Ca2+-dependent K+ currents induced by muscarinic receptor activation in guinea pig adrenal chromaffin cells

The neuronal effects of the metabotropic glutamate receptor agonist (1S,3R)-aminocyclopentane-1,3-dicarboxylic acid have been studied in cultured rat cerebellar granule cells, and compared with those of the endogenous excitotoxin glutamate, and the dietary excitotoxin beta-N-methylamino-L-alanine. Glutamate, beta-N-methylamino-L-alanine, and (1S,3R)-aminocyclopentane-1,3-dicarboxylic acid all caused concentration-dependent cerebellar granule cell death over a 24-h exposure period. The metabotropic antagonist (RS)-alpha-methyl-4-carboxyphenylglycine reduced glutamate-, beta-N-methylamino-L-alanine-, and (1S,3R)-aminocyclopentane-1,3-dicarboxylic acid-induced death by 50, 37, and 90%, respectively. (1S,3R)-Aminocyclopentane-1,3-dicarboxylic acid-induced death was unaffected by the group I antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid, increased by the group II antagonist ethylglutamic acid, and markedly decreased by the group III antagonist (RS)-alpha-methylserine-O-phosphate. Neither (1S,3R)-aminocyclopentane-1,3-dicarboxylic acid nor the group I agonist (RS)-3,5-dihydroxyphenylglycine caused an increase in intracellular free calcium levels. The group III agonist L-(+)-2-amino-4-phosphonobutyric acid also induced concentration-dependent cerebellar granule cell death, and so it was suggested that the group III metabotropic glutamate receptors were responsible for (1S,3R)-aminocyclopentane-1,3-dicarboxylic acid-induced death. Blocking these receptors with (RS)-alpha-methylserine-O-phosphate also prevented a proportion of glutamate- and beta-N-methylamino-L-alanine-induced death.     

Muscarinic receptor activation modulates Ca2+ channels in rat intracardiac neurons via a PTX- and voltage-sensitive pathway

With use of the whole cell patch-clamp technique, effects of the potent muscarinic agonist oxotremorine methiodide (oxo-M) on voltage-activated Ca2+ channel currents were investigated in acutely dissociated adult rat intracardiac neurons. In all tested neurons oxo-M reversibly inhibited the peak Ba2+ current. Inhibition of the peak Ba2+ current by oxo-M was associated with slowing of activation kinetics and was concentration dependent. The concentration of oxo-M necessary to produce a half-maximal inhibition of current and the maximal inhibition were 40.8 nM and 75.9%, respectively. Inhibitory effect of oxo-M was completely abolished by atropine. Among different muscarinic receptor antagonists, methoctramine (100 and 300 nM) significantly antagonized the current inhibition by oxo-M, with a negative logarithm of dissociation constant of 8.3 in adult rat intracardiac neurons. Internal dialysis of neurons with guanosine 5'-(thio)triphosphate (GTPgammaS, 0.5 mM) could mimic the muscarinic inhibition of the peak Ba2+ current and significantly occlude inhibitory effects of oxo-M. In addition, the internal dialysis of guanosine-5'-O-(2-thiodiphosphate) (GDPbetaS, 2 mM) also significantly reduced the muscarinic inhibition of the peak Ba2+ current by oxo-M. Inhibitory effects of oxo-M were significantly abolished by pertussis toxin (PTX, 200 and 400 ng/ml) but not by cholera toxin (400 ng/ml). Furthermore, the bath application of N-ethylmaleimide (50 microM) significantly reduced the inhibition of the peak Ba2+ current by oxo-M. The oxo-M shifted the activation curve derived from measurments of tail currents toward more positive potentials. A strong conditioning prepulse to +100 mV significantly relieved the muscarinic inhibition of peak Ba2+ currents by oxo-M and the GTPgammaS-induced current inhibition. In a series of experiments, changes in intracellular concentration of bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid and protein kinase activities failed to mimic or occlude the current inhibition by oxo-M. The dihydropyridine antagonist nifedipine (10 microM) was not able to occlude any of the inhibitory effects of oxo-M, and oxo-M (3 microM) failed to reduce the slow tail currents induced by the L-type agonist methyl 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylate (FPL 64176; 2 microM). However, omega-conotoxin (omega-CgTX) GVIA (1 microM) significantly occluded the muscarinic inhibition of the Ba2+ currents. In the presence of omega-CgTX GVIA (1 microM) and nifedipine (10 microM), oxo-M could further inhibit approximately 20% of the total Ca2+ current. After complete removal of N-, Q-, and L-type currents with use of omega-CgTX GVIA, omega-agatoxin IVA, and nifedipine, 70% of the R-type current (approximately 6-7% of the total current) was inhibited by oxo-M (3 microM). In conclusion, the M2 muscarinic receptor activation selectively inhibits N-, Q-, and R-type Ca2+ channel currents, sparing L-type Ca2+ channel currents mainly via a PTX- and voltage-sensitive pathway in adult rat intracardiac neurons.     

Effect of KB-2796, a new diphenylpiperazine Ca2+ antagonist, on voltage-dependent Ca2+ currents and oxidative metabolism in dissociated mammalian CNS neurons

The effects of KB-2796, 1-[bis(4-fluorophenyl)methyl]-4-(2,3,4- trimethoxybenzyl)piperazine-2HCl, on the low- and high-voltage activated Ca2+ currents (LVA and HVA ICa, respectively) and on oxidative metabolism were studied in neurons freshly dissociated from rat brain. KB-2796 reduced the peak amplitude of LVA ICa in a concentration-dependent manner with a threshold concentration of 10(-7) M when the LVA ICa was elicited every 30 s in the external solution with 10 mM Ca2+. The concentration for half-maximum inhibition (IC50) was 1.9 x 10(-6) M. At 10(-5) M or more of KB-2796, a complete suppression of the LVA ICa was observed in the majority of neurons tested. There was no apparent effect on the current-voltage (I-V) relationship and the current kinetics. KB-2796 delayed the reactivation and enhanced the inactivation of the Ca2+ channel for LVA ICa voltage- and time-dependently, suggesting that KB-2796 preferentially binds to the inactivated Ca2+ channel. KB-2796 at a concentration of 3.0 x 10(-6) M also decreased the peak amplitude of the HVA ICa without shifting the I-V relationship. In addition, KB-2796 reduced the oxidative metabolism (the formation of reactive oxygen species) of the neuron in a concentration-dependent manner with a threshold concentration of 3 x 10(-6) M. It is suggested that the inhibitory action of KB-2796 on the neuronal Ca2+ influx and the oxidative metabolism, in combination with a cerebral vasodilatory action, may reduce ischemic brain damage.     

Selective activation of Ca2+-activated K+ channels by co-localized Ca2+ channels in hippocampal neurons

Calcium entry through voltage-gated calcium channels can activate either large- (BK) or small- (SK) conductance calcium-activated potassium channels. In hippocampal neurons, activation of BK channels underlies the falling phase of an action potential and generation of the fast afterhyperpolarization (AHP). In contrast, SK channel activation underlies generation of the slow AHP after a burst of action potentials. The source of calcium for BK channel activation is unknown, but the slow AHP is blocked by dihydropyridine antagonists, indicating that L-type calcium channels provide the calcium for activation of SK channels. It is not understood how this specialized coupling between calcium and potassium channels is achieved. Here we study channel activity in cell-attached patches from hippocampal neurons and report a unique specificity of coupling. L-type channels activate SK channels only, without activating BK channels present in the same patch. The delay between the opening of L-type channels and SK channels indicates that these channels are 50-150 nm apart. In contrast, N-type calcium channels activate BK channels only, with opening of the two channel types being nearly coincident. This temporal association indicates that N and BK channels are very close. Finally, P/Q-type calcium channels do not couple to either SK or BK channels. These data indicate an absolute segregation of coupling between channels, and illustrate the functional importance of submembrane calcium microdomains.     

Three kinetically distinct Ca2+-independent depolarization-activated K+ currents in callosal-projecting rat visual cortical neurons

Three kinetically distinct Ca2+-independent depolarization-activated K+ currents in callosal-projecting rat visual cortical neurons. J. Neurophysiol. 78: 2309-2320, 1997. Whole cell, Ca2+-independent, depolarization-activated K+ currents were characterized in identified callosal-projecting (CP) neurons isolated from postnatal day 7-16 rat primary visual cortex. CP neurons were identified in vitro after in vivo retrograde labeling with fluorescently tagged latex microbeads. During brief (160-ms) depolarizing voltage steps to potentials between -50 and +60 mV, outward K+ currents in these cells activate rapidly and inactivate to varying degrees. Three distinct K+ currents were separated based on differential sensitivity to 4-aminopyridine (4-AP); these are referred to here as IA, ID, and IK, because their properties are similar (but not identical) K+ currents termed IA, ID, and IK in other cells. The current sensitive to high (>/=100 mu M) concentrations of 4-AP (IA) activates and inactivates rapidly; the current blocked completely by low (相似文献   

Distribution and activation of intracellular Ca2+ stores in cultured olfactory bulb neurons

The presence and distribution of intracellular Ca2+ release pathways in olfactory bulb neurons were studied in dissociated cell cultures. Histochemical techniques and imaging of Ca2+ fluxes were used to identify two major intracellular Ca2+ release mechanisms: inositol 1, 4,5-triphosphate receptor (IP3R)-mediated release, and ryanodine receptor-mediated release. Cultured neurons were identified by immunocytochemistry for the neuron-specificmarker beta-tubulin III. Morphometric analyses and immunocytochemistry for glutamic acid-decarboxylase revealed a heterogeneous population of cultured neurons with phenotypes corresponding to both projection (mitral/tufted) and intrinsic (periglomerular/granule) neurons of the in vivo olfactory bulb. Immunocytochemistry for the IP3R, and labeling with fluorescent-tagged ryanodine, revealed that, irrespective of cell type, almost all cultured neurons express IP3R and ryanodine binding sites in both somata and dendrites. Functional imaging revealed that intracellular Ca2+ fluxes can be generated in the absence of external Ca2+, using agonists specific to each of the intracellular release pathways. Local pressure application of glutamate or quisqualate evoked Ca2+ fluxes in both somata and dendrites in nominally Ca2+ free extracellular solutions, suggesting the presence of IP3-dependent Ca2+ release. These fluxes were blocked by preincubation with thapsigargin and persisted in the presence of the glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. Local application of caffeine, a ryanodine receptor agonist, also evoked intracellular Ca2+ fluxes in the absence of extracellular Ca2+. These Ca2+ fluxes were suppressed by preincubation with ryanodine. In all neurons, both IP3- and ryanodine-dependent release pathways coexisted, suggesting that they interact to modulate intracellular Ca2+ concentrations.     

P2Y2 nucleotide receptors expressed heterologously in sympathetic neurons inhibit both N-type Ca2+ and M-type K+ currents

The P2Y2 receptor is a uridine/adenosine triphosphate (UTP/ATP)-sensitive G-protein-linked nucleotide receptor that previously has been reported to stimulate the phosphoinositide signaling pathway. Messenger RNA for this receptor has been detected in brain tissue. We have investigated the coupling of the molecularly defined rat P2Y2 receptor to neuronal N-type Ca2+ channels and to M-type K+ channels by heterologous expression in rat superior cervical sympathetic (SCG) neurons. After the injection of P2Y2 cRNA, UTP inhibited the currents carried by both types of ion channel. As previously reported [Filippov AK, Webb TE, Barnard EA, Brown DA (1997) Inhibition by heterologously expressed P2Y2 nuerones. Br J Pharmacol 121:849-851], UTP inhibited the Ca2+ current (ICa(N)) by up to 64%, with an IC50 of approximately 0.5 microM. We now find that UTP also inhibited the K+M current (IK(M)) by up to 61%, with an IC50 of approximately 1.5 microM. UTP had no effect on either current in neurons not injected with P2Y2 cRNA. Structure-activity relations for the inhibition of ICa(N) and IK(M) in P2Y2 cRNA-injected neurons were similar, with UTP >/= ATP > ITP > GTP,UDP. However, coupling to these two channels involved different G-proteins: pretreatment with Pertussis toxin (PTX) did not affect UTP-induced inhibition of IK(M) but reduced inhibition of ICa(N) by approximately 60% and abolished the voltage-dependent component of this inhibition. In unclamped neurons, UTP greatly facilitated depolarization-induced action potential discharges. Thus, the single P2Y2 receptor can couple to at least two G-proteins to inhibit both Ca2+N and K+M channels with near-equal facility. This implies that the P2Y2 receptor may induce a broad range of effector responses in the nervous system.     

Ca2+ currents in compensated hypertrophy and heart failure

Transmembrane voltage-gated Ca2+ channels play a central role in the development and control of heart contractility which is modulated by the concentration of free cytosolic calcium ions (Ca2+). Ca2+ channels are closed at the normal membrane resting potential of cardiac cells. During the fast upstroke of the action potential (AP), they are gated into an open state by membrane depolarisation and thereby transduce the electrical signal into a chemical signal. In addition to its contribution to the AP plateau, Ca2+ influx through L-type Ca2+ channels induces a release of Ca2+ ions from the sarcoplasmic reticulum (SR) which initiates contraction. Because of their central role in excitation-contraction (E-C) coupling, L-type Ca2+ channels are a key target to regulate inotropy [1]. The role of T-type Ca2+ channels is more obscure. In addition to a putative part in the rhythmic activity of the heart, they may be implicated at early stages of development and during pathology of contractile tissues [2]. Despite therapeutic advances improving exercise tolerance and survival, congestive heart failure (HF) remains a major problem in cardiovascular medicine. It is a highly lethal disease; half of the mortality being related to ventricular failure whereas sudden death of the other patients is unexpected [3]. Although HF has diverse aetiologies, common abnormalities include hypertrophy, contractile dysfunction and alteration of electrophysiological properties contributing to low cardiac output and sudden death. A significant prolongation of the AP duration with delayed repolarisation has been observed both during compensated hypertrophy (CH) and in end-stage HF caused by dilated cardiomyopathy (Fig. 1A) [4-8]. This lengthening can result from either an increase in inward currents or a decrease in outward currents or both. A reduction of K+ currents has been demonstrated [6,9]. Prolonged Na+/Ca2+ exchange current may also be involved [9]. In contrast, there is a large variability in the results concerning Ca2+ currents (ICa). The purpose of this paper is to review results obtained in various animal models of CH and HF with special emphasis on recent studies in human cells. We focus on: (i) the pathophysiological role of T-type Ca2+ channels, present in some animal models of hypertrophy; (ii) the density and properties of L-type Ca2+ channels and alteration of major physiological regulations of these channels by heart rate and beta-adrenergic receptor stimulation; and (iii) recent advances in the molecular biology of the L-type Ca2+ channel and future directions.     

A Ca(2+)-sensing receptor mutation causes hypoparathyroidism by increasing receptor sensitivity to Ca2+ and maximal signal transduction

This is a follow-up report of a 14 1/2 year-old boy with Laron syndrome, who received twice daily therapy with IGF-I 120 micrograms/kg for 5 years that resulted in a linear growth of 40 cm. Concomitantly he became very obese which is attributed to IGF-I action via the insulin receptors.     

G-Protein-coupled modulation of presynaptic calcium currents and transmitter release by a GABAB receptor

Presynaptic GABAB receptors play a regulatory role in central synaptic transmission. To elucidate their underlying mechanism of action, we have made whole-cell recordings of calcium and potassium currents from a giant presynaptic terminal, the calyx of Held, and EPSCs from its postsynaptic target in the medial nucleus of the trapezoid body of rat brainstem slices. The GABAB receptor agonist baclofen suppressed EPSCs and presynaptic calcium currents but had no effect on voltage-dependent potassium currents. The calcium current-EPSC relationship measured during baclofen application was similar to that observed on reducing [Ca2+]o, suggesting that the presynaptic inhibition generated by baclofen is caused largely by the suppression of presynaptic calcium influx. Presynaptic loading of the GDP analog guanosine-5'-O-(2-thiodiphosphate) (GDPbetaS) abolished the effect of baclofen on both presynaptic calcium currents and EPSCs. The nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) suppressed presynaptic calcium currents and occluded the effect of baclofen on presynaptic calcium currents and EPSCs. Photoactivation of GTPgammaS induced an inward rectifying potassium current at the calyx of Held, whereas baclofen had no such effect. We conclude that presynaptic GABAB receptors suppress transmitter release through G-protein-coupled inhibition of calcium currents.     

Inwardly rectifying and Ca2+-permeable AMPA-type glutamate receptor channels in rat neocortical neurons

Inwardly rectifying and Ca2+-permeable AMPA-type glutamate receptor channels in rat neocortical neurons. J. Neurophysiol. 78: 2592-2605, 1997. Current-voltage (I-V) relations and Ca2+ permeability of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)type glutamate receptor channels were investigated in neurons of rat neocortex by using the whole cell patch-clamp technique in brain slices. To activate AMPA receptor channels, kainate was used as a nondesensitizing agonist. A patch pipette was filled with solution containing 100 mu M spermine to maintain the inward rectification of Ca2+-permeable AMPA receptor channels. Three types of responses to kainate were observed: type I response with outwardly rectifying I-V relation, type II response with I-V relation of marked inward rectification, and intermediate response with I-V relation of weaker inward rectification. Neurons with type I, type II and intermediate I-V relations were referred to as type I, type II, and intermediate neurons, respectively. Of a total of 223 recorded cells, 90 (40.4%) were type I, 129 (57.8%) intermediate, and 4 (1.8%) type II neurons. Properties of AMPA receptor channels were examined in the former two types of neurons. The value of PCa:PCs, the ratio of the permeability coefficients of Ca2+ and Cs+, was estimated from the reversal potentials of kainate responses in the outside-out patches bathed in Na+-free solution containing 100 mM Ca2+ according to the constant-field equation. They ranged from 0.05 to 0.10 (0.08 +/- 0. 02, mean +/- SD, n = 8) for type I neurons and from 0.14 to 1.29 (0. 60 +/- 0.37, n = 11) for the intermediate neurons. There was a close correlation between the inward rectification and the Ca2+ permeability in AMPA receptor channels in these neurons. Intermediate neurons stained with biocytin were nonpyramidal cells with ellipsoidal-shaped somata. Type I neurons had either triangular- or ellipsoidal-shaped somata. Excitatory postsynaptic currents (EPSCs) recorded in both type I and intermediate neurons had 6-cyano-7-nitroquinoxaline-2,3-dione-sensitive fast and -2-amino-5-phosphonovalerate-sensitiveslow components. The I-V relation of the fast component exhibited inward rectification in the intermediate neuron, whereas that in the type I neuron showed slight outward rectification. The fast component of EPSCs in the intermediate neuron was suppressed more prominently (to 56 +/- 15% of the control, n = 12) than that in the type I neuron (to 78 +/- 6% of the control, n = 6) by bath application of 1 mM spermine. These results indicate that inwardly rectifying and Ca2+-permeable AMPA receptor channels are expressed in a population of neurons of rat neocortex and are involved in excitatory synaptic transmission.     

Targeted disruption of the Ca2+ channel beta3 subunit reduces N- and L-type Ca2+ channel activity and alters the voltage-dependent activation of P/Q-type Ca2+ channels in neurons

In comparison to the well characterized role of the principal subunit of voltage-gated Ca2+ channels, the pore-forming, antagonist-binding alpha1 subunit, considerably less is understood about how beta subunits contribute to neuronal Ca2+ channel function. We studied the role of the Ca2+ channel beta3 subunit, the major Ca2+ channel beta subunit in neurons, by using a gene-targeting strategy. The beta3 deficient (beta3-/-) animals were indistinguishable from the wild type (wt) with no gross morphological or histological differences. However, in sympathetic beta3-/- neurons, the L- and N-type current was significantly reduced relative to wt. Voltage-dependent activation of P/Q-type Ca2+ channels was described by two Boltzmann components with different voltage dependence, analogous to the "reluctant" and "willing" states reported for N-type channels. The absence of the beta3 subunit was associated with a hyperpolarizing shift of the "reluctant" component of activation. Norepinephrine inhibited wt and beta3-/- neurons similarly but the voltage sensitive component was greater for N-type than P/Q-type Ca2+ channels. The reduction in the expression of N-type Ca2+ channels in the beta3-/- mice may be expected to impair Ca2+ entry and therefore synaptic transmission in these animals. This effect may be reversed, at least in part, by the increase in the proportion of P/Q channels activated at less depolarized voltage levels.     

Dopamine D2-receptor isoforms expressed in AtT20 cells inhibit Q-type high-voltage-activated Ca2+ channels via a membrane-delimited pathway

Dopamine D2 receptors both acutely and chronically inhibit high-voltage-activated Ca2+ channels (HVA-CCs). Two alternatively spliced isoforms, D2L (long) and D2S (short), are expressed at high levels in rat pituitary intermediate lobe melanotropes but are lacking in anterior lobe corticotropes. We stably transfected D2L and D2S into corticotrope-derived AtT20 cells. Both isoforms coupled to inhibition of Q-type calcium channels through pertussis toxin-sensitive G proteins. Thus, we have created a model system in which to study the kinetics of D2-receptor regulation of Ca2+ channels. Rapid inhibition of HVA-CCs was characterized using a novel fluorescence video imaging technique for the measurement of millisecond kinetic events. We measured the time elapsed (lag time) between the arrival of depolarizing isotonic 66 mM K+, sensed by fluorescence from included carboxy-X-rhodamine (CXR), and the beginning of increased intracellular Ca2+ levels (sensed by changes in indo 1 fluorescence ratio). The lag time averaged 350-550 ms, with no significant differences among cell types. Addition of the D2-agonist quinpirole (250 microM) to the K+/CXR solution significantly increased the lag times for D2-expressing cells but did not alter the lag time for AtT20 controls. The increased lag times for D2L- and D2S-transfected cells suggest that at least a fraction of the Ca2+ channels was inhibited within the initial 350-550 ms. As this inhibition time is too fast for a multistep second messenger pathway, we conclude that inhibition occurs via a membrane-delimited diffusion mechanism.     

Activation of Ca2+-dependent currents in dorsal root ganglion neurons by metabotropic glutamate receptors and cyclic ADP-ribose precursors

Cultured dorsal root ganglion neurons were voltage clamped at -90 mV to study the effects of intracellular application of nicotinamide adenine dinucleotide (betaNAD+), intracellular flash photolysis of caged 3',5'-cyclic guanosine monophosphate (cGMP), and metabotropic glutamate receptor activation. The activation of metabotropic glutamate receptors evoked inward Ca2+-dependent currents in most cells. This was mimicked both by intracellular flash photolysis of the caged axial isomer of cGMP [P-1-(2-nitrophenyl)ethyl cGMP] and intracellular application of betaNAD+. Whole cell Ca2+-activated inward currents were used as a physiological index of raised intracellular Ca2+ levels. Extracellular application of 10 microM glutamate evoked the activation of Ca2+-dependent inward currents, thus reflecting a rise in intracellular Ca2+ levels. Similar inward currents were also activated after isolation of metabotropic glutamate receptor activation by application of 10 microM glutamate in the presence of 20 microM 6-cyano-7-nitroquinoxaline-2,3-dione and 20 microM dizocilpine maleate (MK 801), or by extracellular application of 10 microM trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid. Intracellular photorelease of cGMP, from its caged axial isomer, in the presence of betaNAD+ was also able to evoke similar Ca2+-dependent inward currents. Intracellular application of betaNAD+ alone produced a concentration-dependent effect on inward current activity. Responses to both metabotropic glutamate receptor activation and cGMP were suppressed by intracellular ryanodine, chelation of intracellular Ca2+ by bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid, and depletion of intracellular Ca2+ stores, but were insensitive to the removal of extracellular Ca2+. Therefore both cGMP, possibly via a mechanism that involves betaNAD+ and/or cyclic ADP-ribose, and glutamate can mobilize intracellular Ca2+ from ryanodine-sensitive stores in sensory neurons.     

Region-specific activity of the plasma membrane Ca2+ pump and delayed activation of Ca2+ entry characterize the polarized, agonist-evoked Ca2+ signals in exocrine cells

The initial release of Ca2+ from the intracellular Ca2+ stores is followed by a second phase during which the agonist-dependent Ca2+ response becomes sensitive to the extracellular Ca2+, indicating the involvement of the plasma membrane (PM) Ca2+ transport systems. The time course of activation of these transport systems, which consist of both Ca2+ extrusion and Ca2+ entry pathways, is not well established. To investigate the participation of these processes during the agonist-evoked Ca2+ response, isolated pancreatic acinar cells were exposed to maximal concentrations of an inositol 1,4,5-trisphosphate-mobilizing agonist (acetylcholine, 10 microM) in different experimental conditions. Following the increase of [Ca2+]i, there was an almost immediate activation of the PM Ca2+ extrusion system, and maximal activity was reached within less than 2s. The rate of Ca2+ extrusion was dependent on the level of [Ca2+]i, with a steep activation at values just above the resting [Ca2+]i and reached a plateau value at 700 nM Ca2+. In contrast, the PM Ca2+ entry pathway was activated with a much slower time course. There was also a delay of 3-4 s between the maximal effective depletion of the intracellular Ca2+ stores and the activation of this entry pathway. By use of digital imaging data, the PM Ca2+ transport systems were also analyzed independently in two regions of the cells, the lumenal and the basal poles. With respect to the activation of the Ca2+ entry pathways, no significant difference existed between these two regions. In contrast, the PM Ca2+ pump displayed a different pattern of activity in these regions. In the basal pole, the pump activity was more sensitive to changes of [Ca2+]i and had a higher maximal activity. Also, in the lumenal pole, the pump became saturated at values of [Ca2+]i around 700 nM, whereas at the basal pole [Ca2+]i had a biphasic effect on the pump activity, and higher [Ca2+]i inhibited the pump. It is argued that these differences in sensitivity to the levels of [Ca2+]i and the different relationship between [Ca2+]i and the rate of extrusion at the two functional poles of the pancreatic acinar cells indicate that the plasma membrane Ca2+ ATPase might play an important role in the polarization of the Ca2+ response.     

Mobilization of Ca2+ from intracellular stores in transfected neuro2a cells by activation of multiple opioid receptor subtypes

In neuronal cell lines, activation of opioid receptors has been shown to mobilize intracellular Ca2+ stores. In this report, we describe the excitatory actions of opioid agonists on murine neuroblastoma neuro2a cells stably expressing either delta, mu, or kappa opioid receptors. Fura-2-based digital imaging was used to record opioid-induced increases in intracellular Ca2+ concentration ([Ca2+]i). Repeated challenges of delta, mu, or kappa opioid receptor expressing cells with 100 nM [D-Ala2,D-Leu5]-enkephalin (DADLE), [D-Ala2,N-Me-Phe4,Gly-ol]-enkephalin (DAMGO), or trans-(+/-)-3,4-dichloro N-methyl-N-(2-[1-pyrollidinyl] cyclohexyl) benzene acetamide (U-50488H), respectively, elicited reproducible Ca2+ responses. Non-transfected neuro2a cells did not respond to opioid agonists. Removal of extracellular Ca2+ from the bath prior to and during agonist challenge did not affect significantly the agonist-evoked increase in [Ca2+]i, indicating that the response resulted from the release of Ca2+ from intracellular stores. Naloxone reversibly inhibited responses in all three cell lines, confirming that they were mediated by opioid receptors. Expression of cloned opioid receptors in neuro2a cells, coupled with digital [Ca2+]i imaging, provides a model system for the study of opioid receptors and opioid-activated signaling processes. The fact that all three receptors coupled to the same intracellular signaling mechanism suggests that the primary functional difference between opioid responses in vivo results from their selective localization.     

Inhibition of M-type K+ and N-type Ca2+ channels by the human gonadotropin-releasing-hormone receptor heterologously expressed in adult neurons

Gonadotropin-releasing hormone (GnRH) controls all aspects of reproductive function. GnRH is secreted by hypothalamic neurons and exerts its effects on the endocrine system through pituitary gonadotropes, while its effects on sexual receptivity are mediated by the central nervous system. The electrophysiological responses of central neurons to GnRH have shown both excitatory and inhibitory responses, but little is known about the mechanisms by which GnRH can change neuronal excitability. The present study addresses the mechanisms whereby stimulation of the human GnRH receptor changes neuronal excitability by using a combination of electrophysiological and heterologous expression techniques. Microinjection of in vitro transcribed cRNA coding for the human GnRH receptor into enzymatically dissociated adult rat superior cervical ganglion neurons resulted in GnRH receptor expression. Activation of the GnRH receptor inhibited both M-type K+ and N-type Ca2+ channels. Inhibition of M-type K+ channels was insensitive to pertussis toxin pretreatment and blocked by intracellular GDPbetaS. Inhibition of Ca2+ channels was slow in onset, voltage independent and insensitive to pertussis toxin. Wash-out of GnRH resulted in an unusual transient reversal of tonic G-protein-mediated Ca2+ channel inhibition. Block of the N-type Ca2+ channel with omega-conotoxin GVIA decreased Ca2+ current inhibition from 43 to 15%, indicating that the N-type Ca2+ channel is an effector target. Ca2+ channel inhibition was completely abolished by including a Ca2+ chelator in the patch pipette. Cell-attached macropatch experiments indicated that Ca2+ channel inhibition is mediated by a diffusible second messenger. These results demonstrate that the human GnRH receptor can inhibit M-type K+ and N-type Ca2+ channels when heterologously expressed in adult rat neurons. Modulation of M-type K+ and N-type Ca2+ channels in central neurons which contain GnRH receptors is likely to contribute to the changes in neuronal excitability elicited by GnRH.