Role of reactive oxygen species in the signalling cascade of cyclosporine A-mediated up-regulation of eNOS in vascular endothelial cells

1. The action of mibefradil was studied on wild type class A calcium (Ca2+) channels and various class A/L-type channel chimaeras expressed in Xenopus oocytes. The mechanism of Ca2+ channel block by mibefradil was evaluated with two microelectrode voltage clamp. 2. Resting-state dependent block (or initial block) of barium currents (IBa) through class A Ca2+ channels was concentration dependent with an IC50 value of 208+/-23 microM. 3. Mibefradil (50 microM) did not significantly affect the midpoint voltage of the steady-state inactivation curve suggesting that inactivation does not promote Ca2+ channel block. Chimaeric class A/L-type Ca2+ channels inactivating with faster or slower kinetics than wild type class A channels were equally well inhibited by mibefradil as wild type class A channels. 4. Frequent Ca2+ channel activation facilitated IBa inhibition by mibefradil (use-dependent block). Recovery from use-dependent block was voltage-dependent, being slower at depolarized membrane potentials (tau = 75+/-15 s at -70 mV, (n=6) vs tau = 20+/-2 s at -100 mV, (n=6), P<0.05). 5. We suggest that use-dependent block of class A Ca2+ channels by mibefradil occurs because of slow recovery from open channel block (SROB) and not because of drug binding to inactivated channels. 6. Voltage-dependent slow recovery from open state-dependent block provides a molecular basis for understanding the cardiovascular profile of mibefradil such as selectivity for vasculature and relative lack of negative inotropic effects.     

Developmental features of human striatal tissue transplanted in a rat model of Huntington''s disease

The actions of the novel calcium (Ca2+) channel antagonist mibefradil (Ro 40-5967), a selective T-type channel blocker in myocardium, were investigated in embryonic rat spinal motoneurones maintained in culture. Whole-cell currents were recorded with the patch-clamp technique. Motoneurones displayed transient, low-voltage-activated (LVA) and, more sustained, high-voltage-activated (HVA) Ca2+ currents. The LVA currents were small and preferentially blocked by amiloride and low doses of nickel. Most of the HVA Ca2+ current flowed through N-type Ca2+ channels, while L-, and P/Q-type channels represented a smaller fraction. Mibefradil caused a rapid and reversible dose-dependent block of inward Ca2+ channel currents. Inhibition was nearly complete at 10 microM, suggesting mibefradil blockade of all subclasses of Ca2+ channels. The IC50 was approximately 1.4 microM on currents measured at 0 mV, from a holding potential of -90 mV. Inhibition of LVA Ca2+ current occurred over the same contraction range. Slow tail currents induced by the dihydropyridine agonist Bay K 8644 were also blocked by mibefradil, although with a slightly lower potency (IC50 = 3.4 microM). These broad inhibitory effects of mibefradil on Ca2+ influx were also supported by the strong inhibition of depolarization-induced intracellular calcium transients, measured from Indo-1 loaded motoneurones imaged with confocal microscopy. We conclude that mibefradil has potent blocking effects on Ca2+ channels in mammalian motoneurones. We hypothesize that therapeutic and pharmacological effects of mibefradil may involve actions on Ca2+ channels other than type T.     

Gonadotropin response to naloxone in the mare: effect of time of year and reproductive status

Effects of a novel dihydropyridine type of antihypertensive drug, cilnidipine, on the regulation of the catecholamine secretion closely linked to the intracellular Ca2+ were examined using nerve growth factor (NGF)-differentiated rat pheochromocytoma PC12 cells. By measuring catecholamine secretion with high-performance liquid chromatography coupled with an electrochemical detector, we showed that high K+ stimulation evoked dopamine release from PC12 cells both before and after NGF treatments. Cilnidipine depressed dopamine release both from NGF-treated and untreated PC12 cells in a concentration-dependent manner. In contrast, inhibition by nifedipine was markedly decreased in the differentiated PC12 cells. With intracellular Ca2+ concentration ([Ca2+]i) measurements using fura 2, the elevation of high K+-evoked [Ca2+]i was separated into nifedipine-sensitive and -resistant components. The nifedipine-resistant [Ca2+]i increase was also blocked by cilnidipine, as well as omega-conotoxin-GVIA. By the use of the conventional whole-cell patch-clamp technique, the compositions of the high-voltage-activated Ca2+ channel currents in the NGF-treated PC12 cells were divided into types: L-type, N-type, and residual current components. It was also estimated that cilnidipine at 1 and 3 micromol/L strongly blocked the N-type current without affecting the residual current. These results suggest that cilnidipine inhibits catecholamine secretion from differentiated PC12 cells by blocking Ca2+ influx through the N-type Ca2+ channel, in addition to its well-known action on the L-type Ca2+ channel.     

Regulation of mouse skeletal muscle L-type Ca2+ channel by activation of the insulin-like growth factor-1 receptor

We investigated the modulation of the skeletal muscle L-type Ca2+ channel/dihydropyridine receptor in response to insulin-like growth factor-1 receptor (IGF-1R) activation in single extensor digitorum longus muscle fibers from adult C57BL/6 mice. The L-type Ca2+ channel activity in its dual role as a voltage sensor and a selective Ca2+-conducting pore was recorded in voltage-clamp conditions. Peak Ca2+ current amplitude consistently increased after exposure to 20 ng/ml IGF-1 (EC50 = 5.6 +/- 1.8 nM). Peak IGF-1 effect on current amplitude at -20 mV was 210 +/- 18% of the control. Ca2+ current potentiation resulted from a shift in 13 mV of the Ca2+ current-voltage relationship toward more negative potentials. The IGF-1-induced facilitation of the Ca2+ current was not associated with an effect on charge movement amplitude and/or voltage distribution. These phenomena suggest that the L-type Ca2+ channel structures involved in voltage sensing are not involved in the response to the growth factor. The modulatory effect of IGF-1 on L-type Ca2+ channel was blocked by tyrosine kinase and PKC inhibitors, but not by a cAMP-dependent protein kinase inhibitor. IGF-1-dependent phosphorylation of the L-type Ca2+ channel alpha1 subunit was demonstrated by incorporation of [gamma-32P]ATP to monolayers of adult fast-twitch skeletal muscles. IGF-1 induced phosphorylation of a protein at the 165 kDa band, corresponding to the L-type Ca2+ channel alpha1 subunit. These results show that the activation of the IGF-1R facilitates skeletal muscle L-type Ca2+ channel activity via a PKC-dependent phosphorylation mechanism.     

Single L-type calcium channels in smooth muscle cells from resistance arteries of spontaneously hypertensive rats

The amplitude of the whole-cell L-type Ca2+ channel current recorded from vascular smooth muscle cells is reportedly greater in spontaneously hypertensive rats (SHR) than in Wistar-Kyoto rats (WKY). However, no study has examined properties of single Ca2+ channels in arterial cells from these strains. To further test the hypothesis that activation of L-type Ca2+ channels in arterial smooth muscle cells would be enhanced in SHR, we recorded single Ca2+ channel currents in resistance mesenteric artery cells from SHR and WKY (8 to 9 weeks of age) using a cell-attached patch clamp technique. With 50 mmol/L Ba2+ in the recording pipette, the depolarizing pulse from a holding potential of -40 mV evoked the single L-type Ca2+ channel current. Opening of the single channels was more frequent in cells from SHR than from WKY. Single-channel conductance (20 pS) and open time (1 ms at 0 mV) did not differ in the two strains. The results suggest that an increased amplitude of the whole-cell current can be attributed to the enhanced opening of single Ca2+ channels in the arterial smooth muscle cells from SHR compared with WKY.     

Primary central nervous system lymphomas in immunocompetent individuals: histology, Epstein-Barr virus genome, Ki-67 proliferation index, p53 and bcl-2 gene expression

We investigated the effects of potassium channel inhibitors on electrical activity, membrane ionic currents, intracellular calcium concentration ([Ca2+]i) and hormone release in GH3/B6 cells (a line of pituitary origin). Patch-clamp recordings show a two-component after hyperpolarization (AHP) following each action potential (current clamp) or a two-component tail current (voltage-clamp). Both components can be blocked by inhibiting Ca2+ influx. Application of D-tubocurarine (dTc) (20-500 microM) reversibly suppressed the slowly decaying Ca2+-activated K+ tail current (I AHPs) in a concentration-dependent manner. On the other hand, low doses of tetraethylammonium ions (TEA+) only blocked the rapidly decaying voltage- and Ca2+-activated K+ tail current (I AHPf). Therefore, GH3/B6 cells exhibit at least two quite distinct Ca2+-dependent K+ currents, which differ in size, voltage- and Ca2+-sensitivity, kinetics and pharmacology. These two currents also play quite separate roles in shaping the action potential. d-tubocurarine increased spontaneous Ca2+ action potential firing, whereas TEA increased action potential duration. Thus, both agents stimulated Ca2+ entry. I AHPs is activated by a transient increase in [Ca2+]i such as a thyrotrophin releasing hormone-induced Ca2+ mobilization. All the K+ channel inhibitors we tested: TEA, apamin, dTC and charybdotoxin, stimulated prolactin and growth hormone release in GH3/B6 cells. Our results show that I AHPs is a good sensor for subplasmalemmal Ca2+ and that dTc is a good pharmacological tool for studying this current.     

Comprehensive study on the membrane currents of porcine granulosa cells in culture

The membrane currents of primary cultured porcine granulosa cells have been studied using the whole-cell configuration of the patch-clamp technique. And effects of K+ channel blockers upon progesterone production of the cells have been also studied. The author has identified and characterized two types of K+ currents, transient outward current (Ito) and a delayed rectifier K+ current (Ik), and Ca2+ current (Ica). Ito and Ik were voltage -and calcium-dependent. Both of the currents were blocked by 4-aminopyridine (4-AP), a K+ channel blocker, but only Ik was sensitive to tetraethylammonium (TEA), another K+ channel blocker. Ica was inactivated within 50 ms of the test pulse. Nifedipine and verapamil, L-type Ca2+ channel blockers, did not suppress Ica even at a concentration of 10 microM. Tetramethrin (1 microM), a T-type Ca2+ channel blocker, decreased Ica. These findings suggested that the current was T-type Ca2+ current. LH and dibutyryl cAMP, potent stimulants of steroid production, attenuated Ito by 13.9 +/- 1.8% (n = 7) and 21.0 +/- 1.5% (n = 4), respectively. However, they did not affect Ik and Ica. These results indicated that LH did not modulate Ca2+ current directly, but it suppressed Ito through cAMP. 4-AP (0.2-5 mM) suppressed basal and LH-induced progesterone production of porcine granulosa cells dose-dependently, but TEA (2-10 mM) did not influence progesterone production. These data suggest that Ito may play a role in steroid secretion or other functions in granulosa cells.     

Activation of calcium channels by cAMP in STC-1 cells is dependent upon Ca2+ calmodulin-dependent protein kinase II

Activation of L-type calcium channels in the neuroendocrine, cholecytstokinin-secreting cell line, STC-1, is vital for secretion of CCK. In the present study, the regulation of L-type Ca2+ channels by cAMP and Ca2+ calmodulin dependent protein kinase II (CaM-KII) in STC-1 cells was investigated. Exposure to 3-isobutyl-1-methylxanthine (IBMX) increased intracellular cAMP levels, whole cell Ca2+ currents and activated Ca2+ channels in cell-attached membrane patches. Furthermore, in Fura-2AM loaded cells, cytosolic Ca2+ levels increased upon exposure to IBMX. By contrast, pretreatment of cells with the CaM-KII inhibitor KN-62, prevented IBMX activation of Ca2+ channels in cell-attached patches or increases in cytosolic Ca2+ levels. Inclusion of the synthetic peptide fragment 290-309 of CaM-KII, a CaM-KII antagonist, in the pipette solution, blocked the activation of whole cell Ca2+ currents upon addition of IBMX. These results indicate a unique mechanism of L-type Ca2+ channel activation involving two phosphorylation events.     

Modulation of big K+ channel activity by ryanodine receptors and L-type Ca2+ channels in neurons

As metabotropic glutamate receptor type 1 (mGluR1) is known to couple L-type Ca2+ channels and ryanodine receptors (RyR, Chavis et al., 1996) in cerebellar granule cells, we examined if such a coupling could activate a Ca2+-sensitive K+ channel, the big K+ (BK) channel, in cultured cerebellar granule cells. We observed that (+/-)-1-amino-cyclopentane-trans-1,3-dicarboxylic acid (t-ACPD) and quisqualate (QA) stimulated the activity of BK channels. On the other hand, (2S, 3S, 4S)-alpha-carboxycyclopropyl-glycine (L-CCG-I) and L-(+)-2-amino-4-phosphonobutyrate (L-AP4) had no effect on BK channels, indicating a specific activation by group I mGluRs. Group I mGluRs stimulation of the basal BK channel activity was mimicked by caffeine and both effects were blocked by ryanodine and nifedipine. Interestingly, carbachol stimulated BK channel activity but through a pertussis toxin (PTX)-sensitive pathway that was independent of L-type Ca2+ channel activity. Our report indicates that unlike the muscarinic receptors, group I mGluRs activate BK channels by mobilizing an additional pathway involving RyR and L-type Ca2+ channels.     

A two-step mechanism for recruitment of Pip by PU.1

Vasoactive effects of soluble matrix proteins and integrin-binding peptides on arterioles are mediated by alphav beta3 and alpha5 beta1 integrins. To examine the underlying mechanisms, we measured L-type Ca2+ channel current in arteriolar smooth muscle cells in response to integrin ligands. Whole-cell, inward Ba2+ currents were inhibited after application of soluble cyclic RGD peptide, vitronectin (VN), fibronectin (FN), either of two anti-beta3 integrin antibodies, or monovalent beta3 antibody. With VN or beta3 antibody coated onto microbeads and presented as an insoluble ligand, current was also inhibited. In contrast, beads coated with FN or alpha5 antibody produced significant enhancement of current after bead attachment. Soluble alpha5 antibody had no effect on current but blocked the increase in current evoked by FN-coated beads and enhanced current when applied in combination with an appropriate IgG. The data suggest that alphavbeta3 and alpha5 beta1 integrins are differentially linked through intracellular signaling pathways to the L-type Ca2+ channel and thereby alter control of Ca2+ influx in vascular smooth muscle. This would account for the vasoactive effects of integrin ligands on arterioles and provide a potential mechanism for wound recognition during tissue injury.     

L-type calcium current activation in cultured human myotubes

The time course of activation of the skeletal muscle L-type calcium channel was studied in voltage-clamped myotubes derived from human satellite cells. The slow L-type current was isolated by inactivating faster calcium current components using appropriate prepulses or by subtracting the currents not blocked by 5 microM nifedipine. The L-type current exhibited a single exponential activation and time constants which showed little voltage dependence in the range +10 to +50mV. Currents blocked by nifedipine could be partially restored by UV-light flash photolysis. When a flash of light was applied during a depolarizing step, the activation time course of the resulting inward current contained a rapid, almost instantaneous component followed by a slower component. The amplitude of the rapid component was different when the flash was applied at different times during the depolarizing step: depolarization first increased and then decreased the fraction of channels which could rapidly be restored from the block by photolysis. Plotted versus time after the onset of the depolarization this fraction closely matched the time course of the L-type current obtained before the block by nifedipine. This indicates that the slow gating recations of the Ca2+ channel remain functional in the nifedipine-blocked state. Large conditioning depolarizations which had been shown to enhance the speed of L-type current activation in frog muscle fibres showed no effect in human myotubes. Numerical simulations using a gating scheme proposed for frog muscle demonstrate that such differences can be caused by changing just a single kinetic parameter.     

Inhibition by mibefradil, a novel calcium channel antagonist, of Ca(2+)- and volume-activated Cl- channels in macrovascular endothelial cells

1. We have studied the effects of mibefradil, a novel calcium antagonist, on the resting potential and ion channel activity of macrovascular endothelial cells (calf pulmonary artery endothelial cells, CPAE). The patch clamp technique was used to measure ionic currents and the Fura-II microfluorescence technique to monitor changes in the intracellular Ca2+ concentration, [Ca2+]i. 2. Mibefradil (10 microM) hyperpolarized the membrane potential of CPAE cells from its mean control value of -26.6 +/- 0.6 mV (n = 7) to -59.8 +/- 1.7 mV (n = 6). A depolarizing effect was observed at higher concentrations (-13.7 +/- 0.6 mV, n = 4, 30 microM mibefradil). 3. Mibefradil inhibited Ca(2+)-activated Cl- currents, ICl,Ca, activated by loading CPAE cells via the patch pipette with 500 nM free Ca2+ (Ki = 4.7 +/- 0.18 microM, n = 8). 4. Mibefradil also inhibited volume-sensitive Cl- currents, ICl,vol, activated by challenging CPAE cells with a 27% hypotonic solution (Ki = 5.4 +/- 0.22 microM, n = 6). 5. The inwardly rectifying K+ channel, IRK, was not affected by mibefradil at concentrations up to 30 microM. 6. Ca2+ entry activated by store depletion, as assessed by the rate of [Ca2+]i-increase upon reapplication of 10 mM extracellular Ca2+ to store-depleted cells, was inhibited by 17.6 +/- 6.5% (n = 8) in the presence of 10 microM mibefradil. 7. Mibefradil inhibited proliferation of CPAE cells. Half-maximal inhibition was found at 1.7 +/- 0.12 microM (n = 3), which is similar to the concentration for half-maximal block of Cl- channels. 8. These actions of mibefradil on Cl- channels and the concomitant changes in resting potential might, in addition to its effect on T-type Ca2+ channels, be an important target for modulation of cardiovascular function under normal and pathological conditions.     

A single non-L-, non-N-type Ca2+ channel in rat insulin-secreting RINm5F cells

Single high-voltage-activated (HVA) Ca2+ channel activity was recorded in rat insulinoma RINm5F cells using cell-attached and outside-out configurations. Single-channel recordings revealed three distinct Ca2+ channel subtypes: one sensitive to dihydropyridines (DHPs)-(L-type), another sensitive to omega -conotoxin (CTx)-GVIA (N-type) and a third type insensitive to DHPs and omega -CTx-GVIA (non-L-, non-N-type). The L-type channel was recorded in most patches between -30 and +30 mV. The channel had pharmacological and biophysical features similar to the L-type channels described in other insulin-secreting cells (mean conductance 21 pS in control conditions and 24 pS in the presence of 5 microM Bay K 8644). The non-L-, non-N-type channel was recorded in cells chronically treated with omega -CTx-GVIA in the presence of nifedipine to avoid the contribution of N- and L-type channels. Channel activity was hardly detectable below -10 mV and was recruited by negative holding potentials (< -90 mV). The channel open probability increased steeply from -10 to + 40 mV. Different unitary current sublevels could be detected and the current voltage relationship was calculated from the higher amplitude level with a slope conductance of 21 pS. Channel activity lasted throughout depolarizations of 300-800ms with little sign of inactivation. Above 0 mV the channel showed a persistent flickering kinetics with brief openings (tau o 0.6 ms) and long bursts (tau burst 60 ms) interrupted by short interburst intervals. The third HVA Ca2+ channel subtype, the N-type, had biophysical properties similar to the non-L-, non-N-type and was best identified in outside-out patches by its sensitivity to omega -CTx-GVIA. The channel was detectable only above -10 mV from a -90 mV holding potential, exhibited a fast flickering behaviour, persisted during prolonged depolarizations and had a slope conductance of about 19 pS. The present data provide direct evidence for a slowly inactivating non-L-, non-N-type channel in insulin-secreting RINm5F cells that activates at more positive voltages than the L-type channel and indicate the possibility of identifying unequivocally single HVA Ca2+ channels in cell-attached and excised membrane patches under controlled pharmacological conditions.     

Cloning and functional expression of a voltage-gated calcium channel alpha1 subunit from jellyfish

Voltage-gated Ca2+ channels in vertebrates comprise at least seven molecular subtypes, each of which produces a current with distinct kinetics and pharmacology. Although several invertebrate Ca2+ channel alpha1 subunits have also been cloned, their functional characteristics remain unclear, as heterologous expression of a full-length invertebrate channel has not previously been reported. We have cloned a cDNA encoding the alpha1 subunit of a voltage-gated Ca2+ channel from the scyphozoan jellyfish Cyanea capillata, one of the earliest existing organisms to possess neural and muscle tissue. The deduced amino acid sequence of this subunit, named CyCaalpha1, is more similar to vertebrate L-type channels (alpha1S, alpha1C, and alpha1D) than to non-L-type channels (alpha1A, alpha1B, and alpha1E) or low voltage-activated channels (alpha1G). Expression of CyCaalpha1 in Xenopus oocytes produces a high voltage-activated Ca2+ current that, unlike vertebrate L-type currents, is only weakly sensitive to 1,4-dihydropyridine or phenylalkylamine Ca2+ channel blockers and is not potentiated by the agonist S(-)-BayK 8644. In addition, the channel is less permeable to Ba2+ than to Ca2+ and is more permeable to Sr2+. CyCaalpha1 thus represents an ancestral L-type alpha1 subunit with significant functional differences from mammalian L-type channels.     

Butanedione monoxime promotes voltage-dependent inactivation of L-type calcium channels in heart. Effects on gating currents

The effect of 20 mM extracellularly applied 2,3-Butanedione monoxime (BDM) on L-type Ca2+ channel charge movement current was studied in whole-cell voltage-clamped guinea-pig ventricular myocytes. Intramembraneous charge movement in response to depolarizing pulses (charge 1), was reduced after the application of BDM. The effect was more pronounced at the OFF of the charge transient (41%) than at the ON (7%). The steady-state availability curve of charge 1 was shifted to the left; the magnitude of the voltage shift was similar to the shift in Ca2+ current availability. Charge movement recorded in the negative voltage range (charge 2) after conditioning depolarizing pulses of different duration, was increased by BDM. For a 300-ms conditioning pulse charge 2 measured during a negative test pulse increased 40% (in Ba2+ external solution) or 35% (in Ca2+ external solution). These results show that BDM promotes voltage-dependent inactivation of L-type Ca2+ channels in parallel with charge interconversion between intramembranous charges 1 and 2. Mechanistically they are consistent either with dephosphorylation or a dihydropyridine-like action, but argue against open channel block as the mechanism of the effect of the drug.     

Multiple mechanisms of activating Ca2+ entry in freshly isolated rabbit aortic endothelial cells

In Fura-2-loaded, freshly isolated rabbit aortic endothelial cells the Ca2+ entry pathway was investigated using the Mn2(+)-quenching technique. Acetylcholine (ACh) interaction with muscarinic receptors activated Mn2+ influx through the plasma membrane. Sarcoplasmic-endoplasmic reticulum Ca2+ ATPase blockers such as cyclopiazonic acid (CPA), thapsigargin and BHQ, which block the endoplasmic reticulum Ca2+ pump and do not interact with receptors, also activated Mn2+ influx. Mn2+ influx activated by either ACh or CPA was blocked by the following agents: SKF96365, a receptor-operated Ca2+ channel (ROC) blocker; NCDC, a PLC and ROC blocker, and genistein, a tyrosine kinase inhibitor. D600, the L-type Ca2+ channel blocker, had no significant effect on Mn2+ influx. Caffeine blocked the ACh-induced Ca2+ release but had no effect on the ACh-induced Mn2+ influx. Similarly dantrolene, which blocked intracellular Ca2+ release induced by ACh, did not affect the ACh-activated Mn2+ influx. These data suggest that ACh can activate Ca2+ influx without depletion of the ACh-sensitive intracellular Ca2+ store. It is concluded (1) that in freshly isolated endothelial cells depletion of the intracellular Ca2+ store is not necessary for ACh-activated Ca2+ influx, and (2) that receptor activation and intracellular Ca2+ store depletion may activate the same Ca2+ entry pathway through parallel mechanisms.     

The presence of Human Papillomavirus (HPV) in microinvasive in situ spinocellular carcinoma of the oral cavity. Preliminary report

To determine whether functional Ca2+ channels are present in vestibular dark cells, changes in intracellular Ca2+ concentration ([Ca2+]i) due to K+ applications were measured using the Ca(2+)-sensitive dye (fura-2) and patchclamp whole-cell recordings were made in dark cells isolated from the ampullae of the semicircular canal of the guinea pig. Exchange of the external solution with a buffer medium containing a high K+ concentration (80 mM K+ or 150 mM K+) caused a concentration-dependent increase in [Ca2+]i in vestibular dark cells. Application of 1 microM nifedipine as a Ca2+ channel antagonist completely blocked the increase in [Ca2+]i. Further treatment with 10 microM BAY K 8644 as a Ca2+ channel agonist caused an increase in [Ca2+]i. In the patch-clamp whole-cell recordings a 1-s depolarizing pulse given into the dark cell in the presence of a high barium concentration (50 mM Ba2+) induced an inward current. In determining the current-voltage relationship, a current was detected at a potential that depolarized at-50 mV and was maximal at +10 mV. This inward current was completely blocked by 1 mM La3+ as a Ca2+ channel antagonist. These findings suggest the presence of voltage-dependent Ca2+ channels in dark cells, which have a presumed function in the regulation of [Ca2+]i in the vestibular endolymph.     

Gadolinium blocks the delayed rectifier potassium current in isolated guinea-pig ventricular myocytes

The effect of Gd3+ on the delayed rectifier potassium current (IK) in single guinea-pig ventricular myocytes was tested using whole-cell patch-clamp techniques. It was found that Gd3+ blocked 70% of the IK tail current at a concentration of 100 microM. The EC50 was 24 microM. Action potential durations were, however, reduced, consistent with a predominant effect on depolarizing L-type Ca2+ current (Ica.L). In the presence of 5 microM nifedipine Gd3+ prolonged the action potential. Using carbon fibres to stretch cells we observed that 10 microM Gd3+ was not effective in reducing a large stretch-activated increase in resting calcium. Modelling studies using the OXSOFT HEART program suggest that this lack of response is influenced by blockade of repolarizing current but is best reproduced by additional blockade of Ca2+ extrusion via the Na(+)-Ca2+ exchanger. When Gd3+ is used as a blocker of stretch-activated channels its actions upon both Ica.L and IK must therefore be accounted for.     

L-type Ca2+ channel is involved in the regulation of spontaneous transmitter release at developing neuromuscular synapses

Involvement of an L-type Ca2+ channel in the regulation of spontaneous transmitter release was studied in Xenopus nerve-muscle cultures. The frequency of spontaneous synaptic currents, which reflects impulse-independent acetylcholine release from the nerve terminals, showed a marked increase in high-K+ medium or after treatment with a phorbol ester, 12-O-tetradecanoyl-phorbol 13-acetate, a drug that activates protein kinase C and depolarizes the presynaptic neuron. The potentiation effect of high K+ and 12-O-tetradecanoyl-phorbol 13-acetate requires Ca2+ influx through the L-type Ca2+ channel in the plasma membrane, since it was significantly reduced by the presence of nifedipine, verapamil or diltiazem and enhanced by Bay K 8644, an L-type Ca2+ channel agonist. It was shown recently that adenosine 5'-triphosphate markedly potentiates the spontaneous acetylcholine release at these synapses through the binding of P2-purinoceptors and the activation of protein kinase C. We found in the present study that potentiation effects of adenosine 5'-triphosphate are inhibited by L-type Ca2+ channel blockers, suggesting that the L-type Ca2+ channel is responsible for the positive regulation of spontaneous acetylcholine secretion by adenosine 5'-triphosphate at the developing neuromuscular synapses. Our data suggest that modulation of the L-type Ca2+ channel in embryonic motor nerve terminals is important for the regulation of spontaneous transmitter release.     

Investigation of the relationship between farrowing environment, sex steroid concentrations and maternal aggression in gilts

The involvement of cAMP-dependent phosphorylation sites in establishing the basal activity of cardiac L-type Ca2+ channels was studied in HEK 293 cells transiently cotransfected with mutants of the human cardiac alpha1 and accessory subunits. Systematic individual or combined elimination of high consensus protein kinase A (PKA) sites, by serine to alanine substitutions at the amino and carboxyl termini of the alpha1 subunit, resulted in Ca2+ channel currents indistinguishable from those of wild type channels. Dihydropyridine (DHP)-binding characteristics were also unaltered. To explore the possible involvement of nonconsensus sites, deletion mutants were used. Carboxyl-terminal truncations of the alpha1 subunit distal to residue 1597 resulted in increased channel expression and current amplitudes. Modulation of PKA activity in cells transfected with the wild type channel or any of the mutants did not alter Ca2+ channel functions suggesting that cardiac Ca2+ channels expressed in these cells behave, in terms of lack of PKA control, like Ca2+ channels of smooth muscle cells.