omega-conotoxin-(conus-magus) and 1-4-dihydropyridine

The inhibition of high-threshold Ca2+ channel currents by activated G-proteins was studied in mouse cerebellar granule cells making use of the hydrolysis-resistant GTP analog GTP-gamma-S. When individual granule cells were internally dialyzed with GTP-gamma-S, the high-threshold Ca2+ current decreased to approximately 20% of its initial value within approximately 2 min. The GTP-gamma-S-resistant current was reduced further by the subsequent addition of either omega-conotoxin or dihydropyridine antagonist, indicating that both N- and L-type Ca2+ channels carried the remaining current. Continuous exposure to the dihydropyridine agonist +(S)-202-791 caused a rapid increase in the GTP-gamma-S-resistant current. The L-type current evoked by the agonist subsequently decreased to the level observed prior to adding the drug following a time course similar to the initial inhibition of the total high-threshold current. A second application of the drug at a later time failed to increase the current a second time, indicating a persistent blockade of the agonist-evoked L-current. Pretreating cells with pertussis toxin prevented the initial inhibition of the total whole-cell Ca2+ channel current as well as the subsequent inhibition of the agonist-evoked L-current. The results show that a pertussis toxin-sensitive G-protein produces a persistent inhibition of L-type Ca2+ channels in these central neurons.

Topics: Animals; Cadmium; Calcium Channel Blockers; Calcium Channels; Cells, Cultured; Cerebellum; Dihydropyridines; Electric Conductivity; Evoked Potentials; Gadolinium; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Kinetics; Mice; Neurons; omega-Conotoxins; Peptides, Cyclic; Pertussis Toxin; Time Factors; Virulence Factors, Bordetella

1. Calcium channel currents were measured with the whole-cell patch clamp technique in cultured, identified mouse motoneurons. Three components of current were operationally defined on the basis of voltage dependence, kinetics, and pharmacology. 2. Test potentials to -50 mV or greater (10 mM external Ca2+) elicited a low-voltage activated T-type current that was transient (decaying to baseline in less than 200 ms) and had a relatively slow time to peak (20-50 ms). A 1-s prepulse to -45 mV produced approximately half-maximal inactivation of this T current. 3. Two high-voltage activated (HVA) components of current (1 transient and 1 sustained) were activated by test potentials to -20 mV or greater (10 mM external Ca2+). A 1-s prepulse to -35 mV produced approximately half-maximal inactivation of the transient component without affecting the sustained component. 4. When Ba2+ was substituted for Ca2+ as the charge carrier, activation of the HVA components was shifted in the hyperpolarizing direction, and the relative amplitude of the transient HVA component was reduced. 5. Amiloride (1-2 mM) caused a reversible, partial block of the T current without affecting the HVA components. 6. The dihydropyridine agonist isopropyl 4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5-nitro-3- pyridine-carboxylate [(+)-SDZ 202-791, 100 nM-1 microM)] shifted the activation of the sustained component of HVA current to more negative potentials and increased its maximal amplitude. Additionally, (+)-SDZ 202-791 caused the appearance of a slowed component of tail current.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amiloride; Animals; Barium; Calcium Channel Blockers; Calcium Channels; Cells, Cultured; Dihydropyridines; Electrophysiology; Female; Mice; Motor Neurons; Nicotinic Acids; omega-Conotoxins; Oxadiazoles; Peptides, Cyclic; Pregnancy; Synaptic Transmission

We have studied the effect of the purified toxin from the funnel-web spider venom (FTX) and its synthetic analog (sFTX) on transmitter release and presynaptic currents at the mouse neuromuscular junction. FTX specifically blocks the omega-conotoxin- and dihydropyridine-insensitive P-type voltage-dependent Ca2+ channel (VDCC) in cerebellar Purkinje cells. Mammalian neuromuscular transmission, which is insensitive to N- or L-type Ca2+ channel blockers, was effectively abolished by FTX and sFTX. These substances blocked the muscle contraction and the neurotransmitter release evoked by nerve stimulation. Moreover, presynaptic Ca2+ currents recorded extracellularly from the interior of the perineural sheaths of nerves innervating the mouse levator auris muscle were specifically blocked by both natural toxin and synthetic analogue. In a parallel set of experiments, K(+)-induced Ca45 uptake by brain synaptosomes was also shown to be blocked or greatly diminished by FTX and sFTX. These results indicate that the predominant VDCC in the motor nerve terminals, and possibly in a significant percentage of brain synapses, is the P-type channel.

Topics: Action Potentials; Animals; Arginine; Cadmium; Calcium; Calcium Channel Blockers; Calcium Channels; Cerebral Cortex; Dihydropyridines; Electric Stimulation; Evoked Potentials; Magnesium; Male; Mice; Neuromuscular Junction; Neurotoxins; Neurotransmitter Agents; omega-Conotoxins; Peptides, Cyclic; Phrenic Nerve; Polyamines; Potassium; Purkinje Cells; Spermidine; Synapses; Synaptosomes

N-type calcium channels are thought to be expressed specifically in neuronal cells and to have a dominant role in the control of neurotransmitter release from sympathetic neurons. But their unitary properties are poorly understood and the separation of neuronal Ca2+ current into components carried by N-type or L-type Ca2+ channels is controversial. Here we show that individual N-type Ca2+ channels in sympathetic neurons can carry two kinetically distinct components of current, one that is rapidly transient and one that is long lasting. The mechanism that gives rise to these two components is unexpected for Ca2+ channels: a test depolarization elicits either a rapidly inactivating, single short burst with an average duration of 40 ms, or sustained, noninactivating channel activity lasting for over 1 s. The switching between inactivating and noninactivating activity is a slow process, the occurrence of each type of unitary kinetic behaviour remaining statistically correlated over several seconds. Variable coupling of inactivation in N-type Ca2+ channels could be an effective mechanism for the modulation of neuronal excitability and synaptic plasticity.

Topics: Animals; Barium; Calcium Channels; Dihydropyridines; Electric Conductivity; Ganglia, Sympathetic; Ion Channel Gating; Kinetics; Neurons; omega-Conotoxins; Peptides, Cyclic; Rats

The stimulation of TSH secretion by TRH involves the phosphatidylinositol second messenger pathway via activation of phospholipase C. This effect is mediated by a GTP-binding protein and leads to a mobilization of intracellular Ca2+ stores and an activation of protein kinase C. However, TRH stimulation also results in an influx of extracellular Ca2+. Since we have previously demonstrated that a non-TRH fragment of the prepro-TRH molecule, the connecting peptide PS4 (prepro-TRH 160-169), was able to potentiate the TRH-induced TSH release in a dose-dependent manner, we attempted to determine whether this potentiation might be due to a Ca(2+)-dependent phenomenon and whether a specific class of voltage-dependent Ca2+ channels, the L type Ca2+ channels, might be involved in the effect of PS4. This was studied by perifusing normal pituitary fragments with medium containing either the Ca2+ ionophore, ionomycin, and Co2+ ions, or organic compounds well known to block L-type Ca2+ channels, and by measuring the TSH response to a pulse of TRH (10 nM) in the presence or absence of PS4 (100 nM).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Dihydropyridines; Male; omega-Conotoxins; Peptide Fragments; Peptides, Cyclic; Perfusion; Pituitary Gland; Protein Precursors; Rats; Rats, Inbred Strains; Thyrotropin; Thyrotropin-Releasing Hormone; Virulence Factors, Bordetella

Integration and processing of electrical signals in individual neurons depend critically on the spatial distribution of ion channels on the cell surface. In hippocampal pyramidal neurons, voltage-sensitive calcium channels have important roles in the control of Ca2(+)-dependent cellular processes such as action potential generation, neurotransmitter release, and epileptogenesis. Long-term potentiation of synaptic transmission in the hippocampal pyramidal cell, a form of neuronal plasticity that is thought to represent a cellular correlate of learning and memory, is dependent on Ca2+ entry mediated by synaptic activation of glutamate receptors that have a high affinity for NMDA (N-methyl(-D-aspartate) and are located in distal dendrites. Stimuli causing long-term potentiation at these distal synapses also cause a large local increase in cytosolic Ca2+ in the proximal regions of dendrites. This increase has been proposed to result from activation of voltage-gated Ca2+ channels. At least four types of voltage-gated Ca2+ channels, designated N, L. T and P, may be involved in these processes. Here we show that L-type Ca2+ channels, visualized using a monoclonal antibody, are located in the cell bodies and proximal dendrites of hippocampal pyramidal cells and are clustered in high density at the base of major dendrites. We suggest that these high densities of L-type Ca2+ channels may serve to mediate Ca2+ entry into the pyramidal cell body and proximal dendrites in response to summed excitatory inputs to the distal dendrites and to initiate intracellular regulatory events in the cell body in response to the same synaptic inputs that cause long-term potentiation at distal dendritic synapses.

Topics: Animals; Antibodies, Monoclonal; Calcium Channels; Dendrites; Dihydropyridines; Hippocampus; Immunoenzyme Techniques; Immunosorbent Techniques; Isradipine; Neurons; omega-Conotoxins; Oxadiazoles; Peptides, Cyclic; Rats; Rats, Inbred Strains; Synaptic Membranes

The major component of whole-cell Ca2+ current in differentiated, neuron-like rat pheochromocytoma (PC12) cells and sympathetic neurons is carried by dihydropyridine-insensitive, high-threshold-activated N-type Ca2+ channels. We show that these channels have unitary properties distinct from those of previously described Ca2+ channels and contribute both slowly inactivating and large sustained components of whole-cell current. The N-type Ca2+ currents are modulated by GTP binding proteins. The snail toxin omega-conotoxin reveals two pharmacological components of N-type currents, one blocked irreversibly and one inhibited reversibly. Contrary to previous reports, neuronal L-type channels are insensitive to omega-conotoxin. N-type Ca2+ channels appear to be specific for neuronal cells, since their functional expression is greatly enhanced by nerve growth factor.

Topics: Acetylcholine; Animals; Calcium; Calcium Channels; Dihydropyridines; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Ion Channel Gating; Mollusk Venoms; Neurons; omega-Conotoxins; Pheochromocytoma; Rats; Thionucleotides; Tumor Cells, Cultured

Changes in membrane potential may influence Ca2+-dependent functions through changes in cytosolic free calcium concentration [( Ca2+]i). This study characterized pharmacologically those voltage-dependent Ca2+ channels in normal rat anterior pituitary cells that are involved in the elevation of [Ca2+]i upon high potassium-induced membrane depolarization. The [Ca2+]i was monitored directly by means of the intracellularly trapped fluorescent indicator fura-2. The addition of K+ (6-100 mM) increased [Ca2+]i in a concentration-dependent manner. The fluorescent signal reached a peak within seconds and then decayed to form a new elevated plateau. K+ at the highest concentration used (100 mM) raised [Ca2+]i by about 450 nM. The K+-induced increase in [Ca2+]i was absent in a Ca2+-free medium. BAY K 8644, a 1,4-dihydropyridine Ca2+ channel agonist, also caused an increase in [Ca2+]i. The maximum response in [Ca2+]i upon stimulation with BAY K 8644 (100 nM) was about 40 nM. The half-maximally effective concentration of BAY K 8644 (100 nM) was about 20 nM. The response in [Ca2+]i upon BAY K 8644-stimulation was abolished in a Ca2+-free medium. Predepolarization with various K+ concentrations enhanced the effect of BAY K 8644 (1 microM) on [Ca2+]i. Pretreatment with BAY K 8644 (1 microM) enhanced the response in [Ca2+]i induced by K+ (25 mM). The addition of Mg2+ (30 mM) and nifedipine (1 microM) lowered the resting [Ca2+]i by about 40 and 20 nM, respectively. Mg2+, nifedipine, nimodipine, Gö 5438, verapamil, and diltiazem inhibited the K+ (25 mM)-induced increase in [Ca2+]i; the order of potency (and half-maximally inhibitory concentrations) were nimodipine = Gö 5438 = nifedipine (approximately 100 nM) greater than verapamil (900 nM) greater than diltiazem (greater than 10 microM) greater than Mg2+ (6 mM). Omega-Conotoxin (100 nM) did not inhibit the K+ (25 mM)-induced increase in [Ca2+]i. These data demonstrate that, over a wide range, membrane depolarization induced by high potassium concentration is indeed associated with increases in [Ca2+]i in normal rat anterior pituitary cells. This elevation of [Ca2+]i is mainly due to an influx of Ca2+ through 1,4-dihydropyridine-sensitive, omega-conotoxin-insensitive calcium channels (L-type).

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Benzofurans; Calcium; Calcium Channel Blockers; Cytosol; Dihydropyridines; Fluorescent Dyes; Fura-2; Ion Channels; Magnesium; Male; Membrane Potentials; Mollusk Venoms; omega-Conotoxins; Pituitary Gland, Anterior; Potassium; Rats; Rats, Inbred Strains