological experiments showed an increased membrane conductance with cisplatin only when Ca(2+) was present extracellularly. Increase of [Ca(2+)](i) was related to the activation of calpain but not caspase-8 and triggered apoptosis in HeLa-S3 but not in U2-OS cells. CONCLUSIONS AND IMPLICATIONS: Our observations on the activation of IP(3)-receptors, calcium entry and apoptotic rate by cisplatin in specific carcinogenic cells might open new possibilities in the treatment of some forms of cancer.

A combined blockade of glycine and calcium-dependent potassium channels abolishes the respiratory rhythm.

In order to test whether glycinergic inhibition is essential for the in vivo respiratory rhythm, we analysed the discharge properties of neurones in the medullary respiratory network after blockade of glycine receptors in the in situ perfused brainstem preparation of mature wild type and oscillator mice with a deficient glycine receptor. In wild type mice, selective blockade of glycine receptors with low concentrations of strychnine (0.03-0.3 microM) provoked considerable changes in neuronal discharge characteristics: The cycle phase relationship of inspiratory, post-inspiratory and expiratory specific patterns of membrane potential changes was altered profoundly. Inspiratory, post-inspiratory and expiratory neurones developed a propensity for fast voltage oscillations that were accompanied by multiple burst discharges. These burst discharges were followed by "after-burst" hyperpolarisations that were capable of triggering secondary burst discharges. Blockade of glycine receptors and the "big" Ca2+-dependent K+-conductance by charybdotoxin (3.3 nM) resulted in loss of the respiratory rhythm, whilst only tonic discharge activity remained. In contrast, rhythmic activity was only weakened, but preserved after the "small" Ca2+-dependent activated K+ conductance was blocked with apamin (8 nM). Also low concentrations of pentobarbital sodium (6 mg/kg) abolished rhythmic respiratory activity after blockade of glycine receptors in the wild type mice and in glycine receptor deficient oscillator mice. The data imply that failure of glycine receptors provokes enhanced bursting behaviour of respiratory neurones, whilst the additional blockade of BKCa channels by charybdotoxin or with pentobarbital abolishes the respiratory rhythm.

Extracellular pH modulates aluminium-blockade of mammalian voltage-activated calcium channel currents.

The pH-dependence of aluminium (Al) blockade of voltage activated calcium channels (VACCs) was investigated. Using cultures of rat dorsal root ganglion (DRG) neurones, whole-cell patch-clamp experiments were performed. Various concentrations of Al were extracellularly applied within solutions of different pH-values. The block of VACC currents was highly pH-dependent. At pH 7.3-7.8, the concentration-response curve shifted slightly to higher concentrations, whereas at pH 6.4-6.9 a pronounced shift to lower concentrations was observed. This effect could be due to changes of the chemical equilibria of the different Al species or to altered properties of the VACCs. Thus, pH-shifts may influence the interactions of Al with VACCs making them more susceptible to the effects of Al and therefore contribute to its toxicity.

Aluminium reduces glutamate-activated currents of rat hippocampal neurones.

The actions of aluminum on glutamate-activated currents of acutely isolated hippocampal neurones were investigated. N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) and glutamate mediated currents were reduced by 50% in the presence of 1.4 micrograms ml-1 aluminium. Higher concentrations (> or = 2.7 micrograms ml-1) inhibited all currents completely and irreversibly. Additionally, successive application of agonists in the presence of 2.7 micrograms ml-1 aluminium resulted in non-specific membrane currents followed by the loss of the seal resistance. Application of aluminium per se had no influence on resting membrane current or voltage-activated sodium currents. The estimation of the concentration-response relationship of the action of aluminium on NMDA-activated currents revealed a threshold concentration < 0.27 micrograms ml-1. Our data indicate that glutamate receptors are putative sites of action in aluminium neurotoxicity.

Mercury (Hg2+) decreases voltage-gated calcium channel currents in rat DRG and Aplysia neurons.

Inorganic mercury (Hg2+) reduced voltage-gated calcium channel currents irreversibly in two different preparations. In cultured rat dorsal root ganglion (DRG) neurons, studied with the whole cell patch clamp technique, a rapid concentration-dependent decrease in the L/N-type currents to a steady state was observed with an IC50 of 1.1 microM and a Hill coefficient of 1.3. T-currents were blocked with Hg2+ in the same concentration range (0.5-2 microM). With increasing Hg2+ concentrations a slow membrane current was additionally activated, most obviously at concentrations over 2 microM Hg2+. This current was irreversible and might be due to the opening of other (non-specific) ion channels by Hg2+. The current-voltage (I-V) relation of DRG neurons shifted to more positive values, suggesting a binding of Hg2+ to the channel protein and/or modifying its gating properties. In neurons of the abdominal ganglion of Aplysia californica, studied with the two electrode voltage clamp technique, a continuous decrease of calcium channel currents was seen even with the lowest used concentration of Hg2+ (5 microM). A steady state was not reached and the effect was irreversible without any change on resting membrane currents, even with high concentrations (up to 50 microM). No shift of the I-V relation of the calcium channel currents was observed. Effects on voltage-activated calcium channel currents with Hg2+ concentrations such low have not been reported before. We conclude that neurotoxic effects of inorganic mercury could be partially due to the irreversible blockade of voltage-activated calcium channels.

Voltage gated calcium channel currents of rat dorsal root ganglion (DRG) cells are blocked by Al3+.

The effects of the trivalent cation aluminum (Al3+) on voltage activated calcium channel currents were examined. Al3+ blocks sustained and transient components of voltage activated calcium channel currents of cultured rat dorsal root ganglion (DRG) cells. Currents were elicited by voltage jumps from -80 to 0 mV. The channel block was use dependent. Steady state blockade occurred after 1 to 5 min, when opening the channel every 10 s. There was little or no recovery after washing. Threshold concentration was about 20 microM Al3+ and total blockade (> 80%) was obtained at 200 microM Al3+; the IC50 was 83 microM and the Hill number was around 3. The degree of blockade was pH dependent, and increased with H+ concentration. The current-voltage relation frequently shifted to depolarised voltages after applying Al3+. The degree of the shift was a function of Al3+ concentration, but the magnitude differed from cell to cell. In the effective concentration range (< 200 microM Al3+) the effect was quite specific to voltage activated calcium channel currents. Voltage activated potassium and sodium channels were reduced less than 15% by 200 microM Al3+. We conclude that Al3+ is a potent and irreversible blocker of voltage activated calcium channel currents in mammalian neurons.

Zinc (Zn2+) blocks voltage gated calcium channels in cultured rat dorsal root ganglion cells.

Dorsal root ganglion cells (DRGs) exhibit 3 types of voltage-dependent calcium channels. We have cultured DRGs from 2- to 4-day-old rat pups and obtained whole-cell patch-clamp recordings of calcium-channel currents after 1-5 days in culture. The calcium-channel currents (carried by barium) were recorded with tetrodotoxin (TTX) in the external solution. A cesium-based solution containing Na-ATP, HEPES and EGTA was used in the recording pipette. Cells were held at -80 mV and calcium channel currents were evoked by stepping to depolarized voltages. The divalent cation zinc (Zn2+) blocked sustained and transient voltage sensitive calcium channel currents. Onset of the blockade was fast and a steady-state was reached within 5-15 min, depending upon the concentration used. The IC50 for inhibition of the peak current evoked by a step depolarization from -80 mV to 0 mV (N plus L channels) for 80 ms was 69 microM Zn2+ and the Hill slope about 1. The calcium current evoked by a voltage step from -80 mV to voltages between -40 mV and -15 mV (T-type current) was more sensitive (> 80% block with 20 microM Zn2+). During wash the effect was only partly reversible in 50% of the neurons. Thus, Zn2+ is a potent blocker of voltage dependent calcium currents in mammalian neurons, especially of T-type currents.

Pb2+ blocks calcium currents of cultured dorsal root ganglion cells.

The divalent cation lead (Pb2+) blocks sustained and transient voltage sensitive calcium channel currents of cultured rat dorsal root ganglion cells. The IC50 for inhibition of the total peak current evoked by a step depolarization from -80 to 0 mV was 0.6 microM, compared to an IC50 of 2.2 microM for Cd2+. The current activated by a depolarization from -40 to 0 mV was inhibited by 50% by 1.0 microM Pb2+. Low threshold currents activated by a step from -100 to -30 mV were blocked by Pb2+ at higher concentrations (IC50 = 6 microM). The block progressed in the absence of channel activation and showed little voltage dependence. Peak sodium current was reduced by 6.6% at 1 microM Pb2+ while at 20 microM the peak current was reduced by 40% with marked slowing of the time course of activation. The potassium rectifier current was reduced by 4.1% at 1 microM Pb2+. Thus, Pb2+ selectively blocks calcium currents at concentrations in the range of those causing toxicity in man.

Coexpression of heat-evoked and capsaicin-evoked inward currents in acutely dissociated rat dorsal root ganglion neurons.

Noxious heat is able to activate heat-sensitive nociceptors in the skin very rapidly, but little is known about the mechanisms by which heat is transduced. We used the whole-cell patch-clamp technique to study the effects of noxious heat and capsaicin on freshly dissociated rat dorsal root ganglion neurons in vitro. Using temperatures between 41 degrees C and 53 degrees C, 8 of 19 small neurons (phi < or = 30 microm) exhibited a heat-evoked inward current. All heat-sensitive neurons tested were also capsaicin-sensitive. Moreover, the heat response tended to be enhanced after capsaicin (360 +/- 150 pA versus 125 +/- 45 pA, P < 0.1, n = 7). Two of five heat-insensitive neurons were excited by capsaicin; both neurons developed a heat response after capsaicin. Large neurons (phi > 30 microm) did not respond to heat (0/7), and were not sensitive to capsaicin either. These findings indicate that heat stimuli may directly activate capsaicin-sensitive primary nociceptive afferents.

Zn2+ blocks the voltage activated calcium current of Aplysia neurons.

We have investigated the effect of Zn2+ on voltage-activated calcium currents of Aplysia neurons, using conventional two-electrode voltage-clamp techniques. The peak of these currents was reversibly reduced by Zn2+ (50% reduction at 3.75 mM; total block at 20 mM), while the current-voltage relation and the activation and inactivation curves were shifted to depolarized voltages. The effects of Zn2+ were concentration-dependent. The Hill coefficient was 1.62. The high concentrations required, the shift of the current-voltage relation and the effects on activation and inactivation are best explained by a charge-screening effect combined with a specific binding site for Zn2+ near the entrance of the channel.

The respiratory rhythm in mutant oscillator mice.

Since glycinergic inhibition is important for respiratory rhythm generation in mature mammals, we tested the hypothesis that the loss of glycine receptors during postnatal development (P17-P23) of homozygous mutant oscillator mice (spd(ot)/spd(ot)) may result in serious impairment of respiratory rhythm. We measured breathing in a plethysmographic recording chamber on conscious oscillator mice and used an in situ perfused brainstem preparation to record phrenic nerve activity, as well as membrane properties of respiratory neurones. The deletion of glycinergic inhibition did not result in failure of respiratory rhythm: homozygous mutant oscillator mice continue to generate a disturbed respiratory rhythm until death. Postsynaptic activity and membrane potential trajectories of respiratory neurones revealed a persistence of GABAergic inhibition and changes in respiratory rhythm and pattern generation.

Effects of (+/-)-kavain on voltage-activated inward currents of dorsal root ganglion cells from neonatal rats.

Kava pyrones extracted from pepper Piper methysticum are pharmacologically active compounds. Since kava pyrones exhibit anticonvulsive, analgesic and centrally muscle relaxing properties, the influence of a synthetic kava pyrone, (+/-)-kavain, on voltage-dependent ion channel currents was studied. Effects of (+/-)-kavain on voltage-activated inward currents were analysed in cultured dorsal root ganglion cells derived from neonatal rats. Voltage-activated Ca2+ and Na+ currents were elicited in the whole-cell configuration of the patch clamp technique. Extracellularly applied (+/-)-kavain dissolved in hydrous salt solutions reduced voltage-activated Ca2+ and Na+ channel currents within 3-5 min. As the solubility of (+/-)-kavain in hydrous solutions is low, dimethyl sulfoxide (DMSO) was added to the saline as a solvent for the drug in most experiments. When (+/-)-kavain was dissolved in DMSO, the drug induced a fast and pronounced reduction of both Ca2+ and Na+ currents, which partly recovered within 2-5 min even in the presence of the drug. The present study indicates that (+/-)-kavain reduces currents through voltage-activated Na+ and Ca2+ channels.

Methyl mercury reduces voltage-activated currents of rat dorsal root ganglion neurons.

Methyl mercury (MeHg) is a widespread toxicant with major actions on the nervous system. Since the function of neurons depends on voltage gated ion channels, we examined the effects of micromolar concentrations of methyl mercury on voltage-activated calcium, potassium and sodium channel currents of cultured rat dorsal root ganglion (DRG) neurons. The cells, which were obtained from 2-4 day old rat pups, were whole-cell patch-clamped. Currents were separated by selective intra- and extracellular solutions as well as specific depolarizing voltage steps. We did not distinguish between different calcium, potassium or sodium channel subtypes. All three types of voltage-activated currents were irreversibly reduced by MeHg in a concentration dependent manner. Voltage-activated calcium and potassium channel currents were more sensitive to MeHg (Calcium: IC50 = 2.6 +/- 0.4 microM; Potassium: IC50 = 2.2 +/- 0.3 microM) than voltage-activated sodium channels (IC50 = 12.3 +/- 2.0 microM). The Hill coefficients for the reduction of the currents were calculated as approximately 1 for calcium and potassium channel currents and as 1.7 for sodium currents. In the cases of the voltage-activated calcium and sodium channel currents the reduction was clearly use dependent. Higher concentrations of MeHg (> or = 5 microM) resulted in a biphasic change in the holding membrane current at the potential of -80 mV in approximately 25% of the cases.

Pb2+ modulates the NMDA-receptor-channel complex.

The actions of Pb2+ on NMDA channel currents of acutely dissociated hippocampal CA1- and CA3-neurones from adult rats activated by aspartate plus glycine (asp/gly) were examined. A fast reversible and a slow irreversible response to Pb2+ were found. Pb2+ applied simultaneously with asp/gly decreased an inward current. The threshold concentration was below 2 microM, the current was reduced > 90% at concentrations over 100 microM. The decrease of the asp/gly activated current showed no voltage dependence. Opening of NMDA channels was not necessary for Pb(2+)-action, as preincubation in 50 microM Pb(2+)-containing external solution for several seconds dramatically reduced the response to asp/gly/Pb2+. This effect was reversed within 2 to 5 s of wash. Presence of Pb2+ or asp/Pb2+ or glycine/Pb2+ in the external solution did not prevent recovery of the NMDA receptor/channel complex from desensitization. Prolonged perfusion of a cell with the asp/gly/Pb(2+)-containing external solution resulted in an irreversible decrease of the asp/gly current, whereas the amplitude of the asp/gly/Pb2+ response did not change over the duration of an experiment. We conclude that Pb2+ modulates NMDA channel activity via interaction with the NMDA/glycine receptor: as a result the channel current decreases.

Effects of inorganic and triethyl lead and inorganic mercury on the voltage activated calcium channel of Aplysia neurons.

Using conventional two electrode voltage clamp techniques we have studied the effects of Pb2+, triethyl lead (TEL) and Hg2+ on voltage-activated calcium channels of Aplysia neurons and found that all three metals are potent inhibitors at micromolar concentrations. However, the time course of current reduction or block and its reversibility vary when comparing Pb2+ to TEL and Hg2+. With application of Pb2+ the calcium current decreases immediately and a steady state is reached within three to seven minutes, depending upon the concentration of Pb2+ (IC50 = 61 microM). The block was easily reversed upon wash out of Pb2+ with a time course similar to that of onset. Perfusion with either TEL (5 to 50 microM) or Hg2+ (5 to 200 microM) resulted only in a small reduction of current when the substances reached the cell membrane but with clear reduction within 2 min. The decrease continued at about the same speed for the total duration of the application. Upon washing there was no recovery of the response. At the onset of washing the rate of current decline stopped for several minutes, but then the current continued to decline at a slower rate in the absence of toxicant. Our data suggest that Pb2+ acts by a direct and reversible blockade of the calcium channel. In contrast TEL and Hg2+ act slowly and irreversibly to block calcium channels at concentrations which do not greatly affect membrane potential or resistance. In spite of the slow time course these substances are probably acting directly on the calcium channel.

Blockade of mammalian and invertebrate calcium channels by lead.

We have compared the effects of Pb2+ on voltage-dependent calcium channels of the marine mollusc Aplysia, studied with a two electrode voltage clamp, with those on calcium channels in cultured rat dorsal root ganglion (DRG) neurons studied with whole cell patch clamp. In both preparations Pb2+ was a potent blocker of calcium channel currents at concentrations that did not significantly affect potassium and sodium currents. The blockade was concentration dependent and the percentage of blockade was reduced when the concentration of the charge carrier was elevated. In Aplysia the threshold Pb2+ concentration was about 1 microM, and the Hill coefficient near 1.0 under all conditions. Pb2+ did not significantly change inactivation but shifted the voltage dependence of activation to hyperpolarized voltages in a dose-dependent manner. The blockade of calcium currents by Pb2+ was highly voltage dependent and increased with depolarization. Rat dorsal root ganglion cells exhibit three different types of voltage-dependent calcium channels (N, L and T) which can be distinguished by the potential at which the channel activates or inactivates and by their sensitivity to pharmacologic antagonists. The IC50 for blockade of the L current was 1.03 microM with a Hill slope of 1.15. Currents elicited by voltage steps which activate N plus L currents had an IC50 of 0.64 microM and a Hill slope of 1.16. T currents were less sensitive, having an IC50 of 6 microM. Sodium and potassium currents were relatively unaffected in both preparations at concentrations at which the calcium channel was blocked more than 60% (1 microM or 200 microM respectively). The blockade in DRG neurons was less voltage-dependent and reversible than that in the invertebrate model system. These observations indicate that Pb2+ is a potent, reversible and selective blocker of voltage-dependent calcium channels at low concentrations.

Voltage-activated calcium channel currents of rat DRG neurons are reduced by mercuric chloride (HgCl2) and methylmercury (CH3HgCl).

The actions of bath applied mercuric chloride (HgCl2) and methylmercury (CH3HgCl) on voltage-activated calcium channel currents (VACCCs) were tested, using the whole cell patch clamp recording technique with cultured dorsal root ganglion (DRG) neurons from 2-4 day old rat pups. Both metal compounds reduced the current irreversibly in a concentration dependent fashion, reaching a new (lower) steady state within 3 to 5 min after application. Inorganic mercury was more effective in reducing the VACCCs with an IC50 of 1.3 microM, while the IC50 for methylmercury was 2.6 microM. But the threshold concentrations were below 0.25 microM for both metal compounds and the calcium channel currents were reduced by more than 90% with concentrations of 5 microM and 20 microM, respectively. The Hill coefficient for both dose-response relationship was calculated as approximately 1. Calcium channel currents were reduced over the entire voltage range, but the current-voltage relation shifted to more positive potentials in a concentration dependent manner, the effect being more pronounced with HgCl2 than with CH3HgCl (1 microM HgCl2: 10 mV shift, 5 microM CH3HgCl: 5 mV shift). At higher concentrations (> or = 2 microM for HgCl2, and > or = 10 microM for CH3HgCl) an unidentified membrane current was observed. The inorganic mercury caused an inward current, while the organic mercury compound generated a biphasic current with a transient inward and a long lasting outward component. Our results suggest that mercury compounds affect the electrical properties of neurons and thereby decrease cognitive and motor performance.

Calcium channels as target sites of heavy metals.

Zinc (Zn), aluminium (Al), mercury (Hg), methylmercury (MeHg) and lead (Pb) extracellulary applied reduce voltage-activated calcium channel currents (VACCCs); Pb and Al also reduce N-methyl-D-aspartate (NMDA)-activated channel currents (NACCs). Pb is most effective in reducing VACCCs, with an IC50 of 0.46 microM, followed by Hg (IC50 = 1.1 microM) and MeHg (IC50 = 2.6 microM). Zn and Al were less potent (IC50 = 69 and 84 microM, respectively). Al acts on channels in the open state; its effect is pH dependent. The effects of Pb were specific for VACCCs and NACCs. Hg, Al and Zn had only minor effects on voltage-activated potassium and sodium channels, while MeHg reduced potassium channel currents (IC50 = 2.2 microM) and, at higher concentrations, sodium channel currents (IC50 = 12.3 microM). Al also reduced other receptor-activated channel currents. These results demonstrate that a variety of metal species produce different actions at the level of the cell membrane.

Voltage-activated calcium channel currents of rat dorsal root ganglion cells are reduced by trimethyl lead.

Using the conventional whole-cell patch-clamp recording technique with cultured neurones of rat dorsal root ganglions (DRG), we analysed the effects of trimethyl lead (TML) on voltage-activated calcium channel currents. TML reduces voltage-activated calcium channel currents in a dose-dependent manner, with a threshold concentration below 0.5 microM and a total reduction of the current ( > or =80% of the control current) at concentrations above 50 microM. Half of the current is abolished at TML concentrations between 1 and 5 microM. The action is irreversible and is not voltage dependent. After application of TML the current decreases with each activation of the channel until a steady state is reached after 8-12 min, when the channel was activated every 10 s. The channel had to be in the open state for TML to act. TML is a potent compound for reducing voltage activated calcium channel currents. These effects of TML must be taken into account in explaining the neurotoxic effects of this organic metal compound.