Influence of external pH on two types of low-voltage-activated calcium currents in primary sensory neurons of rats.

The influence of extracellular pH (pH(o)) on low-voltage-activated calcium channels of acutely isolated DRG neurons of rats was examined using the whole cell patch-clamp technique. It has been found that in the neurons of middle size with capacitance C=60+/-4.8 pF (mean+/-S.E., n=8) extracellular acidification from pH(o) 7.35 to pH(o) 6.0 significantly and reversibly decreased LVA calcium current densities by 75+/-3.7%, shifted potential for half-maximal activation to more positive voltages by 18.7+/-0.6 mV with significant reduction of its voltage dependence. The half-maximal potential of steady-state inactivation shifted to more positive voltages by 12.1+/-1.7 mV (n=8) and also became less voltage dependent. Dose-response curves for the dependence of maximum values of LVA currents on external pH in neurons of middle size have midpoint pK(a)=6.6+/-0.02 and hill coefficient h=0.94+/-0.04 (n=5). In small cells with capacitance C=26+/-3.6 pF (n=5), acidosis decreased LVA calcium current densities only by 15.3+/-1.3% and shifted potential for half-maximal activation by 5.5+/-1.0 mV with reduction of its voltage dependence. Half-maximal potential of steady-state inactivation shifted to more positive voltages by 10+/-1.6 mV (n=4) and also became less voltage dependent. Dose-response curves for the dependence of maximum values of LVA currents on external pH in neurons of small size have midpoint pK(a)=7.9+/-0.04 and hill coefficient h=0.25+/-0.1 (n=4). These two identified types of LVA currents besides different pH sensitivity demonstrated different kinetic properties. The deactivation of LVA currents with weak pH sensitivity after switching off depolarization to -30 mV had substantially longer decay time than do currents with strong pH sensitivity (tau(d) approximately 5 ms vs. 2 ms respectively). It was found that the prolongation of depolarization steps slows the subsequent deactivation of T-type currents in small DRG neurons. Deactivation traces in these neurons were better described by the sum of two exponentials. Thus, we suppose that T-type channels in small DRG neurons are presented mostly by alpha1I subunit. We suggest that these two types of LVA calcium channels with different sensitivity to external pH can be differently involved in the origin of neuropathic changes.

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.

Effect of streptozotocin-induced diabetes on the activity of calcium channels in rat dorsal horn neurons.

We have previously found that spinal dorsal horn neurons from streptozotocin-diabetic rats, an animal model for diabetes mellitus, show the prominent changes in the mechanisms responsible for [Ca2+]i regulation. The present study aimed to further characterize the effects of streptozotocin-induced diabetes on neuronal calcium homeostasis. The cytoplasmic Ca2+ concentration ([Ca2+]i) was measured in Fura-2AM-loaded dorsal horn neurons from acutely isolated spinal cord slices using fluorescence technique. We studied Ca2+ entry through plasmalemmal Ca2+ channels during potassium (50 mM KCl)-induced depolarization. The K+-induced [Ca2+]i elevation was inhibited to a different extent by nickel ions, nifedipine and omega-conotoxin suggesting the co-expression of different subtypes of plasmalemmal voltage-gated Ca2+ channels. The suppression of [Ca2+]i transients by Ni2+ (50 microM) was the same in control and diabetic neurons. On the other hand, inhibition of [Ca2+]i transients by nifedipine (50 microM) and omega-conotoxin (1 microM) was much greater in diabetic neurons compared with normal animals. These data suggest that under diabetic conditions the activity of N- and L- but not T-type voltage-gated Ca2+ channels substantially increased in dorsal horn neurons.

Changes in calcium signalling in dorsal horn neurons in rats with streptozotocin-induced diabetes.

Intracellular calcium signalling was studied in the dorsal horn from neurons of rats with streptozotocin-induced diabetes versus control animals. The cytoplasmic Ca2+ concentration ([Ca2+]i) was measured in Fura-2 acetoxymethyl ester-loaded dorsal horn neurons from acutely isolated spinal cord slices using a fluorescence technique. The recovery of depolarization-induced [Ca2+]i increase was delayed in diabetic neurons compared with normal animals. In normal neurons, [Ca2+]i after the end of KCl depolarization recovered to the basal level monoexponentially with a time constant of 8.0+/-0.5 s (n = 23), while diabetic neurons showed two exponentials in the [Ca2+]i recovery. The time constants of these exponentials were 7.2+/-0.5 and 23.0+/-0.6 s (n = 19), respectively. The amplitude of calcium release from caffeine-sensitive endoplasmic reticulum calcium stores became significantly smaller in diabetic neurons. The amplitudes of [Ca2+]i transients evoked by 30 mM caffeine were 268+/-29 nM (n = 13) and 31+/-9 nM (n = 17) in control and diabetic neurons, respectively. We conclude that streptozotocin-induced diabetes is associated with prominent changes in the mechanisms responsible for [Ca2+]i regulation, which presumably include a slowdown of Ca2+ elimination from the cytoplasm by the endoplasmic reticulum.