E. P. Kostyuk

Disruption of Calcium Homeostasis in Alzheimer’s Disease

Alzheimer’s disease is accompanied by a number of pathological modifications, from those at the subcellular level to the impairment of cognitive cerebral functions. Abnormal accumulation of a specific protein, β-amyloid, in brain neurons plays a central role in the pathogenesis of this neurodegenerative disease. In this case, one significant pathogenetic factor is disruption of calcium homeostasis in cerebral cells. In this review, we describe changes in the intracellular calcium signalling related to Alzheimer disease, namely disorders of the functioning of main intracellular calcium stores (mitochondria and endoplasmic reticulum), as well as of those of calcium channels of the plasma membrane.

Effects of Gabapentin on Calcium Transients in Neurons of the Rat Dorsal Root Ganglia

In our experiments on rat dorsal root ganglia (DRG) neurons, we studied the effects of an antiepileptic agent, gabapentin, on calcium transients evoked by depolarization of the membrane using the fluorescence calciumsensitive dye Fura-2/AM. Application of gabapentin to neurons with large-diameter somata practically did not change the characteristics of calcium transients. In mid-sized neurons, the amplitude of transients decreased, on average, by 27% with respect to the control, while in small-sized neurons the transients changed insignificantly (on average, less than by 7%). The mid-sized neurons were additionally subjected to the capsaicin test, which allowed us to differentiate primary nociceptive neurons of this group where TRPV1-type channels are expressed. In capsaicin-sensitive neurons, application of gabapentin led to a decrease in the amplitude of calcium transients, on average, by 37%, while such a decrease was only 16% in capsaicininsensitive neurons. Based on our own data and findings of other researchers on the ability of gabapentin to demonstrate affine binding with the accessory α2δ subunit of voltage-dependent calcium channels and also on the peculiarities of expression of these channels in somatosensory neurons of the corresponding types, we discuss the probable pattern of expression of subunits of the α2δ-1 subtype in DRG cells of different sizes. We demonstrated that the effects of gabapentin on calcium transients in nociceptive and hypothetically nonnociceptive mid-sized DRG neurons are selective (the effects in neurons involved in the sensation of acute pain are probably more intense).

β-amyloid-induced changes in calcium homeostasis in cultured hippocampal neurons of the rat

A two-wave technique of calciometry with the use of a fluorescence dye, fura-2/AM, was applied for examination of the effect of a protein, β-amyloid (the main component of senile plaques in Alzheimer’s disease), on calcium homeostasis in cultured neurons of the rat hippocampus; β-amyloid was added to the culture medium. In most neurons, the effect of β-amyloid appeared as a more than twofold increase in the basic calcium concentration, as compared with the control (153.4 ± 11.5 and 71.7 ± 5.4 nM, respectively; P < 0.05). The characteristics of calcium transients induced by application of hyperpotassium solution also changed; the amplitude of these transients decreased, and the duration of a part corresponding to calcium release from the cell (rundown of the transient) increased. The mean amplitude of calcium transients under control conditions was 447.5 ± 20.1 nM, while after incubation in the presence of β-amyloid this index dropped to 278.4 ± 22.6 nM. Under control conditions, the decline phase of calcium transients lasted, on average, 100 ± 6 sec, while after incubation of hippocampal cell cultures in the presence of β-amyloid this phase lasted 250 ± 10 sec. Therefore, an excess of β-amyloid influences significantly calcium homeostasis in the nerve cells by disturbing functions of the calcium-controlling systems, such as voltage-operated calcium channels of the plasma membrane and calcium stores of the mitochondria and endoplasmic reticulum.

Peculiarities of Ion Channels and Modulation of Their Functions in Neurons Belonging to the Nociceptive System

This review deals with the questions related to the composition of ion channels in the membranes of neurons belonging to the nociceptive system; special attention is focused on channels belonging to the TRP family.The factors (in particular, genetically determined) influencing the activity of these channels are discussed. The roles of certain enzymes (protein kinases, phospholipases, etc.) in modulation of the functioning of channel structures typical of nociceptive neurons are reviewed. The roles of calcium transmembrane currents and the state of cellular calcium-controlling compartments in transmission of nociceptive signals are also discussed. Special attention is paid to long-lasting modulatory changes in the activity of different ion channels responsible for the development of stable shifts of sensitive abilities of the nociceptive system (hyperalgesia, hypoalgesia, and allodynia) typical of certain neurological disorders.

Transmission of nociceptive signalling and mechanisms underlying its modulation

Specific features of calcium signalling in neurons of the nociceptive system, in particular in primary afferent (dorsal root ganglion) and secondary dorsal horn spinal units, are described. The roles of different types of calcium channels and intracellular Ca stores (those of the mitochondria and endoplasmic reticulum) and interactions between these cellular structures in the norm and under pathological conditions (in particular in diabetic neuropathy) are discussed.

Characteristics of store-operated calcium channels activated by depletion of the endoplasmic reticulum ryanodine-sensitive calcium stores in rat dorsal root ganglion neurons

In neurons of the rat dorsal root ganglia (DRG), using a patch-clamp technique in the whole-cell configuration, we studied the characteristics of calcium channels activated by depletion of the ryanodine-sensitive calcium stores of the endoplasmic reticulum. Current-voltage (I-V) relationships of these store-operated calcium channels were obtained by subtraction of the integral I-V characteristics after application of caffeine from the integral I-V characteristics of calcium channels in the control. Currents through store-operated calcium channels could be induced by application of a series of hyperpolarization current pulses to the cell under conditions of replacement of a calcium-free solution containing caffeine by a caffeine-free solution containing 2 mM Ca2+. In this case, the following two main conditions were abserved: Voltage-operated calcium channels were inactivated, while a gradient of the electrochemical potential for calcium ions was increased, which made easier passing of these currents through store-operated calcium channels. Therefore, we found that in DRG neurons, despite the presence of great numbers of both voltage-operated and receptor-dependent calcium channels, one more mechanism underlying the entry of calcium through store-operated channels does exist.

Effects of Gabapentin on Different Neuronal Populations in the Dorsal-Root Ganglia of Rats with Streptozotocin-Induced Diabetes Mellitus

We studied the effect of gabapentin (an agent similar, in its molecular structure, to gamma-aminobutyric acid, GABA) on depolarization-evoked calcium transients in small, mid-sized, and large (diameter of the soma up to 25, 25 to 35, and 35 μm or more, respectively) neurons of the dorsal-root ganglia (DRGs) of rats with experimental streptozotocin-induced diabetes mellitus. These transients were measured using a calcium-sensitive fluorescent dye, Fura 2/AM. The amplitude of calcium transients in rats with diabetes was somewhat higher than that in healthy animals (in large and mid DRG neurons by nearly 12% and in small cells by about 8%, on average). The development of diabetes led to a dramatic increase in the total duration of such transients. In large, mid, and small DRG neurons, the values of this parameter in animals with diabetes were, respectively, about 260, 430, and 250% as compared with the norm. The duration of transients at the level of 50% amplitude (Т0.5) in diabetes changed to a significantly smaller extent. Applications of gabapentin (25 μM) led to a decrease in the amplitude of calcium transients, their full duration, and Т0.5. The effects of gabapentin were the strongest in large DRG neurons where the amplitude of calcium transients dropped by nearly 36%, while the total duration demonstrated a more than threefold decrease. Upon the action of gabapentin, the parameter Т0.5 changed moderately (in all groups of DRG neurons, the decrease varied from 8 to 12%). Gabapentin-induced decreases in the amplitude of calcium transients differed in various subgroups of DRG neurons. Among neurons with mid-sized somata, the decrease in this parameter in capsaicin-positive cells was 16.3%, while that in capsaicin-negative cells reached 36.7%. The obtained data are indicative of the ability of gabapentin to normalize, to a certain extent, the parameters of diabetes-modified calcium transients in DRG neurons. This ability is more clearly pronounced in large neurons (we hypothesize that a part of such cells in animals with diabetes are, probably, abnormally involved in transmission of nociceptive influences) and also in a part of mid-sized DRG neurons participating in the formation of acute pain sensation.

Activity of TRPV1 Channels in Primary Nociceptive Neurons of Rats: Modulation by Changes in Intracellular Calcium Level

In rat neurons of the dorsal root ganglia (DRG) with mid- (35 to 25 μm) and small-sized (less than 25 μm) somata, we studied calcium transients induced by application of capsaicin (selective agonist of TRPV1 channels) under conditions of the development of other calcium transients caused by preliminary depolarization of the plasma membrane of these neurons. The above transients in rat DRG neurons were measured using the calcium-sensitive fluorescent dye Fura 2/AM. At delays of 3, 7, and 10 sec with respect to the beginning of preliminary potassium depolarization, the amplitudes of capsaicin-induced responses were smaller, as compared with the control, on average, by 26.8, 22.1, and 4.5%, respectively, in the population of mid-sized neurons and by 35.3, 21.1, and 22.4% in small neurons. Under such conditions, we observed noticeable delays of reactions to applications of capsaicin and a certain decrease in the level of intracellular calcium at the moment of beginning of development of these reactions with respect to the corresponding values in isolated depolarization-induced transients. We conclude that excitation of primary nociceptive neurons and activation of voltage-operated calcium channels result in noticeable modulation of the activity of TRPV1 channels and change their role during pain reception.

Potassium permeability of voltage-operated calcium channels of dorsal root ganglion neurons in a calcium-free medium

In freshly isolated neurons of the rat spinal ganglia, we studied the behavior of voltage-operated calcium channels of these cells under conditions of the absence of calcium ions in the extracellular solution; a patch-clamp technique in the whole-cell configuration was used. We found that such channels in a part of the studied neurons lose their selectivity in a calcium-free potassium-containing solution and become capable of passing an inward potassium current. This current was inhibited by blockers of voltage-operated calcium channels, nifedipine and nickel, and also was to some extent inhibited by caffeine. The latter effect is realized, perhaps, due to calcium-dependent inactivation of calcium channels induced by the action of calcium ions released from the endoplasmic reticulum upon caffeine-induced activation of ryanodine receptors. The peculiarities of current-voltage relationships and characteristics of activation/inactivation of calcium channels modified in calcium-free medium and the possible mechanisms of such modification are discussed.

Intracellular mechanisms participating in the formation of neuronal calcium signals

Ion and metabolic processes in the endoplasmic reticulum, mitochondria, plasma membrane, etc. providing calcium signaling in the cells of excitable and nonexcitable tissues are discussed.