I. A. Kruglikov

Role of Ca2+,Mg2+-ATPases in Diabetes-Induced Alterations in Calcium Homeostasis in Input Neurons of the Nociceptive System

In a rat model of streptozotocin (STZ)-induced diabetes, we earlier showed that under these conditions the concentration of free cytosolic Ca2+ in input neurons of the nociceptive system increases, Ca2+ signals are prolonged, while Ca2+ release from intracellular calcium stores decreases. The aim of our study was to test the hypothesis that changes in the activities of Ca2+,Mg2+-ATPases of the endoplasmic reticulum (SERCA) and plasmalemma (PMCA) could be responsible for diabetes-induced disorders of calcium homeostasis in nociceptive neurons. We measured the Ca2+,Mg2+-ATPase activities in microsomal fractions obtained from tissues of the dorsal root ganglia (DRG) and spinal dorsal horn (DH) of control rats and rats with experimentally induced diabetes. The integral specific Ca2+,Mg2+-ATPase activity in microsomes from diabetic rats was lower than that in the control group. The activity of SERCA in samples of DRG and DH of diabetic rats was reduced by 50 ± 8 and 48 ± 12%, respectively, as compared with the control (P < 0.01). At the same time, the activity of PMCA decreased by 63 ± 6% in DRG and by 60 ± 9% in DH samples (P < 0.01). We conclude that diabetic polyneuropathy is associated with the reduction of the rate of recovery of the Ca2+ level in the cytosol of DRG and DH neurons due to down-regulation of the SERCA and PMCA activities.

Metabotropic purinoreceptors in rat dorsal horn neurones: predominant dendritic location.

Elevations of the cytosolic free Ca2+ concentration ([Ca2+]i) in rat dorsal horn neurons induced by addition of ATP to the medium were compared in spinal cord slices and after isolation of the neurons. In slices, application of ATP results in an increase in the [Ca2+]i by 201 ± 12 nM, on the average; in a Ca2+ -free external solution the respective rise was 156 ± 14 nM (n = 45 of 76 examined cells), which indicate the presence of active purinergic metabotropic receptors in about 59% of the neurons. In freshly isolated neurons with absent dendrites, we found no metabotropic responses. Thus, the results confirm the conclusion on the localization of metabotropic postsynaptic purinoreceptors mostly on the dendritic tree of dorsal horn neurons.

Processes Maintaining Calcium Homeostasis in Acinar Cells of the Rat Submandibular Salivary Gland

Using a Ca2+-sensitive fluorescent indicator, fura-2/AM, we recorded calcium transients in secretory cells of isolated acini of the rat submandibular salivary gland; these transients were induced by hyperpotassium-induced depolarization (after an increase in [K+] e up to 50 mM) of the plasma membrane of the above cells. Calcium transients were significantly suppressed by 50 μM nifedipine. Addition of 10 μM carbonyl cyanide m-chlorophenylhydrazone to the normal extracellular solution was accompanied by a rise in [Ca2+]i, whereas when hyperpotassium solution is used the effect was less expressed. Blockers of CA2+-ATPase in the cellular membrane and in the endoplasmic reticulum, eosin Y (5 μM) and cyclopiazonic acid (CPA, 5 μM), respectively, evoked a significant increase in [Ca2+]i and a decrease in the K+-depolarization-induced calcium transient. Extracellular application of caffeine (2, 10, or 30 mM) was accompanied by a concentration-dependent rise in [Ca2+]i. Therefore, potassium depolarization of the plasma membrane of acinar cells of the rat submandibular salivary gland activates both the voltage-dependent Ca2+ influx and Ca2+-induced Ca2+ release from the endoplasmic reticulum; the initial level of [Ca2+]i was restored at the joint involvement of Ca2+-ATPases in the plasma membrane and the membranes of the endoplasmic reticulum and mitochondria.

Caffeine-induced calcium release from the endoplasmic reticulum of acinar cells of the submandibular salivary gland

Ryanodine receptors (RyRs) play a key role in the generalization and spreading of calcium waves in excitable cells; however, the question of the existence of functionally active RyRs in nonexcitable cells demonstrating the capacity for exocytosis (e.g., salivary gland acini) remains open. We studied changes in the total amount of calcium stored in the endoplasmic reticulum (ER) of acinar cells of the submandibular salivary gland of rats and changes in the concentration of ionized Ca2+ inside the ER ([Ca2+]ER) using, respectively, a metallochrome dye, arsenazo III, and a low-affinity fluorescent dye, mag-fura 2/AM. In permeabilized cells, caffeine caused dose-dependent decreases in the total amount of calcium and concentration of ionized calcium. The effective concentration of caffeine providing a 50% drop in the [Ca2+]ER (EC50) was, on average, 7.3 ± 1.1 mM. The caffeine-induced drop in the [Ca2+]ER was insensitive to heparin; in addition, it was blocked by high concentrations (100 µM) of ryanodine, potentiated by ryanodine applied in mild concentrations (10 µM), and also demonstrated a bell-shaped dependence on the concentration of cytoplasmic Ca2+. Such peculiarities are typical characteristics of the RyR-mediated reaction. Therefore, functional RyRs whose activation results in a transient release of calcium from the ER are present in acinar cells of the submandibular salivary gland.

Mechanisms Underlying Leakage of Calcium from the Endoplasmic Reticulum of Acinar Cells of the Submandibular Salivary Gland

In the resting state, the Ca2+ concentration in agonist-sensitive intracellular stores reflects the balance between active uptake of Ca2+, which is mediated by Ca2+-ATPase (SERCA), and passive leakage of Ca2+. The mechanisms underlying such a leakage in cells of the submaxillary salivary gland were not studied. In our experiments, we examined possible pathways of passive leakage of Ca2+ from the endoplasmic reticulum (ER) of acinar cells obtained from the rat submaxillary salivary gland; direct measurements of the concentration of Ca2+ in the ER ([Ca2+]ER) using a low-affinity calcium-sensitive dye, mag-fura 2/AM, were performed. The cellular membrane was permeabilized with the help of β-escin (40 μg/ml); the Ca2+ concentration in the cytoplasm ([Ca2+]i) was clamped at its level typical of the resting state (∼100 nM) using an EGTA/Ca2+ buffer. Incubation of permeabilized acinar cells in a calcium-free intracellular milieu, as well as application of thapsigargin, resulted in complete inhibition of the uptake of Ca2+ with the involvement of SERCA. This effect was observed 1 min after the beginning of superfusion of the cells with the corresponding solutions and was accompanied by the leakage of Ca2+ from the ER; this is confirmed by a gradual drop in the [Ca2+]ER. Such a leakage of Ca2+ remained unchanged in the presence of thapsigargin, heparin, and ruthenium red; therefore, it is not mediated by SERCA, inositol 1,4,5-trisphosphate-sensitive receptors (InsP3R), or ryanodine receptors (RyRs). At the same time, an antibiotic, puromycin (0.1 to 1.0 mM), which disconnects polypeptides from the ER-ribosome translocon complex, caused intensification of passive leakage of Ca2+ from the ER. This effect did not depend on the functioning of SERCA, InsP3R, or RyR. Therefore, passive leakage of Ca2+ from the ER in acinar cells of the submaxillary salivary gland is realized through pores of the translocon complex of the ER membrane.

Changes in the Functioning of Ca2+-ATPases of Rat Exocrine Cells in Experimental Diabetes Mellitus

It is obvious that disruption of functions of the nervous system in diabetes mellitus is to a great extent related to the changes of synthesis or exocytosis of neurotransmitters. Since the mechanisms underlying exocytosis are similar in cells of different types, it may be assumed that studying these mechanisms in secretory cells will allow experimenters to obtain information on ways to control this process in neurons. Based on the supposition that changes in the activity of Ca2+-controlling systems in exocrine cells play an important role in functional disorders in the salivary glands in diabetes mellitus, we demonstrated, using the fura-2/AM dye, that the intracellular calcium concentration ([Ca2+]i) in secretory cells of the above glands in rats with streptozotocin-induced diabetes mellitus (being in the resting state) is significantly increased (on average, by 65%). In our study, we showed that Ca2+-ATPases play an important role in the control of calcium homeostasis in secretory cells of salivary glands in diabetes mellitus. In particular, we demonstrated that the kinetic parameters of microsomal Ca2+-ATPases decreased: V0, by 50 ± 7, and Pmax, by 52 ± 6%, on average. In diabetes mellitus, Vmax of Ca2+-ATPases also dropped significantly, by 47 ± 8 and 79 ± 9%, on average, for PMCA and SERCA, respectively. The decrease in KATP was 71 ± 11% for SERCA and that in KCa was 92 ± 3% for PMCA. We concluded that the activity of Ca2+-ATPases of secretory cells in diabetes mellitus is suppressed because of a decrease in the turnover and/or in the specific number of active molecules of the enzyme.

Imaging of [Ca2+] in the Intracellular Calcium Stores of Permeabilized Cells

1 Franko National University, Lviv, Ukraine. 2 Bogomolets Institute of Physiology, National Academy of Sciences of Ukraine, Kyiv, Ukraine. Correspondence should be addressed to N. Fedirko (e-mail: n_fedirko@franko.lviv.ua). A common feature of both smooth muscle and secretory cells (SMC and SC, respectively) is their ability to produce prolonged Ca-dependent and Caregulated functional responses (like tonic contraction of SMC and spontaneous secretion of SC). Stimulusresponse coupling in these cells considerably depends on the functioning of the intracellular calcium stores, but the mechanisms of such regulation have not been completely interpreted. Thus, studying the intracellular Ca-dependent mechanisms underlying prolonged secretion may be useful for understanding the regulation of smooth muscle contraction. The main pitfalls in biochemical studies of Ca-regulated functional responses and Ca imaging of intracellular stores are the inaccessibility of the secretory apparatus and diffi culties for nonpermeable substrates to access the cytoplasmic space in intact cells. Several techniques have been employed to gain access to the intracellular milieu. In particular, cell permeabilization with mild non-ionic detergents (digitonin and saponin) was proposed. Digitonin can be used to selectively permeabilize the plasma membrane in a wide variety of the cells without signifi cantly affecting the gross structure and function of Ca-sequestering organelles, such as the endoplasmic reticulum (ER) and mitochondria. Chemically controlled permeabilization allows experimenters to access the cell interior and enables them to control intracellular events. Such permeabilized cells open up extensive opportunities for the advancement of our understanding of the mechanisms involved in stimulusresponse coupling. The objective of our study was to elaborate the technique for chemical permeabilization of secretory cells of the salivary gland. We tried to characterize their intracellular calcium stores and the process of Са-dependent protein secretion. Experiments were carried out on isolated cells of the rat submandibular salivary gland. The total intracellular Ca content and protein content were measured using arsenazo III and Lowry techniques, respectively. The fluorescent low-affinity Ca indicator mag-fura 2/AM was used to measure the free Ca concentration in the intracellular stores of permeabilized salivary cells. It was shown that digitonin-permeabilized acinar cells perform Са-dependent protein secretion, whose level reaches its maximum at a concentration of digitonin of 20 μg/ml (10.2 ± 1.3%, n = 7; P 100 μg/ml) and longer exposures (>5 min) caused drops in both secretion and total calcium content. We suppose that the effects of high digitonin concentrations are mediated by destruction of the membranes of intracellular organelles resulting in loss of proteins and cal-

Neuronal Control of Exocytosis and Calcium Homeostasis

We showed that 5 μM acetylcholine (ACh) and 100 μM norepinephrine (NE) cause increases in the total Ca2+ content in acinar cells by 30 and 87% and in the exocytosis intensity by 15 and 20%, respectively. Application of 5 μM ACh and 100 μM NE increased the free cytosolic Ca2+ concentration ([Ca2+]i) by 87 ± 2 and 140 ± 7 nM, respectively. Application of ACh and NE in a Ca2+-free external solution caused a [Ca2+]i increase that was 40 and 67% lower than in physiological solution. We postulate that the exocytosis developing upon neural stimulation of the gland results from generation of Ca2+ transients that are spreading from the basal to the apical region of the exocrine cell, where secretory granules are concentrated.

Calcium Release From the Internal Stores as a Possible Target for Antinociceptive Treatment in Rats with Experimentally-Induced Diabetes

Distal neuropathy is the most common complication of diabetes mellitus, and it is highly important to reveal the cellular mechanisms leading to its development. In our experiments, neurons of control and streptozotocin-treated diabetic rats were examined. Changes in the intracellular free calcium concentrations ([Ca2+]i) were fluorometrically measured in primary and secondary nociceptive (dorsal root ganglion, DRG, and dorsal horn, DH, respectively) neurons. The [Ca2+]i elevation was induced by different agents, which can release calcium from the endoplasmic reticulum (ER) calcium stores. The amplitudes of calcium elevation induced by application of caffeine and ionomicine in DRG and DH neurons of diabetic rats were significantly lower, as compared with the control. Application of ATP and glutamate to a Ca-free extracellular solution induced calcium release from the IP3-sensitive store in DH neurons. Release of calcium from the IP3-sensitive ER calcium stores became significantly smaller in neurons from diabetic rats. Taken together, these data indicate that significant changes in the calcium regulating mechanisms of the ER develop under diabetes conditions.

Participation of Intracellular Ca2+ Stores in Ca2+ Signalling in Neurons of the Thalamic Laterodorsal Nucleus of Rats

Experiments were carried out on isolated neurons of the thalamic nucleus lateralis dorsalis (LD) from 12-day-old rats. According to the morphological characteristics, LD neurons were classified as relay thalamo-cortical units and interneurons. The concentration of free Ca2+ ions in the cytoplasm ([Ca2+]i) was measured by a fluorescent calcium indicator, fura-2AM. Application of 30 mM caffeine caused a transient change in the [Ca2+]i in 8 of 15 and in 6 of 11 of the thalamo-cortical units and interneurons under study, respectively. After stimulation of a cell with application of 50 mM KCl, a caffeine-induced increase in the [Ca2+]i was observed in all tested neurons. To study the contribution of Ca2+-induced Ca2+ release (CICR) to the calcium transient evoked by depolarization of the neuronal membrane, caffeine in a subthreshold concentration was pre-applied. After 50 mM KCl had been added to the medium following pre-application of 0.5 mM caffeine, the calcium transient amplitude in thalamo-cortical neurons increased by 51 ± 7% (n = 16). In interneurons this effect was not observed (n = 11). The data obtained allow us to hypothesize that CICR contributes to the depolarization-evoked calcium transient only in the relay (thalamo-cortical) neurons. Differences in the pattern of calcium signalling, which were detected in two types of neurons of the thalamic LD, can be a factor determining distinctions in the physiological characteristics of these neurons.