Effects of pertussis toxin on the affinity of exocytosis for Ca2+ in bovine adrenal chromaffin cells

Effects of pertussis toxin (islet-activating protein, IAP) on the secretory function of bovine adrenal chromaffin cells in culture were studied. Treatment of chromaffin cells with IAP resulted in an increase in both basal release of catecholamine and evoked-release by either acetylcholine (ACh) or high K+. In the dose-response curve for ACh-evoked release, IAP treatment produced an increase of the maximal response without affecting the half-maximal concentration of ACh. When the cells were permeabilized with digitonin after IAP-pretreatment, Ca2(+)-dependent exocytosis was markedly increased where the affinity of exocytosis for Ca2+ was augmented. These findings suggest that IAP-sensitive GTP-binding protein (or proteins) directory controls the Ca2(+)-triggered process in the machinery of exocytosis by modulating the affinity for Ca2+ of its unknown target.     

Pertussis toxin attenuates clonidine inhibition of catecholamine release in adrenal chromaffin cells

Characteristics of the inhibitory action of clonidine on catecholamine release in bovine adrenal chromaffin cells were investigated. Clonidine at 3 x 10(-5) M inhibited acetylcholine (ACh)-evoked release by about 50%, but not catecholamine release evoked by high K+. Another alpha 2-agonist alpha-methyladrenaline was ineffective at inhibiting ACh-evoked release. The inhibition by clonidine of ACh-evoked release was not reversed by alpha 2-antagonists. Treatment of these cells with pertussis toxin reversed the inhibitory effect of clonidine, while it did not affect the inhibitory action of hexamethonium and of nifedipine. Therefore, clonidine inhibition of catecholamine release in these cells seems not to be mediated by the alpha 2-adrenoceptor, but might be mediated by a specific receptor for clonidine.     

Nicotinic receptor-mediated intracellular calcium release in cultured bovine adrenal chromaffin cells

When cultured bovine adrenal chromaffin cells were stimulated by a nicotinic agonist, carbamylcholine (0.3 mM) or 1,1-dimethyl-4-phenylpiperazinium (50 microM), in the Ca2+-free medium containing 0.1 mM ethyleneglycoltetraacetic acid, intracellular free Ca2+ concentration ([Ca2+]i) rose from approximately 90 to 149 nM. High K+ (56 mM) and veratridine (50 microM) had no effect on the [Ca2+]i in Ca2+-free medium. The carbamylcholine-evoked rise in [Ca2+]i was blocked by hexamethonium (0.1 mM) but not by atropine (1 microM). Furthermore, the carbamylcholine-evoked rise in [Ca2+]i was inhibited by an intracellular Ca2+ antagonist, 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxy-benzoate hydrochloride (10 microM) but not by a calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (20 microM). These results show the existence of intracellular Ca2+ store sites, from which Ca2+ is released upon nicotinic receptor stimulation, in cultured adrenal chromaffin cells.     

Neuropeptide Y inhibits nicotinic cholinergic currents but not voltage-dependent calcium currents in bovine chromaffin cells

The effects of neuropeptide Y (NPY; 1–36) and NPY fragment (16–36) on nicotinic currents (I _ACh) and voltage-dependent calcium currents (I _Ca) were studied in bovine chromaffin cells using the whole-cell patchclamp technique. The peak amplitude of inward nicotinic currents was markedly depressed by both NPY(1–36) and NPY (16–36). In contrast, I _Ca was unaffected by either NPY(1–36) or NPY(16–36). Both pertussis toxin pretreatment and including GDP[-S] in the patch pipette solution completely abolished the inhibitory effect of NPY on I _ACh It is concluded that inhibition of I _ACh probably represents the mechanism by which NPY decreases catecholamine release from adrenal medulla. This effect appears to be mediated by a G-protein-coupled Y₂ receptor.     

Differential modulation of voltage-dependent Ca2+ currents by EGTA and BAPTA in bovine adrenal chromaffin cells

Whole-cell patch-clamp recordings were made to examine the effects of the Ca2+ chelators EGTA and BAPTA on the biophysical properties of voltage-operated Ca2+ currents in bovine adrenal chromaffin cells. Ca2+ currents in the presence of either EGTA or BAPTA over a concentration range of 0.1-60 mM were recorded under otherwise identical conditions. Analysis of current-voltage relationships yielded unexpected differences in several important parameters such as the voltage dependence of activation, kinetics, slope, and reversal potential, which seemed to be unrelated to the Ca(2+)-binding properties of these chelators. Increasing concentrations of BAPTA augmented the peak Ca2+ current amplitude while current amplitudes in the presence of EGTA remained constant over the entire concentration range tested. Increasing concentrations of BAPTA shifted the voltage sensitivity of Ca2+ currents by about 15 mV towards positive voltages. EGTA, over the same concentration range, did not affect the voltage sensitivity. The shift in voltage sensitivity observed with BAPTA was unrelated to its faster Ca92+)-binding kinetics, as it was also observed when substituting Ca2+ with Ba2+ as the charge carrier. The mechanism by which BAPTA affects Ca2+ channel voltage dependence also seems unrelated to kinase-mediated modulation of Ca2+ channels, since the protein-kinase-C- (PKC-) specific drugs bisindolylmaleimide and phorbol ester (PMA) neither mimicked nor prevented the action of BAPTA. The less specific kinase inhibitor staurosporine, however, augmented Ca2+ currents similarly to BAPTA, but without affecting the voltage sensitivity. The BAPTA-mediated shift in voltage sensitivity was partially suppressed by non-hydrolysable analogs of GTP (GDP[beta-S] and GTP[gamma-S]). Lowering [Mg2+]i mimicked the BAPTA-induced shift in voltage sensitivity and prevented further shifts in voltage sensitivity by BAPTA. The results demonstrate that BAPTA and EGTA, despite their similarities in terms of Ca2+ buffering, have disparate effects on the voltage dependence of Ca2+ channels and careful selection of the chelator is required to quantitatively assess Ca2+ currents.     

Effects of enflurane on the voltage-gated membrane currents of bovine adrenal chromaffin cells.

The effects of the volatile anesthetic enflurane on voltage-gated ionic currents of bovine adrenal chromaffin cells were studied using the patch clamp technique. Bath application of 3.5% (1.7 mM) enflurane decreased the outward Ca(2+)-dependent K+ current (IK(Ca)) 'hump' by 88 +/- 6% (mean +/- S.E.M., n = 5 cells) and the peak inward Ca2+ current by 60 +/- 3% (n = 5), whereas the Ca(2+)-independent K+ current fell by only 34 +/- 3% (n = 5) and peak inward Na+ current was unchanged. Exposure of excised patch 'BK' Ca(2+)-dependent K+ channels to 3.5% enflurane revealed that the anesthetic directly suppressed the channel probability of opening by 68 +/- 10% (n = 4) with no effect on open state conductance. The differential sensitivity of depolarizing and hyperpolarizing current pathways may contribute to the biphasic response, excitation and depression, observed in certain neuronal systems in response to this inhalational anesthetic.     

Modulation of calcium current by ATP in guinea-pig adrenal chromaffin cells

The effects of extracellular adenosine 5-triphosphate (ATP) on voltage-dependent Ca²⁺ currents were examined using the whole-cell voltage-clamp technique in guinea-pig isolated adrenal chromaffin cells. ATP (500 M) reversibly suppressed Ca²⁺ currents in the presence of 5 mM Ca²⁺ in the extracellular solution. The inhibitory effect of ATP on Ca²⁺ currents tended to increase with increases in the peak amplitude of ATP-evoked current when the intracellular solution contained 0.1 or 1 mM ethylenebis(oxonitrilo)tetraacetate(EGTA). Using the intracellular solution containing 10 mM EGTA, on the other hand, the inhibitory efftect did not change regardless of the amplitude of current responses to ATP In the presence of 10 mM Ba²⁺, ATP (100 mol/l). reduced Ba²⁺ currents in a manner similar to Ca²⁺ currents. This reduction was decreased by dialysis of cells with the internal solution containing guanosine 5-O-(2-thiodiphosphate) (GDP [-S]; 1 mM) or guanosine 5-O-(3-thiotriphos-phate) (GTP [-S]; 100 mol/l). A depolarizing prepulse channels. In addition, ATP seems to modulate Ca²⁺ channels via the pathway related to G-protein. Adenine nucleotides and adenosine may play a role in controlling secretory activity in guinea-pig adrenal chromaffin cells.     

Voltage-dependent potassium currents during fast spikes of rat cerebellar Purkinje neurons: inhibition by BDS-I toxin

We characterized the kinetics and pharmacological properties of voltage-activated potassium currents in rat cerebellar Purkinje neurons using recordings from nucleated patches, which allowed high resolution of activation and deactivation kinetics. Activation was exceptionally rapid, with 10-90% activation in about 400 mus at +30 mV, near the peak of the spike. Deactivation was also extremely rapid, with a decay time constant of about 300 mus near -80 mV. These rapid activation and deactivation kinetics are consistent with mediation by Kv3-family channels but are even faster than reported for Kv3-family channels in other neurons. The peptide toxin BDS-I had very little blocking effect on potassium currents elicited by 100-ms depolarizing steps, but the potassium current evoked by action potential waveforms was inhibited nearly completely. The mechanism of inhibition by BDS-I involves slowing of activation rather than total channel block, consistent with the effects described in cloned Kv3-family channels and this explains the dramatically different effects on currents evoked by short spikes versus voltage steps. As predicted from this mechanism, the effects of toxin on spike width were relatively modest (broadening by roughly 25%). These results show that BDS-I-sensitive channels with ultrafast activation and deactivation kinetics carry virtually all of the voltage-dependent potassium current underlying repolarization during normal Purkinje cell spikes.     

Voltage-dependent calcium currents in dissociated granule cells from rat cerebellum.

Voltage-dependent calcium currents were investigated by the patch-clamp technique in whole-cell recording configuration in cultures from 8-day-old rat cerebella, which contained greater than or equal to 90% granule cells. In solutions designed to minimize the sodium and potassium conductances and in 20 mM barium, an inward current activated around -25 mV, reached a peak amplitude at +20 mV and reversed around +80 mV. In 20 mM calcium, this current was approximately 50% of that in barium. From one to three days in vitro only 16% of the cells tested (n = 20) had a current exceeding 50 pA in maximum amplitude, while after four days in vitro the current reached 100 pA in all neurons tested (n greater than 70). Verapamil (50-100 microM) reversibly depressed this current. The dihydropyridine agonist Bay K 8644 (1 microM) enhanced the maximum conductance by 25 +/- 8% (n = 4), caused a negative shift in the activation of 21 +/- 5 mV and a prolongation of the deactivation time course as the voltage was stepped back from +20 to -80 mV. The GABAB agonist baclofen (50 microM) reversibly depressed the current by 27 +/- 8% in 80% of the cells. The effect was similar to that of GABA (10 microM), when the GABAA response (chloride current) was partially blocked by bicucculine. This current can be classified as a dihydropyridine-sensitive high-voltage-activated calcium current. The modulation by GABAB agonists is likely to be significant for presynaptic inhibition.     

Histamine promotes excitability in bovine adrenal chromaffin cells by inhibiting an M-current

The current study has investigated the electrophysiological responses evoked by histamine in bovine adrenal chromaffin cells using perforated-patch techniques. Histamine caused a transient hyperpolarization followed by a sustained depolarization of 7.2 ± 1.4 mV associated with an increase in spontaneous action potential frequency. The hyperpolarization was abolished after depleting intracellular Ca²⁺ stores with thapsigargin (100 n m ), and was reduced by 40 % with apamin (100 n m ). Membrane resistance increased by about 60 % during the histamine-induced depolarization suggesting inhibition of a K⁺ channel. An inward current relaxation, typical of an M-current, was observed in response to negative voltage steps from a holding potential of −30 mV. This current reversed at −81.6 ± 1.8 mV and was abolished by the M-channel inhibitor linopirdine (100 μ m ). During application of histamine, the amplitude of M-currents recorded at a time corresponding with the sustained depolarization was reduced by 40 %. No inward current rectification was observed in the range −150 to −70 mV, and glibenclamide (10 μ m ) had no effect on either resting membrane potential or the response to histamine. The results show that an M-current is present in bovine chromaffin cells and that this current is inhibited during sustained application of histamine, resulting in membrane depolarization and increased discharge of action potentials. These results demonstrate for the first time a possible mechanism coupling histamine receptors to activation of voltage-operated Ca²⁺ channels in these cells.     

Catecholamine uptake into isolated adrenal chromaffin cells: inhibition of uptake by acetylcholine

We have investigated the process of catecholamine uptake in guinea-pig chromaffin cells. Isolated guinea-pig chromaffin cells accumulate [3H]norepinephrine and [3H]epinephrine by a saturable transport system. Catecholamine uptake is dependent upon temperature, energy, and extracellular Na+. The apparent KmS for norepinephrine and epinephrine transport are approximately 1 and 3.5 microM, respectively; the transport maximum (Vmax) for both compounds is about 100 pmol/min/mg protein. The uptake of norepinephrine into chromaffin cells is inhibited by imipramine (Ki = 50 nM) and by desmethylimipramine (IC50 = 20 nM). In both its substrate specificity and its sensitivity to pharmacological inhibition, the catecholamine uptake system in chromaffin cells is similar to the catecholamine transport system previously described in sympathetic neurons. Decreasing external Na+ from 130 to 19 mM increases the apparent Km for norepinephrine to 2.8 microM. Decreasing external norepinephrine increases the Na+ concentration required for half-maximal transport. Agents that depolarize chromaffin cells, such as acetylcholine and veratridine, significantly inhibit [3H]norepinephrine uptake. This decrease in uptake is due to an increase in the apparent Km for norepinephrine. The inhibition of [3H]norepinephrine uptake by depolarizing agents cannot be accounted for by the preferential release of newly-accumulated [3H]norepinephrine, or by the competitive inhibition of [3H]norepinephrine uptake by secreted catecholamines. The inhibition of catecholamine uptake by depolarizing agents suggests that the transport system may be regulated by the membrane potential. Norepinephrine and epinephrine that are spontaneously released from the adrenal medulla may be recaptured in vivo. The inhibition of transport by acetylcholine may prevent the re-uptake of catecholamine released during the physiological stimulation of secretion.     

Calcium permeability of nicotinic acetylcholine receptor channels in bovine adrenal chromaffin cells

The fractional contribution of Ca to current flow through neuronal-type nicotinic acetylcholine receptor channels was determined by quantitative fluorescence microfluorimetry using fura-2. The method, which has been applied already to several types of cells and channels is described in detail here. At –70 mV and 2 mM external Ca concentration it was found that Ca contributes 2.5% to the net current. The fractional contribution was found to be voltage dependent, increasing at negative potentials e-fold for a 110 mV potential difference. Total non-specific cation current was found to have a bell-shaped dependence on external Ca concentration peaking at 2 mM.     

Autocrine/paracrine modulation of calcium channels in bovine chromaffin cells

 Applying 10-s pulses of 10 mM Ba²⁺ to resting or K⁺-depolarized (70 mM) bovine adrenal chromaffin cells superfused with a nominal 0Ca²⁺ solution produced a large catecholamine secretory peak. In contrast, pulses of 10 mM Sr²⁺ or Ca²⁺ did not induce secretion from polarized resting cells, and induced smaller and narrower secretory peaks from depolarized cells; the areas of the secretory peaks from depolarized cells were 1.87, 3.06 and 27.4 nA s, respectively, for Ca²⁺, Sr²⁺ and Ba²⁺. Ca²⁺ channel currents in isolated cells or in cells surrounded by other unpatched cells (cell cluster) were studied with either the continuous-flow or the flow-stop method. When applied to an isolated cell, flow-stop reduced the amplitude of I _Ca by 19%, I _Sr by 31%, and I _Ba by 53%, compared with the current amplitude measured under continuous-flow conditions. This decrease in current amplitude was accompanied by a pronounced slowing down of current activation and could be largely relieved by applying strong depolarizing prepulses (facilitation). Under continuous-flow conditions, 10 μM exogenous ATP reduced (about 50%) I _Ca, I _Sr and I _Ba similarly. On the other hand, the use of Na⁺ as a charge carrier through Ca²⁺ channels, or intracellular dialysis with 1 mM BAPTA prevented the modulation of current by flow-stop. In cell clusters, activating secretion from unpatched cells, by either 10 mM Ba²⁺, 100 μM acetylcholine or 70 mM K⁺, caused a pronounced slowing down of current activation, as well as a decrease of its magnitude in the voltage-clamped cell immersed in the cluster. Such modulation of isolated cells was not observed. These data are compatible with the idea that the secretory activity of adrenal medullary chromaffin cells ”in situ” controls the activity of their Ca²⁺ channels through autocrine/paracrine mechanisms. Received: 29 June 1998 / Received after revision: 20 August 1998 / Accepted: 1 September 1998     

Voltage-dependent GABA-induced modulation of calcium currents in chick sensory neurons

Externally applied gamma-aminobutyric acid (GABA) quickly and reversibly reduces by 60% voltage activated Ca2+ currents in chick dorsal root ganglion cells. This action is antagonized by depolarization, with characteristic time and voltage requirements. Intracellular perfusion with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) mimicks and blocks the GABA effect, respectively. A 3-state model describing the reactions involved is proposed.     

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 (GABA_b) receptor activation on K⁺-depolarization-induced signals and on voltage-activated Ca²⁺ currents were investigated. Both GABA (20 M) plus bicuculline (20 M) and (-)baclofen (20–100 M), 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, 1g/ml, for 4–6h at 37°C). Baclofen (50 M) 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 S (100 M), 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 GTPS-free solution, the Ca²⁺ current amplitude decreased by only about 30% for 5–6 min. It is concluded that bovine chromaffin cells have functional GABA_b receptors the activation of which, mediated by a PTX-sensitive GTP-binding protein, inhibits the evoked increase in cytosolic free Ca²⁺. The small size of the effect on Ca²⁺ current in whole-cell mode as compared to that on the Ca signal in intact cells may be explained by washout of some regulatory element during cell dialysis, or by a relatively small contribution of the normal voltage-activated Ca²⁺ current to the Ca signal. Alternatively, it might indicate GABA_b effects on mechanisms other than Ca²⁺ channels.     

Cytoplasmic organelles determine complexity and specificity of calcium signalling in adrenal chromaffin cells

Complex and coordinated fluctuations of intracellular free Ca2+ concentration ([Ca2+]c) regulate secretion of adrenaline from chromaffin cells. The physiologically relevant intracellular Ca2+ signals occur either as localized microdomains of high Ca2+ concentrations or as propagating Ca2+ waves, which give rise to global Ca2+ elevations. Intracellular organelles, the endoplasmic reticulum (ER), mitochondria and nuclear envelope, are endowed with powerful Ca2+ transport systems. Calcium uptake and Ca2+ release from these organelles determine the spatial and temporal parameters of Ca2+ signalling events. Furthermore, the ER and mitochondria form close relations with the sites of plasmalemmal Ca2+ entry, creating 'Ca2+ signalling triads' which act as elementary operational units, which regulate exocytosis. Ca2+ ions accumulating in the ER and mitochondria integrate exocytotic activity with energy production and protein synthesis.     

Inflammatory activation of neutrophils by Helicobacter pylori; a mechanism insensitive to pertussis toxin

Chronic active gastritis of the antral mucosa is a characteristic feature of infection with Helicobacter pylori and interactions between bacterial components and inflammatory cells are believed to play an important pathogenic role. Neutrophils stimulated with H. pylori sonicate were demonstrated to release L-selectin (CD62L) expressed on the cellular surface, with a subsequent up-regulation of the beta2-integrins CD11b and CD11c, both in a dose- and time-dependent manner, reaching maximum levels after 45-60 min of stimulation. No changes were observed for the CD11a receptor upon stimulation. The activating properties of H. pylori sonicates on neutrophils were heat-labile and susceptible to protease attack, indicating the protein nature of the activating factor. After size fractionation, the major neutrophil-inducing activity was detected in the high molecular weight fraction exhibiting urease activity. Pertussis toxin was unable to inhibit neutrophil activation by the H. pylori protein(s). We conclude that proteins from H. pylori have a potent inflammatory effect on the surface membrane molecules CD62L, CD11b and CD11c essential for transendothelial migration of neutrophils to areas of inflammation. The neutrophil-activating protein(s) act via a pertussis toxin-insensitive mechanism.