Tolbutamide enhances potential-dependent inactivation of calcium channels in snail neurons

A two-microelectrode voltage-clamp method was used to measure a high-threshold calcium current (I_Ca) on isolated snail neurons. Tolbutamide (1–5 mmole/liter) and H-8 (1–30 µmole/liter), inhibitors of kinase A, caused a decrease in the peak amplitude and accelerated I_Ca decay during a depolarizing stimulus. The half-life of the "slow" time constant of I_Ca decay decreased in the presence of tolbutamide, and was two to three times stronger than the half-life of the "fast" time constant. I_Ca inactivation curves plotted in a double-stimulus experiment have shown that after tolbutamide application, I_Ca inactivation elicited by the application of high-amplitude depolarizing pre-stimuli preferentially rises (V_c=+30 to +70 mV). The results suggest that dephosphorylation of Ca²⁺ channels enhances a potential-dependent component of the inactivation process.Scientific-Research Institute of the Brain, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 23, No. 5, pp. 515–519, September–October, 1991.     

Restoration of voltage-dependent antibrain antibody-inhibited calcium current by cyclic adenosine monophosphate

A dual-microelectrode voltage clamp technique was used for recording voltage-dependent calcium current (I_ca) in unidentified neurons isolated fromHelix pomatia. Neither intracellular injection of cyclic adenosine monophosphate (cAMP; 10 nA, 5 min) nor intracellular application of dibutyril-cAMP (dcAMP; 1 mM, 10–20 min) induced a change in normal I_ca or produce a reversible 10–20% reduction in amplitude. Adding S-100 protein fraction antibodies to the external medium led to the onset of calcium-dependent inactivation of I_ca, bringing amplitude of I_ca down to 15±12% of its initial level. Either cAMP or dcAMP then restored inhibited I_ca to 50±11% of its original level. It was found that the effects of cAMP on I_ca of intact neurons depend on level of cytoplasmic Ca²⁺.Institute for Brain Research, All-Union Center for Mental Health Research, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 21, No. 2, pp. 247–252, March–April, 1989.     

Mechanisms of the inhibitory effect of cyclic adenosine monophosphate on calcium current in intact mollusk neurons

Inhibitory effects of cyclic adenosine monophosphate (cAMP) on calcium current (I_Ca) were investigated in experiments on unidentified neurons isolated fromHelix pomatia by means of voltage clamping techniques using two microelectrodes. Intracellular level of cAMP was raised by intracellular injection of this substance or by extracellular application of dibutyryl-cAMP or isobutylmethyl-xanthine. A set of neurons showing inhibitory effects of cAMP on I_Ca was used. Effects on barium current (I_Ba) of an equal extent were also revealed. Injection of cGMP through a double-barreled microelectrode into these neurons produced an increase in amplitude of I_Ca. Intracellular application of phorbol ester had no effect on this current, however. Intracellular injection of EGTA led to enhancement of I_Ca amplitude, but the inhibitory effect of cAMP persisted following the action of EGTA. Tolbutamide and H-8 (but to a lesser extent) inhibited I_Ca. The inhibitory effects of tolbutamide and dibutyryl cAMP were not found to be cumulative in six out of twelve instances. These findings would imply that the inhibitory action of cAMP on I_Ca is unassociated with activation of cAMP-dependent protein kinase, cGMP-dependent protein kinase or protein kinase C; nor does it depend on level of intracellular Ca²⁺. The possibility of direct interaction between cAMP and channel-forming protein is considered.Institute of Brain Research, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 22, No. 1, pp. 54–61, January–February, 1990.     

Effects of cAMP on calcium currents in mollusk neurons differing in the sensitivity of their calcium conductance to serotonin action

The effects of cAMP and serotonin (5-HT) on calcium current (I_Ca) were investigated inHelix pomatia neurons using voltage clamp and intracellular perfusion techniques. Three types of neuronal response to extracellular application of 5-HT (1–10 µM) were found: reversible blockage of calcium conductance, absence of response, and increase in I_Ca amplitude. Intracellular application of exogenous cAMP was also found to produce an increase in I_Ca in cells stimulated by 5-HT action. Effects of 5-HT and cAMP were non-additive under these circumstances and were potentiated equally by cyclic nucleotide phosphodiesterase inhibitor. Applying cAMP led to no noticeable increase in I_Ca amplitude in cells with calcium conductance unchanged or blocked by 5-HT. Findings would indicate that the stimulating action of 5-HT is mediated by a rise in intracellular level of cAMP. It is postulated that two types of calcium channels differing in their dependence on cAMP metabolism exist; the presence of cAMP-dependent calcium channels at the neuronal membrane fits in with a certain type of 5-HT receptor also present in the cell, moreover. A new approach is suggested for research on isolated neurons, i.e., that of functional identification.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 22, No. 5, pp. 605–512, October–September, 1990.     

Effects of quinine on calcium current in mollusk neurons

The effects of quinine on the peak amplitude and the decay of calcium currents (I_Ca) were investigated in nonidentified neurons isolated fromHelix pomatia. A concentration of 1×10^–5–5×10^–4 M quinine was found to produce a reversible dose-dependent deceleration in the decline of I_Ca ("lead" effect) and a reversible, slowly evolving dose-dependent reduction in I_Ca amplitude ("lag" effect). A reduction in amplitude down to half control level is observed at a quinine concentration of 6 ×10^–5 M, while the current-voltage relationship of I_Ca shifts by 5–10 mV towards negative potentials. Results show that quinine successfully blocks calcium channels inHelix pomatia neurons.Institute of Brain Research, All-Union Mental Health Research Center, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 19, No. 3, pp. 413–417, May–June, 1987.     

Depressing action of nitroglycerin on voltage-activated calcium current in isolated smooth muscle cells of the guinea pig gut

The effect of nitroglycerin (NG) on inward voltage-activated calcium current (I _Ca) was studied in isolated smooth muscle cells (SMC) of the guinea pigtaenia coli with the voltage clamp technique in an intracellular dialysis mode. Addition of NG (10^–7 to 10^–4 M) to the extracellular solution reduced theI _Ca amplitude and increased theI _Ca inactivation rate. The maximum inhibition ofI _Ca (on the average, by 41.7 ± 4.8%,n=13) was produced by 10^–4 M NG; the inhibition was dose-dependent. No shift of theI _Ca current-voltage curve under the NG influence was observed. Application of dibutyril-cGMP (2·10^–4 M), a membrane-penetrating analog of cyclic 3,5-guanosine monophosphate (cGMP), likewise decreased theI _Ca amplitude and increased its inactivation rate. The results obtained suggest that the NG inhibitory effect onI _Ca is related to a cGMP-dependent modulation of the voltage-activated calcium channels of L-type in the SMC membrane in the guinea pigtaenia coli.Neirofiziologiya/Neurophysiology, Vol. 26, No. 3, pp. 218–222, May–June, 1994.     

Mechanism of action of nitric oxide donors on voltage-activated calcium channels in vascular smooth muscle cells

The effects of nitric oxide (NO) donors on inward barium current (I _Ba) in freshly isolated smooth muscle cells (SMC) of the guinea pig mesenteric artery and on inward calcium current (I _Ca) in SMC of the canine coronary artery were studied using a patch-clamp recording technique in whole-cell configuration. The inward current in SMC of the guinea pig artery was shown to flow through a single type of calcium channels, which have characteristics of high-threshold slowly inactivated channels of L-type. Nitroglycerin (NG) and sodium nitroprusside (NP) reversibly inhibitedI _Ba in a dose-dependent manner. Effects of NO donors onI _Ba were related to the changes in voltage-dependent properties of calcium channels. In particular, NG and NP accelerated the current inactivation, and their blocking effects increased with the membrane depolarization. Methylene blue, the guanylate cyclase inhibitor, decreased the inhibitory action of NG onI _Ba by a factor of 5. 8-Bromo-cGMP, the membrane-permeant cGMP analog, evokedI _Ba inhibition similar to that caused by NO donors. In the canine coronary artery, NO donors also inhibitedI _Ca flowing through the L-type calcium channels. It has been concluded that NO originating from NG and NP inhibits activity of L-type calcium channels in vascular SMC; it is possible that cGMP-dependent processes are involved.Neirofiziologiya/Neurophysiology, Vol. 28, No. 6, pp. 296–304, November–December, 1996.     

Changes in the ionic mechanisms of electrical excitability in the somatic membrane of rat dorsal root ganglion neurons during ontogenesis. Relationship between calcium channels and intracellular metabolism

It was found during experiments on rat sensory neurons that the relationship between high-threshold calcium channels and the system of intracellular cyclic nucleotide metabolism declined in the course of postnatal ontogenesis. Intracellullar administration of the cAMP-ATP-Mg²⁺ complex led to restoration after dialysis-induced decline in peak amplitude of high-threshold calcium currents in 70% of cells belonging to the first age group of 5–9-day-old animals, as against 26% of those examined in the 2nd (45-day-old) and only 10% of all those investigated in the third (90-day-old) group. Kinetics and voltage-dependence of high-threshold calcium current in the neuronal soma were identical in rats of all three age groups. The effect of recovery in calcium conductivity produced by intracellular application of the cAMP-ATP-Mg²⁺ complex was different in neurons with different inward current combinations. This recovery did not occur in cells with "fast" sodium and high-threshold calcium currents only. Conventional effects of intracellular cAMP application were seen in neurons mainfesting a "slow" TTX-resistant sodium inward current together with the two main inward currents.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vo.. 18, No. 6, pp. 827–832, November–December, 1986.     

Electrophysiological and Theoretical Analysis of Depolarization-Dependent Outward Currents in the Dendritic Membrane of an Identified Nonspiking Interneuron in Crayfish

Depolarization-dependent outward currents were analyzed using the single-electrode voltage clamp technique in the dendritic membrane of an identified nonspiking interneuron (LDS interneuron) in situ in the terminal abdominal ganglion of crayfish. When the membrane was depolarized by more than 20 mV from the resting potential (65.0 ± 5.7 mV), a transient outward current was observed to be followed by a sustained outward current. Pharmacological experiments revealed that these outward currents were composed of 3 distinct components. A sustained component (I _s) was activated slowly (half rise time > 5 msec) and blocked by 20 mM TEA. A transient component (I _t1) that was activated and inactivated very rapidly (peak time < 2.5 msec, half decay time < 1.2 msec) was also blocked by 20 mM TEA. Another transient component (I _t2) was blocked by 100 M 4AP, activated rapidly (peak time < 10.0 msec) and inactivated slowly (half decay time > 131.8 msec). Two-step pulse experiments have revealed that both sustained and transient components are not inactivated at the resting potential: the half-maximal inactivation was attained at –21.0 mV in I _t1, and –38.0 mV in I _t2. I _s showed no noticeable inactivation. When the membrane was initially held at the resting potential level and clamped to varying potential levels, the half-maximal activation was attained at –36.0 mV in I _s, –31.0 mV in I _t1 and –40.0 mV in I _t2. The activation and inactivation time constants were both voltage dependent. A mathematical model of the LDS interneuron was constructed based on the present electrophysiological records to simulate the dynamic interaction of outward currents during membrane depolarization. The results suggest that those membrane conductances found in this study underlie the outward rectification of the interneuron membrane as well as depolarization-dependent shaping of the excitatory synaptic potential observed in current-clamp experiments.     

Investigation of the effect of intracellular calcium on the cAMP-mediated increase in the calcium current

The experiments were conducted on intracellularly persfused neurons of the molluskHelix pomatia, in which a serotonin (5-HT)-induced increase in the calcium current (I_Ca), mediated by cAMP, is observed. It was established that desensitization of the cell to the action of 5-HT is due to an increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i). At [Ca²⁺]_i=10^–7 and 10^–6 M, the effect of 5-HT constituted 60 and 32% of this value in the control (in the presence of 10 mM EGTA in the intracellular solution), respectively; at [Ca²⁺]_i=10^–5 M, there was no effect of 5-HT at all. The addition of the calmodulin antagonist trifluoperazine (5·10^–5 mM) or blockers of phosphodiesterase (5 mM theophylline or 10^–4 M IBMX) to the perfusate sharply weakened the ability of calcium to inhibit the effect of 5-HT; at [Ca²⁺]_i=10^–5 M, in the presence of one of these compounds, the effect constituted 60–70% of its control value. It is concluded that the calcium-calmodulin-dependent phosphodiesterase is a key link in the interaction of the two-signal transduction pathway — Ca²⁺ and cAMP in modulation of the calcium-channel activity.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 3, pp. 306–313, May–June, 1991.     

Effects of substituting barium for calcium ions during research into inward currents in mammalian neurons

The effect of Ca²⁺ by Ba²⁺ in artificial extracellular fluid on high-threshold inward calcium current at the neuronal somatic membrane was investigated in rat spinal ganglia using techniques of intracellular dialysis and voltage clamping. An increase in the Ba²⁺ conductance of high-threshold calcium channels was found, assessed by the increase in maximum current amplitude. The fall in the latter occurring during dialysis and connected with washout of intracellular contents was considerably retarded when Ca²⁺ was replaced by Ba²⁺, probably due to the removal of the blocking effect of intracellular Ca²⁺ on the high-threshold calcium channels. It was discovered that the connection between high-threshold calcium channels and cyclic nucleotide metabolism remained unimpaired when Ca²⁺ is replaced by Ba²⁺.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 3, pp. 313–318, May–June, 1986.     

Effects of pressure overload-induced hypertrophy on TTX-sensitive inward currents in guinea pig left ventricle

We investigated the effects of pressure overload hypertrophy on inward sodium (I _Na) and calcium currents (I _Ca) in single left ventricular myocytes to determine whether changes in these current systems could account for the observed prolongation of the action potential. Hypertrophy was induced by pressure overload caused by banding of the abdominal aorta. Whole-cell patch clamp experiments were used to measure tetrodotoxin (TTX)-sensitive inward currents. The main findings were that I _Ca density was unchanged whereas I _Na density after stepping from –80 to –30 mV was decreased by 30% (–9.0 ± 1.16 pA pF^–1 in control and –6.31 ± 0.67 pA pF^–1 in hypertrophy, p < 0.05, n= 6). Steady-state activation/inactivation variables of I _Na, determined by using double-pulse protocols, were similar in control and hypertrophied myocytes, whereas the time course of fast inactivation of I _Na was slowed (p < 0.05) in hypertrophied myocytes. In addition, action potential clamp experiments were carried out in the absence and presence of TTX under conditions where only Ca²⁺ was likely to enter the cell via TTX-sensitive channels. We show for the first time that a TTX-sensitive inward current was present during the plateau phase of the action potential in hypertrophied but not control myocytes. The observed decrease in I _Na density is likely to abbreviate rather than prolong the action potential. Delayed fast inactivation of Na⁺ channels was not sustained throughout the voltage pulse and may therefore merely counteract the effect of decreased I _Na density so that net Na⁺ influx remains unaltered. Changes in the fast I _Na do not therefore appear to contribute to lengthening of the action potential in this model of hypertrophy. However, the presence of a TTX-sensitive current during the plateau could potentially contribute to the prolongation of the action potential in hypertrophied cardiac muscle. (Mol Cell Biochem 261: 217–226, 2004)     

Changes produced in intracellular calcium concentration and transmembrane currents by iontophoretic injection of cAMP in Helix pomatia neurons

Transmembrane currents and changed [Ca²⁺]_in produced by iontophoretic injection of cAMP were investigated in voltage clampedHelix pomatia neurons. The Fura-2 fluorescence probe technique was used to measure [Ca²⁺]_in. Injection of cAMP was found to produce a protracted rise in the latter at a membrane potential range of –40 to –100 mV in conjunction with transmembrane inward current. Duration of the changes in [Ca²⁺]_in largely dependent on neuronal size and varied between 50 and 500 sec (parameters for neurons with somata of around 100 and 40 µm respectively). In a medium with Ca²⁺ replaced by Mg²⁺ (as well as after addition of EDTA, a calcium chelator) both transmembrane current and the pattern of increase in [Ca²⁺]_in remained unchanged. Inward current usually declined substantially but degree of change in [Ca²⁺]_in remained the same when Na⁺ was eliminated from the solution by replacing its Tris⁺. Addition of 2 mM Cd²⁺ to the external medium hardly affected current level and increase in [Ca²⁺]_in. Neither procaine, a local anesthetic, nor ryanodine (which inhibits release of calcium from the intracellular store) changed the cAMP effects observed. A concentration of 1 mM La³⁺ depressed both inward current and the [Ca²⁺]_in increase. Findings would imply the occurrence of cAMP-dependent release of calcium from the intracellular store in the neurons tested.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 3, pp. 396–402, May–June, 1989.     

Apparent loss of calcium-activated potassium current in internally perfused snail neurons is due to accumulation of free intracellular calcium

Summary Internal perfusion ofHelix neurons with a solution containing potassium aspartate, MgCl₂, ATP, and HEPES causes the calcium-activated potassium current (I _K(Ca)) evoked by depolarizing voltage steps to decrease with time. When internal free Ca⁺⁺ is strongly buffered to 10^–7 m by including 0.5mm EGTA and 0.225mm CaCl₂ in the internal solution,I _K(Ca) remains constant for up to 3 hours of perfusion. In cells whereI _K(Ca) is small at the start of perfusion, perfusion with the strongly buffered 10^–7 m free Ca⁺⁺ solution produces increases inI _K(Ca) which ultimately saturate. In cells perfused with solutions buffered to 10^–6 m free Ca⁺⁺,I _K(Ca) is low and does not change with perfusion. These results lead us to conclude thatI _K(Ca) is stable in perfusedHelix neurons and that the apparent loss ofI _K(Ca) seen initially with perfusion is due to accumulation of cytoplasmic calcium. Since the calcium current (I _Ca) provides the Ca⁺⁺ which activatesI _K(Ca) during a depolarizing pulse,I _Ca is also stable in perfused cells when free intracellular Ca⁺⁺ is buffered.Perfusion with 1 m calmodulin (CaM) produces no effect onI _K(Ca) with either 10^–7 or 10^–6 m free internal calcium. Inhibiting endogenous CaM by including 50 m trifluoperazine (TFP) in both the bath and the internal perfusion solution also produces no effect onI _K(Ca) with 10^–7 m free internal calciu. It is concluded that CaM plays no role inI _K(Ca) activation.     

Slow low-threshold potassium current inHelix pomatia neurons

A low-threshold outward current was studied in the neurons ofHelix pomatia at –70 to –30 mV using a two-electrode voltage clamp technique. In addition to the well-known A current (I _A), a slower outward current calledI _As (slow) was revealed. Activation and inactivation times ofI _As at –40 mV ranged from 90 to 120 msec and from 3 to 5 sec, respectively. The current recovered within 2 to 5 sec after inactivation at –120 mV. Analysis of changes in the reversal potential ofI _As caused by an increase in external potassium concentration suggests a potassium origin forI _As. The curves ofI _As stationary activation and inactivation fit the Boltzmann equation. Deriving from an activation curve, the activation potential for a half-maximum current,, is –40 mV, and the slope factor,k, is –9.8 mV, while those values for the inactivation curve are –84 mV (a half-maximum inactivation) and 7.5 mV.I _As is blocked by 4-aminopyridine (1–30 µM), tetraethylammonium (1 mM), and Ba²⁺ (1 mM), but is resistant to Cs⁺ (1 mM). PeakI _As is not affected either by substitution of external Ca²⁺ for Mg²⁺ or by application of Cd²⁺ (0.5–1.0 mM). The results suggest that activation ofI _As does not require Ca²⁺ entry into the cell.Neirofiziologiya/Neurophysiology, Vol. 25, No. 6, pp. 427–432, November–December, 1993.     

Expression of low voltage-activated calcium channels inXenopus oocytes

Calcium channels were expressed inXenopus oocytes by means of messenger RNA extracted from the rat thalamo-hypothalamic complex, mRNA(h). Inward barium currents,I _Ba, were recorded in Cl^–-free extracellular solution with 40 mM Ba²⁺ as a charge carrier, using two-microelectrode technique. Depolarizations from a very negative holding potential (V _h=–120 mV) began to activateI _Ba at about –80 mV; this current peaked at –30 to –20 mV and reversed at +50 mV, indicating that I _Ba may be transferred through the low voltage-activated (LVA) calcium channels. The time-dependent inactivation of the current during a prolonged depolarization to –20 mV was quite slow, followed a single exponential decay with a time constant of 1550 msec, and contained a residual component constituting 30% of the maximum amplitude. The current could not be completely inactivated at any holding potential. As expected for LVA current, a steady-state inactivation curve was shifted towards negative potentials. It could be described by the Boltzmann's equation with the half-inactivation potential of –78 mV, slope factor of 15 mV, and residual level of 0.3. ExpressedI _Ba could be blocked by flunarizine (K _d=0.42 µM), nifedipine (K _d=10 µM), and amiloride at a 500 µM concentration. Among the inorganic Ca²⁺ channel blockers, the most potent was La³⁺ (K _d=0.48 µM), while Cd²⁺ and Ni²⁺ were not very selective and almost thousand-fold less effective (K _d=0.52 mM andK _d=0.62 mM, respectively) than La³⁺. Our data show that mRNA(h) induces expression in the oocytes of almost exclusively LVA Ca²⁺ channels with voltage-dependent and pharmacological properties very similar to those observed for T-type Ca²⁺ current in native hypothalamic neurons, though kinetic properties of the expressed and natural currents are somewhat different.Neirofiziologiya/Neurophysiology, Vol. 27, No. 3, pp. 183–189, May–June, 1995.     

Ionic mechanisms for the antiarrhythmic action of cinnamophilin in rat heart

We investigated the electrophysiological effect and antiarrhythmic potential of cinnamophilin (Cinn), a thromboxane A₂ antagonist isolated fromCinnamomum philippinense, on rat cardiac tissues. Action potential and ionic currents in single rat ventricular cells were examined by current clamp or voltage clamp in a whole-cell configuration. In 9 episodes of ischemia-reperfusion arrhythmia, 10 µM Cinn converted 6 of them to normal sinus rhythm. Cinn suppressed the maximal rate of rise of the action potential upstroke (V_max) and prolonged the action potential duration at 50% repolarization (APD₅₀). Voltage clamp study showed that the suppression of V_max by Cinn was associated with an inhibition of sodium inward current (I_Na, IC₅₀=10.0 ± 0.4 µM). At 30 µM, V_1/2 for the steady-state inactivation curve of I_Na was shifted from –84.1 ± 0.2 to –93.0 ± 0.5 mV. Cinn also reduced calcium inward current (I_Ca) dose-dependently with an IC₅₀ value of 9.5 ± 0.3 µM. Cinn (10 µM) reduced the I_Ca with a negative shift of V_1/2 for the steady-state inactivation curve of I_Ca from –32.2 ± 0.3 to –50.7 ± 0.4 mV. The prolongation of APD₅₀ was associated with an inhibition of the integral of potassium outward current with IC₅₀ values between 4.8 and 7.1 µM. At 10 µM, Cinn reduced I_Na without a negative shift of its voltage-dependent steady-state inactivation curves. The inhibition of transient outward current (I_to) by Cinn (3–30 µM) was associated with an acceleration of its time constant of inactivation and negative shift of its potential-dependent steady-state inactivation curves. The equilibrium dissociation constant (K_d) of Cinn to inhibit open state I_to channels, as calculated from the time constant of developing block, was 18.3 µM. The time constant of recovery of I_to from inactivation state was unaffected by Cinn. The rate constant for the relief from the depolarization-dependent block of I_to was calculated to be 23.9 ms. As compared with its effect on I_to, Cinn exerted about half the potency to block I_Na and I_Ca. These results indicate that the inhibition of I_Na, I_Ca and I_to may contribute to the antiarrhythmic activity of Cinn against ischemia-reperfusion arrhythmia.     

Phenylalanine intraperitoneal injection effect on high-voltage calcium channels in rat hippocampal neurons

Neurons from the hippocampus of one-day-old (controls) rats intraperitoneally injected with phenylalanine were isolated and cultured [3, 8]. The potential-dependent calcium input channel (I_Ca) in hippocampal neurons of controls after five-seven days of culture was made up of two components, low and high voltage, when the membrane potential was fixed under intracellular perfusion. The high-voltage channel was 69±13% of the total calcium channel at Vt = –10 mV. After rats were injected with phenylalanine, the high-voltage I_Ca significantly decreased to 32±14% of the total channel in the neurons at the same Vt. The low-voltage I_Ca did not change significantly.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 1, pp. 98–104, January–February, 1991.     

Bisphenol A Modulates Calcium Currents and Intracellular Calcium Concentration in Rat Dorsal Root Ganglion Neurons

The endocrine-disrupting chemical bisphenol A (BPA) is used to manufacture plastics including food containers, and it may leach into these containers. Consumption of BPA that has leached out of plastics may be harmful as recent research highlighted that BPA can induce alterations in the nervous system. In the present work, we studied the effects of BPA on Ca²⁺ channels in dorsal root ganglion (DRG) neurons. Using whole-cell patch-clamp recordings, we found that I _Ca could be reduced by BPA in a concentration-dependent manner. Additionally, BPA shifted the activation curve of calcium currents toward a depolarizing direction and increased the slope factor of the curve. The inactivation curve for the currents was also assessed, and the curve shifted toward the depolarizing direction, although it was not significant. Moreover, inhibitory effects of BPA on the increments of intracellular Ca²⁺ concentrations ([Ca²⁺]_i) induced by 50 mM KCl were observed in DRG neurons using a laser scanning confocal microscopy assay. Further work revealed that the PKA and PKC pathways may be involved in the inhibitory effects of BPA since the PKA antagonist GÖ-6983 and the PKC antagonist H-89 significantly alleviated the inhibitory effects of BPA on I _Ca. As such, the results of the present study provide direct evidence that BPA decreases I _Ca and impairs calcium homeostasis, which may be involved in any toxic effects of BPA on DRG neurons.     

Effect of intracellular administration of L-tyrosine and L-phenylalanine upon voltage-dependent calcium current in PC 12 pheochromocytoma cells

Using the voltage clamp technique under conditions of intracellular perfusion, we investigated changes in high-threshold voltage-dependent calcium current (I_Ca) in the surface membrane of PC 12 cells caused by intracellular administration of the aromatic amino acids L-tyrosine and L-phenylalanine. Administration of L-tyrosine (20 mmole/1) prevented decrease in I_Ca caused by perfusion of the cell with an artificial saline solution and had a transient restorative effect upon the cell. Administration of L-phenylalanine (20 mM) quickened the decrease in amplitude of I_Ca observed in the control. These effect of aromatic amino acids are maintained when ATP (2 mM) in the intracellular solution is replaced by an equivalent quantity of ADP. The tyrosine hydroxylase blocker -methyl D,L-tyrosine (20 MM) had an effect upon I_Ca analogous to that of L-tyrosine.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 1, pp. 105–111, January–February, 1991.