Effects of VPAC2 receptor activation on membrane excitability and GABAergic transmission in subparaventricular zone neurons targeted by suprachiasmatic nucleus.

The hypothalamic suprachiasmatic nucleus (SCN) harbors the master circadian pacemaker. SCN neurons produce the amino acid gamma-aminobutyric acid (GABA) and several peptide molecules for coordination and communication of their circadian rhythms. A subpopulation of SCN cells synthesizes vasoactive intestinal polypeptide (VIP) and provides a dense innervation of the subparaventricular zone (SPZ), an important CNS target of the circadian pacemaker. In this study, using patch-clamp recording techniques and rat brain slice preparations, the contribution of VIP to SCN efferent signaling to SPZ was evaluated by examining membrane responses of SPZ neurons to exogenous VIP receptor ligands. In approximately 50% of the SPZ neurons receiving monosynaptic GABAA receptor-mediated inputs from SCN, bath-applied VIP (0.5-1 microM) resulted in a membrane depolarization caused by tetrodotoxin-resistant inward currents reversing at approximately -23 mV. These data suggest the existence of postsynaptic receptors that activate a nonselective cationic conductance. In addition, a subset of SPZ neurons showed an increase in the amplitude of SCN-evoked GABAergic inhibitory postsynaptic currents (IPSCs) and a decrease in their paired-pulse ratios. This, together with an increase in frequency of spontaneous and miniature IPSCs, implies the presence of presynaptic receptors that facilitate GABA release from SCN and possibly other synaptic terminals. The effects occurred in separate neurons and could be mimicked by the selective VPAC2 receptor agonist BAY 55-9837 (0.2-0.5 microM) and partially blocked by the VIP receptor antagonist VIP(6-28) (5 microM). The results indicate that VIP acts via both post- and presynaptic VPAC2 receptors to differentially modulate SCN GABAergic signaling to distinct subpopulations of SPZ neurons.

Acid-sensitive TASK-like K+ conductances contribute to resting membrane potential and to orexin-induced membrane depolarization in rat thalamic paraventricular nucleus neurons.

Orexin (hypocretin) peptides are known to depolarize rat thalamic paraventricular nucleus (PVT) neurons by suppression of one or more undefined potassium conductances. Here, we investigated a contribution of TWIK-related acid-sensitive K(+) (TASK) channels to the resting membrane potential and orexin-induced depolarization of PVT neurons, using patch clamp recording techniques in brain slice preparations. Upon exposure to an acidic (pH 6.3) extracellular solution, PVT neurons displayed membrane depolarization. Under voltage-clamp and in the presence of tetrodotoxin (TTX, 0.5 microM), low pH solutions induced an inward shift in baseline membrane current, accompanied by a net decrease in membrane conductance, reversing close to the potassium equilibrium potential. By contrast, exposure to alkaline (pH 8.3) solutions resulted in membrane hyperpolarization, induced an outward shift in baseline membrane current and an increase in net conductance that reversed close to the potassium equilibrium potential. A local anesthetic bupivacaine (20-40 microM) and the endocannabinoid anandamide (5-10 microM) mimicked the effects of the acidic solution. Exposure to the volatile anesthetic isoflurane (0.2-0.5 mM) induced changes in resting membrane potential, baseline current and membrane conductance similar to those caused by the alkaline solution. Although responsiveness to orexins was preserved under each of the above conditions, the amplitude of the orexin B (0.5 microM)-induced inward current was depressed in the acidic solution and in the presence of anandamide, remained largely unchanged in the alkaline solution, and was enhanced by isoflurane when compared with that in normal artificial cerebrospinal solution. We conclude that pH-sensitive potassium channels, TASK-1 and TASK-3 channels, contribute substantially to the resting membrane conductance(s) and excitability in PVT neurons. The observations that orexin-induced currents were affected by putative TASK-specific drugs in a manner predictable from their effects on TASK channels also suggest that the orexin-induced excitation in PVT neurons is mediated by closure of TASK channels.

Orexin-induced modulation of state-dependent intrinsic properties in thalamic paraventricular nucleus neurons attenuates action potential patterning and frequency.

The thalamic paraventricular nucleus (PVT) receives a dense innervation from orexin-synthesizing lateral hypothalamic neurons. Since PVT neurons display state-dependent tonic or low threshold spike-driven burst firing patterns, we examined how the response to exogenously applied orexins might modulate these features. Data were obtained with whole-cell patch clamp recording techniques in rat brain slices prepared during the subjective lights-on period. PVT neurons displayed a mean resting membrane potential of -61+/-6 mV and input conductance of 1.3+/-0.1 nS (n=60). The majority (90/107) of cells tested responded to orexin A and/or orexin B peptides (100-1000 nM), each inducing similar slowly rising and prolonged membrane depolarizations. We next evaluated associated changes in firing patterns and action potential frequency. Of 17 spontaneously silent neurons, 5 were induced into tonic firing and 4 into burst firing modes. Of nine spontaneously bursting neurons, three displayed an increase in burst frequency and in the number of action potentials within a burst. By contrast, another six cells were induced into tonic firing mode, with a marked decrease in instantaneous firing frequency and a shift in their excitatory postsynaptic potential-evoked responses from burst firing patterns to single action potentials. Under voltage clamp, orexins induced inward current (-21.8+/-2.4 pA at -60 mV) in 20/22 cells. In 13 cells, current-voltage (I-V) plots revealed a decrease in net conductance and reversal at -110+/-9 mV, while 3 cells displayed an increase in net conductance that reversed at -26+/-8 mV. These observations imply suppression of potassium and/or induction of nonselective cationic conductances in orexin-induced depolarization in PVT neurons, permitting these peptides to modulate intrinsic state-dependent properties. In vivo, such changes in firing patterns and frequency of action potential discharges could influence neurotransmission through PVT and activity-dependent synaptic plasticity at target sites of these neurons.

Vasopressin induces depolarization and state-dependent firing patterns in rat thalamic paraventricular nucleus neurons in vitro.

The thalamic midline paraventricular nucleus (PVT) is prominently innervated by vasopressin-immunoreactive neurons from the suprachiasmatic nucleus (SCN), site of the brain's biological clock. Using patch-clamp recordings in slice preparations taken from Wistar rats during the subjective day, we examined 90 PVT neurons for responses to bath-applied AVP (0.5-2 microM; 1-3 min). In current clamp at resting membrane potentials (-65 +/- 1 mV), PVT neurons displayed low-threshold spikes (LTSs) and burst firing patterns. In 50% of cells tested, AVP induced a slowly rising, prolonged membrane depolarization and tonic firing, returning to burst firing upon recovery. AVP modulated hyperpolarization-activated LTSs by decreasing the time to the initial sodium spike at the onset of LTS, also increasing the duration of the afterdepolarization. Responses were blockable with a V(1a) receptor antagonist (Manning compound). Under voltage clamp, AVP induced a TTX-resistant, slowly rising, and prolonged (approximately 15 min) inward current (<40 pA). Current-voltage relationship (I-V) analyses of the AVP responses revealed a decrease in membrane conductance to 73.1 +/- 6.2% of control, with net AVP current reversing at -106 +/- 4 mV, and decreased inward rectification at negative potentials. These observations are consistent with an AVP-induced closure of an inwardly rectifying potassium conductance. On the basis of these in vitro observations, we suggest that the SCN vasopressinergic innervation of PVT is excitatory in nature, possibly releasing AVP with circadian rhythmicity and contributing to state-dependent firing patterns in PVT neurons over the sleep-wake cycle.

[Modified anteriolateral retroperitoneal approach for surgical treatment of patients with compression of the cauda equina and lumbar nerve roots (topographic anatomical study)]. / Modifitsirovannyi perednebokovoi retroperitoneal'nyi dostup dlia operativnogo lecheniia bol'nykh s kompressiei konskogo khvosta i spinno-mozgovykh koreshkov poiasnichnogo otdela pozvonochnika (topograficheskoe issledovanie).

Mini-invasive techniques have been increasingly used in lumbar surgery in the past decade, most commonly during operations on the intervertebral disks. However, mini-invasive interventions may be used as a modified traditional approach to treating more serious and challenging pathology, such as tumors of the lumbar vertebrae, metastatic disease, as well as lumbar fractures of different origin. In the literature, there are only several papers on the mini-invasive anterolateral retroperitoneal approach (ALMIRA) to corpectomy with repair of the lumbar spine. This paper deals with the autopsy of 15 cadavers (aged 55-83 years) and 5 blocks of the lumbar spine. The approach is described with illustrations being given. All the results are analyzed and shown in a table. Fifteen corpectomies of L2, L3, and L4 vertebrae were performed by ALMURA, using a self-retaining retractor. The duration of a procedure from skin incision to complete preparation of a spinal specimen for retractor's attachment averaged 50 min (30-70 min). The corpses were rotated in the axial plane at 45, 35, and 25 degrees for corpectomy of L2, L3, L4, respectively. The procedure was easier-to-use when convex binding of the spine was performed after the approach and before insertion of a retractor, in obese cases in particular. Damage to the peritoneum occurred in 5 cases, that to the ascending lumbar vein took place in 4 (during L4 corpectomy) and m. psoas in 2 cases, both in a cachectic cadaver. In the authors' opinion, the method is effective, but before applying it in the clinical theater, spinal surgeons should be well trained under experimental conditions.

Cell cycle-related changes in regulatory volume decrease and volume-sensitive chloride conductance in mouse fibroblasts.

Cell cycle-related changes in the ability to regulate cell volume following hyposmotic swelling were studied in mouse fibroblasts using videomicroscopy and the whole-cell patch clamp technique. Regulatory volume decrease (RVD) and volume-sensitive Cl- conductance (G(Cl,vol)) were measured: (1) in proliferating cells of different sizes; (2) in cells arrested in defined phases of the cell cycle (G1, G1/S, S, and M phases) using mevastatin, mimosine, hydroxyurea, aphidicolin, cytosine beta-D-arabinofuranoside, and taxol; and (3) in serum-starved cells (G(0) state). Cells in all groups were able to undergo RVD, although the cells approaching mitosis (i.e., the largest cells in proliferating cultures and the taxol-treated cells) had the lowest rates of shrinkage during RVD. In agreement with this finding, the density of G(Cl,vol) was stable in proliferating and cell cycle-arrested cells for most of the cell cycle, with the exception of the cells approaching mitosis and the new daughter cells where the density was decreased to half. The impairment of RVD was greatest in serum-starved cells which also had the lowest density of G(Cl,vol). We conclude that proliferating cells maintain an ability to recover from osmotic swelling as they progress through the cell cycle, although this ability may be compromised during mitosis.

Dual role of ATP in supporting volume-regulated chloride channels in mouse fibroblasts.

The effects of inhibitors of protein tyrosine kinases (PTKs) on the Cl(-) current (I(Cl(vol))) through volume-regulated anion/chloride (VRAC) channels whilst manipulating cellular ATP have been studied in mouse fibroblasts using the whole-cell patch clamp technique. Removal of ATP from the pipette-filling solution prevented activation of the current during osmotic cell swelling and when the volume of patched cells was increased by the application of positive pressure through the patch pipette to achieve rates exceeding 100%/min. Equimolar substitution of ATP in the pipette solution with its non-hydrolyzable analogs, adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) or adenylyl-(beta,gamma-methylene)-diphosphonate (AMP-PCP), not only supported activation of the current but also maintained its amplitude. The PTK inhibitors, tyrphostins A25, B46, 3-amino-2,4-dicyano-5-(4-hydroxyphenyl)penta-2,4-dienonitrile++ + and genistein (all at 100 microM), inhibited I(Cl(vol)) in a time-dependent manner. Tyrphostin A1, which does not inhibit PTK activity, did not affect the current amplitude. The PTK inhibitors also inhibited I(Cl(vol)) under conditions where ATP in the pipette was substituted with ATPgammaS or AMP-PCP. We conclude that in mouse fibroblasts ATP has a dual role in the regulation of the current: it is required for protein phosphorylation to keep VRAC channels operational and, through non-hydrolytic binding, determines the magnitude of I(Cl(vol)). We also suggest that tyrosine-specific protein kinases and phosphatases exhibit an interdependent involvement in the regulation of VRAC channels.

Protein phosphotyrosine phosphatase inhibitors suppress regulatory volume decrease and the volume-sensitive Cl- conductance in mouse fibroblasts.

The effects of the protein tyrosine phosphatase (PTP) inhibitors, pervanadate, monoperoxo(picolinato)- oxo-vanadate(V) [mpV(pic)] and dephostatin, on regulatory volume decrease (RVD) and the volume-sensitive Cl- current in mouse L-fibroblasts were studied with the aid of video microscopy and the whole-cell patch-clamp technique. The RVD induced by the hyposmotic shift from 300 to 150 mosmol/l, was strongly suppressed in cells that had been pre-incubated in pervanadate (25 microM) or in mpV(pic) (10 microM), or subjected to extracellular application of dephostatin (20 microM). The acceleration in RVD caused by gramicidin (0.5 microM) was also slowed down by pervanadate pre-treatment, suggesting that the PTP inhibitors affected the volume-sensitive Cl- conductance. Inhibition of the volume-sensitive Cl- current by pervanadate (25 microM) pre-treatment and by acutely applied dephostatin (20 microM) was confirmed in the whole-cell experiments (by @70% and by @50%, respectively). Both pervanadate and dephostatin inhibited the outward and inward Cl- currents equally, which suggests that only the number of open channels was affected. The amplitude of the Cl- current decreased slowly during application of dephostatin and did not recover after its termination. We conclude that in mouse L-fibroblasts, similar to bovine chromaffin cells, inhibition of PTPs results in the suppression of both RVD and the volume-sensitive Cl- current.

High intracellular chloride delays the activation of the volume-sensitive chloride conductance in mouse L-fibroblasts.

1. The relationship between cell volume and volume-sensitive Cl- conductance during hyposmotic cell swelling of patched cells and the effects of intracellular chloride on the conductance have been studied in mouse L-fibroblasts. To this end, swelling-activated current and cell volume were measured simultaneously in cells dialysed with low-Cl- (16 mM) or high-Cl- (130 mM) solutions using the whole-cell patch-clamp technique and videomicroscopy. 2. The increase in cell volume of patched cells and the volume-sensitive conductance saturated during a 4-5 min exposure to mildly hyposmotic solutions (15-20 % less than isosmotic). The swelling of patched cells varied considerably and was greater than the swelling of intact cells. No correlation between the maximal values of the volume-sensitive conductance and the maximal volumes of swollen cells was evident for cells dialysed with the low-Cl- solutions. 3. The amplitude of the volume-sensitive conductance decreased with a reduction in either extracellular or intracellular Cl- concentration; the size of the maximal conductance was not modulated by intracellular Cl- ions. 4. The activation of the volume-sensitive conductance was slower in high-Cl- cells than in low-Cl- cells whether it was induced by hypotonic cell swelling or by cell inflation; in low-Cl- cells the conductance saturated before the cell volume had reached its maximal value. 5. It is concluded that in patched cells an increase in cell volume triggers activation of the volume-sensitive Cl- conductance but does not determine its amplitude and that the rate of activation of the conductance is affected by the intracellular Cl- concentration.

Pervanadate inhibits volume-sensitive chloride current in bovine chromaffin cells.

The role of protein tyrosine phosphorylation in the activation of the volume-sensitive Cl- current in bovine chromaffin cells was investigated by studying the effects of inhibitors of protein tyrosine kinases (PTKs) and phosphatases (PTPs). The whole-cell current was induced by intracellular guanosine-5'-0-(3-thiotriphosphate) (GTP-[gamma-S], 100-250 microM), the nonhydrolysable GTP analogue, or by cell inflation through the patch pipette under voltage-clamp conditions. PTK inhibitors tyrphostin B46 (5-50 microM) and genistein (200 microM) did not inhibit the volume-sensitive Cl- current nor did they induce it in the absence of other stimuli. In contrast, the PTP inhibitor pervanadate (200 microM) applied intracellularly prevented activation of the current. Voltage-activated Na+ and Ca2+ currents were unaffected by pervanadate. Neither sodium orthovanadate nor hydrogen peroxide alone mimicked the action of pervanadate. Other PTP inhibitors tested, i.e. ammonium molybdate (10-100 microM), phenylarsine oxide (10 microM), and ZnCl2 (500 microM), as well as the serine/threonine protein phosphatases inhibitor okadaic acid (200 nM) failed to inhibit the volume-sensitive Cl- current. It is suggested that the inhibitory action of pervanadate indicates the involvement of protein tyrosine phosphorylation in the regulation of volume-sensitive Cl- channels in bovine chromaffin cells. The possibility of pervanadate acting via a pathway unrelated to protein phosphorylation is also discussed.

Routes of Cl- transport across the trophectoderm of the mouse blastocyst.

The blastocyst stage of embryo development is characterized by a fluid-filled cavity called the blastocoel. Blastocoel formation requires vectorial Na+ and Cl- transport and the accompanying osmotic accumulation of fluid. We found under conditions of low external Cl- that inhibitors of Cl- transport mechanisms inhibited blastocoel expansion, indicating a possible transcellular route for Cl- uptake across the outer epithelial layer (the trophectoderm). Using the Cl--sensitive fluorophore, N-(6-methoxyquinolyl)acetoethyl ester, we found that Cl- efflux from the blastocoel can occur via pathways with properties that resemble both HCO-3/Cl- exchange and Cl- channels, as well as by another yet uncharacterized pathway. In contrast, Cl- re-uptake into Cl--depleted blastocoels (the physiologically relevant direction for Cl- transport during blastocoel expansion) occurred only via the channel-like mechanism. Patch-clamp recordings detected a component of current carried by apical Cl- channels. Intracellular pH measurements during external Cl- removal detected HCO-3/Cl- exchange activity in collapsed blastocysts but little in intact blastocysts, suggesting predominantly basolateral HCO-3/Cl- exchange activity. This was corroborated by the immunolocalization of the AE2 isoform of HCO-3/Cl- exchanger to the basolateral surface of the trophectoderm. Thus, it appears that Cl- transport into the blastocoel may occur via apical Cl- channels, while efflux also involves a basolateral HCO-3/Cl- exchanger.

Pharmacologically distinct presynaptic calcium channels in cerebellar excitatory and inhibitory synapses.

We have used whole-cell patch clamp recordings and pharmacological blockers of Ca channels to compare the pharmacology of Ca channels that mediate synaptic transmission at the three types of synapses innervating Purkinje cells in rat cerebellar slices. Both parallel fiber and climbing fiber excitatory synapses were sensitive to the P-type Ca channel blocker, omega-AgaIVA and the P/Q/N-type channel blocker, omega-conotoxin MVIIC. Transmission at inhibitory interneuronal synapses was not suppressed by these toxins, or by the N-type (omega-conotoxins GVIA and MVIIA) or L-type (nimodipine) channel blockers. Inhibitory transmission could be inhibited by Ni2+ and amiloride, but only at concentrations (IC50 approximately 300 microM) that affect other types of Ca channels. These results indicate that excitatory and inhibitory presynaptic terminals of the cerebellar cortex possess different types of voltage-gated Ca channels. The excitatory terminals contain P-type, Q-type and N-type Ca channels, with P-type channels playing the most prominent role. The inhibitory terminals possess quite different type(s) of Ca channel. The heterogeneous distribution of Ca channel types should impart unique properties to transmitter release from the excitatory and inhibitory terminals.

A functional role for GTP-binding proteins in synaptic vesicle cycling.

The squid giant synapse was used to test the hypothesis that guanosine-5'-triphosphate (GTP)-binding proteins regulate the local distribution of synaptic vesicles within nerve terminals. Presynaptic injection of the nonhydrolyzable GTP analog GTP gamma S irreversibly inhibited neurotransmitter release without changing either the size of the calcium signals produced by presynaptic action potentials or the number of synaptic vesicles docked at presynaptic active zones. Neurotransmitter release was also inhibited by injection of the nonhydrolyzable guanosine diphosphate (GDP) analog GDP beta S but not by injection of AIF4-. These results suggest that a small molecular weight GTP-binding protein directs the docking of synaptic vesicles that occurs before calcium-dependent neurotransmitter release. Depletion of undocked synaptic vesicles by GTP gamma S indicates that additional GTP-binding proteins function in the terminal at other steps responsible for synaptic vesicle replenishment.

Volume-sensitive chloride conductance in bovine chromaffin cell membrane.

1. Bovine chromaffin cells were inflated by pressure applied through a pipette or swollen during intracellular perfusion with hypertonic solutions. Effects of such procedures on electrical properties of the membrane were studied by a combination of the tight-seal whole-cell patch-clamp technique and Fura-2 fluorescence measurements of free intracellular calcium concentration ([Ca2+]i). 2. Application of air pressure (about +5 cmH2O or 490 Pa) through the patch pipette caused an increase in the cell volume and concomitant development of an inwardly directed transient current at the holding potential of -60 mV. The current gradually increased to a peak value and subsequently decayed almost to its initial level within 5-10 min. A short pulse of pressure (5-10 s) was sufficient to elicit the whole sequence of events. 3. Intracellular free Ca2+ ion concentration, [Ca2+]i, steeply increased at the beginning of the pressure pulse to about 0.2 microM and either stayed at this level or decayed back to the more usual value of approximately 0.1 microM. 4. Similar changes in the transmembrane current and [Ca2+]i were observed during intracellular perfusion of cells with hypertonic solutions (30-50 mosM difference relative to the bath solution) or during extracellular application of hypotonic solution. 5. Swelling of non-perfused cells by extracellular application of hyposmotic solution caused the appearance of inward currents in cell-attached membrane patches held at a fixed potential -30 mV relative to the cell's resting potential. The kinetics of the current resembled those of the whole-cell current. 6. Intracellular introduction of guanosine triphosphate (GTP, 300 microM) significantly prolonged the duration (from 62 +/- 10 s, n = 5, to 98 +/- 8 s, n = 4, when measured at the level of half-amplitude), while introduction of the non-hydrolysable analogue of guanosine diphosphate (GDP), guanosine 5'-O-(2-thiodiphosphate) (GDP beta S, 300 microM), decreased the maximal rate of increase (from 11.4 +/- 2.6 pA/s, n = 6, to 3.2 +/- 2.1 pA/s, n = 10) of the current activated by pressure. 7. Lowering of the intracellular free Ca2+ ion concentration by introduction of 10 mM-EGTA did not significantly affect the current amplitude or time course. However, a rapid increase in the [Ca2+]i to micromolar levels (by activation of the voltage-operated calcium channels during membrane depolarization) could terminate development of the current activated by pressure and cause its fast decay to zero-current level.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of serotonin and cAMP on calcium currents in different neurones of Helix pomatia.

Effects of application of serotonin (5-HT) and intracellular administration of cyclic adenosine monophosphate (cAMP) on voltage-gated calcium current (ICa) were studied in isolated, intracellularly perfused Helix pomatia neurones. Two types of the effects of 5-HT (1-10 microM) were observed in different neurones: reversible inhibition (by about 20%) or reversible potentiation (up to 50%) of the current amplitude. Some cells did not respond to 5-HT application. In cells with the potentiating effect of 5-HT, ICa could also be increased by intracellular introduction of cAMP (100 microM), but not the guanosine analogue, cGMP (50-100 microM). These effects were not additive and could be potentiated by theophylline (5 mM) and 3-isobutyl-1-methylxanthine (IBMX, 100-500 microM); they could be mimicked by forskolin (10-50 microM) and abolished by tolbutamide (1-5 mM) or protein kinase inhibitor (500 micrograms/ml), indicating that cAMP-dependent phosphorylation mediates the potentiating action of 5-HT on ICa. In neurones showing inhibitory effect of 5-HT, neither cAMP nor forskolin increased ICa. Methiothepin (10-50 microM), a 5-HT1,2 receptor antagonist, irreversibly inhibited the potentiating effect of 5-HT, while antagonists of 5-HT2 receptors cyproheptadine (10-50 microM) or ketanserine (10-60 microM) and of 5-HT3 receptors ISC 205-930 (10-50 microM) or cocaine (5-25 microM) had no effect on ICa and its enhancement by 5-HT. It is suggested that in certain snail neurones the possibility of cAMP-dependent up-regulation of ICa correlates with the presence of 5-HT1-like receptors in the neuronal membrane.

Pertussis-toxin-sensitive inhibition by (-) baclofen of Ca signals in bovine chromaffin cells.

Ca signals in bovine adrenal chromaffin cells were studied both in Fura-2/AM-loaded intact cells, and in voltage-clamped cells under whole-cell patch-clamp conditions. The effects of gamma-aminobutyric acid b subtype (GABAb) receptor activation on K(+)-depolarization-induced signals and on voltage-activated Ca2+ currents were investigated. Both GABA (20 microM) plus bicuculline (20 microM) and (-)baclofen (20-100 microM), effectively inhibited the Ca signal in intact cells. The effects caused by baclofen continued to develop during the time interval between two successive stimuli. The restoration of the Ca signal during washout of baclofen was also delayed and continued in some experiments for 10-20 min. The inhibitory effect of baclofen on the Ca signal was eliminated by pre-treatment of the cells with pertussis toxin (PTX, 1 micrograms/ml, for 4-6 h at 37 degrees C). Baclofen (50 microM) inhibited Ca2+ current in whole-cell mode by at most 20%. The effect developed quickly and was reversible. Infusion into the cells of a non-hydrolyzable analogue of guanosine 5'-triphosphate GTP gamma S (100 microM), led to complete inhibition of the Ca2+ conductance and of voltage-evoked intracellular Ca ([CA]i) transients within 2 min. In paired cells intracellularly perfused with GTP gamma S-free solution, the Ca2+ current amplitude decreased by only about 30% for 5-6 min. It is concluded that bovine chromaffin cells have functional GABAb receptors the activation of which, mediated by a PTX-sensitive GTP-binding protein, inhibits the evoked increase in cytosolic free Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Novel chloride conductance in the membrane of bovine chromaffin cells activated by intracellular GTP gamma S.

1. The effects of introducing the non-hydrolysable GTP analogue guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) into perfused bovine chromaffin cells were studied by a combination of the tight-seal whole-cell patch-clamp technique and Fura-2 fluorescence [Ca2+]i measurements. 2. GTP gamma S (5-300 microM) induced a slowly developing transient current (inwardly directed at the holding potential -60 to -70 mV) and [Ca2+]i oscillations. The current activated with a 10-50 s delay after the start of whole-cell dialysis, peaked at 70-120 s and decayed almost to its initial level during the next 150-300 s. Calcium oscillations were observed within the first 100-150 s of cell perfusion. 3. GTP competitively lowered the probability of current activation by GTP gamma S. At low GTP gamma S/GTP ratio (5 and 300 microM, respectively) activation of the current was observed only rarely. 4. The activation of the current was accompanied by an increase in conductance but not by changes in the current reversal potential. The changes in the conductance did not depend on the membrane potential; no time-dependent relaxation of the current was induced by steps in the membrane voltage. 5. The current reversal potential was close to the Cl- equilibrium potential; changes in the extracellular Cl- concentration induced corresponding changes in the current amplitude and shifted its reversal potential. The permeability to larger anions--aspartate, glutamate and isethionate--was about one-tenth of that for chloride. 6. Single-channel conductance, estimated from the ratio of the mean current and its variance, was about 1-2 pS. 7. The current could be reversibly blocked by 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS, 10 microM), chlorpromazine (5 microM) and tolbutamide (0.5-5 mM). 8. It is suggested that the GTP gamma S-induced increase in the permeability to Cl- ions is due to a G protein-mediated production of an as yet unidentified second messenger.

Second messengers mediating activation of chloride current by intracellular GTP gamma S in bovine chromaffin cells.

1. Intracellular mechanisms and second messengers involved in chloride current activation by intracellular GTP gamma S (guanosine 5'-O-(3-thiotriphosphate] in bovine chromaffin cells were studied using the whole-cell patch-clamp technique combined with measurements of intracellular calcium [Ca2+]i. 2. No correlation between the time of current activation and the appearance of [Ca2+]i transients was observed; intracellular introduction of sufficient EGTA (10 mM) to suppress the [Ca2+]i transients did not affect the current activation by GTP gamma S. 3. The cyclic nucleotides, cyclic AMP or cyclic GMP, did not activate the current when introduced intracellularly (50-250 microM). The ability of GTP gamma S to activate the current decreased when cyclic GMP (250 microM), together with MgATP (2 mM), was added to the perfusate. 4. Neomycin (0.5-1 mM), a presumed inhibitor of phospholipase C effectively prevented the current activation by GTP gamma S but it did not prevent [Ca2+]i transients. 5. Modulation of protein kinase C activity using specific inhibitors (H-7, 300 microM; polymyxin B, 400 U/ml) or activators (phorbol ester PMA, 100 nM, 20-90 min at 37 degrees C) did not affect the current activation by GTP gamma S nor did it cause current activation in the absence of GTP gamma S. 6. Activation of the current by GTP gamma S could be prevented by incubating the cells for 10-15 min with 2.5 microM p-bromophenacyl bromide (p-BPB), an inhibitor of phospholipase A2 activity. Exogenous arachidonic acid (5-10 microM), applied extracellularly or intracellularly, neither activated the current itself nor did it interfere with its activation by GTP gamma S. 7. Activation of the current by GTP gamma S could also be prevented by incubating the cells with 1 microM-nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, but not with indomethacin (2 microM), an inhibitor of cyclo-oxygenase pathway of arachidonic acid metabolism. 8. It is suggested that chloride current activation by GTP gamma S in bovine chromaffin cells involves G protein-mediated stimulation of phospholipase A2 activity and subsequent formation of lipoxygenase product(s) of arachidonic acid metabolism.