Diabetes-induced changes in calcium homeostasis and the effects of calcium channel blockers in rat and mice nociceptive neurons.

Aims/hypothesis: Distal neuropathy is the most common complication of diabetes mellitus, making it important to reveal the cellular mechanisms leading to its development, one of which might be the alteration in intracellular calcium homeostasis in primary and secondary nociceptive neurons. We aimed to investigate these possible changes.

Methods: Control and streptozotocin-treated diabetic rats and mice were used.

Simultaneous measurements of changes in sarcoplasmic reticulum and cytosolic.

1. The role of the sarcoplasmic reticulum (SR) was investigated in spontaneous and agonist-induced uterine Ca2+ transients, by combining low- (mag-fluo-4) and high-affinity (fura-2) indicators to measure intraluminal SR ([Ca2+]L) and cytosolic ([Ca2+]i) calcium concentration, simultaneously, in single smooth muscle cells from pregnant rat uterus. 2.

The endoplasmic reticulum and mitochondria as elements of the mechanism of intracellular signaling in the nerve cell.

Experimental data obtained in our laboratory from studies of intracellular signals arising within nerve cells during excitation are summarized. Measurements of transmembrane ion currents in conditions of fixed membrane potential and intracellular free Ca ion concentrations, using fluorescent probes, yielded the time and spatial characteristics of transient elevations in the Ca concentration (the "calcium signal") in various types of mouse and rat neurons. These studies showed that intracellular structures-the endoplasmic reticulum and mitochondria-had significant roles in forming these signals; these structures can take up Ca from the cytosol and liberate Ca into the cytosol; the contribution of these processes was extremely variable, depending on the internal organization of different functional types of neurons.

The role of the sarcoplasmic reticulum as a Ca2+ sink in rat uterine smooth muscle cells.

1. The mechanisms responsible for removing calcium ions from the cytoplasm were investigated in single rat uterine myocytes using indo-1. 2.

[Endoplasmic reticulum and mitochondria as elements of mechanism of intracellular signaling in neurons].

The temporal and spatial characteristics of a transitory increase in free Ca2+ ("calcium signals") concentration were determined in various types of the mice and rat neurones. Intracellular structures: endoplasmatic reticulum and mitochondria, were shown to play a major part in formation of these signals, the structures being able to absorb the Ca2+ ions from cytosol and release them back. The contribution of these processes proves rather varying depending on internal organisation and functional assignment of a neurone.

Carboxyeosin decreases the rate of decay of the [Ca2+]i transient in uterine smooth muscle cells isolated from pregnant rats.

In myometrial smooth muscle cells the rate of decline of intracellular calcium ([Ca2+]i) is determined by Ca2+ extrusion from the cell and uptake into intracellular stores. The relative quantitative contribution of these processes however, has not been established. We therefore examined the effect of the sarcolemmal Ca2+ pump inhibitor, carboxyeosin, on the rate of the [Ca2+]i transient decline in myocytes isolated from pregnant rat uterus.

Properties of voltage-activated [Ca2+]i transients in single smooth muscle cells isolated from pregnant rat uterus.

1. The intracellular calcium concentration ([Ca2+]i) was measured at 35 degrees C using the fluorescent indicator indo-1 in patch-clamped, single uterine myocytes from pregnant rats to investigate the relationship between depolarization, Ca2+ current (ICa) and [Ca2+]i. 2.

InsP3-induced Ca2+ release in dorsal root ganglion neurones.

The intracellular calcium signalling was studied on subpopulation of freshly isolated adult mouse dorsal root ganglia (DRG) neurones with large somatas (30-45 microns in diameter). The cytoplasmic Ca2+ concentration ([Ca2+]i) was measured using indo-1 based microfluorimetry. The extracellular application of ATP (100 microM) triggered both inward current and [Ca2+]i elevation.

ATP induces Ca2+ release from IP3-sensitive Ca2+ stores exclusively in large DRG neurones.

PURINORECEPTOR-MEDIATED intracellular Ca2+ release was studied in freshly isolated adult mouse dorsal root ganglia (DRG) neurones. The cytoplasmic Ca2+ concentration ([Ca2+]i) was measured using indo-1-based microfluorimetry. The application of 100 microM ATP in Ca(2+)-free solution triggered an increase in [Ca2+]i in 93% of large DRG neurones but in no small ones.

Gradual caffeine-induced Ca2+ release in mouse dorsal root ganglion neurons is controlled by cytoplasmic and luminal Ca2+.

Cytosolic free calcium concentration ([Ca2+]c) was recorded from acutely isolated mouse dorsal root ganglion neurons loaded with Ca(2+)-indicator indo-1. The initiation of intracellular Ca2+ release by low (1-5 mM) caffeine concentrations failed to completely empty the caffeine-sensitive stores; subsequent challenge with higher doses of caffeine produced an additional [Ca2+]c elevation. This indicates a gradual Ca2+ release from caffeine-sensitive stores.

Calcium-induced calcium release in neurones.

Neurones express several subtypes of intracellular Ca2+ channels, which are regulated by cytoplasmic calcium concentration ([Ca2+]c) and provide the pathway for Ca(2+)-induced Ca2+ release (CICR) from endoplasmic reticulum Ca2+ stores. The initial studies of CICR which employed several pharmacological tools (and in particular caffeine and ryanodine) demonstrated that: (i) caffeine induces intracellular calcium release in various peripheral and central neurones; and (ii) inhibition of CICR affects the parameters of depolarization-triggered [Ca2+]c responses. Experiments with caffeine demonstrated also that Ca2+ release from internal pools was incremental, suggesting the coexistence of several subpopulations of Ca2+ release channels with different sensitivity to caffeine.

Calcium-induced calcium release in rat sensory neurons.

1. In isolated dorsal root ganglion cells (DRG neurons), changes in the concentration of global cytosolic Ca2+ (delta [Ca2+]c) were measured by the fluorescence of K5-indo-1. Depolarizations from -60 to 0 mV (500 ms) and Ca2+ influx through Ca2+ channels (ICa) increased [Ca2+]c by 480 +/- 113 nM, the peak occurring 542 +/- 76 ms (mean +/- S.

Changes of pH affect calcium currents but not outward potassium currents in rat myometrial cells.

Spontaneous contraction of uterine smooth muscle is enhanced by alkalinization and depressed by acidification. We have investigated the ionic currents responsible for this in single myometrial cells. Intracellular acidification (20 mM butyrate) at constant external pH depressed the magnitude of the calcium current to 58+/-6% of control, but had little effect on outward currents.

Calcium signal prolongation in sensory neurones of mice with experimental diabetes.

Depolarization-induced Ca2+ transients were studied in dorsal root ganglion neurones of different size (large, 30-45 microns; small, 18-25 microns in diameter) from normal and diabetic mice. Whereas in large neurones no definite changes in the amplitude and time course of the transients were observed, in small neurones the decay of transient became substantially prolonged during streptozotocin-induced and spontaneously occurring diabetes. As small and large neurones differ substantially in their mechanisms of Ca2+ transient termination, we conclude that the prolongation of Ca2+ transients, probably induced by chronic hyperglycaemia, is specific only for small sensory neurones (transmitting mostly nociceptive signals) and may be a cause of the increased pain sensitivity often accompanying this disease.

Dual action of thapsigargin on calcium mobilization in sensory neurons: inhibition of Ca2+ uptake by caffeine-sensitive pools and blockade of plasmalemmal Ca2+ channels.

The action of thapsigargin on intracellular calcium homeostasis and voltage-activated calcium currents was studied on freshly isolated adult mouse dorsal root ganglia neurons. The cytoplasmic Ca2+ concentration ([Ca2+]i) was measured using indo-1-based microfluorimetry; transmembrane Ca2+ currents were recorded under voltage-clamp in the whole-cell configuration of the patch-clamp technique. Extracellular applications of thapsigargin at concentrations of 20-2000 nM did not cause substantial changes of basal [Ca2+]i level in the majority of neurons studied.

Role of caffeine-sensitive Ca2+ stores in Ca2+ signal termination in adult mouse DRG neurones.

The role of calcium stores in cytoplasmic calcium signal termination was studied in acutely isolated small and large DRG neurones from mice. Cytoplasmic calcium concentration ([Ca2+]in) was recorded using indo-1 microfluorometry and membrane potential was monitored by 'perforated' whole-cell patch-clamp. Depolarization-induced [Ca2+]in transients recovered significantly faster in large neurones than in small neurones.

Different properties of caffeine-sensitive Ca2+ stores in peripheral and central mammalian neurones.

Using indo-1 based microfluorometry for measuring the cytoplasmic free calcium concentration ([Ca2+]i), the properties of caffeine-induced Ca2+ release from internal stores were studied in rat cultured central and peripheral neurones, including dorsal root ganglia (DRG) neurones, neurones from nucleus cuneatus, CA1 and CA3 hippocampal region and pyramidal neocortical neurones. Under resting conditions the Ca2+ content of internal stores in DRG neurones was high enough to produce caffeine-triggered [Ca2+]i transients. Caffeine-induced Ca2+ release depleted internal stores in DRG neurones, but they refilled themselves spontaneously up to 81.

Caffeine-induced calcium release from internal stores in cultured rat sensory neurons.

Free intracellular calcium concentration ([Ca2+]in) was recorded at 22 degrees C by means of Indo-1 or Fura-2 single-cell microfluorometry in cultured dorsal root ganglion neurons obtained from neonatal rats. The resting [Ca2+]in in dorsal root ganglion neurons was 73 +/- 21 nM (mean +/- S.D.

[The functional status of the small intestine in rats in the dynamics of a bone marrow form of acute radiation sickness].

A study was made of 75Se-methionine absorption rate and contraction and choline-reactivity of smooth muscle cells of rat jejunum longitudinal layer. Whole-body X-irradiation with a dose of 5.8 Gy (LD50/30) caused a sharp decrease in the rate of absorption of 75Se-methionine by the blood from the intestine on days 3--4 after irradiation followed by an incomplete recovery of absorption by the 14th day.

[Effect of neurotensin and substance P on the active state of the gastric smooth muscles].

Activating effect of neurotensin applied to muscle strips taken from fundal and antral portions of the guinea pig stomach was found to be due to depolarization of the muscle cells membrane and activation of the electrically controlled calcium channels. Activating effect of substance P seems to be associated with the activation of the chemo-controlled calcium channels as the contraction following the substance P application develops with no apparent changes in the membrane potential. Substance P increases the amplitude of the potassium contracture tonic component.