Changes in functioning of rat submandibular salivary gland under streptozotocin-induced diabetes are associated with alterations of Ca2+ signaling and Ca2+ transporting pumps.

Xerostomia and pathological thirst are troublesome complications of diabetes mellitus associated with impaired functioning of salivary glands; however, their cellular mechanisms are not yet determined. Isolated acinar cells were loaded with Ca2+ indicators fura-2/AM for measuring cytosolic Ca2+ concentration ([Ca2+]i) or mag-fura-2/AM-inside the endoplasmic reticulum (ER). We found a dramatic decrease in pilocarpine-stimulated saliva flow, protein content and amylase activity in rats after 6 weeks of diabetes vs. healthy animals. This was accompanied with rise in resting [Ca2+]i and increased potency of acetylcholine (ACh) and carbachol (CCh) but not norepinephrine (NE) to induce [Ca2+]i transients in acinar cells from diabetic animals. However, [Ca2+]i transients mediated by Ca2+ release from ER stores (induced by application of either ACh, CCh, NE, or ionomycin in Ca2+-free extracellular medium) were decreased under diabetes. Application of inositol-1,4,5-trisphosphate led to smaller Ca2+ release from ER under the diabetes. Both plasmalemma and ER Ca2+-ATPases activity was reduced and the latter showed the increased affinity to ATP under the diabetes. We conclude that the diabetes caused impairment of salivary cells functions that, on the cellular level, associates with Ca2+ overload, increased Ca2+-mobilizing ability of muscarinic but not adrenergic receptors, decreased Ca2+-ATPases activity and ER Ca2+ content.

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.

Calcium signalling in granule neurones studied in cerebellar slices.

The cytoplasmic free calcium concentration ([Ca2+]i) was studied in Fura-2/AM loaded granule neurones in acutely prepared cerebellar slices isolated from neonatal (6 days old) and adult (30 days old) mice. Bath application of elevated (10-50 mM) KCl-containing extracellular solutions evoked [Ca2+]i rise which was dependent on extracellular Ca2+. The K(+)-induced [Ca2+]i elevation was inhibited to different extends by verapamil, nickel and omega-conotoxin suggesting the coexpression of different subtypes of plasmalemmal voltage-gated Ca2+ channels. Bath application of caffeine (10-40 mM) elevated [Ca2+]i by release of Ca2+ from intracellular stores. Caffeine-induced [Ca2+]i elevation was inhibited by 100 microM ryanodine and 500 nM thapsigargin. Depletion of internal Ca2+ stores by caffeine, or blockade of Ca2+ release channels by ryanodine, did not affect depolarization-induced [Ca2+]i transients, suggesting negligible involvement of Ca(2+)-induced Ca2+ release in [Ca2+]i signal generation following cell depolarization. External application of 100 microM glutamate, but not acetylcholine (1-100 microM), carbachol (10-100 microM) or (1S,3R)-ACPD (100-500 microM) evoked [Ca2+]i elevation. Part of glutamate-triggered [Ca2+]i transients in neurones from neonatal mice was due to Ca2+ release (presumably via inositol-(1,4,5)-trisphosphate-sensitive mechanisms) from internal Ca2+ stores. In adult animals, glutamate-triggered [Ca2+]i elevation was exclusively associated with plasmalemmal Ca2+ influx via both voltage-gated and glutamate-gated channels.

Calcium clamp in single nerve cells.

Free calcium concentration in isolated single neurons was clamped using a new technical approach based on a feed-back connection between the Fura-2 fluorescence signal measuring the intracellular Ca2+ concentration ([Ca2+]i) and iontophoretic current injecting Ca2+ into the cell. Beginning of [Ca2+]i clamping at a level above the basal one triggered fast (few seconds) current transients equal to injection of 36 +/- 20 microM Ca2+ (for a 0.1 microM change of [Ca2+]i), representing the filling of a fast cytosolic buffer. Continuation of clamping required very small clamping currents (corresponding to injection of 0.39 +/- 0.20 microM.s-1 Ca2+). This value increased proportionally to the magnitude of the change of [Ca2+]i above basal level, indicating the activation of calcium-dependent mechanisms for Ca2+ removal from the cytosol. The described approach allowed measurement, under physiological conditions, of the capacitative and kinetic properties of different Ca-regulating systems functioning in a single nerve cell as well as other types of cells.

Role of mitochondria in intracellular calcium signaling in primary and secondary sensory neurones of rats.

The participation of different calcium-regulated mechanisms in the generation of cytosolic Ca(2+) transients during neuronal excitation has been compared in isolated large and small primary (dorsal root ganglia (DRG)) and secondary (spinal dorsal horn (DH)) rat sensory neurones. As it was shown before in murine primary sensory neurones the application of mitochondrial protonophore CCCP by itself induced only small elevation of [Ca(2+)](i). However, its preceding application substantially increased the peak amplitude of depolarization-induced transients. Application of CCCP immediately after termination of the depolarizing pulse induced in both types of primary neurones a massive release of Ca(2+) from mitochondria into the cytosol. In secondary neurones application of CCCP by itself induced a substantial release of Ca(2+) from the mitochondria, but its preceding application resulted in only an insignificant increase in the peak amplitude of depolarization-triggered calcium transients. Application of CCCP immediately after termination of depolarization elicited a small release of Ca(2+), which became more pronounced when the application was delayed. Preceding application of CCCP increased the amplitude of the transients induced by caffeine-triggered Ca(2+) release from the endoplasmic reticulum in secondary neurones and did not affect those in large primary neurones. These findings may be explained by substantial differences in the density and distribution of mitochondria in the cytosol of primary and secondary sensory neurones. This suggestion was confirmed electronmicroscopically, showing a much lower density of mitochondria near plasmalemma in secondary sensory neurones and predominant clustered location of mitochondria beneath the plasmalemma in the primary cells. The possible functional importance of these differences is discussed.

Low-voltage activated calcium channels: achievements and problems.

Low-voltage activated Ca2+ channels, which possess unique properties quite different from those of common (high-voltage activated) channels, were discovered 15 years ago but the first alpha1 subunit has only recently been identified which might provide their structural basis. However, simultaneously, extensive data are being accumulated on the functional diversity of low-voltage activated Ca2+ currents with regard to their pharmacological sensitivity, ionic selectivity, activation and inactivation kinetics. Such diversity corresponds to equally prominent heterogeneity in the location and function of the channels. This commentary summarizes the data available in an attempt to predict a possibly wider structural subdivision of low-voltage activated Ca2+ channels into subtypes.

Diversity of calcium ion channels in cellular membranes.

Successful introduction of techniques for separation of different ionic currents and recording of single channel activity has demonstrated the diversity of membrane structures responsible for generation of calcium signal during various forms of cellular activity. In excitable cells the electrically-operated calcium channels have been separated into two types functioning in different membrane potential ranges (low- and high-threshold ones). The low-threshold channels are ontogenetically primary and may play a role in regulation of cell development and differentiation. A similar function may also be characteristic of chemically-operated channels in some highly specialized cells (lymphocytes). The high-threshold channels in excitable cells generate an intracellular signal coupling membrane excitation and intracellular metabolic processes responsible for specific cellular reactions (among them retention of traces of previous activity in neurons--"learning"--being especially important). Chemically-operated N-methyl-D-aspartate-channels also participate in this function. The calcium signal can be potentiated by activation of calcium-operated channels in the membranes of intracellular structures, resulting in the liberation of calcium ions from the intracellular stores. Although different types of calcium channels have some common features in their structure which may indicate their genetic similarity, their specific properties make them well suited for participation in a wide range of cellular mechanisms.

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. However, 5-10 min incubation of neurons with 20 nM thapsigargin completely and almost irreversibly inhibited caffeine-mediated Ca2+ release from intracellular pools. This inhibition was associated with deceleration of the recovery of depolarization-induced [Ca2+]i transients, presumably due to the inhibition of Ca2+ uptake by intracellular calcium stores. At concentrations between 200 and 2000 nM, thapsigargin markedly depressed the amplitudes of depolarization-triggered [Ca2+]i transients due to the inhibition of transmembrane Ca2+ entry through voltage-activated Ca2+ channels. We found that thapsigargin discriminates between low- and high-voltage-activated Ca2+ channels: 2000 nM of thapsigargin decreased the amplitudes of high-voltage-activated currents by 60%, while the amplitudes of low-voltage-activated Ca2+ currents were reduced by only 25%. Thus, thapsigargin exerts a dual action on [Ca2+]i handling mechanisms in mouse sensory neurons: at low concentrations (< 50 nM) it inhibits Ca2+ accumulation by endoplasmic reticulum pools, whereas at higher concentrations (200-2000 nM) thapsigargin blocks high-voltage-activated Ca2+ currents, reducing Ca2+ entry into the cell.

Spatial heterogeneity of caffeine- and inositol 1,4,5-trisphosphate-induced Ca2+ transients in isolated snail neurons.

Inositol 1,4,5-trisphosphate- and caffeine-induced Ca2+ release was examined in neurons isolated from the mollusc Helix pomatia using Ca2+ indicator fura-2 and fluorescent digital-imaging microscopy technique. Extracellular application of caffeine caused a fast and pronounced augmentation of [Ca2+]i whose amplitude and kinetics differ in the centre of the cell and near its membrane. Mean values of caffeine-induced increase of [Ca2+]i were 0.97 +/- 0.11 microM at the periphery and 0.53 +/- 0.13 microM in the centre. The rates of rise and relaxation of caffeine-evoked [Ca2+]i transients were faster near the membrane. Pressure injection of inositol, 1,4,5-trisphosphate into the same neurons produced an abrupt and significant increase of [Ca2+]i in the centre (mean value of inositol 1,4,5-trisphosphate-induced elevation = 0.55 +/- 0.11 microM) while the response was smaller or even absent near the cellular membrane. Inositol 1,4,5-trisphosphate- and caffeine-induced Ca2+ transients did not affect each other. The data obtained indicate that in snail neurons these two calcium pools are not overlapping and at least some part of the caffeine-sensitive store is located close to the cellular membrane and that the inositol 1,4,5-trisphosphate-sensitive one is located in the centre of the cell.

Polarization of primary afferent terminals of lumbosacral cord elicited by the activity of spinal locomotor generator.

The changes of electrical polarization of primary afferent terminals in the lumbosacral spinal cord have been investigated on immobilized, decorticated and spinal cats during fictive locomotion. Fictive locomotion was spontaneous or provoked by stimulation of either dorsal root or dorsal funiculi in the lumbar segments. The activation of the locomotor generator and appearance of fictive locomotion were always associated with a sustained dorsal root hyperpolarization. On the background of this positivity periodic negative dorsal root potential oscillations appeared synchronously with efferent discharges in motor hind-limb nerves. These periodic waves of primary afferent depolarization occurred in phase in different ipsilateral lumbosacral segments. On the contralateral side the periodic changes in dorsal root potential were out of phase during fictive stepping and in phase during fictive galloping. The use of Wall's technique has shown that tonic and periodic changes in dorsal root potential reflect the changes occurring in polarization of central terminals of cutaneous and muscle (Ia and Ib) groups of afferent fibres. It is concluded that the level of electrical polarization of primary afferent terminals is determined directly by the activity of the spinal locomotor generator; activation of the generator is followed by hyperpolarization of primary afferent terminals. By so modulating the polarization of afferent terminals, the locomotor generator can perform tonic and phase-dependent selection of afferent information.

Potassium outward current dependent on extracellular calcium in snail neuronal membrane.

In experiments on isolated intracellularly dialysed neurons of the snail Helix pomatia a component of delayed inactivating potassium outward current depending on the presence of Ca2+ ions in the extracellular medium has been distinguished, which differs from the already known potassium current sensitive to intracellular calcium ions. This component decreases with a decrease in extracellular calcium (in the range of 10(-2) - 10(-5) M); it is not affected or even increased by intracellular introduction of ethyleneglycolbis(aminoethylether)tetra-acetate (10 mM) or fluoride ions (77 mM) and can be blocked by addition of 1.5 mM cobalt ions to the extracellular solution. Contrary to the slow rising potassium current dependent on intracellular calcium, this current has a fast rising phase (several milliseconds) and time-dependent inactivation. The inactivation depends on extracellular potassium ions: it slowed down when [K+]out is increased in the range of 1-10 mM. Extracellular application of calmodulin blockers calmidazolium (6.5 X 10(-7) M) and chlorpromazine (2.5 X 10(-6) M) selectively inhibits the potassium current dependent on intracellular calcium but does not affect that dependent on external calcium. Tetraethylammonium (10 mM) depresses the latter current on both intra- and extracellular application, the former being more effective. The existence of a special type of potassium channel sensitive to extracellular calcium ions is postulated.

Role of mitochondrial dysfunction in calcium signalling alterations in dorsal root ganglion neurons of mice with experimentally-induced diabetes.

The role of mitochondrial dysfunction in alterations of calcium signalling in primary sensory neurons has been studied in mice with streptozotocin-induced and genetically predisposed diabetes mellitus before and after additional treatment with insulin infusions. Cytosolic calcium transients triggered by membrane depolarization were measured using a membrane-permeable form of fluorescent indicator indo-1, and their changes after application of mitochondrial uncoupler carbonyl cyanide m-chlorphenylhydrazone were compared in cells of control and diabetic animals. Considerable prolongation of residual elevation of cytosolic calcium after termination of membrane depolarization was observed in diabetic mice, which was expressed mainly in small-sized (nociceptive) neurons. This correlated with the level of hyperglycemia, which was maximal in cells from streptozotocin-treated mice. Insulin partly reversed these changes. Carbonyl cyanide m-chlorophenylhydrazone application to neurons of control mice enlarged the peak of calcium transients and decreased residual calcium elevations, indicating that mitochondria in physiological conditions participate in shaping of these transients by diminishing their peak due to rapid uptake of calcium ions and by prolonging them due to subsequent slow calcium release back into the cytosol. Depression of the calcium accumulating function of mitochondria by carbonyl cyanide m-chlorophenylhydrazone eliminated these changes. The prolonged residual elevation of cytosolic calcium characteristic for neurons of diabetic animals was also eliminated by carbonyl cyanide m-chlorophenylhydrazone, confirming the suggestion that such elevation is determined mainly by mitochondrial dysfunction, the latter being dependent on the level of hyperglycemia. Predominant expression of such changes in small-sized neurons can be explained by the absence in them of effective calcium-buffering by the endoplasmic reticulum. Possible role of the described calcium signalling changes in the origin of neuropathic syndromes is discussed.

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. The sensitivity of Ca2+ stores to caffeine was strongly influenced by endoplasmic reticulum luminal Ca2+ concentration ([Ca2+]i) as an increase in [Ca2+]i produced by a conditioning depolarization-induced Ca2+ entry, caused a several fold decrease of caffeine EC50. By elevating [Ca2+]c a threshold concentration (about 350 nM) can be reached, at which low doses of caffeine (2-5 mM) produced a regenerative Ca2+ release, that depletes the Ca2+ stores almost completely, indicating that all-or-nothing Ca(2+)-induced Ca2+ release can be generated in nerve cells.

Glucocorticoid modulation of calcium currents in growth hormone 3 cells.

Clonal malignant pituitary growth hormone 3 cells were used for the analysis of the influence of hydrocortisone and dexamethasone on voltage-gated calcium currents and hormone secretion. The whole-cell patch-clamp technique was used. The presence of low-threshold inactivating and high-threshold persisting components in the total calcium current was shown; they could be separated at less negative holding potential level. Some increase in current densities of both components was observed as early as 30 min after treatment with 10(-6) mol/l glucocorticoids. The increase was maximal for both types of currents after 2 h of incubation; however, the high-threshold component was affected much more strongly (current density increased by more than four-fold) than the low-threshold one (current density increased by about a three-fold). Potentiation of currents was blocked by actinomycine D (10(-4) M), suggesting that protein synthesis was required. A substantial increase in growth hormone secretion (measured by radioimmunoassay method) was observed in the same cells after 2 h of incubation with hydrocortisone, while the secretion of prolactin remained even slightly depressed.

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., n = 94). Fast application of 20 mM caffeine evoked [Ca2+]in transient which reached a peak of 269 +/- 64 nM within 5.9 +/- 1.1 s. After reaching the peak the [Ca2+]in level started to decline in the presence of caffeine and for 87.2 +/- 10.6 s cytoplasmic calcium returned to an initial resting value. In 40% of neurons tested [Ca2+]in decreased to subresting levels following the washout of caffeine (the so-called post-caffeine undershoot). On average, the undershoot level was 19 +/- 2.5 nM below the resting [Ca2+]in value. Prolonged exposure of caffeine depleted the caffeine-sensitive stores of releasable Ca2+; the degree of this depletion depended on caffeine concentration. The depletion of the caffeine-sensitive internal stores to some extent was linked to calcium extrusion via La(3+)-sensitive plasmalemmal Ca(2+)-ATPases. The stores could be partially refilled by the uptake of cytoplasmic Ca2+, but the complete recovery of releasable Ca2+ content of the caffeine-sensitive pools required the additional calcium entry via voltage-operated calcium channels. Caffeine-evoked [Ca2+]in transients were effectively blocked by 10 microM ryanodine, 5 mM procaine, 10 microM dantrolene or 0.5 mM Ba2+, thus sharing the basic properties of the Ca(2+)-induced-Ca2+ release from endoplasmic reticulum. Pharmacological manipulation with caffeine-sensitive stores interfered with the depolarization-induced [Ca2+]in transients. In the presence of low caffeine concentration (0.5-1 mM) in the extracellular solution the rate of rise of the depolarization-triggered [Ca2+]in transients significantly increased (by a factor 2.15 +/- 0.29) suggesting the occurrence of Ca(2+)-induced Ca2+ release. When the caffeine-sensitive stores were emptied by prolonged application of caffeine, the amplitude and the rate of rise of the depolarization-induced [Ca2+]in transients were decreased. These facts suggest the involvement of internal caffeine-sensitive calcium stores in the generation of calcium signal in sensory neurons.

Ionic mechanisms of electrical excitability in rat sensory neurons during postnatal ontogenesis.

Inward currents in the somatic membrane of rat dorsal root ganglion neurons have been studied in three groups of animals (8-10, 45-50 and 90-100 days of postnatal development) by intracellular perfusion and voltage clamp techniques. Altogether, 228 neurons have been examined (76 in each age group). Four components have been identified in the inward current: fast tetrodotoxin-sensitive (IfNa) and slow tetrodotoxin-insensitive (IsNa) sodium, low threshold (IlCa) and high threshold (IhCa) calcium currents. The existence of certain types of inward currents in the somatic membrane allowed the heterogeneous population of all the investigated neurons to be divided into six homogeneous subpopulations. A significant decrease in the percentage of neurons was found, demonstrating four types of inward currents (IfNa, IsNa, IlCa, IhCa) during postnatal ontogenesis. The percentage of cells in the subpopulation showing only IfNa and IhCa increased from 26% in the first age group to 43% in the second and 62% in the third age groups. Correlations between densities of various types of inward currents were studied. A linear relationship was found between the densities of IhCa and IsNa in subpopulations of neurons with IsNa. An inverse dependence was observed between the densities of IfNa and IhCa in cells showing only two current components. In all cells studied a "washout" of IhCa was observed during intracellular dialysis with saline solutions. Recovery of calcium conductance produced by intracellular application of an cAMP-ATP-Mg2+ complex was different in neurons with various inward current combinations. Intracellular introduction of cAMP-ATP-Mg2+ failed to restore IhCa in most cells of the second and third age groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative analysis of ionic currents in the somatic membrane of embryonic and newborn rat sensory neurons.

Inward currents in the somatic membrane of dissociated rat dorsal root ganglion neurons have been studied in two groups of animals (17 days of embryonic development and the first day after birth) by suction pipette (whole-cell configuration) and voltage-clamp techniques. Altogether 157 neurons were examined. Four components in the inward currents have been identified: fast tetrodotoxin-sensitive (INaf) and slow tetrodotoxin insensitive (INas) sodium, low-(ICal) and high-threshold (ICah) calcium currents. The percentage of neurons demonstrating four types of inward currents INaf, INas, ICal, ICah increased from 21% in embryo to 61% in newborn. The percentage of neurons with INaf, INas and ICah increased from 4% in embryonic to 14% in the first day after birth. The percentage of cells with INaf, ICal and ICah (without tetrodotoxin-insensitive INas) decreased from 56 to 11% in embryo and newborn rats, respectively. A statistically significant linear correlation was found between the densities of INaf and ICah currents for both ages. A correlation also occurred between the densities of INas and ICah. A reciprocal relation between the densities of both types of calcium currents and the size of cell soma was found in the neurons with all four types of inward currents from newborn animals. A comparison of these data with previous study of inward currents during postnatal development indicates that the most dramatic changes in their distributions and mean densities takes place some time after the birth of the animals.

Long-term depolarization changes morphological parameters of PC12 cells.

It is well known that neuronal differentiation is strongly dependent on the intracellular level of free calcium ions ([Ca2+]i). In the present study the morphological and intracellular free calcium concentration changes were compared on PC12 pheochromocytoma cells cultured in control conditions and in a medium with high KCl level. Culturing PC12 cells in a medium with 20-30 mM KCl deprived of nerve growth factor supported cell proliferation and rapid growth of small neurite-like processes. However, their lengths did not increase with prolongation of the time of culturing. During culturing with 40 mM KCl the growth of these processes became blocked; the cells stopped proliferating and showed signs of degeneration. Measurements of [Ca2+]i level during the first days of PC12 cells culturing in a hyperpotassium medium indicate that such changes in this level could be an important factor in the induction of the observed morphological alterations; however, other effects induced by membrane depolarization may also be responsible for them.

Modulation of calcium current by calmodulin antagonists.

The short-term effects of bath applied calmodulin antagonists--chlorpromazine, trifluoperazine and calmidazolium (R24571)--on potential-dependent calcium channels in the membrane of intracellularly perfused snail neurons were studied in voltage clamp conditions. All the drugs affected the calcium inward current peak value, the effects being reversible and dependent on the concentration used. Submicromolar concentrations (0.1-1 microM) increased the current amplitude (the maximal effect was on the average 20% at 0.5 microM), whereas higher concentrations inhibited the current. Analysis of the dose-effect curve for the blockade suggests positive cooperativity in the interaction of the drugs with the channel; experimental data on chlorpromazine action (10-100 microM) are well approximated by a binding curve for two molecules with the effective Kd = 70 microM. The efficiency of the blockade depended neither on the current-carrying cations (calcium or barium) nor on the intracellular introduction of 10 mM EGTA. The presence of calmodulin antagonists influenced the blockade of the calcium current by inorganic blockers: 50 microM chlorpromazine decreased the Kd value from 90 to 50 microM for the current blockade by Cd ions. It is suggested that calmodulin antagonists interact with two sites in the calcium channel, with high and low binding affinity (responsible for enhancement and inhibition of the current, respectively). The interaction induces changes in binding of penetrating cations in the channel, thereby producing modulation of the calcium current amplitude.

Nifedipine-induced morphological differentiation of rat pheochromocytoma cells.

Extension of neuronal processes is fundamental for the establishment of the intricate network of the nervous system. The rat PC12 pheochromocytoma cell line is a well-known model system for the study of neuronal differentiation. In the present study the effect of a high-voltage-activated calcium channel blocker, nifedipine, on the cell differentiation was investigated. In cells cultured in a modified medium containing nifedipine (at 2.5 microM or 5.0 microM) a decrease of mitotic activity took place on the third day of culturing. An increase of total length and number of processes occurred on the fifth day. A higher concentration of nifedipine (10.0 microM) considerably suppressed vital functions of the cells. Retraction of processes together with blocking of proliferation activity was observed. In parallel with these changes, a reduction of [Ca2+]i from 100 +/- 6 nM to 50 +/- 2 nM was detected on the second day of cultivation in 2.5 microM and 5.0 microM nifedipine containing medium. At 10.0 microM concentration of nifedipine in the medium a decrease in [Ca2+]i was observed on the first day only (to 76 +/- 8 nM); then the level of free calcium concentration rose dramatically. The data obtained allow us to suggest that the decrease of [Ca2+]i during first two days of PC12 cells culturing in a nifedipine-containing medium could be a factor which stimulated their morphological differentiation.