Catecholamine secretion by chemical hypoxia in guinea-pig, but not rat, adrenal medullary cells: differences in mitochondria.

The effects of mitochondrial inhibitors (CN(-), a complex IV inhibitor and CCCP, protonophore) on catecholamine (CA) secretion and mitochondrial function were explored functionally and biochemically in rat and guinea-pig adrenal chromaffin cells. Guinea-pig chromaffin cells conspicuously secreted CA in response to CN(-) or CCCP, but rat cells showed a little, if any, secretory response to either of them. The resting metabolic rates in rat adrenal medullae did not differ from those in guinea-pig adrenal medullae. On the other hand, the time course of depolarization of the mitochondrial membrane potential (ΔΨm) in guinea-pig chromaffin cells in response to CN(-) was slower than that in rat chromaffin cells, and this difference was abolished by oligomycin, an F1F0-ATPase inhibitor. The extent of CCCP-induced decrease in cellular ATP in guinea-pig chromaffin cells, which was indirectly measured using a Mg(2+) indicator, was smaller than that in rat chromaffin cells. Relative expression levels of F1F0-ATPase inhibitor factor in guinea-pig adrenal medullae were smaller than in rat adrenal medullae, and the opposite was true for F1F0-ATPase α subunit. The present results indicate that guinea-pig chromaffin cells secrete more CA in response to a mitochondrial inhibitor than rat chromaffin cells and this higher susceptibility in the former is accounted for by a larger extent of reversed operation of F1F0-ATPase with the consequent decrease in ATP under conditions where ΔΨm is depolarized.

Modeling of stimulation-secretion coupling in a chromaffin cell.

We constructed a chromaffin cell model for analysis of stimulation-secretion coupling in computer simulation studies. The model includes mechanisms involved in the excitatory synapse, voltage-dependent Na(+), K(+) and Ca(2+) channels, Ca(2+)-activated K(+) channels (SK type), buffered Ca(2+) diffusion, Ca(2+) extrusion, fluorescent Ca(2+) indicators and Ca(2+)-triggered exocytosis. Calculations of the modeled mechanisms were carried out using the NEURON simulation environment (Hines and Carnevale, Neural Computation 9:1179-1209, 1997). A set of parameter values was determined so as to fit basic experimental results reported in the literature. The model was also applied to simulate our experimental results obtained from chromaffin cells in the perfused rat adrenal medulla. Observed profiles of Ca(2+)responses induced by electrically stimulating the splanchnic nerve with various frequencies (1-50 Hz) were adequately simulated with minor readjustments of parameter values for Ca(2+)influx and extrusion. Secretory responses measured at the same time as the Ca(2+)responses were also simulated with consideration of a time constant to detect catecholamines in the experiment. Similarly, model simulations reproduced both Ca(2+)responses and secretory responses evoked by elevations of the extracellular K(+) concentration for different periods. The results suggest that the presented model provides a useful tool for analyzing and predicting quantitative relations in various events occurring in stimulation-secretion coupling in chromaffin cells.

Modes of secretagogue-induced [Ca(2+)](i) responses in individual chromaffin cells of the perfused rat adrenal medulla.

Chromaffin cells in the perfused rat adrenal medulla were loaded with indo-1 for confocal image analyses. Resting levels of [Ca(2+)](i) in chromaffin cells were similar and were stable with time. This is in contrast to the situation in isolated rat chromaffin cells, in which spontaneous oscillations of [Ca(2+)](i) are known to occur. When chromaffin cells were stimulated for 3-4 min by high K(+) or nicotine, [Ca(2+)](i) increased to a peak in 20-30 s and then declined rather smoothly. In contrast, chromaffin cells stimulated by muscarine or low pH (6.5) commonly exhibited irregular oscillations in [Ca(2+)](i). This provides additional evidence supporting the previous claim that muscarine and low pH evoke catecholamine secretion using partly shared mechanisms. Although muscarine and low pH were speculated to produce weaker responses in noradrenaline-secreting cells due to their selective stimulation of adrenaline secretion, no clear indications for segregation of cell types from [Ca(2+)](i) responses to these stimulants were found. The perfused adrenal medulla loaded with Indo-1 was also employed for simultaneously monitoring integrated changes in [Ca(2+)](i)(Ca responses) by conventional microfluorometry and in catecholamine secretion from a whole medulla (secretory responses). When the profiles of secretory responses were approximated by the kth power of the profiles of Ca responses, the k-values were estimated to be 2.2 and 2.3 for high-K(+)- and nicotine-elicited responses, respectively, whereas a k-value of 1.4 was obtained for both muscarine- and low-pH-elicited responses. An analysis showed that the significant difference in the k-value with these two classes of stimulants is accounted for by the stimulant-dependent patterns of [Ca(2+)](i) responses found in confocal image analysis.

Protein kinase C-dependent supply of secretory granules to the plasma membrane.

To elucidate the mechanism for supplying secretory granules to the cell membrane, chromaffin cells isolated from the bovine adrenal medulla were observed by the evanescent wave microscopy after staining their granules with acridine orange. The secretory granules showed only a very small fluctuation, indicating their docking to the plasma membrane. The rate and range of movement increased greatly by application of botulinum toxin A or C. The number of secretory granules docked to the plasma membrane significantly decreased by botulinum toxin C. Conversely, the number increased greatly by activation of protein kinase C with phorbol 12,13-dibutyrate (PDBu). In the presence of an anti-actin reagent cytochalasin D, no increasing effect of PDBu on the number of docked granules was observed. While in the presence of an anti-mitotic reagent, colchicine, a clear increasing effect of PDBu was observed. The final step for supplying granules to the plasma membrane in endocrine cells is concluded to be mediated by a phosphorylation-dependent and actin-based transport system.

Mechanism by which wortmannin and LY294002 inhibit catecholamine secretion in the rat adrenal medullary cells.

The effects of wortmannin and LY294002, inhibitors of PI(3)-kinase, in secretagogue-stimulated rat adrenal chromaffin cells loaded with Calcium Green-1 were studied by simultaneously measuring changes in the fluorescence intensity of the indicator (Ca-response) and in the release of catecholamine (secretory response). Before application of these agents, the profile of the secretory response evoked by a 10-min stimulation with 30 mM K(+)] was approximated by the k th (2.6 on average) power of that of the Ca-response. Both agents dose-dependently inhibited the high-K(+)-elicited Ca-response and secretory response in a similar mode to which the k th power relation was preserved despite the occurrence of profound changes in the shapes and sizes of these two responses. The L-type Ca(2+)-channel blocker PN200-110 inhibited the high-K(+)-evoked responses in a similar fashion. Thus, it is likely that wortmannin and LY294002 inhibit high-K(+)-evoked CA secretion by inhibiting a Ca(2+)-influx through voltage-dependent Ca(2+)channels. Although regulation of L-type Ca(2+)channel activity via PI(3)-kinase has been reported in vascular myocytes, this possibility may be limited in the present case since the doses of LY294002 and wortmannin used to inhibit the secretory response are much higher than IC(50)'s for inhibition of PI(3)-kinase with these agents. Compared with the high-K(+)-elicited responses, muscarine-evoked Ca-responses and secretory responses were more strongly inhibited by wortmannin, but less affected by LY294002. The differential effects suggest that the inhibition of the muscarine-evoked secretion by these agents i s not associated with the inhibition of PI(3)-kinase.

Mechanism of wortmannin-induced inhibition of secretory responses in rat adrenal medullary cells.

The effect of wortmannin (WT), an inhibitor of myosin light chain kinase (MLCK) as well as PI3-kinase, on catecholamine (CA) secretion in the perfused rat adrenal medulla was studied. After a 35-min application of 10 microM WT, secretory response to repetitive stimulation with 30 mM extracellular K+ was reduced to 30% of that obtained with intact medullae, and the response to 200 nM bradykinin (BK) was almost completely abolished. Aiming to identify the target for the WT effect, the WT derivative KT7692, which retains the same potency to inhibit MLCK as that of WT but its potency to PI3-kinase is one-hundredth that of WT, was used. Unlike WT, KT7692 at 10 microM did not affect the high-K+-evoked secretion and slightly potentiated the BK-evoked secretion. These results oppose the notion that WT inhibits the secretory responses through inhibition of MLCK. However, the alternative idea, that PI3-kinase is a target for WT, is also difficult to accept since WT concentrations required for the inhibition of the secretions are much higher than those needed to inhibit PI-3-kinase by WT.

Light-evoked recovery from wortmannin-induced inhibition of catecholamine secretion and synaptic transmission.

Wortmannin (WT) is known to inhibit catecholamine (CA) secretion in chromaffin cells. This effect was found to be sensitive to UV light in experiments designed to perform simultaneous monitoring of changes in [Ca2+]i and CA secretion in perfused rat adrenal medullas. When the change in [Ca2+]i was measured using calcium green-1 (490 nm excitation), a 35-min treatment with 10 microM WT caused a 69% inhibition of CA secretion evoked by excess (30 mM) extracellular K+ and a moderate inhibition of the [Ca2+]i response. In contrast, the same treatment of fura-2-loaded cells with WT caused only an 11% inhibition of the high-K+-evoked secretion and no significant attenuation of the [Ca2+]i response. However, during interruption of fluorometry with fura-2, the inhibitory effect of WT developed at a rate similar to that exhibited in calcium green-1-loaded cells. The WT-induced inhibition of high-K+- or bradykinin-evoked secretory responses, which was otherwise irreversible, was reversed by exposing WT-treated chromaffin cells to 380-nm light. When WT was reapplied to the cells of which the secretory ability had been restored by light irradiation, the secretory response was inhibited with a time course similar to that shown during the initial treatment with WT. The photosensitive effect of WT was also demonstrated using bullfrog sympathetic ganglia in which WT-induced inhibition of synaptic transmission was reversed by irradiation with 380-nm light. These results suggest that UV light removes the inhibitory effects of WT by disrupting the covalent bond formed between WT and a target molecule which remains to be determined, although myosin light chain kinase has been reported as the target molecule in both cases examined in this study.

Modulations of early and late secretory processes by activation of protein kinases in the rat adrenal medulla.

Modulatory effects of the activation of either protein kinase C (PKC) by phorbol 12,13-dibutyrate (PDBu) or protein kinase A (PKA) by forskolin on stimulant-evoked secretory processes in the perfused rat adrenal medulla were studied. PDBu or forskolin was applied during repetitive stimulation (30 s each at 10-min intervals) with nicotine, bradykinin, muscarine or histamine, and changes in [Ca2+]i (fura-2 microfluorometry) and catecholamine secretions (electrochemical detection) were simultaneously measured. PDBu markedly potentiated the nicotine-evoked secretion without altering the [Ca2+]i response. PDBu partially inhibited the muscarine-evoked secretion and almost completely blocked the histamine-evoked secretion, concomitantly with extensive suppressions of the [Ca2+]i responses to these stimulants. The bradykinin-evoked secretion was enhanced by PDBu despite a slight attenuation of the [Ca2+]i response. PDBu reduced bradykinin-induced intracellular Ca2+ release in a Ca2+-free medium but enhanced the secretion associated with the released Ca2+. These results suggest that PDBu-activated PKC modulates secretory processes at, at least, two different stages. An early-stage modulation may downregulate receptor/G protein systems, which accounts for the inhibitory effect of PDBu on the muscarine- and histamine-evoked responses. A late-stage modulation may generally promote Ca2+-triggered exocytosis after elevation of [Ca2+]i, which explains the potentiation of the nicotine-evoked secretion by PDBu. The late-stage modulation may counteract the early-stage modulation in bradykinin-stimulated cells. Forskolin potentiated the secretory responses to the four secretagogues without increasing the [Ca2+]i responses. PKA may modulate secretory process at a step(s) distal to the rise in [Ca2+]i as is the case with the late-stage modulation by PKC.

Involvement of protein kinase C in homologous desensitization of histamine-evoked secretory responses in rat chromaffin cells.

The secretory responses in rat adrenal chromaffin cells to histamine H1 receptor stimulation desensitize during repetitive stimulation. The rate of development of this desensitization was slowed by Ro 31-8220, a protein kinase C (PKC) inhibitor. Ro 31-8220 also reversed part of the desensitization which had been induced by earlier histamine stimulation. Phorbol 12,13-dibutyrate (PDBu), an activator of protein kinase C, inhibited histamine-evoked catecholamine (CA) secretion almost completely. The inhibitory effect of PDBu on the H1-receptor-mediated secretory response was antagonized by Ro 31-8220. Histamine induced [Ca2+]i increases due to Ca2+ entry and Ca2+ release from intracellular Ca2+ stores in fura-2-loaded adrenal medullary cells. These [Ca2+]i increases were abolished in PDBu-treated cells. These results suggest that the activation of PKC following histamine H1 receptor stimulation plays a significant role in the process of homologous desensitization of histamine-evoked secretory responses in rat chromaffin cells, through modulation by PKC of H1 receptors and/or GTP-binding proteins coupled with H1 receptors.

Potentiation by indomethacin of receptor-mediated catecholamine secretion in rat adrenal medulla.

Effects of indomethacin on catecholamine secretion evoked by receptor agonists, muscarine, bradykinin or histamine, in rat adrenal chromaffin cells were studied. Indomethacin at 200 microM increased a sustained component of secretion during stimulation with muscarine, bradykinin and histamine by a factor of 2.3, 2.1 and 2.9, respectively, whereas it did not significantly alter basal, high-K(+)- and nicotine-evoked secretions. Although indomethacin at above 400 microM dose-dependently increased basal secretion, the amount of secretion induced by indomethacin alone was much smaller than that in muscarine-evoked secretion as compared at the same concentration of indomethacin applied. Bradykinin-evoked secretion and its potentiation by indomethacin were not inhibited by 20 microM nifedipine but were suppressed by 0.5 mM Ni2+. The cyclooxygenase inhibitor, ibuprofen (200 microM) did not mimic the effect of indomethacin; prostaglandin E2 (20 microM) and arachidonic acid (100 microM) did not significantly alter either bradykinin-evoked secretion itself or its potentiation by indomethacin. Bradykinin increased the intracellular free Ca2+ concentration, [Ca2+]i, in cells loaded with indo-1, and this response was enhanced in the presence of indomethacin. These results suggest that indomethacin may promote Ca2+ entry to potentiate agonist-evoked catecholamine secretions through a novel action that is not directly related to the inhibition of cyclooxygenase activity with indomethacin.

Properties of intracellular calcium stores and their role in receptor-mediated catecholamine secretion in rat adrenal chromaffin cells.

Simulation of rat adrenal chromaffin cells either with receptor agonists such as muscarine, bradykinin and histamine or with caffeine in Ca(2+)-free Krebs solution induced a brief increase in the intracellular free Ca2+ concentration, [Ca2+]i, which terminated within 90 s. Both the agonist- and caffeine-induced [Ca2+]i responses were abolished in cells which had been treated with either 500 nM thapsigargin (TG) or 20 microM ryanodine, suggesting that inositol trisphosphate and caffeine cause Ca2+ release either from the same Ca2+ store or from distinct stores which exchange Ca2+ rapidly. In normal Krebs solution, these agonists evoked catecholamine (CA) secretion which showed an initial transient followed by a sustained component. Neither component of the secretion was significantly affected by TG or ryanodine. In the medium containing 16 microM Ca2+ or no Ca2+, CA secretion evoked by 30-second stimulation with 100 microM muscarine was 59 or 7%, respectively, of that evoked in the normal medium containing 2 mM Ca2+. In TG-treated cells, the CA secretion at 16 microM Ca2+ was reduced to 23% and that in the Ca(2+)-free medium was completely abolished. These results suggest that the receptor-mediated Ca2+ entry solely determines the rate of CA secretion in rat chromaffin cells when stimulated by receptor agonists in the normal medium, whereas intracellular Ca2+ release and Ca2+ entry may cooperatively support the secretion when cells are stimulated in media containing low concentrations of Ca2+.

Characterization of low pH-induced catecholamine secretion in the rat adrenal medulla.

Catecholamine (CA) secretion was evoked when the isolated rat adrenal gland was perfused with HEPES-buffered Krebs solution acidified by the addition of HCl or by gassing with 95% O2/5% CO2. The secretion was detectable at pH 7.0 and increased with decreasing pH until at approximately 6.4. The low pH-induced CA secretion consisted of two phases, an initial transient response followed by a sustained phase. An intracellular Ca2+ antagonist, 3,4,5-trimethoxybenzoic acid 8-(N,N-diethylamino)octyl ester, selectively inhibited the initial phase of secretion. Both of the responses were resistant to nifedipine, a blocker of voltage-gated Ca2+ channel, but were completely inhibited in Ca(2+)-free (1 mM EGTA containing) solution. Adrenaline was an exclusive component in CAs released by low pH. The time course and extent of intracellular acidification caused either by low pH in the external medium or by the offset of a transitory NH4Cl application had no correlation with those of the secretory responses in the corresponding period. These results suggest that extracellular acidification preferentially activates adrenaline secretive cells to evoke CA secretion and that this low pH-induced CA secretion may be mediated by dihydropyridine-insensitive Ca2+ influx. Furthermore, the initial transient phase of the low pH-induced CA secretion might be caused by a Ca2+ release from intracellular stores, which is also induced by the Ca2+ influx.

Effects of corticosteroids on cytosolic free calcium concentration of the rat adrenal zona glomerulosa cell.

In the perfused cells isolated from the rat adrenal zona glomerulosa, deoxycorticosterone, aldosterone and dexamethasone (10 microM-1 mM) significantly inhibited elevation of the cytosolic concentration of calcium ion [Ca2+]c caused by a preceding stimulation with angiotensin II (ANG II) or high K+. The inhibition of [Ca2+]c was observed within a few minutes after application of the corticosteroids. The resting level of [Ca2+]c was reduced by aldosterone, whereas it was not significantly altered by deoxycorticosterone or dexamethasone. Since it is well established that the elevation of [Ca2+]c is required for the secretory responses to ANG II or high K+, the present results are compatible with a view that aldosterone secretion from the cortical cells in response to the secretagogues may, in turn, be attenuated by corticosteroids, including aldosterone itself.

Changes in intracellular pH of mouse hippocampal slices responding to hypoxia and/or glucose depletion.

Effects of hypoxia and/or glucose depletion on apparent intracellular pH, (pHi)app, were studied in hippocampal slices of mouse brain using the fluorescence pH indicator BCECF (2',7'-bis(carboxyethyl)carboxyfluorescein). In the presence of glucose (11 mM), (pHi)app of the CA1 pyramidal cell layer decreased with hypoxia but rapidly recovered with reoxygenation. The response in (pHi)app to hypoxia was consistent with changes in lactate content. By superfusion with a hypoxic and glucose-devoid solution, (pHi)app decreased in the first 5 min, but then increased in two phases: during a 5- to 7-min period and again during the latter period of superfusion. BCECF fluorescence rapidly diminished in this latter period. These changes in (pHi)app could not be explained only by the lactate content in the slices. Some other mechanisms may also regulate the (pHi)app in the slices during and after hypoxia and glucose depletion.

Differential effects of protein kinase C activation on catecholamine secretions evoked by stimulations of various receptors in the rat adrenal medulla.

Catecholamine secretions during continuous receptor stimulations by histamine, muscarine and bradykinin in the rat adrenal medulla commonly consisted of two phases, a transient initial secretion followed by a sustained secretion. On activating protein kinase C (PKC) by phorbol dibutyrate (PDBu), both phases of histamine-evoked secretion were inhibited whereas the initial phase alone was inhibited with muscarine. In contrast, bradykinin-evoked secretion as a whole was potentiated. Similar modes of modulations were exhibited when the secretions with these agonists were elicited in muscarine- or bradykinin-pretreated medullae in which PKC had been activated by endogenous processes. It is suggested that PKC may selectively affect the receptors or/and GTP-binding proteins to cause the differential effects on the secretory response in the rat adrenal medulla.

Functional expression from cloned cDNAs of glutamate receptor species responsive to kainate and quisqualate.

The complete amino acid sequences of two mouse glutamate receptor subunits (GluR1 and GluR2) have been deduced by cloning and sequencing the cDNAs. Xenopus oocytes injected with mRNA derived from the GluR1 cDNA exhibit current responses both to kainate and to quisqualate as well as to glutamate, whereas oocytes injected with mRNA derived from the GluR2 cDNA show little response. Injection of oocytes with both the mRNAs produces current responses larger than those induced by the GluR1-specific mRNA and the dose-response relations indicate a positively cooperative interaction between the two subunits. These results suggest that kainate and quisqualate can activate a common glutamate receptor subtype and that glutamate-gated ionic channels are hetero-oligomers of different subunits.

Binding properties of sea anemone toxins to sodium channels in the crayfish giant axon.

1. Effects of four different sea anemone toxins from Anthopleura (AP-A and AP-C), Anemonia (ATX II) and Parasicyonis (PaTX), and a scorpion toxin from Leiurus (LqTX) on crayfish giant axons were studied. 2. These toxins slowed the Na channel inactivation process, inducing a maintained Na current during a depolarizing pulse. 3. The binding rates for these toxins markedly decreased under depolarization. The decrease in AP-A binding was mainly derived from an increased dissociation rate under depolarization whereas that in PaTX binding from a reduced association rate. 4. The potential-dependent toxin binding kinetics seemed to be related to the gating mechanism of the Na channel. 5. Competitive bindings between these toxins were demonstrated.

Potential-dependent action of Anemonia sulcata toxins III and IV on sodium channels in crayfish giant axons.

Effects of toxins III and IV (ATX III and IV) from the sea anemone Anemonia sulcata on the Na current of crayfish giant axons were studied. Both toxins slowed the inactivation of Na channels, producing a maintained Na current during a depolarizing voltage pulse. Using the intensity of the toxin-induced maintained current as an index for the fraction of Na channels to which toxin is bound, the toxin association and dissociation kinetics were analyzed. The dissociation rate of ATX III was increased by two orders of magnitudes by depolarizing the membrane from -70 to -40 mV. This increase of the dissociation rate caused a marked decrease in the binding rate of ATX III to Na channels in the same potential range. ATX IV exhibited association and dissociation kinetics that had a potential dependency quite similar to that of ATX III in spite of different ionic charge distribution in these two toxins. The results support the view that the potential-dependent kinetics of these toxins are not due to an electrostatic interaction between the ionic charges of toxins and the membrane potential but result from a modulation of the binding energy depending on the gate configuration of the Na channel.

Effects of histrionicotoxin derivatives on ion channels and acetylcholine receptor-channel complexes in bullfrog sympathetic ganglia.

1. Four synthetic histrionicotoxin derivatives (H8-, H12-, C4H10- and C5H10-HTX) were applied to bullfrog (Rana catesbeiana) sympathetic ganglia and their effects were compared electrophysiologically. 2. The derivatives (60-100 microM) blocked both acetylcholine receptor-channel complex (ACh RC complex) and Na+ channel to cause a transmission failure. They also blocked K+ and Ca2+ channels. 3. Although all 4 derivatives exhibited similar effects, their potencies on respective ionic channels differed from one another. 4. Two types of (presumably subsynaptic and extrasynaptic) ACh RC complexes in ganglion cells were distinguished based on their differential sensitivities to HTX derivatives.