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1.
J Physiol ; 526 Pt 1: 99-108, 2000 Jul 01.
Article in English | MEDLINE | ID: mdl-10878103

ABSTRACT

We employed the whole-cell recording technique in conjunction with fluorometry to measure cytosolic Ca(2+) concentration ([Ca(2+)](i)) and exocytosis (capacitance measurement) in single, identified rat gonadotrophs. Direct activation of G-protein (via intracellular dialysis of non-hydrolysable analogues of GTP, but not of GDP) triggered a slow rise in capacitance even in the presence of a fast intracellular Ca(2+) chelator. The broad-spectrum kinase inhibitors H7 and staurosporine did not prevent this Ca(2+)-independent exocytosis, ruling out the involvement of the cAMP and PKC pathways. AlF(4)(-), a potent stimulator of heterotrimeric G-proteins, failed to stimulate any exocytosis when the intracellular Ca(2+) store was depleted, implicating the involvement of AlF(4)(-)-insensitive G-protein(s). Maximal stimulation of Ca(2+)-independent exocytosis by GTP analogues did not reduce the number of readily releasable granules that were available subsequently for Ca(2+)-dependent release. The last finding raises the possibility that the G-protein-stimulated Ca(2+)-independent exocytosis may regulate a pool of granules that is distinct from the Ca(2+)-dependent pool.


Subject(s)
Calcium/metabolism , Egtazic Acid/analogs & derivatives , Exocytosis/physiology , GTP-Binding Proteins/metabolism , Pituitary Gland, Anterior/metabolism , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , Aluminum Compounds/pharmacology , Animals , Calcium-Transporting ATPases/antagonists & inhibitors , Cyclic AMP/metabolism , Cytoplasmic Granules/drug effects , Cytoplasmic Granules/metabolism , Cytosol/metabolism , Egtazic Acid/pharmacology , Enzyme Inhibitors/pharmacology , Exocytosis/drug effects , Fluorides/pharmacology , Fluorometry , GTP-Binding Proteins/pharmacology , Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology , Guanylyl Imidodiphosphate/pharmacology , Male , Patch-Clamp Techniques , Photolysis , Pituitary Gland, Anterior/cytology , Pituitary Gland, Anterior/drug effects , Protein Kinase C/antagonists & inhibitors , Rats , Staurosporine/pharmacology
2.
Bioessays ; 21(10): 861-5, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10497336

ABSTRACT

The release of Ca(2+) from intracellular stores is an important trigger for secretion in many cell types. Depending on the spatial relationship between the intracellular Ca(2+) stores and the site of exocytosis, the Ca(2+) signal can be very local or spread throughout the entire cell. Here, we review how the release of Ca(2+) from inositol trisphospate (IP(3))-sensitive stores contributes differently to the stimulus-secretion coupling in three types of secretory cells (acinar cells of the pancreas, gonadotrophs, and corticotrophs of the anterior pituitary gland). We propose that in both pancreatic acinar cells and pituitary gonadotrophs the IP(3)-sensitive stores may be in close proximity to the sites of exocytosis such that the concentration of Ca(2+) at these sites are transiently much higher than the average cytosolic Ca(2+) concentration. In contrast, the local Ca(2+) gradient is less prominent in pituitary corticotrophs. Finally, some recent technical developments that may contribute significantly to future investigations of local Ca(2+) signals are discussed.


Subject(s)
Calcium Signaling/physiology , Exocytosis/physiology , Adrenocorticotropic Hormone/metabolism , Animals , Gonadotropins, Pituitary/metabolism , Humans , Pancreas/cytology , Pancreas/physiology , Pituitary Gland, Anterior/cytology , Pituitary Gland, Anterior/physiology
3.
J Biol Chem ; 274(27): 19095-102, 1999 Jul 02.
Article in English | MEDLINE | ID: mdl-10383412

ABSTRACT

The fungal metabolite, brefeldin A (BFA), is known to inhibit guanine nucleotide exchange on the ADP-ribosylating factors that are involved in vesicle membrane trafficking. Here, we investigated the action of BFA on Ca2+-regulated exocytosis in single rat adrenal chromaffin cells. Incubation of chromaffin cells with BFA (1 or 10 microM) for 2 h effectively disrupted the Golgi membranes but did not affect the pattern of catecholamine release triggered by high extracellular K+, which was monitored with carbon fiber amperometry along with cytosolic Ca2+ measurement. The BFA treatment, however, increased the mean quantal size of catecholamine-containing vesicles and the occurrence of amperometric events with a "foot" or "stand alone" signal (which reflects sluggish or incomplete dilation of the fusion pore). To examine whether BFA altered the Ca2+-dependence of exocytosis, we employed the whole-cell recording technique in conjunction with the capacitance measurement to measure exocytosis evoked from the entire cell during voltage-gated Ca2+ entry. Our results suggested that BFA treatment did not alter either the initial rate of capacitance increase or the total amount of capacitance increase. Therefore, in chromaffin cells, BFA treatment affects Ca2+-regulated exocytosis predominantly by increasing the quantal size and by slowing the fusion kinetics of some vesicles.


Subject(s)
Brefeldin A/pharmacology , Catecholamines/metabolism , Chromaffin Cells/physiology , Protein Synthesis Inhibitors/pharmacology , Animals , Calcium/metabolism , Cells, Cultured , Chromaffin Cells/drug effects , Electrophysiology , Exocytosis/drug effects , Golgi Apparatus/drug effects , Golgi Apparatus/physiology , Kinetics , Male , Potassium/metabolism , Rats
4.
Endocrinology ; 140(7): 3025-33, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10385394

ABSTRACT

Alpha-latrotoxin (LTX) from the black widow spider venom, stimulates neurotransmitter release from neuronal cells via Ca2+ -dependent as well as Ca2+ -independent mechanisms. In some peptide-secreting endocrine cells, however, LTX stimulates hormone release mainly via a Ca2+ -independent mechanism. Here we investigated the action of LTX in rat pituitary gonadotropes that secrete the peptide, LH. Using the patch-clamp technique in conjunction with the fluorescent Ca2+ indicator (indo-1) to simultaneously measure the cytosolic Ca2+ concentration ([Ca2+]i) and ionic current, we showed that LTX elicited bursts of inward current that were accompanied by [Ca2+]i elevations. In the presence of a physiological concentration of extracellular Ca2+, the unitary conductance of the LTX-induced current was about 300 pS, and only about 6.4% of the current was carried by Ca2+. The LTX-induced current was occasionally followed by intracellular Ca2+ release. At [Ca2+]i of 1 microM or more, exocytosis (detected by membrane capacitance measurement) was consistently triggered, and it was frequently followed by endocytosis. Thus, LTX triggers Ca2+ -dependent exocytosis in gonadotropes via extracellular Ca2+ entry as well as intracellular Ca2+ release. In approximately 25% of the cells, LTX could also trigger a slow exocytosis in the absence of [Ca2+]i elevation. Therefore, LTX has both Ca2+ -dependent and Ca2+ -independent actions in gonadotropes.


Subject(s)
Calcium/metabolism , Cytosol/metabolism , Exocytosis/drug effects , Gonadotropins, Pituitary/metabolism , Pituitary Gland, Anterior/physiology , Spider Venoms/pharmacology , Animals , Cations/metabolism , Electric Conductivity , Extracellular Space/metabolism , Ion Channels/metabolism , Male , Osmolar Concentration , Pituitary Gland, Anterior/cytology , Pituitary Gland, Anterior/metabolism , Rats , Rats, Sprague-Dawley
5.
J Physiol ; 512 ( Pt 2): 385-93, 1998 Oct 15.
Article in English | MEDLINE | ID: mdl-9763629

ABSTRACT

1. The patch clamp technique was used in conjunction with a fluorescent Ca2+ indicator (indo-1, or indo-1FF) to measure simultaneously cytosolic Ca2+ concentration ([Ca2+]i), ionic current and changes in membrane capacitance in single rat corticotrophs identified with the reverse haemolytic plaque assay. 2. Application of the adrenocorticotropin (ACTH) secretagogue noradrenaline (NA; norepinephrine), triggered [Ca2+]i oscillation in corticotrophs via alpha-adrenergic receptors and the guanosine trisphosphate (GTP) binding protein-coupled phosphoinositide pathway. 3. Simultaneous measurement of [Ca2+]i and capacitance shows that exocytosis was triggered during the first cycle of NA-induced [Ca2+]i oscillation and the mean increase in cell membrane surface area was 1.4 +/- 0.3 % (n = 6). 4. When Ca2+ was directly released from the inositol 1,4, 5 trisphosphate (IP3)-sensitive store via flash photolysis of caged IP3, the mean increase in cell surface area was 1.5 +/- 0.5 % (n = 6). Thus, NA-stimulated ACTH secretion in rat corticotrophs is closely coupled to intracellular Ca2+ release. 5. Large and rapid elevation of [Ca2+]i (>15 microM) via flash photolysis of caged Ca2+ triggered two phases of exocytosis: a rapid exocytic burst that was complete in approximately 100 ms and a slow burst that continued for many seconds. 6. The rapid exocytic burst reflected the exhaustion of a pool of readily releasable granules and, on average, increased the cell surface by 2.8 +/- 0.1 % (n = 14). 7. We suggest that the relatively weak exocytic response in corticotrophs during intracellular Ca2+ release may be partially attributed to a smaller pool of readily releasable granules.


Subject(s)
Adrenergic alpha-Agonists/pharmacology , Calcium/metabolism , Cytosol/metabolism , Exocytosis/drug effects , Pituitary Gland/metabolism , Adrenocorticotropic Hormone/metabolism , Animals , Calcium Signaling/drug effects , Calcium Signaling/physiology , Cytosol/drug effects , Cytosol/radiation effects , Electric Stimulation , Electrophysiology , Exocytosis/radiation effects , GTP-Binding Proteins/metabolism , In Vitro Techniques , Inositol 1,4,5-Trisphosphate/metabolism , Male , Membrane Potentials/physiology , Norepinephrine/pharmacology , Patch-Clamp Techniques , Photolysis , Pituitary Gland/cytology , Pituitary Gland/drug effects , Rats , Rats, Sprague-Dawley
6.
Neuron ; 18(1): 121-32, 1997 Jan.
Article in English | MEDLINE | ID: mdl-9010210

ABSTRACT

Exocytosis and the cell-averaged cytosolic [Ca2+], [Ca2+]i, were tracked in single gonadotrophs. Cells released 100 granules/s at 1 microM = [Ca2+]i when gonadotropin-releasing hormone (GnRH) activated IP3-mediated Ca2+ release from internal stores, but only 1 granule/s when [Ca2+]i was raised uniformly to 1 microM by other means. Strong exocytosis was then seen only at higher [Ca2+]i (half-maximal at 16 microM). Parallel second messengers did not contribute to GnRH-induced exocytosis, because IP3 alone was as effective as GnRH, and because even GnRH failed to trigger rapid exocytosis when the [Ca2+]i rise was blunted by EGTA. When [Ca2+]i was released from stores, exocytosis depended on [Ca2+]i rising rapidly, as if governed by Ca2+ flux into the cytosol. We suggest that IP3 releases Ca2+ selectively from subsurface cisternae, raising [Ca2+] near exocytic sites 5-fold above the cell average.


Subject(s)
Calcium/metabolism , Cytoplasmic Granules/metabolism , Exocytosis , Gonadotropin-Releasing Hormone/pharmacology , Inositol 1,4,5-Trisphosphate/pharmacology , Pituitary Gland, Anterior/physiology , Acetates/pharmacology , Animals , Chelating Agents/pharmacology , Cytoplasmic Granules/drug effects , Cytoplasmic Granules/ultrastructure , Cytosol/metabolism , Egtazic Acid/pharmacology , Ethylenediamines/pharmacology , In Vitro Techniques , Kinetics , Membrane Potentials , Patch-Clamp Techniques , Photolysis , Pituitary Gland, Anterior/drug effects , Pituitary Gland, Anterior/ultrastructure , Rats , Rats, Sprague-Dawley , Time Factors
7.
Pflugers Arch ; 430(5): 645-52, 1995 Sep.
Article in English | MEDLINE | ID: mdl-7478915

ABSTRACT

In rat pituitary gonadotropes, gonadotropin-releasing hormone (GnRH) stimulates rhythmic release of Ca2+ from stores sensitive to inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], which in turn induces an oscillatory activation of apamin-sensitive Ca2+-activated K+ current, IK(Ca). Since GnRH also activates protein kinase C (PKC), we investigate the action of PKC while simultaneously measuring intracellular Ca2+ concentration ([Ca2+]i) and IK(Ca). Stimulation of PKC by application of phorbol 12-myristate 13-acetate (PMA) did not affect basal [Ca2+]i. However, PMA or phorbol 12,13-dibutyrate (PdBu), but not the inactive 4alpha-phorbol 12,13-didecanoate (4alpha-PDD), reduced the frequency of GnRH-induced [Ca2+]i oscillation and augmented the IK(Ca) induced by any given level of [Ca2+]i. The slowing of oscillations and the enhancement of IK(Ca) were mimicked by synthetic diacylglycerol (1,2-dioctanoyl-sn-glycerol) and could be induced during ongoing oscillations that had been initiated irreversibly in cells loaded with guanosine 5'-O-(3-thiotriphosphate) (GTP-[gammaS]). In contrast, when oscillations were initiated by loading cells with Ins(1,4,5)P3, phorbol esters enhanced IK(Ca) without affecting the frequency of oscillation. The protein kinase inhibitor, staurosporine, reduced IK(Ca) without affecting [Ca2+]i and partially reversed the phorbol-ester-induced slowing of oscillation. Therefore, activation of PKC has two rapid effects on gonadotropes. It slows [Ca2+]i oscillations probably by actions on phospholipase C, and it enhances IK(Ca) probably by a direct action on the channels.


Subject(s)
Apamin/pharmacology , Calcium/metabolism , Gonadotropin-Releasing Hormone/biosynthesis , Pituitary Gland/metabolism , Potassium Channels/metabolism , Alkaloids/pharmacology , Animals , Calcium/pharmacology , Diglycerides/pharmacology , Electrophysiology , Enzyme Inhibitors/pharmacology , Gonadotropin-Releasing Hormone/pharmacology , In Vitro Techniques , Male , Membrane Potentials/drug effects , Membrane Potentials/physiology , Patch-Clamp Techniques , Phorbol Esters/pharmacology , Pituitary Gland/cytology , Pituitary Gland/drug effects , Potassium Channels/drug effects , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Rats , Rats, Sprague-Dawley , Staurosporine , Type C Phospholipases/antagonists & inhibitors , Type C Phospholipases/metabolism
10.
Proc Natl Acad Sci U S A ; 91(21): 9750-4, 1994 Oct 11.
Article in English | MEDLINE | ID: mdl-7937885

ABSTRACT

Gonadotropin-releasing hormone induces oscillatory release of Ca2+ from inositol trisphosphate-sensitive stores of gonadotropes. Simultaneously with electrophysiological measures of cytoplasmic [Ca2+], corresponding changes in [Ca2+] within intracellular stores were monitored with a fluorescent dye, mag-indo-1. Each cycle of oscillation released only 10% of the detectable stored Ca2+. Some Ca2+ was recovered by the stores using a mechanism sensitive to inhibitors of intracellular Ca2+ ATPases, and much of the remainder was temporarily and rapidly pumped into other intracellular compartments or out of the cell. The dynamics of Ca2+ oscillations are thus more complex than a repeated emptying and refilling of a single compartment. The free concentrations measured show that intracellular Ca2+ store compartments contain strong Ca2+ buffers.


Subject(s)
Calcium/metabolism , Gonadotropin-Releasing Hormone/pharmacology , Pituitary Gland, Anterior/metabolism , Animals , Calcium-Transporting ATPases/antagonists & inhibitors , Hydroquinones/pharmacology , In Vitro Techniques , Kinetics , Male , Oscillometry , Pituitary Gland, Anterior/cytology , Pituitary Gland, Anterior/drug effects , Rats , Rats, Sprague-Dawley , Terpenes/pharmacology , Thapsigargin , Time Factors
11.
J Physiol ; 477 ( Pt 3): 511-25, 1994 Jun 15.
Article in English | MEDLINE | ID: mdl-7932239

ABSTRACT

1. Whole-cell voltage clamp was used in conjunction with the fluorescent Ca2+ indicator indo-1 to measure extracellular Ca2+ entry and intracellular Ca2+ concentrations ([Ca2+]i) in rat gonadotrophs identified with the reverse haemolytic plaque assay. 2. Depolarizations to potentials more positive than -40 mV elicited inward Ca2+ current (ICa) and transient elevations of [Ca2+]i. 3. The relationship between [Ca2+]i elevations and Ca2+ entry with different Ca2+ buffer concentrations in the pipette showed that endogenous Ca2+ buffers normally bind approximately 99% of the Ca2+ entering the cell. 4. With [Ca2+]i elevations less than 500 nM, decay of [Ca2+]i could be approximated by an exponential whose time constant increased with the concentration of exogenous Ca2+ buffers. 5. Inhibitors of intracellular Ca(2+)-ATPases, thapsigargin, cyclopiazonic acid (CPA) and 2,5-di-(tert-butyl)-1,4-benzohydroquinone (BHQ), caused [Ca2+]i to rise. Application of BHQ during [Ca2+]i oscillations induced by gonadotrophin-releasing hormone (GnRH) terminated the oscillation in a slowly decaying elevation. BHQ slowed the decay of depolarization-induced [Ca2+]i elevations about 3-fold. 6. Taking into account the Ca2+ buffering properties of the cytoplasm permitted estimation of the fluxes and rate constants for Ca2+ movements in gonadotrophs. The intracellular store is a major determinant of Ca2+ homeostasis in gonadotrophs.


Subject(s)
Calcium/metabolism , Gonadotropins, Pituitary/metabolism , Pituitary Gland, Anterior/metabolism , Animals , Buffers , Calcium-Transporting ATPases/metabolism , Cell Membrane Permeability/physiology , Electrophysiology , Homeostasis , Intracellular Membranes/metabolism , Male , Osmolar Concentration , Pituitary Gland, Anterior/cytology , Rats , Rats, Sprague-Dawley , Time Factors
14.
J Cell Biol ; 121(3): 543-52, 1993 May.
Article in English | MEDLINE | ID: mdl-8486735

ABSTRACT

We have investigated the mechanism of cell fusion mediated by HA, the fusogenic hemagglutinin of the Influenza viral envelope. Single erythrocytes (RBCs) were attached to fibroblasts expressing the HA on their cell surface, and fusion of the paired cells was triggered by rapid acidification. The RBC membrane was stained with fluorescent lipid, and the fusion-induced escape of lipid into the fibroblast was observed by quantitative image analysis. At the same time, the formation of an aqueous connection (i.e., the fusion pore) between the two cells was monitored electrically. Within minutes after acidification, an electrical conductance between the two cells appeared abruptly as the fusion pore opened, and then increased gradually as the pore dilated. Later, fluorescent lipid diffused into the fibroblast, approaching equilibrium over the next 5-20 min. No lipid flux was seen while the pore conductance remained 0.5 nS or less. Evidently lipid flux requires a threshold pore size. Our finding suggests that the smallest and earliest fusion pores are surrounded by a ring of protein. A fusion pore expands by breaking this ring and recruiting lipid into its circumference.


Subject(s)
Cell Fusion/drug effects , Cell Membrane/drug effects , Hemagglutinins, Viral/pharmacology , Lipid Metabolism , Viral Envelope Proteins/pharmacology , 3T3 Cells , Animals , Erythrocytes , Hemagglutinin Glycoproteins, Influenza Virus , Humans , Hydrogen-Ion Concentration , Mice , Orthomyxoviridae
15.
Science ; 260(5104): 82-4, 1993 Apr 02.
Article in English | MEDLINE | ID: mdl-8385366

ABSTRACT

In pituitary gonadotropes, gonadotropin-releasing hormone (GnRH) induces the rhythmic release of Ca2+ from an inositol 1,4,5-trisphosphate (IP3)-sensitive store. Simultaneous measurement of the concentration of cytosolic free Ca2+ ([Ca2+]i) and exocytosis in single identified gonadotropes showed that each elevation of [Ca2+]i induced a burst of exocytosis. These phenomena were largely suppressed by buffering of [Ca2+]i but persisted in the absence of extracellular Ca2+. Activation of voltage-gated Ca2+ channels by brief depolarizations seldom supplied enough Ca2+ for exocytosis, but [Ca2+]i elevations induced by photolysis of caged IP3 did trigger exocytosis, confirming that GnRH-stimulated gonadotropic hormone secretion is closely coupled to intracellular Ca2+ release. Agonist-induced oscillations of [Ca2+]i in secretory cells may be a mechanism to optimize the secretory output while avoiding the toxic effects of sustained elevation of [Ca2+]i.


Subject(s)
Calcium/metabolism , Exocytosis/drug effects , Gonadotropin-Releasing Hormone/pharmacology , Pituitary Gland/physiology , Action Potentials , Animals , Calcium Channels/drug effects , Calcium Channels/physiology , Cytoplasmic Granules/drug effects , Cytoplasmic Granules/physiology , Electrophysiology , Gonadotropin-Releasing Hormone/administration & dosage , Inositol 1,4,5-Trisphosphate/pharmacology , Male , Periodicity , Photolysis , Pituitary Gland/drug effects , Pituitary Gland/ultrastructure , Rats , Rats, Sprague-Dawley
16.
J Neurosci ; 12(5): 1781-8, 1992 May.
Article in English | MEDLINE | ID: mdl-1374462

ABSTRACT

Hippocampal astrocytes were acutely isolated by papain treatment and mechanical trituration. Astrocytes were identified by their distinctive stellate morphology and immunocytochemical staining for glial fibrillary acidic protein. The electrophysiological properties of these cells were investigated using whole-cell voltage-clamp techniques. Three kinetically and pharmacologically distinct voltage-activated K+ currents were identified in most cells; they resembled the neuronal A-current, delayed rectifier, and inward rectifier. The activation threshold of the A-current was -40 mV with a time to peak that ranged from 10 msec at -20 mV to 6 msec at 100 mV. Steady-state inactivation was observed when the holding potential was positive to -100 mV. The current was half-inactivated at -60 mV and totally inactivated at -20 mV. The A-current was suppressed by 4-aminopyridine (4-AP). The delayed rectifier was activated by depolarizing pulses more positive than -40 mV and had a half time of activation that ranged from 18 msec at -20 mV to 10 msec at potentials more positive than 40 mV. This current did not inactivate during a 100 msec pulse and was suppressed by extracellular tetraethylammonium (TEA). An inwardly rectifying current was elicited by hyperpolarizing pulses more negative than -80 mV. This current was not blocked by extracellular TEA or 4-AP and was never observed in the presence of external Ba2+. Voltage-activated inward Na+ currents were never observed. Voltage-activated K+ channels may enhance the local K+ spatial buffering capabilities of the astrocyte syncytium when extracellular [K+] increases during neuronal activity.


Subject(s)
Astrocytes/physiology , Hippocampus/physiology , Potassium/physiology , Animals , Astrocytes/cytology , Cell Separation/methods , Electrophysiology , Hippocampus/cytology , Ion Channels/physiology
17.
J Comp Neurol ; 304(1): 135-46, 1991 Feb 01.
Article in English | MEDLINE | ID: mdl-2016409

ABSTRACT

Synaptic terminals of excitatory and inhibitory neurons supplying muscle fibers in leg muscles of crabs (Pachygrapsus crassipes and Hyas areneus) were investigated with light and electron microscopy. Terminals responsible for large excitatory postsynaptic potentials (EPSPs) at low frequencies of activation had a compact configuration with clusters of terminal boutons radiating from the main axon branch. Terminals responsible for small EPSPs had a more diffuse organization, with boutons often arranged in series along thin axon branches. Inhibitory neurons, when activated, produced both presynaptic and postsynaptic inhibitory effects, with the former being more potent at low frequencies of activation. Presynaptic inhibition was variable in magnitude but was generally strong in fibers with large EPSPs. Representative terminals from regions of strong and weak presynaptic inhibition were identified by activity-dependent uptake of horseradish peroxidase, serially sectioned, and reconstructed from electron micrographs. Both regions were found to contain axo-axonal synapses from inhibitory to excitatory terminals, with a larger number in the region of strong presynaptic inhibition. In addition, axo-axonal synapses were more uniformly distributed in the latter region. The number of inhibitory presynaptic dense bars (active zones) was somewhat higher in the region of weak inhibition, but larger individual dense bars occurred in the region of strong inhibition. Possible factors contributing to the differences in strength of inhibition include: (1) morphology and electrical properties of terminals; and (2) high probability of transmission at a relatively small number of inhibitory synapses during low frequency activation in the region of strong inhibition.


Subject(s)
Brachyura/anatomy & histology , Neuromuscular Junction/ultrastructure , Action Potentials , Animals , Hindlimb/innervation , Microscopy, Electron , Nerve Endings/ultrastructure , Synapses/ultrastructure
18.
Neuroscience ; 43(2-3): 429-36, 1991.
Article in English | MEDLINE | ID: mdl-1681465

ABSTRACT

We employed the whole cell patch-clamp technique to examine the ionic currents induced via activation of kainate/quisqualate receptors on striatal neurons in primary culture when N-methyl-D-aspartate receptors were blocked by selective antagonists. Bath perfusion of 10 microM-1 mM each of quisqualate, glutamate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (a selective quisqualate agonist) or kainate, induced only a sustained current, but more rapid application by pressure ejection of each of the first three agonists (but not kainate) also activated a rapidly desensitizing current. The current induced by a near-saturating concentration of kainate (1 mM) was, on average, 16-fold larger than the maximum sustained current induced by quisqualate (10 microM), or 7.5-fold larger than that induced by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (100 microM) or glutamate (100 microM). When kainate (100 microM-10 mM) was co-applied with each of the agonists (1 microM-1 mM), the sustained current was not the algebraic sum of the currents activated by kainate or the other agonist alone; rather, the kainate-induced current was increasingly occluded by co-application with increasing concentrations of another agonist. The potency to occlude kainate-induced current had a rank order of quisqualate greater than alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate approximately glutamate; although at sufficiently high concentrations all three agonists could occlude the kainate-induced current completely. When kainate and quisqualate were co-applied during the continued presence of quisqualate, the onset of the kainate-induced sustained current was dramatically slowed. However, the steady-state occlusion by quisqualate could be abolished when the ratio kainate to quisqualate was raised to 100:1; therefore, the occlusion appears to involve a competition between kainate and quisqualate at some shared receptor binding sites which have a higher affinity for quisqualate than kainate.


Subject(s)
Corpus Striatum/physiology , Kainic Acid/pharmacology , Neurons/physiology , Quisqualic Acid/pharmacology , Amino Acids/physiology , Animals , Cells, Cultured , Corpus Striatum/cytology , Drug Interactions , Electrophysiology , Glutamates/pharmacology , Glutamic Acid , Ibotenic Acid/analogs & derivatives , Ibotenic Acid/pharmacology , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid
20.
Mol Pharmacol ; 38(2): 229-36, 1990 Aug.
Article in English | MEDLINE | ID: mdl-2166903

ABSTRACT

The pharmacological properties and modulation by lithium of the kainate (KA) receptor system coupled to the evoked release of [3H]-gamma-aminobutyric acid [( 3H]GABA) from purified populations of striatal neurons in primary culture were examined. KA evoked a dose-dependent (EC50, 100 microM) and saturable increase in [3H]GABA release from striatal neurons that was unaffected by the removal of extracellular calcium and resistant to the actions of tetrodotoxin. The release of [3H]GABA evoked by 100 microM KA was attenuated in a dose-dependent manner by the following excitatory amino acid antagonists (IC50):6-cyano-2, 3-dihydroxy-7-nitroquinoxaline (2 microM),2,3-dihydroxy-6,7-dinitroquinoxaline (2 microM), kynurenate (0.3 mM), and gamma-D-glutamylglycine (2 mM). The antagonist properties of 6-cyano-2,3-dihydroxy-7-nitroquinoxaline, kynurenate, and gamma-D-glutamylglycine were competitive in nature, inducing parallel rightward shifts of the KA dose-response curves. At concentrations at which it did not significantly increase basal levels of [3H]GABA release, quisqualate attenuated in a dose-dependent manner (IC50, 10 microM) the release due to 100 microM KA. The quisqualate receptor agonist alpha-amino-3-hydroxyisoxazolepropionic acid (AMPA), however, exerted a biphasic effect on 100 microM KA-evoked release of [3H]GABA. At lower concentrations of AMPA (0.1-10 microM), the release due to 100 microM KA was potentiated 25-50%; at higher concentrations (greater than 10 microM) AMPA induced a dose-dependent (IC50, 100 microM) attenuation of KA-evoked release. The release of [3H]GABA due to 100 microM KA was significantly potentiated by the replacement of sodium with lithium in the extracellular medium. A significant potentiation (20-30%) was detected with as little as 5-10 mM lithium, and maximal effects (100-110% increase) were obtained with 50-75 mM lithium. Replacement of sodium with choline or N-methyl-D-glucamine could not mimic the actions of lithium. Lithium (25 mM) also induced a 4-fold increase in the levels of endogenous GABA release due to 100 microM KA. Whole-cell voltage-clamp recordings of these striatal neurons indicated that the 100 microM KA-induced inward current was not significantly altered in the presence of 25 mM lithium. Lithium attenuated vasoactive intestinal polypeptide-stimulated cyclic AMP formation by 50%, with a dose dependence similar to that of its actions on KA-evoked release. The results of this study demonstrate a distinct pharmacological profile for the KA receptor system coupled to the evoked release of [3H]GABA from striatal neurons.(ABSTRACT TRUNCATED AT 400 WORDS)


Subject(s)
Corpus Striatum/drug effects , Kainic Acid/pharmacology , Lithium/pharmacology , Receptors, Neurotransmitter/metabolism , gamma-Aminobutyric Acid/metabolism , Animals , Cells, Cultured , Corpus Striatum/metabolism , Dose-Response Relationship, Drug , Electrophysiology , Mice , Neurons/drug effects , Neurons/metabolism , Receptors, Kainic Acid
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