David Bleakman

Calcium buffering properties of calbindin D28k and parvalbumin in rat sensory neurones.

1. We have examined the ability of the Ca(2+)‐binding proteins (CABP) calbindin D28k and paravalbumin to modulate increases in the intracellular free Ca2+ concentration ([Ca2+]i), produced by brief depolarizations, in rat dorsal root ganglion (DRG) neurones. 2. In order to obtain good voltage control, we replated DRG neurones prior to performing these experiments. Immunocytochemical staining of these cells revealed that approximately 10% stained for CABPs. 3. Using fluorescently labelled parvalbumin, we demonstrated that in the whole‐cell voltage clamp mode the protein freely entered the cell soma with a mean half‐life t0.5 of 6 min 22 s +/‐ 54 s. 4. Analysis of the effects of calbindin D28k (370 microM) and parvalbumin (1 mM) on Ca2+ currents in the whole‐cell voltage clamp mode, revealed that neither protein changed the rate of inactivation of the Ca2+ current or its rate of run‐down. 5. Introducing either calbindin D28k (370 microM) or parvalbumin (1 mM) into the cell soma did not significantly alter the basal [Ca2+]i when compared to control cells. 6. Compared to control cells, both CABPs significantly reduced the peak [Ca2+]i obtained for a Ca2+ influx of an equivalent charge density, whereas lysozyme (1 mM), a protein with low affinity for Ca2+, failed to do so. 7. Calbindin D28k caused an 8‐fold decrease in the rate of rise in [Ca2+]i and altered the kinetics of decay of [Ca2+]i to a single slow component. Parvalbumin also slowed the rate of rise in [Ca2+]i. Parvalbumin selectively increased a fast component in the decay of the Ca2+ signal. 8. These data demonstrate that both calbindin D28k and paravalbumin effectively buffer Ca2+ in a cellular environment and may therefore regulate Ca(2+)‐dependent aspects of neuronal function.

Effects of neuropeptide Y on the electrical properties of neurons

Neuropeptide Y, one of the scions of the pancreatic polypeptide family, is found throughout the nervous system. Based on its abundance alone, one would expect neuropeptide Y to play an important role in the regulation of neuronal activity, and indeed many pharmacological studies have demonstrated neuromodulatory effects of neuropeptide Y. Here, William F. Colmers and David Bleakman review the known actions of neuropeptide Y on the electrical properties of nerve cells. Neuropeptide Y inhibits Ca2+ currents, and modulates transmitter release in a highly selective manner. Neuropeptide Y might be quite important in the regulation of neuronal state, as exemplified by its actions in the hippocampus and the dorsal raphé nucleus.

Capsaicin-induced neurotoxicity in cultured dorsal root ganglion neurons: Involvement of calcium-activated proteases

We examined the mechanism by which capsaicin produces its toxic effects on cultures of rat sensory neurons. Capsaicin caused a robust increase in [Ca2+]i in a subpopulation of cultured rat dorsal root ganglion neurons. Similarly, a brief exposure to capsaicin resulted in delayed degeneration of a subpopulation of the cells. This subpopulation (about 35% of the cells present) was characterized by a capsaicin-induced uptake of Co2+, which could be detected cytochemically. Both capsaicin-induced Co2+ uptake and capsaicin-induced cell death were blocked by the capsaicin antagonist Ruthenium Red. Cell death was also prevented by removal of external calcium or by inhibiting calcium-activated proteases such as calpain. Evidence that calpain activity was increased was provided by examining the amount of degradation of the preferred calpain substrate alpha-spectrin. Capsaicin treatment produced a significant increase in the levels of the 150,000 molecular weight spectrin breakdown product. Furthermore, applying the protease inhibitors E64 or MDL 28,170 reduced capsaicin-mediated cell death. It is concluded that capsaicin kills a subpopulation of sensory neurons by activating a receptor-operated channel. The consequent Ca2+ ion influx causes large increases in [Ca2+]i and subsequent activation of Ca(2+)-sensitive proteases. This model provides support for the role of [Ca2+]i as the orchestrator of delayed neuronal degeneration.

Tachykinins potentiate N-methyl-D-aspartate responses in acutely isolated neurons from the dorsal horn.

Abstract: Substance P and neurokinin A both potentiated N‐methyl‐d‐aspartate (NMDA)‐induced currents recorded in acutely isolated neurons from the dorsal horn of the rat. To elucidate the mechanism underlying this phenomenon, we measured the effects of tachykinins and glutamate receptor agonists on [Ca2+]i in these cells. Substance P, but not neurokinin A, increased [Ca2+]i in a subpopulation of neurons. The increase in [Ca2+]i was found to be due to Ca2+ influx through voltage‐sensitive Ca2+ channels. Substance P and neurokinin A also potentiated the increase in [Ca2+]i produced by NMDA, but not by α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid, kainate, or 50 mM K+. Phorbol esters enhanced the effects of NMDA and staurosporine inhibited the potentiation of NMDA effects by tachykinins. It is concluded that activation of protein kinase C may mediate the enhancement of NMDA effects by tachykinins in these cells. However, the effects of tachykinins on [Ca2+]i can be dissociated from their effects on NMDA receptors.

The effect of capsaicin on voltage‐gated calcium currents and calcium signals in cultured dorsal root ganglion cells

1 The effects of capsaicin on voltage‐gated Ca2+ currents (ICa), and intracellular Ca2+ concentrations ([Ca2+]i) in cultured dorsal root ganglion (DRG) neurones of the rat were examined in vitro by use of combined patch clamp‐microfluorometric recordings. 2 Under voltage‐clamp conditions, capsaicin (0.1–10 μm) caused a concentration‐dependent decrease in the magnitude of the ICa, an elevation in the holding current (Ih) and a concomitant rise in the [Ca2+]i in most cells examined. Repeated application of capsaicin produced marked desensitization. 3 Some decrease in the ICa produced by capsaicin was also observed when the rise in [Ca2+]i was buffered with EGTA or BAPTA and when Ba2+ was used as the charge carrier; under these conditions the desensitization previously observed was smaller. 4 The decrement in voltage‐gated current was smaller in Ba2+ containing solutions than in Ca2+ containing solutions suggesting that the capsaicin‐induced influx of Ca2+ partially mediated the observed decrease in the voltage‐gated current. In cells which showed a marked response to capsaicin an outward (positive) current was sometimes observed upon depolarization from −80 to 0mV. This effect was consistent with an outward movement of cations through the capsaicin conductance pathway which may also account, in part, for the apparent reduction in ICa by capsaicin. 5 The effects of capsaicin under voltage‐clamp conditions were prevented by ruthenium red (1 μm). 6 Under current clamp conditions, capsaicin depolarized and caused a rise in [Ca2+]i in the majority of DRG cells examined. Both of these effects could be prevented by ruthenium red (500 nm). 7 It is concluded that capsaicin reduces the ICa of rat DRG neurones primarily by indirect mechanisms.

Neuropeptide Y inhibits Ca2+ influx into cultured dorsal root ganglion neurones of the rat via a Y2 receptor

1 The identity of the neuropeptide Y (NPY) receptor associated with the observed inhibition of neuronal Ca2+ currents (ICa) in rat dorsal root ganglion (DRG) cells has been established on the basis of agonist responses to analogues and carboxy terminal (C‐terminal) fragments of the NPY molecule. 2 Whole cell barium currents (IBa) in DRG cells were reversibly inhibited by 100 nm NPY, 100 nm PYY and C‐terminal fragments of NPY in a manner that correlated with the length of the NPY fragments (for inhibition of the IBa NPY = PYY > NPY2–36 > NPY13–36 > NPY16–36 > NPY18–36 ≫ NPY25–36). 3 C‐terminal fragments of NPY were also effective in reversibly reducing the ICa, the associated increase in the intracellular Ca2+ concentration ([Ca2+]i) and the increased [Ca2+]i produced by evoked action potentials in the DRG cells. In addition, a Ca2+‐activated Cl− conductance was also reversibly reduced by NPY fragments only when accompanied by a reduction in Ca2+ entry. 4 We conclude that the Y2 receptor for neuropeptide Y is coupled to inhibition of Ca2+ influx via voltage‐sensitive calcium channels in DRG cells.

Calcium homeostasis in rat septal neurons in tissue culture

Septal neurons from embryonic rats were grown in tissue culture. Microfluorimetric and electrophysiological techniques were used to study Ca2+ homeostasis in these neurons. The estimated basal intracellular free ionized calcium concentration ([Ca2+]i) in the neurons was low (50-100 nM). Depolarization of the neurons with 50 mM K+ resulted in rapid elevation of [Ca2+]i to 500-1,000 nM showing recovery to baseline [Ca2+]i over several minutes. The increases in [Ca2+]i caused by K+ depolarization were completely abolished by the removal of extracellular Ca2+, and were reduced by approximately 80% by the L-type Ca2+ channel blocker, nimodipine (1 microM). [Ca2+]i was also increased by the excitatory amino acid L-glutamate, quisqualate, AMPA and kainate. Responses to AMPA and kainate were blocked by CNQX and DNQX. In the absence of extracellular Mg2+, large fluctuations in [Ca2+]i were observed that were blocked by removal of extracellular Ca2+, by tetrodotoxin (TTX), or by antagonists of N-methyl D-aspartate (NMDA) such as 2-amino 5-phosphonovalerate (APV). In zero Mg2+ and TTX, NMDA caused dose-dependent increases in [Ca2+]i that were blocked by APV. Caffeine (10 mM) caused transient increases in [Ca2+]i in the absence of extracellular Ca2+, which were prevented by thapsigargin, suggesting the existence of caffeine-sensitive ATP-dependent intracellular Ca2+ stores. Thapsigargin (2 microM) had little effect on [Ca2+]i, or on the recovery from K+ depolarization. Removal of extracellular Na+ had little effect on basal [Ca2+]i or on responses to high K+, suggesting that Na+/Ca2+ exchange mechanisms do not play a significant role in the short-term control of [Ca2+]i in septal neurons. The mitochondrial uncoupler, CCCP, caused a slowly developing increase in basal [Ca2+]i; however, [Ca2+]i recovered as normal from high K+ stimulation in the presence of CCCP, which suggests that the mitochondria are not involved in the rapid buffering of moderate increases in [Ca2+]i. In simultaneous electrophysiological and microfluorimetric recordings, the increase in [Ca2+]i associated with action potential activity was measured. The amplitude of the [Ca2+]i increase induced by a train of action potentials increased with the duration of the train, and with the frequency of firing, over a range of frequencies between 5 and 200 Hz. Recovery of [Ca2+]i from the modest Ca2+ loads imposed on the neuron by action potential trains follows a simple exponential decay (tau = 3-5 s).

Investigations into neuropeptide Y‐mediated presynaptic inhibition in cultured hippocampal neurones of the rat

1 We have examined the effects of neuropeptide Y (NPY) on synaptic transmission and [Ca2+]i signals in rat hippocampal neurones grown in culture. [Ca2+]i in individual neurones displayed frequent spontaneous fluctuations often resulting in an elevated plateau [Ca2+]i. These fluctuations were reduced by tetrodotoxin (1 μm) or combinations of the excitatory amino acid antagonists 6‐cyano‐7‐dinitroquinoxaline (CNQX) (10 μm) and aminophosphonovalerate (APV) (50 μm), indicating that they were the result of glutamatergic transmission occurring between hippocampal neurones. 2 [Ca2+]i fluctuations were also prevented by Ni2+ (200 μm), by the GABAB receptor agonist, baclofen (10 μm) and by NPY (100 nm) or Y2 receptor‐selective NPY agonists. Following treatment of cells with pertussis toxin, NPY produced only a brief decrease in [Ca2+]i fluctuations which rapidly recovered. 3 Perfusion of hippocampal neurones with 50 mm K+ produced a large rapid increase in [Ca2+]i. This increase was slightly reduced by NPY or by a combination of CNQX and APV. The effects of CNQX/APV occluded those of NPY. NPY had no effect on Ba2+ currents measured in hippocampal neurones under whole cell voltage‐clamp even in the presence of intracellular GTP‐γ‐S. On the other hand, Ba2+ currents were reduced by both Cd2+ (200 μm) and baclofen (10 μm). 4 Current clamp recordings from hippocampal neurones demonstrated the occurrence of spontaneous e.p.s.ps and action potential firing which were accompanied by increases in [Ca2+]4. This spontaneous activity and the accompanying [Ca2+]i signals were prevented by application of NPY (100 nm). When hippocampal neurones were induced to fire trains of action potentials in the absence of synaptic transmission, these were accompanied by an increase in cell soma [Ca2+]i. NPY (100 nm) had no effect on these cell soma [Ca2+]i signals. NPY (100 nm) also had no effect on inward currents generated in hippocampal neurones by micropipette application of glutamate (50 μm). 5 Thus, NPY is able to abolish excitatory neurotransmission in hippocampal cultures through a pertussis toxin‐sensitive mechanism. However, no effect of NPY on Ca2+ influx into the cell soma of these hippocampal neurones could be discerned. These results are consistent with a localized presynaptic inhibitory effect of NPY on glutamate release in hippocampal neurones in culture.

Characteristics of a human N-type calcium channel expressed in HEK293 cells

The human alpha 1B-1 alpha 2b beta 1-2 Ca2+ channel was stably expressed in HEK293 cells producing a human brain N-type voltage-dependent calcium channel (VDCC). Whole cell voltage-clamp electrophysiology and fura-2 based microfluorimetry have been used to study its characteristics. Calcium currents (ICa) recorded in transfected HEK293 cells were activated at potentials more depolarized than -20 mV with peak currents occurring at approx + 10 mV in 5 mM extracellular CaCl2. ICa and associated rises in intracellular free calcium concentrations ([Ca2+]i) were sensitive to changes in both the [Ca2+]o and holding potential. Steady-state inactivation was half maximal at a holding potential of -60 mV. Ba2+ was a more effective charge carrier than Ca2+ through the alpha 1B-1 alpha 2b beta 1-2 Ca2+ channel and combinations of both Ba2+ and Ca2+ as charge carriers resulted in the anomalous mole fraction effect. Ca2+ influx into transfected HEK293 cells was irreversibly inhibited by omega-conotoxin-GVIA (omega-CgTx-GVIA; 10 nM-1 microM) and omega-conotoxin-MVIIA; 100 nM-1 microM) whereas 1 microM) whereas no reductions were seen with agents which block P or L-type Ca2+ channels. The inorganic ions, gadolinium (Gd3+), cadmium (Cd2+) and nickel (Ni2+) reduced the ICa under voltage-clamp conditions in a concentration-dependent manner. The order of potency of the three ions was Gd3+ > Cd2+ > Ni2+. These experiments suggest that the cloned and expressed alpha 1B-1 alpha 2b beta 1-2 Ca2+ channel subunits form channels in HEK293 cells that exhibit properties consistent with the activity of the native-N-type VDCC previously described in neurons.

ω-Conotoxin MVIIC reversibly inhibits a human N-type calcium channel and calcium influx into chick synaptosomes

We have investigated the effects of omega-CmTX MVIIC on the recombinant alpha 1B-mediated calcium channel expressed in HEK 293 cells and on the predominantly N-type calcium channel in chick synaptosomes. omega-CmTX MVIIC potently and reversibly inhibited the calcium current through alpha 1B-mediated calcium channels and inhibited KCl-evoked increases in [Ca2+]i in chick synaptosomes in a concentration-dependent manner.