Neurotransmitter release from bradykinin-stimulated PC12 cells. Stimulation of cytosolic calcium and neurotransmitter release.

The effect of bradykinin on intracellular free Ca2+ and neurotransmitter secretion was investigated in the rat pheochromocytoma cell line PC12. Bradykinin was shown to induce a rapid, but transient, increase in intracellular free Ca2+ which could be separated into an intracellular Ca2+ release component and an extracellular Ca2+ influx component. The bradykinin-induced stimulation of intracellular free Ca2+ displayed a similar time course, concentration dependencies and extracellular Ca2+ dependence as that found for neurotransmitter release, indicating an association between intracellular free Ca2+ levels and neurotransmitter secretion. The selective BK1-receptor antagonist des-Arg9,[Leu8]BK (where BK is bradykinin) did not significantly affect the stimulation of intracellular free Ca2+ or neurotransmitter release. In contrast, these effects of bradykinin were effectively blocked by the selective BK2-receptor antagonist [Thi5,8,D-Phe7]BK, and mimicked by the BK2 partial agonist [D-Phe7]BK in a concentration-dependent manner. The stimulation of intracellular free Ca2+ and neurotransmitter release induced by bradykinin was shown not to involve voltage-sensitive Ca2+ channels, since calcium antagonists had no effect on either response at concentrations which effectively inhibit depolarization-induced responses. These results indicate that bradykinin, acting through the interaction with the BK2 receptor, stimulates an increase in intracellular free Ca2+ leading to neurotransmitter secretion. Furthermore, bradykinin-induced responses involve the release of intracellular Ca2+ and the influx of extracellular Ca2+ that is not associated with the activation of voltage-sensitive Ca2+ channels.     

Voltage-dependent inactivation of T-tubular skeletal calcium channels in planar lipid bilayers

Single-channel properties of dihydropyridine (DHP)-sensitive calcium channels isolated from transverse tubular (T-tube) membrane of skeletal muscle were explored. Single-channel activity was recorded in planar lipid bilayers after fusion of highly purified rabbit T-tube microsomes. Two populations of DHP-sensitive calcium channels were identified. One type of channel (noninactivating) was active (2 microM +/- Bay K 8644) at steady-state membrane potentials and has been studied in other laboratories. The second type of channel (inactivating) was transiently activated during voltage pulses and had a very low open probability (Po) at steady-state membrane potentials. Inactivating channel activity was observed in 47.3% of the experiments (n = 84 bilayers). The nonstationary kinetics of this channel was determined using a standard voltage pulse (HP = -50 mV, pulse to 0 mV). The time constant (tau) of channel activation was 23 ms. During the mV). The time constant (tau) of channel activation was 23 ms. During the pulse, channel activity decayed (inactivated) with a tau of 3.7 s. Noninactivating single-channel activity was well described by a model with two open and two closed states. Inactivating channel activity was described by the same model with the addition of an inactivated state as proposed for cardiac muscle. The single-channel properties were compared with the kinetics of DHP-sensitive inward calcium currents (ICa) measured at the cellular level. Our results support the hypothesis that voltage-dependent inactivation of single DHP-sensitive channels contributes to the decay of ICa.     

Receptor-mediated regulation of calcium channels and neurotransmitter release

Ca2+ influx into the nerve terminal is normally the trigger for the release of neurotransmitters. Many neurons possess presynaptic receptors whose activation results in changes in the quantity of neurotransmitter released by an action potential. This paper reviews studies that show that presynaptic receptors can regulate the activity of Ca2+ channels in the nerve terminal, resulting in changes in the influx of Ca2+ and in neurotransmitter release. Neurons possess several different types of voltage-sensitive Ca2+ channels. Ca2+ influx through N-type channels appears to trigger transmitter release in many instances. In other cases Ca2+ influx through L channels can influence transmitter release. Neurotransmitters can inhibit N channels through a G protein-mediated transduction mechanism. The G proteins are frequently pertussis toxin substrates. Inhibition of N channels appears to involve changes in their voltage dependence. Neurotransmitters can also regulate neuronal K+ channels. Activation of these K+ channels can lead to a reduction in Ca2+ influx and neurotransmitter release; these effects are also mediated by G proteins. Thus neurotransmitters may often regulate both presynaptic Ca2+ and K+ channels. These two effects may be synergistic mechanisms for the regulation of Ca2+ influx and neurotransmitter release.     

Voltage-dependent calcium channels in pituitary cells in culture. II. Participation in thyrotropin-releasing hormone action on prolactin release

Depolarization of membrane potential by high external K+ activates Ca2+ influx via voltage-dependent Ca2+ channels in GH4C1 cells (Tan, K.-N., and Tashjian, A. H., Jr. (1983) J. Biol. Chem. 258, 418-426). The involvement of this channel in thyrotropin-releasing hormone (TRH) action on prolactin (PRL) release was assessed by comparing the pharmacological characteristics of TRH-induced PRL release with PRL release due to high K+. Two components of TRH-stimulated PRL release were detected. The major component (approximately equal to 75%) was dependent on external Ca2+ concentration and was inhibited by voltage-dependent Ca2+ channel blockers in a manner quantitatively similar to high K+-stimulated PRL release. The minor component (approximately equal to 25%) of TRH-stimulated PRL release was insensitive to voltage-dependent Ca2+ channel blockers and could occur in the presence of low external Ca2+ (10(-5)-10(-7) M). Neither voltage-dependent Ca2+ channel blockers nor depletion of medium Ca2+ prevented the action of TRH on mobilizing cell-associated 45Ca2+ from GH4C1 cells. Divalent cations that permeate voltage-dependent Ca2+ channels (Sr2+ and Ba2+) substituted for Ca2+ in supporting high K+- and TRH-stimulated PRL release while Mg2+, a nonpermeant cation, did not. We conclude that TRH stimulates PRL release by increasing [Ca2+]i through at least two mechanisms: one requires only low [Ca2+]o, the second involves Ca2+ influx via voltage-dependent Ca2+ channels. This latter mechanism accounts for approximately equal to 75% of maximum TRH-induced PRL release.     

Voltage-dependent calcium channels in young and old human red cells

Presence of subtypes of voltage-dependent Ca channels was investigated in young and old human red cells, employing immunological and flux-kinetics methods. Western blots showed specific reaction toward polyclonal rabbit antibodies raised against a highly conserved residue of α_1C, subunit of high-voltage activated Ca channels (pan α₁) and against conserved residues of α_1C and α_1E subunits. No specific reaction was detected with antibodies against conserved residues of α_1A, α_1B, or α_1D subunits. Only a single band (approx 260 kDa) was revealed on anti-pan α_1A or anti-α_1E blots, whereas two bands (200 and 230 kDa) were detected by α_1C exposure, Blots from old cells always showed diminished band intensity. Channel activity was assessed by studying the effect of voltage-dependent Ca channels blockers' under conditions likely to alter the red cell membrane potential, through incubation in media of different composition. In a 150 mM NaCl+5 mM KCl medium, blockers of L-, R-, and Q-type caused a 15–50% reductions of ⁴⁵Ca influx into cells, which had the Ca pump inactivated by either exhaustive adenosine triphosphate depletion or presence of vanadate plus substrates. Additionally, some P/Q-and N-type blockers also reduced Ca influx to various extents (25–60%). Old cells were generally insensitive to L-type but not to non-L-type, blockers. Raising external K to about 70–80 mM reduced by 50–100% inhibition by L-type blockers. Incubation in a gluconate medium containing 150 mM Na+5 mM K practically abolished the action of L-type blockers, but only slightly reducing that by non-L-type. The results, clearly demonstrate presence of L- and R-type Ca channels, apparently occurring in different functional states in young and old cells. Other non-L-type channels were also demonstrated only by pharmacological means. A possible physiological role for these channels is discussed.     

Muscarinic stimulation of calcium influx and norepinephrine release in PC12 cells

Muscarinic cholinergic receptor stimulation evokes catecholamine secretion from some cell types, but the mechanism has not been well characterized. Using pheochromocytoma (PC12) cells, we show that the muscarinic agonist methacholine stimulates 45Ca2+ influx and [3H]norepinephrine release in a dose-dependent manner. Experiments performed in Na+-free medium or with inhibitors of voltage-dependent Ca2+ channels suggest the involvement of a receptor-activated Ca2+ channel which differs significantly from the voltage-dependent Ca2+ channel involved in nicotinic receptor-stimulated release. Furthermore, both influx and release were inhibited by pertussis toxin (0.5-2.0 ng/ml, 21 h) with a dose dependency which paralleled the dose dependency of pertussis toxin-dependent in vivo ADP-ribosylation of a 41-kDa protein. These experiments provide the first evidence that muscarinic stimulation evokes neurotransmitter secretion by opening a receptor-activated Ca2+ channel which is controlled by a pertussis toxin-sensitive protein.     

Voltage-dependent inactivation of slow calcium channels in intact twitch muscle fibers of the frog

Inactivation of slow Ca2+ channels was studied in intact twitch skeletal muscle fibers of the frog by using the three-microelectrode voltage-clamp technique. Hypertonic sucrose solutions were used to abolish contraction. The rate constant of decay of the slow Ca2+ current (ICa) remained practically unchanged when the recording solution containing 10 mM Ca2+ was replaced by a Ca2+-buffered solution (126 mM Ca-maleate). The rate constant of decay of ICa monotonically increased with depolarization although the corresponding time integral of ICa followed a bell-shaped function. The replacement of Ca2+ by Ba2+ did not result in a slowing of the rate of decay of the inward current nor did it reduce the degree of steady-state inactivation. The voltage dependence of the steady-state inactivation curve was steeper in the presence of Ba2+. In two-pulse experiments with large conditioning depolarizations ICa inactivation remained unchanged although Ca2+ influx during the prepulse greatly decreased. Dantrolene (12 microM) increased mechanical threshold at all pulse durations tested, the effect being more prominent for short pulses. Dantrolene did not significantly modify ICa decay and the voltage dependence of inactivation. These results indicate that in intact muscle fibers Ca2+ channels inactivate in a voltage-dependent manner through a mechanism that does not require Ca2+ entry into the cell.     

Differential coupling of alpha7 and non-alpha7 nicotinic acetylcholine receptors to calcium-induced calcium release and voltage-operated calcium channels in PC12 cells

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation channels that can modulate various neuronal processes by altering intracellular Ca(2+) levels. Following nAChR stimulation Ca(2+) can enter cells either directly, through the intrinsic ion channel, or indirectly following voltage-operated Ca(2+) channel (VOCC) activation; Ca(2+) levels can subsequently be amplified via Ca(2+)-induced Ca(2+) release from intracellular stores. We have used subtype-selective nAChR agonists to investigate the Ca(2+) sources contributing to alpha7 and non-alpha7 nAChR-mediated increases in intracellular Ca(2+) in PC12 cells. Application of the alpha7 nAChR positive allosteric modulator PNU 120596 (10 mum), in conjunction with the alpha7 nAChR agonist, compound A [(R)-N-(1-azabicyclo[2.2.2]oct-3-yl)(5-(2-pyridyl)thiophene-2-carboxamide), 10 nm], produces a rapid increase in fluo-3 fluorescence that is prevented by the selective alpha7 nAChR antagonist alpha-bungarotoxin. The non-alpha7 nAChR agonist 5-Iodo-A-85380 produces alpha-bungarotoxin-insensitive increases in intracellular Ca(2+) (EC(50) = 11.2 mum). Using these selective agonists or KCl in conjunction with general and selective VOCC inhibitors, we demonstrate that the primary route of Ca(2+) entry following either non-alpha7 nAChR activation or KCl stimulation is via L-type VOCCs. In contrast, the alpha7 nAChR-mediated response is unaffected by VOCC blockers but is inhibited by modulators of intracellular Ca(2+) stores. These results indicate that alpha7 and non-alpha7 nAChRs are differentially coupled to Ca(2+)-induced Ca(2+) release and VOCCs, respectively.     

Functional interactions between presynaptic calcium channels and the neurotransmitter release machinery

In vertebrates, the physical coupling between presynaptic calcium channels and synaptic vesicle release proteins enhances the efficiency of neurotransmission. Recent evidence indicates that these synaptic proteins may feedback directly on synaptic release by negatively regulating calcium entry, and indirectly through pathways involving second messenger molecules. Studies of individual neurons from both vertebrates and invertebrates have provided novel insights into the roles of scaffolding proteins in calcium channel targeting and neurotransmitter release. These studies require us to expand current models of synaptic transmission.     

Protein kinase C evokes quantal catecholamine release from PC12 cells via activation of L-type Ca2+ channels

Application of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to PC12 cells under resting conditions evoked quantal catecholamine secretion, as detected amperometrically. This effect was not mimicked by 4alpha-phorbol-12,13-didecanoate, another phorbol ester, which is inactive with respect to protein kinase C activation, and was prevented by the protein kinase C inhibitor bisindolylmaleimide. TPA also caused a rise of [Ca(2+)](i) in Fura-2-loaded PC12 cells, and again this was not mimicked by 4alpha-phorbol-12,13-didecanoate and could be blocked by bisindolylmaleimide. TPA-evoked secretion was entirely dependent on extracellular Ca(2+) and was fully abolished by nifedipine, as were TPA-induced rises of [Ca(2+)](i). Resting membrane potential, monitored using perforated patch recordings, was unaffected by TPA. However, a small (6-8 mV) hyperpolarizing shift in the voltage dependence of Ca(2+) channel currents (determined using whole-cell patch clamp recordings) was induced by TPA, and this could be fully prevented by nifedipine. In contrast to results with depolarizing stimuli, which evoke exocytosis because of Ca(2+) influx through N-type channels in these cells, the present results indicate that protein kinase C activation leads directly to quantal catecholamine secretion in the absence of depolarizing stimuli via a selective shift in the activation of L-type Ca(2+) channels.     

Multiple calcium channels and kinases mediate alpha7 nicotinic receptor neuroprotection in PC12 cells

alpha7 Nicotinic receptors are calcium permeant and provide neuroprotection against many insults. We investigated the roles of intracellular calcium ions and downstream calcium channels in this protection. The alpha7 agonist GTS-21 prevented pheochromocytoma cell death induced by nerve growth factor + serum deprivation over a 3-day interval. This effect was blocked by the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid in a manner that did not appear to involve changes in receptor density. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid blocked GTS-21-induced protein kinase C activation, a necessary process for protection. The insositol triphosphate calcium-channel blocker xestospongin C and the phospholipases C inhibitor U-73122 blocked protection, ryanodine partially attenuated protection, but the L-type channel antagonist nifedipine had no effect. ERK1/2 but not JNK and p38 were activated by GTS-21, and the ERK phosphorylation inhibitors PD98059 and U0126 blocked protection.     

Voltage-dependent calcium release in human malignant hyperthermia muscle fibers.

Malignant hyperthermia (MH) results from a defect of calcium release control in skeletal muscle that is often caused by point mutations in the ryanodine receptor gene (RYR1). In malignant hyperthermia-susceptible (MHS) muscle, calcium release responds more sensitively to drugs such as halothane and caffeine. In addition, experiments on the porcine homolog of malignant hyperthermia (mutation Arg615Cys in RYR1) indicated a higher sensitivity to membrane depolarization. Here, we investigated depolarization-dependent calcium release under voltage clamp conditions in human MHS muscle. Segments of muscle fibers dissected from biopsies of the vastus lateralis muscle of MHN (malignant hyperthermia negative) and MHS subjects were voltage-clamped in a double vaseline gap system. Free calcium was determined with the fluorescent indicator fura-2 and converted to an estimate of the rate of SR calcium release. Both MHN and MHS fibers showed an initial peak of the release rate, a subsequent decline, and rapid turn-off after repolarization. Neither the kinetics nor the voltage dependence of calcium release showed significant deviations from controls, but the average maximal peak rate of release was about threefold larger in MHS fibers.     

Inhibition by protein kinase C activation of melittin-induced arachidonic acid release in PC12 pheochromocytoma cells.

In rat PC12 pheochromocytoma cells, melittin, a phospholipase A2 activator, stimulated the release of arachidonic acid in a dose-dependent manner in the range between 0.1 and 1 microM. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a protein kinase C-activating phorbol ester, inhibited the melittin-induced release of arachidonic acid dose-dependently in the range between 0.1 nM and 0.1 microM, whereas 4 alpha-phorbol 12, 13-didecanoate, which is inactive for protein kinase C, was ineffective in this capacity. Staurosporine, a protein kinase C inhibitor, recovered the inhibitory effect of TPA on the melittin-induced release of arachidonic acid. These results suggest that the activation of protein kinase C inhibits phospholipase A2 activity in PC12 pheochromocytoma cells.     

Regulation of nicotine-evoked dopamine release from PC12 cells.

The nature of second messengers involved in the nicotine-evoked release of dopamine from PC12 cells was examined. Calmidazolium, a calmodulin inhibitor, abolished the nicotine-evoked release. A23187, a Ca2+ ionophore, enhanced dopamine release, and this was inhibited by calmidazolium. Further, 2', 5'-dideoxyadenosine abolished both the nicotine- and A23187-evoked release. Forskolin, dibutyryl-cyclic AMP, and rolipram (a cyclic AMP phosphodiesterase inhibitor) all enhanced dopamine release. 1, 9-Dideoxyforskolin, a forskolin analog which does not activate adenylate cyclase, did not alter dopamine release. These results suggest an obligatory role for Ca2+ and calmodulin-sensitive adenylate cyclase in the nicotine-evoked release process.     

Calcium channels in undifferentiated PC12 rat pheochromocytoma cells

Undifferentiated rat pheochromocytoma PC12 cells were voltage clamped using the whole cell technique. After blockade of outward currents, calcium currents were elicited from -40 and -100 mV. A subpopulation of cells displayed only one current component activated at -10 mV and slowly decaying. In other cells this current coexisted with a component activated around -40 mV and decaying with a faster time constant. We conclude that undifferentiated PC12 cells can express two types of calcium channels, L (long-lasting) and N (neuronal)-type channels.     

Voltage-dependent modulation of T-type calcium channels by protein tyrosine phosphorylation.

A T-type Ca2+ channel is expressed during differentiation of the male germ lineage in the mouse and is retained in sperm, where is it activated by contact with the the egg's extracellular matrix and controls sperm acrosomal exocytosis. Here, we examine the regulation of this Ca2+ channel in dissociated spermatogenic cells from the mouse using the whole-cell patch-clamp technique. T currents were enhanced, or facilitated, after strong depolarizations or high frequency stimulation. Voltage-dependent facilitation increased the Ca2+ current by an average of 50%. The same facilitation is produced by antagonists of protein tyrosine kinase activity. Conversely, antagonists of tyrosine phosphatase activity block voltage-dependent facilitation of the current. These data are consistent with the presence of a two-state model, in which T channels are maintained in a low (or zero) conductance state by tonic tyrosine phosphorylation and can be activated to a high conductance state by a tyrosine phosphatase activity. The positive and negative modulation of this channel by the tyrosine phosphorylation state provides a plausible mechanism for the control of sperm activity during the early stages of mammalian fertilization.     

Two distinct mechanisms underlie the stimulation of neurotransmitter release by phorbol esters in clonal rat pheochromocytoma PC12 cells

Phorbol ester treatment induces the phosphorylation of SNAP-25 at Ser(187) and the potentiation of Ca(2+)-induced dopamine (DA) and acetylcholine (Ach) release from PC12 cells. In order to evaluate the functional consequences of phosphorylation, quantitative analysis was carried out using an anti-phosphopeptide antibody that specifically recognizes SNAP-25 phosphorylated at Ser(187). DA and ACh release, assayed in low-K(+) as well as high-K(+) solution, increased by treating the cells with phorbol-12-myristate-13-acetate (PMA); however, the stimulation of high-K(+)-dependent release occurred at lower concentrations and with shorter exposures to PMA than that of the basal release in low-K(+)-solution. The PMA-induced phosphorylation of SNAP-25 did not correlate with the potentiation of high-K(+)-dependent neurotransmitter release. The potentiation of high-K(+)-dependent DA release by phorbol 12,13-diacetate (PDA), a water soluble phorbol ester, almost completely disappeared within 1 min after washing PDA in the presence of okadaic acid, conditions under which the phosphorylation of SNAP-25 persisted for at least 15 min. PMA-induced phosphorylation of SNAP-25 was inhibited by staurosporine, however, the potentiation of high-K(+)-dependent DA release was suppressed only partially. These results indicate that protein kinase activation does not account for a large fraction of the phorbol ester-induced potentiation of depolarization-dependent neurotransmitter release from PC12 cells.     

Voltage-dependent calcium channels in cultivated neurons of the rat hippocampus

Calcium currents through the somatic membrane of cultivated (a low-density culture) hippocampal neurons of rats were studied with the use of a patch-clamp technique in the whole-cell configuration. Low- and high-threshold components of calcium currents were found in the somata of all studied cells. Low-threshold currents were activated at a membrane potential of about−75 mV and reached the maximum amplitude at −45±4 mV, while the maximum amplitude of high-threshold currents was observed at 17±6 mV. Low-threshold calcium currents differed from high-threshold current in weak suppression by low Cd²⁺ concentration (10–20 μM), while Ni²⁺ inhibited both types of calcium currents to an equal extent. Experiments with organic channel blockers showed that in most neurons at least four channel types were expressed: these were L, N, P, and channels insensitive to the used blockers (presumably, R-type). A blocker of L-type calcium channels, nifedipine (10 μM), blocked, on the average, 22.7±5.2%; a blocker of N-type channels, ω-CTx-GVIA (1.0 μM), blocked 30.0±5.0% and a blocker of P/Q channels, ω-Aga-IVA (200 nM), blocked 37.2±13.3% of the integral high-threshold current. A resistive component equalled 15.7±5.1% of the latter current. It is concluded that hippocampal neurons cultivated with a low density express a pharmacologically heterogeneous population of calcium channels, and the relative proportions of different type channels are close to the earlier described channel type composition in rat hippocampal slices. Our study shows that the low-density culture can be used as an adequate model for studying calcium channels in the somatic membrane of hippocampal neurons.     

Voltage-dependent calcium channels in skeletal muscle transverse tubules. Measurements of calcium efflux in membrane vesicles

Transverse tubule membranes isolated from rabbit skeletal muscle consist mainly of sealed vesicles that are oriented primarily inside out. These membranes contain a high density of binding sites for 1,4-dihydropyridine calcium channel antagonists. The presence of functional voltage-dependent calcium channels in these membranes has been demonstrated by their ability to mediate 45Ca2+ efflux in response to changes in membrane potential. Fluorescence changes of the voltage-sensitive dye, 3,3'-dipropyl-2,2'-thiadicarbocyanine, have shown that transverse tubule vesicles may generate and maintain membrane potentials in response to establishing potassium gradients across the membrane in the presence of valinomycin. A two-step procedure has been developed to measure voltage-dependent calcium fluxes. Vesicles loaded with 45Ca2+ are first diluted into a buffer designed to generate a membrane potential mimicking the resting state of the cell and to reduce the extravesicular Ca2+ to sub-micromolar levels. 45Ca2+ efflux is then measured upon subsequent depolarization. Flux responses are modulated with appropriate pharmacological specificity by 1,4-dihydropyridines and are inhibited by other calcium channel antagonists such as lanthanum and verapamil.