Restoration of the potential-dependent L-phenylalanine-inhibited calcium current by L-tyrosine in cultured hippocampal neurons]

Calcium currents were recorded in cultured (5-7 days) hippocampal neurons isolated from one-day rats. The animals were intraperitoneally injected L-phenylalanine which induced in their brain biochemical changes typical of phenylketonuria. It has been found that in neurons from injected animals the amplitude of the high-threshold calcium current was substantially inhibited and amounted to 40 +/- 30% at Vt = +20 mV (amplitude of calcium currents at Vt = -10 mV taken as 100%). Addition of L-tyrosine to the cultivation medium (50 mumol/l) restored the high-voltage calcium current, its relative amplitude reaching 280 +/- 57%.     

Inhibition of the high-threshold calcium current in hippocampal neurons of rats subjected to intraperitoneal injection of phenylalanine

Effect of intraperitoneal injection of phenylalanine on the calcium current of hippocampal neurons of rats has been studied by the voltage clamp method of the whole-cell recordings. Calcium currents in hippocampal neurons of control animals after 5-7 days in culture can be separated into two components: low and high voltage-activated ones. The value of high voltage-activated calcium current was 69 +/- 13% at Vt = -10 mV from total calcium inward current in these neurons. High voltage-activated Ica in neurons of phenylalaninemic rats was significantly depressed and its value was 32 +/- 14%, the Vt value being the same. Low voltage-activated calcium current was resistant to intraperitoneal injection of L-phenylalanine.     

Three types of voltage-dependent calcium current in cultured rat hippocampal neurons

Voltage-dependent calcium (Ca2+) currents in cultured rat hippocampal neurons were studied with the whole-cell recording mode of the patch-clamp technique. On the basis of the voltage-dependence of activation, kinetics of inactivation and pharmacology, 3 types of Ca2+ currents were distinguished. The low-threshold Ca2+ current (Il) was activated at -60 mV, and completely inactivated during a 100-ms depolarization to -40 mV (time constant: tau = 16 +/- 1 ms). The high-threshold currents (Ih), which were activated at -20 mV, could be separated into two types. The high-threshold, fast inactivating current (Ih,f) decayed quickly during a maintained depolarization (tau = 33 +/- 3 ms at 0 mV), whereas the high-threshold, slowly inactivating current (Ih,s) decayed with a much slower time constant (tau = 505 +/- 42 ms at 0 mV). The inactivations of Ih,f and Ih,s exhibited different time- and voltage-dependencies. Nickel ions (Ni2+, 25 microM) markedly suppressed Il, but little affected Ih. Cadmium ions (Cd2+, 10 microM) almost completely suppressed Ih, but left a small amount of Il. Lanthanum ions (La3+, 10 microM) almost completely suppressed both Il and Ih. Ih,s was sensitive to block by the dihydropyridine antagonist nicardipine (10 microM).     

Single channels of low and high-threshold calcium channels of the membrane of sensory neurons in the mouse

Single calcium channels of cultured dorsal root ganglion cells from mouse embryos were studied using patch clamp method in its cell-attached configuration. Two types of activity of unitary calcium channels were found. The first one which arose at membrane potentials near--50-40 mV was characterized by unitary current amplitude of 0.37 +/- 0.04 pA with 40 mmol/l Ca2+ in the pipette solution, mean open time of 0.6 ms and intraburst mean shut time of 1.2 ms. It displayed voltage- and time-dependent inactivation. The corresponding values for the second one which required much more positive depolarization to be activated (approximately 0 mV) and did not express noticeable inactivation were: 0.53 +/- 0.04 pA, 0.8 ms and 0.8 ms. It is concluded that the recorded types of unitary activity are associated respectively with low- and high-threshold calcium currents which have been found earlier while studying whole cell currents.     

Voltage-gated influx ion channels expressed in Xenopus oocytes after the administration of brain mRNA]

Expression of "fast", TTX-sensitive sodium and high-threshold calcium channels in the membrane of Xenopus oocytes following mRNA injection from the rat brain has been detected using two microelectrode voltage clamp technique. Barium current through expressed calcium channels was blocked by 200 mumol/l Cd2+ and was insensitive to D-600 (20 mumol/l) and nitrendipine (50 mumol/l). Expressed barium current was inhibited within 20-40 min by omega-conotoxin, a peptide neurotoxin known to block high-threshold calcium channels of the neuronal membrane, in 1 mumol/l concentration. A steady-state inactivation curve for this current could be fitted by the Boltzmann relation with V1/2 = -50 mV and k = 14 mV. Voltage-dependent and pharmacological properties of calcium channels which appeared in the oocyte membrane following mRNA injection from the mammalian brain resembled most of all those of high-threshold inactivating (HTI- or N-type) calcium channels of neurons in spite they did not demonstrate prominent time-dependent inactivation. Evidences in favour of expressed calcium channels heterogeneity were not obtained.     

Multiple types of high-threshold calcium channels in rabbit sensory neurons: high-affinity block of neuronal L-type by nimodipine.

Whole-cell and cell-attached patch recording have been used to characterize multiple types of voltage-dependent calcium channels in neurons freshly dispersed from rabbit dorsal root ganglia. In whole-cell patch recordings, high-threshold current, strongly resistant to inactivation by depolarized holding potentials (L-type; V1/2 = -27.2 mV), was potently inhibited by nimodipine. Assuming 1:1 binding, the dissociation constant for nimodipine binding to the inactivated state of the L-type calcium channel (KI) was 5.3 nM (n = 8). In contrast, a second type of high-threshold current less resistant to inactivation by depolarized holding potentials (N-type; V1/2 = -56.9 mV) was not blocked by nimodipine. Nimodipine-resistant N-type calcium current was inhibited by omega-conotoxin (5 microM). Cell-attached patch recordings of single calcium channel currents demonstrated the existence of three different unitary conductances; 7.4 pS, 13.1 pS, and 24.1 pS. The 24.1 pS high-threshold channel was enhanced by (-) BAY K 8644 and inhibited by nimodipine in a concentration- and voltage-dependent manner. Hyperpolarization reversed this block. These results demonstrate that, as in cardiac and smooth muscle, there is a component of neuronal high-threshold current corresponding to the L-type calcium channel that can be blocked with high affinity by nimodipine.     

Characterization of a fast transient outward current in neocortical neurons from epilepsy patients

A-type currents powerfully modulate discharge behavior and have been described in a large number of different species and cell types. However, data on A-type currents in human brain tissue are scarce. Here we have examined the properties of a fast transient outward current in acutely dissociated human neocortical neurons from the temporal lobe of epilepsy patients by using the whole-cell voltage-clamp technique. The A-type current was isolated with a subtraction protocol. In addition, delayed potassium currents were reduced pharmacologically with 10 mM tetraethylammonium chloride. The current displayed an activation threshold of about -70 mV. The voltage-dependent activation was fitted with a Boltzmann function, with a half-maximal conductance at -14.8 +/- 1.8 mV (n = 5) and a slope factor of 17.0 +/- 0.5 mV (n = 5). The voltage of half-maximal steady-state inactivation was -98.9 +/- 8.3 mV (n = 5), with a slope factor of -6.6 +/- 1.9 mV (n = 5). Recovery from inactivation could be fitted monoexponentially with a time constant of 18.2 +/- 7.5 msec (n = 5). At a command potential of +30 mV, application of 5 mM 4-aminopyridine or 100 microM flecainide resulted in a reduction of A-type current amplitude by 35% or 22%, respectively. In addition, flecainide markedly accelerated inactivation. Current amplitude was reduced by 31% with application of 500 microM cadmium. All drug effects were reversible. In conclusion, neocortical neurons from epilepsy patients express an A-type current with properties similar to those described for animal tissues.     

Changes in the ionic mechanisms of the electro-excitability of the somatic membrane of rat sensory neurons during ontogenesis. Relation between calcium channels and intracellular metabolism

A change in the relationship between high-threshold calcium channels and intracellular metabolism of cyclic nucleotides during postnatal development was found in experiments on rat dorsal root ganglion neurons. In the first age group (5-9 postnatal days) intracellular administration of cAMP-ATP-Mg2+ complex has resulted in restoration of the maximal amplitude of high threshold calcium current for 70% of neurons, whereas in the second (45 days) and third (90 days) age groups this effect was observed only in 26% and 10% of neurons, respectively. Kinetic and voltage-dependent characteristics of high-threshold calcium current in these three age groups were identical. The effect of introduction of cAMP-ATP-Mg2+ complex was different for neurons with different combination of inward currents. Neurons which possessed only two types of inward currents--"fast" sodium and high-threshold calcium ones, show no effect. Conventional effect of the intracellular cAMP injection occurred always in neurons which had exhibited a "slow" (TTX-resistant) sodium inward current together with the two main inward currents.     

Action of omega-conotoxin on calcium currents of the rat pituitary GH3 cell line]

Whole-cell modification of the patch clamp method was used to examine the action of omega-CgTX on calcium currents in GH3 pituitary cells. Two quite distinct components of inward calcium currents were observed in the presence of 15 mmol/l of calcium in the external solution. One was activated from the holding potential -80 mV by testing pulses more positive than -50 mV. The shift of the holding potential to -40 mV resulted in the stationary inactivation of this low voltage activated current component. It was found that omega-CgTX activated both low-threshold and high-threshold calcium currents at the first moment of application, but low-threshold current component increased more significantly. Full effect was developed for less than 30 s. Then time decay of currents was comparable with that of the "wash-out" process. Incubation of cells in the growth medium that contained 5 mumol/l omega-CgTX during 2 hour induced an increase in density of both types of calcium currents, then it fell after 2 hours of incubation in the same medium.     

Effects of insulin-like growth factor 1 on voltage-gated ion channels in cultured rat hippocampal neurons

Insulin-like growth factor 1 (IGF-1) has important functions in the brain, including metabolic, neurotrophic, neuromodulatory, and neuroendocrine actions, and it is also prevents amyloid beta-induced death of hippocampal neurons. However, its functions on the voltage-gated ion channels in hippocampus remain uncertain. In the present study, we investigated the effects of IGF-1 on voltage-gated potassium, sodium, and calcium channels in the cultured rat hippocampal neurons using the whole-cell patch clamp recordings. Following incubation with different doses of IGF-1 for 24 h, a block of the peak transient A-type K+ currents amplitude (IC50: 4.425 ng/ml, Hill coefficient: 0.621) was observed. In addition, after the application of IGF-1, the amplitude of high-voltage activated Ca2+ currents significantly increased but activation kinetics did not significantly alter (V1/2: -33.45 +/- 1.32 mV, k = 6.16 +/- 1.05) compared to control conditions (V1/2: -33.19 +/- 2.28 mV, k = 7.26 +/- 1.71). However, the amplitude of Na+, K+, and low-voltage activated Ca2+ currents was not affected by the application of IGF-1. These data suggest that IGF-1 inhibits transient A-type K+ currents and enhances high-voltage-activated Ca2+ currents, but has no effects on Na+ and low-voltage-activated Ca2+ currents.     

Differential effects of apamin on neuronal excitability in the nucleus tractus solitarii of rats studied in vitro

We demonstrated previously that microinjection of the calcium-dependent potassium channel antagonist, apamin, into the nucleus tractus solitarius (NTS) in vivo potentiated the baroreceptor reflex mediated bradycardia but attenuated the cardiopulmonary reflex. The latter result was surprising since, intuitively, potassium channel blockade would be expected to increase neuronal excitability leading to reflex potentiation. The aim of this in vitro study was to investigate possible neuronal mechanisms to explain our in vivo observations. Transverse brainstem slices of rat were cut at the level of area postrema and recordings were made from 36 NTS neurones in whole-cell mode. The neurones were classified into three groups, based on their response to apamin (10 nM). Each group had a similar resting membrane potential (RMP; -55 +/- 1 mV; n = 36) and input resistance (404 +/- 20 M omega; n = 36). (1) In 15/36 neurones, apamin decreased the number of spikes evoked during injection of positive current by 37 +/- 6%; this was associated with a concomitant fall in input resistance of 12 +/- 2% (P < 0.05). Stimulation of the ipsilateral tractus solitarius evoked EPSP-IPSP complexes in nine of the 12 neurones tested; the inhibitory components were increased in amplitude, at a holding potential of -46 mV, from -1.7 +/- 0.4 to -3.2 +/- 0.6 mV (P < 0.01) in the presence of apamin, while the EPSPs were unaffected. All three of these effects were bicuculline (10 microM) sensitive. (2) In 8/36 neurones, apamin increased the number of spikes evoked during injection of positive current by 27 +/- 8%, but affected neither RMP nor input resistance. Only one of five neurones tested demonstrated a synaptically evoked EPSP-IPSP complex. The remaining four neurones displayed a single evoked EPSP, the amplitudes of which were unaffected by apamin. (3) In the remaining neurones (13/36), apamin affected neither responsiveness to positive current injection, RMP, nor input resistance. Six of 12 neurones demonstrated synaptically evoked EPSP-IPSP complexes; at a holding potential of -46 mV, apamin increased the IPSP component from -2.6 +/- 1 to -3.6 +/- 0.8 mV (P < 0.05), while the EPSPs were unaffected. In conclusion, apamin can both increase and decrease NTS neuronal excitability: the former reflecting blockade of channels on the recorded neurone; the latter may possibly result from an increase in GABA release by interneurones impinging onto the recorded neurone. The possibility of a differential distribution of apamin-sensitive channels in sub-populations of NTS neurones subserving different reflexes is discussed.     

Differential properties of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels in rat dorsal root ganglion neurons.

TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) sodium channel currents were analyzed in acutely dissociated dorsal root ganglion (DRG) neurons isolated from 3-12-d-old and adult rats. Currents were recorded using the whole-cell patch-clamp technique. TTX-R current was more likely to be present in younger animals (3-7 d), whereas TTX-S current was more common in older animals (7-10 d), although TTX-R current was recorded from adult rat DRG neurons. The TTX-R and TTX-S currents differed in their steady-state inactivation, with 50% inactivation voltage at -40 +/- 5 mV (n = 10) for TTX-R currents and -70 +/- 4 mV (n = 10) for TTX-S currents. These current types also differed in their activation kinetics, with 50% activation values of -15 +/- 5 mV (n = 5) for TTX-R currents and -26 +/- 6 mV (n = 5) for TTX-S currents. The interactions of TTX-R and TTX-S channels with various pharmacological agents and divalent cations were studied. The Kd values for TTX-S and TTX-R currents were estimated to be 0.3 nM and 100 microM for TTX, 0.5 nM and 10 microM for saxitoxin, and 50 microM and 200 microM for lidocaine, respectively. TTX-S channels did not exhibit a marked use-dependent block by lidocaine, whereas lidocaine significantly decreased TTX-R current in a use-dependent manner at frequencies ranging from 1 to 33.3 Hz. Several external divalent cations exerted different effects on these current types.(ABSTRACT TRUNCATED AT 250 WORDS)     

Separation of the sodium and potassium channels in the surface membrane of mollusk nerve cells]

Characteristics of transmembrane ionic currents under controlled changes in ionic composition of extra-and intracellular medium were studied by means of intracellular dialysis and voltage clamp in isolated neurons from the molluscs Helix pomatia and Limnea stagnalis. The outward potassium currents were eliminated by replacement of intracellular potassium by Tris and the pure inward current could be measured. Replacement of the Ringer solution by NA-free or Ca-free solutions in the extracellular medium made it possible to separate the inward current into additive components, one of which is carried by sodium ions, and the other, by calcium ions. The sodium and calcium inward currents are shown to have different kinetics and potential dependence: taumNa = 1+/-0.5 ms, taumCa = = 3+/-1 ms, tauhNa = 8+/-2 ms, tauhCa = 115+/-10 ms when Vm = 0, GNa = 0.5 when Vm==-21+/-2 mV, GCa = 0.5 when Vm=-8+/-2 mV. Both currents were not altered by tetrodoxin (TTX), however calcium current is specifically blocked by externally applied calcium ions (2 X 10(-3) M), verapamil, D = 600 as well as by fluoride while introduced inside a cell. These data prove the existence of separate systems of sodium and calcium ion-conducting channels in the somatic membrane.     

Mechanisms of calcium current decay acceleration induced by cyclic AMP]

In isolated snail neurones the level of cyclic AMP was increased either by intracellular injection of cAMP or by extracellular application of dibutyryl-cAMP and the change of high-threshold calcium current (ICa) decay was investigated. In 20 from 38 neurones it was obtained that both fast and slow phases of ICa decay were accelerated 2-2.5 times as affected by cAMP. The effect did not depend on the test-pulse potential and was displayed on the IBa. In the double-pulse experiments it was shown that cAMP enhanced the influence of depolarized prepulses (Vc) on the ICa tested (It). Analysis of the It-Vc curve showed that cAMP enhanced both Ca(2+)-dependent and voltage-dependent inactivation of ICa. The experiments where the intervals between Vc and Vt varied have shown that cAMP slowed down the rate of Ca(2+)-channels recovery from inactivation. The results suggest that cAMP increases the affinity of the Ca(2+)-channel inactivating substrate for Ca(2+)-ions.     

Capsaicin differentially modulates voltage-activated calcium channel currents in dorsal root ganglion neurones of rats

It is discussed whether capsaicin, an agonist of the pain mediating TRPV1 receptor, decreases or increases voltage-activated calcium channel (VACC) currents (I(Ca(V))). I(Ca(V)) were isolated in cultured dorsal root ganglion (DRG) neurones of rats using the whole cell patch clamp method and Ba2+ as charge carrier. In large diameter neurones (>35 micorm), a concentration of 50 microM was needed to reduce I(Ca(V)) (activated by depolarizations to 0 mV) by 80%, while in small diameter neurones (< or =30 microm), the IC50 was 0.36 microM. This effect was concentration dependent with a threshold below 0.025 microM and maximal blockade (>80%) at 5 microM. The current-voltage relation was shifted to the hyperpolarized direction with an increase of the current between -40 and -10 mV and a decrease between 0 and +50 mV. Isolation of L-, N-, and T-type calcium channels resulted in differential effects when 0.1 microM capsaicin was applied. While T-type channel currents were equally reduced over the voltage range, L-type channel currents were additionally shifted to the hyperpolarized direction by 10 to 20 mV. N-type channel currents expressed either a shift (3 cells) or a reduction of the current (4 cells) or both (3 cells). Thus, capsaicin increases I(Ca(V)) at negative and decreases I(Ca(V)) at positive voltages by differentially affecting L-, N-, and T-type calcium channels. These effects of capsaicin on different VACCs in small DRG neurones, which most likely express the TRPV1 receptor, may represent another mechanism of action of the pungent substance capsaicin in addition to opening of TRPV1.     

Electrophysiological properties of neuroendocrine cells of the intact rat pars intermedia: multiple calcium currents

Intracellular recordings for current and voltage clamping were obtained from 130 neuroendocrine cells of the pars intermedia (PI) in intact pituitaries maintained in vitro. Spontaneous and evoked action potentials were blocked by TTX or by intracellular injection of a local anesthetic, QX-222. After potassium (K+) currents were blocked by tetraethylammonium (TEA), 4-aminopyridine, and intracellular cesium (Cs+), 2 distinct calcium (Ca2+) spikes were observed which were differentiated by characteristic thresholds, durations, and amplitudes. Both Ca2+ spikes were blocked by cobalt (Co2+) but were unaffected by TTX or QX-222. The low-threshold spike (LTS) had a smaller amplitude and inactivated when membrane potential was depolarized past -40 mV or when evoked at a fast rate (greater than 0.5 Hz). The high-threshold spike (HTS) typically had a larger amplitude and longer duration, was not inactivated at potentials which inactivated the LTS, and could be evoked at rates of up to 10 Hz. Single-electrode voltage-clamp analysis revealed that 3 distinct components of the Ca2+ current were present in most cells. From a negative holding potential (-90 mV), 2 separate peak inward currents were observed; a low-threshold transient current, similar to a T-type Ca2+ current, activated at -40 mV, whereas a large-amplitude inactivating current activated above -20 mV. This large inactivating Ca2+ current was significantly inactivated at a holding potential of -40 mV or by brief prepulses to positive potentials, and was similar to an N-type Ca2+ current. A sustained Ca2+ current (L-type) was observed which was not altered by different holding potentials.(ABSTRACT TRUNCATED AT 250 WORDS)     

The potential dependence of calcium current deactivation via the somatic membrane of sensory neurons in the mouse

Potential dependence of calcium inward current deactivation kinetics was studied in the somatic membrane of mouse dorsal root ganglion neurons by intracellular dialysis technique. The decay of the high-threshold calcium current upon repolarization was reasonably described by single-exponential process with the time constant tau less than or equal to 130 microseconds (V = = -80 mV), when the intracellular solution contained tris-PO4, and by two-exponential process (tau congruent to 0.1 and tau = 0.8 divided by ms, V = -80 mV), when the intracellular solution contained Cs-aspartate and EGTA. Both time constants were strongly voltage dependent. The amplitude of the fast component of the tail current had sigmoidal voltage dependence, and the slow component had V-shaped voltage dependence. The low-threshold calcium current deactivation occurs more slowly with high voltage dependent kinetics (tau = 1.1 divided by 1.2 ms, V = -160 mV). A dependence of low-threshold current deactivation time constant on the type of penetrating cation was observed. A kinetic model of calcium current deactivation was proposed considering three types of calcium channels presented in the somatic membrane of the neurons studied.     

Excitatory synaptic inputs on myenteric Dogiel type II neurones of the pig ileum

The synaptic input on myenteric Dogiel type II neurones (n = 63) obtained from the ileum of 17 pigs was studied by intracellular recording. In 77% of the neurones, electrical stimulation of a fibre tract evoked fast excitatory postsynaptic potentials (fEPSPs) with an amplitude of 6 +/- 5 mV (mean +/- S.D.) and lasting 49 +/- 29 ms. The nicotinic nature of the fEPSPs was demonstrated by superfusing hexamethonium (20 microM). High-frequency stimulation (up to 20 Hz, 3 seconds) did not result in a rundown of the fEPSPs, and did not evoke slow excitatory or inhibitory postsynaptic potentials. The effects of neurotransmitters, possibly involved in these excitatory responses, were investigated. Pressure microejection of acetylcholine (10 mM in pipette) resulted in a fast nicotinic depolarisation in 67%(18/27) of the neurones (13 +/- 9 mV, duration 7.0 +/- 7.2 seconds) as did 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) application (10 mM; 14 +/- 10 mV, duration 4.1 +/- 2.8 seconds) in 76% of the cells. The fast nicotinic response to acetylcholine was sometimes (6/27) followed by a slow muscarinic depolarisation (8 +/- 4 mV; duration 38.7 +/- 10.8 seconds). Immunostaining revealed 5-hydroxytryptamine hydrochloride (5-HT)- and calcitonin gene-related peptide (CGRP)-positive neuronal baskets distributed around and in close vicinity to Dogiel type II neuronal cell bodies. Microejection of 5-HT (10 mM) resulted in a fast nicotinic-like depolarisation (12 +/- 6 mV, duration 3.0 +/- 1.3 seconds) in 4 of 8 neurones tested, whereas microejection of CGRP (20 mM) gave rise to a slow muscarinic-like depolarisation (6 +/- 2 mV, duration 56.0 +/- 27.5 seconds) in 8 of 12 neurones tested. In conclusion, myenteric Dogiel type II neurones in the porcine ileum receive diverse synaptic input. Mainly with regard to the prominent presence of nicotinic responses, these neurones behave contrary to their guinea pig counterparts.