Lysophospholipids elevate [Ca2+]i and trigger exocytosis in bovine chromaffin cells

Sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) are responsible for many physiological functions, including angiogenesis, neuronal survival, and immunity. However, little is known about their effects in modulating the stimulus-secretion coupling in bovine chromaffin cells. The result of PCR showed that at least two receptors (S1P(3) and LPA(1)) were expressed in bovine chromaffin cells. The elevation of [Ca(2+)](i) by S1P was fast and sustaining; but the elevation by LPA was slow and transient. The EC(50) for S1P and LPA in elevating the [Ca(2+)](i) were 0.55+/-0.01 and 0.54+/-0.40microM, respectively. This elevation could be totally blocked by thapsigargin, 2-APB, and U73122. Pertussis toxin pretreatment inhibited about half of the elevation in [Ca(2+)](i) suggesting the involvement of G(i) and other G-proteins. Repetitive [Ca(2+)](i) elevations elicited by S1P, but not LPA, were inhibited by ryanodine. S1P was more effective than LPA in triggering exocytosis as measured by the changes in membrane capacitance. The whole-cell Ca(2+) current was inhibited by both lysophospholipids but Na(+) current was inhibited by S1P only. These results suggest the differential effects of LPA and S1P in releasing Ca(2+) from the intracellular Ca(2+) stores and modulating the stimulus-secretion coupling in bovine chromaffin cells.     

Lack of Ca2+- and ATP-dependent priming stage in caffeine-induced exocytosis in bovine adrenal chromaffin cells: comparison with Ca2+

1. Caffeine (20-40 mM) secreted catecholamines from beta-escin-permeabilized bovine adrenal chromaffin cells in the presence or absence of 2 mM MgATP. The caffeine-induced catecholamine secretion in the presence of MgATP was to the same extent as that in the absence of MgATP. 2. Ca2+ (0.1-10 microM) induced a significantly greater secretion of catecholamines in the presence of MgATP than in the absence of MgATP. 3. ML-9 (100 microM) and ML-7 (100 microM), myosin light chain kinase inhibitors, and W-7 (100 microM) and trifluoperazine (TFP; 30 microM), calmodulin antagonists, inhibited the Ca2+-induced catecholamine secretion in the presence of MgATP but not in the absence of MgATP. They did not inhibit the caffeine-induced catecholamine secretion in the presence of MgATP. 4. The ATP-independent phase in Ca2+-dependent exocytosis is thought to be associated with the final step that ultimately leads to fusion, while the ATP-dependent phase is thought to be associated with a vesicle priming reaction. Therefore, these results suggest that the ATP-requiring priming stage is lacking in the process of caffeine-induced exocytosis in bovine adrenal chromaffin cells.     

Antimigraine dotarizine blocks P/Q Ca2+ channels and exocytosis in a voltage-dependent manner in chromaffin cells

The mechanism of blockade of P/Q Ca(2+) channels by antimigraine, dotarizine, was studied in voltage-clamped bovine adrenal chromaffin cells. Inward currents through P/Q channels were pharmacologically isolated by superfusing the cells with omega-conotoxin GVIA (1 microM) plus nifedipine (3 microM). Dotarizine (10-30 microM) blocked the P/Q fraction of I(Ba) and promoted current inactivation. Thus, dotarizine caused a greater blockade of the late I(Ba), compared with blockade of the early peak I(Ba). This effect was more prominent, the longer was the duration of the depolarising pulse. The blockade of I(Ba) was also greater at more depolarising holding potentials (i.e. -60 mV), than was the blockade produced at more hyperpolarising holding potentials (i.e. -80 or -110 mV). Catecholamine secretory responses to brief pulses (2 s) of a Krebs-HEPES solution containing 100 mM K(+) and 2 mM Ca(2+) was blocked by 3 microM dotarizine. Blockade was faster and greater when dotarizine was applied on cells that were previously depolarised with Krebs-HEPES deprived of Ca(2+) and containing increasing concentrations of K(+). This voltage-dependent blockade of P/Q channels and exocytosis might be the underlying mechanism explaining the dotarizine prophylaxis of migraine attacks.     

Histamine-induced Ca2+ release in bovine adrenal chromaffin cells

The histamine-induced biphasic increase of the intracellular free [Ca2+] ([Ca2+]i) was studied in bovine adrenal chromaffin cells using fura-2 microfluorimetry and the whole-cell patch-clamp technique. Both the rapid, transient Ca2+ rise and the sustained plateau component of elevated [Ca2+]i were independent of extracellular Ca2+. Incubation with the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) blocker thapsigargin diminished histamine-induced changes in [Ca2+]i. When Ca2+ release was either stimulated by IP3 or blocked with the competitive inhibitor heparin, histamine was unable to elicit the typical Ca2+ rise. Ryanodine, tetracaine and ruthenium red, all blockers of Ca2+ release from caffeine-sensitive stores, had only minor effects on the agonist-induced Ca2+ changes. The contribution of mitochondria in shaping the histamine-induced Ca2+ increase was studied using ruthenium red and the two proton ionophores carbonylcyanide m-chlorophenylhydrazone (CCCP) and carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP). Both mitochondrial uncouplers reversibly increased [Ca2+]i and induced an inward current leading to cell membrane depolarisation. In summary, these results indicate that Ca2+ from IP3-sensitive stores is essential for the generation of both the transient increase and secondary elevation in [Ca2+]i.     

Failure of endothelin-1 to activate store-operated Ca2+ channels by lack of mobilization from intracellular Ca2+ stores in cultured bovine adrenal chromaffin cells

We have recently shown that in addition to L-type voltage-operated Ca2+ channel (VOC), endothelin-1 (ET-1) stimulation opens two types of Ca2+-permeable nonselective cation channels [designated nonselective cation channel-1 (NSCC-1) and NSCC-2]. However, in this Ca2+ entry, the involvement of store-operated Ca2+ channel (SOCC), which is suggested to exist in chromaffin cells, was unclear. Those NSCCs as well as SOCC can be pharmacologically discriminated using Ca2+ channel blockers such as SK&F 96365 and LOE 908. To clarify whether SOCC should actually exist and play a role in Ca2+ entry in chromaffin cells stimulated with ET-1, we examined the effects of removal of extracellular Ca2+, thapsigargin (TG, an inhibitor of endoplasmic reticulum Ca2+-ATPase), LOE 908 and SK&F 96365 on cytosolic free Ca2+ concentrations ([Ca2+]i) in cultured bovine adrenal chromaffin cells. After the cells were exposed to Ca2+-free medium followed by exposure to TG to deplete Ca2+ from the intracellular Ca2+ store, restoration of extracellular Ca2+ caused a gradual increase in [Ca2+]i (to about 200% of control). The increase was unaffected by LOE 908, but completely abolished by SK&F 96365. In the Ca2+-free medium, no increase in [Ca2+]i by ET-1 was observed, but the subsequent restoration of extracellular Ca2+ induced a rapid increase in [Ca2+]i (to the same level of [Ca2+]i as that evoked by ET-1 in the normal medium (1.0 mM Ca2+)). Since SK&F 96365 is also a blocker of SOCC, these results indicate that in bovine adrenal chromaffin cells, Ca2+ entry through SOCC (Ca2+ influx through the capacitative Ca2+ entry system) occurs but is comparably weak, and that it virtually does not work on the stimulation of ET-1.     

Ca(2+) influx stimulated phospholipase C activity in bovine adrenal chromaffin cells: responses to K(+) depolarization and histamine

The role of Ca(2+) influx in activating phospholipase C in bovine adrenal chromaffin cells has been investigated. Phospholipase C activity in response to K(+) depolarization (56 mM) was blocked by the L-type Ca(2+) channel antagonist nifedipine and partially inhibited by the omega-conotoxins GVIA and MVIIC. In contrast, phospholipase C activity in response to histamine receptor activation was unaffected by omega-conotoxin GVIA and partially inhibited by omega-conotoxin MVIIC or nifedipine. This response was however markedly inhibited by the non-selective Ca(2+) channel antagonists La(3+) or 1-[beta-[3-(4-Methoxyphenyl)propoxy]-4-methoyphenethyl]-H-imidazol e (SKF-96365). Despite this Ca(2+) dependence phospholipase C activity was not increased during periods of "capacitative" Ca(2+) inflow generated by histamine-, caffeine- or thapsigargin-mediated depletion of internal Ca(2+) stores. Thus, while Ca(2+) influx in response to K(+) depolarization or G-protein receptor activation can increase phospholipase C activity in these cells, in the latter case it appears to be ineffective unless there is concurrent agonist occupation of the receptor.     

Pharmacological characterization of receptor-mediated Ca2+ entry in endothelin-1-induced catecholamine release from cultured bovine adrenal chromaffin cells

To clarify the mechanism for the endothelin-1 (ET-1)-induced release of catecholamines from the adrenal gland, we examined the effects of removal of extracellular Ca2+, blockers of L-, N-, P- and Q-types of voltage-operated Ca2+ channels (VOCC) such as nifedipine (L-type), omega-conotoxin GVIA (N-type), omega-agatoxin IVA (P-type) and omega-conotoxin MVIIC (Q-type) and blockers of voltage-independent Ca2+ entry channel such as SK&F 96365 and LOE 908 on release of catecholamines, the cytosolic free Ca2+ concentration ([Ca2+]i), and 45Ca2+ uptake in cultured bovine adrenal chromaffin cells. ET-1 but not ET-3 induced increases in release of catecholamines, [Ca2+]i, and 45Ca2+ uptake. The responses to ET-1 were abolished by the antagonist for ET(A) receptors, BQ-123, but not by the antagonist for ET(B) receptors, BQ-788, and they were abolished by removal of extracellular Ca2+. The increases were only partially inhibited (to about 65% of control) by nifedipine but unaffected by any of the omega-toxins. The nifedipine-resistant increase was inhibited by SK&F 96365 (to about 40%) and abolished by LOE 908 alone. These results indicate that ET-1 augments the release of catecholamines from adrenal chromaffin cells through ET(A) receptors, by activating two types of Ca2+ entry channels in addition to L-type VOCC: one (nonselective cation channel-1; NSCC-1) is sensitive to LOE 908 but resistant to SK&F 96365, whereas the other (NSCC-2) is sensitive to both LOE 908 and SK&F 96365.     

Sensitivity of G-protein involved in endothelin-1-induced Ca2+ influx to pertussis toxin in porcine endothelial cells in situ.

1. We designed a new method to determine quantitatively the intracellular Ca2+ concentration ([Ca2+]i) in endothelial cells in situ, using front-surface fluorometry and fura-2-loaded porcine aortic valvular strips. Using this method, we investigated the characteristics of the G-protein involved in endothelin-1 (ET-1)-induced changes in [Ca2+]i of endothelial cells in situ. 2. Endothelial cells were identified by specific uptake of acetylated-low density lipoprotein labelled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI-Ac-LDL). Double staining with DiI-Ac-LDL and fura-2 showed that the valvular strip was covered with a monolayer of endothelial cells and that the cellular component which contributed to the fura-2 fluorescence, [Ca2+]i signal, was exclusively endothelial cells. 3. ET-1 (10(-7) M) induced an elevation of [Ca2+]i consisting of two components: the first was a rapid and transient elevation to reach a peak, followed by a second, sustained elevation (the second phase). The first phase was composed of extracellular Ca(2+)-independent and -dependent components, while the second phase was exclusively extracellular Ca(2+)-dependent. The extracellular Ca(2+)-independent component of the first phase was due to the release of Ca2+ from intracellular storage sites. The second phase and part of the first phase of [Ca2+]i elevation were attributed to the influx of extracellular Ca2+. The Ca2+ influx component was completely inhibited by 10(-3) M Ni2+ but was not affected by 10(-5) M diltiazem.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic ADP-ribose requires FK506-binding protein to regulate intracellular Ca2+ dynamics and catecholamine release in acetylcholine-stimulated bovine adrenal chromaffin cells

The present study was undertaken to elucidate whether cyclic ADP-ribose (cADPR) mediates the amplification of Ca2+ signaling and catecholamine release via the involvement of FK506-binding proteins (FKBPs)/ryanodine receptor (RyR) in bovine adrenal chromaffin cells. cADPR induced Ca2+ release in digitonin-permeabilized chromaffin cells and this was blocked by FK506 and rapamycin, ligands for FKBPs; 8Br-cADPR, a competitive antagonist for cADPR; and antibody for FKBP12/12.6, while it was enhanced by cyclosporin A. Ryanodine-induced Ca2+ release was not affected by 8Br-cADPR and was remarkably enhanced by FK506, rapamycin, cyclosporin A, and cADPR. FK506 binds to FKBP12.6 and removes it from RyRs, but cADPR did not affect the binding between FKBP12.6 and RyR. In intact chromaffin cells, 8Br-cADPR, FK506, and rapamycin, but not cyclosporin A attenuated the sustained intracellular free Ca2+ concentration ([Ca2+]i) rise induced by acetylcholine (ACh). 8Br-cADPR, FK506, and SK&F 96365 reduced the Mn2+ entry stimulated with ACh only when Ca2+ was present in the extracellular medium. 8Br-cADPR, FK506, and rapamycin concentration-dependently inhibited the ACh-induced catecholamine (CA) release. Here, we present evidence that FKBP12.6 associated with RyR may be required for Ca2+ release induced by cADPR in bovine adrenal chromaffin cells. cADPR-mediated Ca2+ release from endoplasmic reticulum in ACh-stimulated chromaffin cells is coupled with Ca2+ influx through the plasma membrane which is essential for ACh-stimulated CA release.     

Release of nonmitochondrial sequestered Ca2+ from permeabilized muscle cells in culture

Activation of alpha 1-adrenergic receptors in BC3H-1 muscle cells results in the rapid elevation of intracellular Ca2+, accompanied by an unusually slow and small increase in inositol 1,4,5-trisphosphate (IP3) formation [J. Biol. Chem. 263: 1952-1959 (1988); Mol. Pharmacol. 32: 376-383 (1987)]. To further assess the role of IP3 in receptor-stimulated Ca2+ mobilization, we have examined Ca2+ disposition in saponin-permeabilized BC3H-1 cells. Permeabilized cells loaded with tracer 45Ca2+ in a buffer containing 100 nM free Ca2+ accumulated greater than 75% of their Ca2+ into an ATP-sensitive compartment and were insensitive to inhibitors of mitochondrial Ca2+ uptake. Application of IP3 resulted in a rapid increase in 45Ca2+ efflux. Under isotopic equilibrium, approximately 90% of the total membrane-enclosed 45Ca2+ was released by 10 microM IP3 within 30 sec. Maximally and half-maximally effective concentrations of IP3 were 22 microM and 0.9 microM, respectively. Application of 10 microM GTP, but not guanine triphosphate-gamma-sulfate, resulted in a slight increase in 45Ca2+ efflux, which reflected a loss in total cellular Ca2+. The GTP-mediated response was slower and of far smaller magnitude than that mediated by IP3. A Ca2+-triggered Ca2+ release mechanism appears not to amplify the receptor response in BC3H-1 cells, inasmuch as 45Ca2+ efflux was not appreciably increased by elevated concentrations of free Ca2+. Furthermore, caffeine and ryanodine had no effect on basal, IP3-mediated, or alpha 1-adrenergic-stimulated Ca2+ release from intact or permeabilized cells. In conclusion, BC3H-1 cells, although showing small and slow increases in IP3 formation upon agonist stimulation, exhibit normal sensitivity to IP3-elicited release of Ca2+ and low sensitivity to other candidate Ca2+-mobilizing agents. The IP3-sensitive Ca2+ stores may be localized within specialized compartments and may play a greater role in the maintenance of elevated cytosolic Ca2+ than in the initial response to receptor activation.     

Cannabidiol-induced intracellular Ca2+ elevations in hippocampal cells

The phytocannabinoid cannabidiol (CBD) is at the forefront of therapeutic cannabinoid research due to its non-psychotropic properties. Research supports its use in a variety of disorders, yet the cellular mechanisms of its action remain unclear. In this study, the effect of CBD upon Ca2+ homeostasis in hippocampal cells was characterised. CBD (1 microM) elevated intracellular Ca2+ ([Ca2+]i) by approximately +45% of basal Ca2+ levels in both glia (77% responders) and neurones (51% responders). Responses to CBD were reduced in high excitability HEPES buffered solution (HBS), but not affected in low excitability/low Ca2+ HBS. CBD responses were also significantly reduced (by 50%) by the universal Ca2+ channel blocker cadmium (50 microM) and the L-type specific Ca2+ channel blocker nifedipine (20 microM). Interestingly, intracellular store depletion with thapsigargin (2 microM) had the most dramatic effect on CBD responses, leading on average to a full block of the response. Elevated CBD-induced [Ca2+]i responses (>+100%) were observed in the presence of the CB1 receptor antagonist, AM281 (1 microM), and the vanilloid receptor antagonist, capsazepine (CPZ, 1 microM). Overall, our data suggest that CBD modulates hippocampal [Ca2+]i homeostasis via intracellular Ca2+ stores and L-type VGCC-mediated Ca2+ entry, with tonic cannabinoid and vanilloid receptor signalling being negatively coupled to this pathway.     

Mode of action of alpha-latrotoxin: role of divalent cations in Ca2(+)-dependent and Ca2(+)-independent effects mediated by the toxin

The potent neurotoxin alpha-latrotoxin (alpha LTx), from black widow spider venom, induces neurotransmitter release in both Ca2(+)-containing and Ca2(+)-free medium, following interaction with a specific cell surface receptor. Binding studies revealed two populations of alpha LTx binding sites in bovine synaptosomal membranes, showing the same high affinity (Kd, 0.3 x 10(-10) M) for alpha LTx, with approximately 50% of the sites being Ca2+ sensitive and the rest being Ca2+ insensitive. In contrast, in PC12 cells alpha LTx binding was completely unaffected by the removal of extracellular Ca2+ (Kd, 5 x 10(-10) M). The use of La3+ as an inhibitor of alpha LTx action, previously shown in synaptosomes, was extended to PC12 cells. In this system, La3+ (100 microM) was shown to inhibit Ca2+ influx, both Ca2(+)-dependent and -independent dopamine release, and polyphosphoinositide (PPI) hydrolysis induced by alpha LTx. At the same time, La3+ did not block alpha LTx binding or dopamine release evoked by either the ionophore ionomycin (0.5 microM) or the phorbol ester tetradecanoylphorbol acetate (100 nM). La3+ also blocked the influx of Mn2+ ions through the alpha LTx-induced cation channel, as measured by quenching of fura-2 fluorescence. In this PC12 cell line, PPI hydrolysis could also be induced by ionomycin, but only when it was present at concentrations that caused an elevation of free intracellular Ca2+ ([Ca2+]i) that was not transient but was as persistent as that evoked by alpha LTx. Our conclusions with regard to the mode of action of alpha LTx are as follows. (i) All the effects of alpha LTx in PC12 cells (dopamine release, PPI hydrolysis, and Ca2+ influx) can be mediated via a single, Ca2(+)-insensitive alpha LTx receptor. (ii) alpha LTx-induced PPI hydrolysis is most likely due to the activation of a Ca2(+)-sensitive phospholipase C following the persistent rise in [Ca2+]i elicited by the toxin in Ca2(+)-containing medium, and not via direct coupling of the alpha LTx receptor to the enzyme. (iii) Toxin-evoked Ca2(+)-independent dopamine release can be blocked by La3+ at the extracellular level, most likely by prevention of the entry of divalent cations.     

Effects of extracellular Ca2+ influx on endothelin-1-induced intracellular mitogenic cascades in C6 glioma cells

We have recently shown that endothelin-1 activates two types of Ca2+-permeable nonselective cation channels (NSCC-1 and NSCC-2) in C6 glioma cells. These channels can be distinguished by their sensitivity to blockers of the receptor-operated Ca2+ channel, 1-[b-(3-[4-methoxyphenyl]propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride (SK&F 96365) and (R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di-[2-(2,3,4-trimethoxyphenyl)ethyl]-acetamide (LOE 908). NSCC-1 is sensitive to LOE 908 and resistant to SK&F 96365, whereas NSCC-2 is sensitive to both LOE 908 and SK&F 96365. Moreover, extracellular Ca2+ influx through these channels plays an essential role in endothelin-1-induced mitogenesis in C6 glioma cells. The purpose of the present study was to investigate the effects of extracellular Ca2+ influx on intracellular pathways of endothelin-1-induced mitogenic responses in C6 glioma cells. We focused on extracellular signal-regulated kinase 1 and 2 (ERK1/2) in this context. An inhibitor of mitogen-activated protein kinase, 2-[2-amino-3-methoxyphenyl]-4H-1-benzopyran-4-one (PD 98059), abolished the endothelin-1-induced increase in ERK1/2 activity, but only partially suppressed the mitogenic response. ERK1/2 activation by endothelin-1 was partially suppressed in the absence of extracellular Ca2+. On the basis of the sensitivity to LOE 908 and SK&F 96365, Ca2+ influx through NSCC-1 and NSCC-2 plays an essential role in the extracellular Ca2+-dependent component of ERK1/2 activity. In contrast, Ca2+ influx through NSCC-2 is involved in the ERK1/2-independent component of endothelin-1-induced mitogenesis. These results indicate that (1) the endothelin-1-induced mitogenic response involves both ERK1/2-dependent and -independent mechanisms, (2) ERK1/2 activation by endothelin-1 involves an extracellular Ca2+ influx-dependent cascade as well as an extracellular Ca2+ influx-independent cascade, (3) because endothelin-1-induced mitogenesis is completely dependent on extracellular Ca2+ influx, extracellular Ca2+ influx also plays an important role in mitogenic pathways downstream of ERK1/2, (4) extracellular Ca2+ influx through NSCC-1 and NSCC-2 has an important role in the extracellular Ca2+ influx-dependent component of ERK1/2-dependent mitogenesis, (5) extracellular Ca2+ influx through NSCC-2 has an important role in ERK1/2-independent mitogenesis, and (6) Ca2+ influx through each Ca2+ channel may play a distinct role in intracellular mitogenic cascades.     

Neomycin blocks dihydropyridine-insensitive Ca2+ influx in bovine adrenal chromaffin cells

There is evidence that bovine adrenal chromaffin cells are provided with both dihydropyridine-sensitive and -resistant voltage-sensitive Ca²⁺ influx pathways. Although recent electrophysiological work indicates that the dihydropyridine-resistant pathway is partially mediated by w-conotoxin-sensitive and -insensitive Ca²⁺ channels, the pharmacological sensitivity of the latter channels remains elusive. We have now found that combined incubations with nitrendipine (1 μM) and neomycin (0.5 mM) reduced high K⁺ (50 mM)-evoked intracellular Ca²⁺ concentration ([Ca²⁺]_i) transients to a larger extent than each drug separately. [Ca²⁺]_i was measured using the fluorescent intracellular Ca²⁺ indicator fura-2. Neomycin (0.05−2 mM) reduced high K⁺-evoked ⁴⁵Ca²⁺ uptake in a dose-dependent manner (IC₅₀ = 0.09 mM). In the presence of nitrendipine (1 μM), the minimal neomycin concentration necessary for total blockade of ⁴⁵Ca²⁺ uptake was reduced to 0.3 mM. Moreover, in the absence of nitrendipine the ⁴⁵Ca²⁺ uptake remaining in 0.3 mM neomycin (26% of maximum) was similar to the fractional inhibition by nitrendipine alone (29%). Neomycin (0.05−2 mM) inhibited the [Ca²⁺]_i transient induced by the L-type Ca²⁺ channel agonist Bay K 8644 (1 μM) much more extensively at 2 mM than at 0.3 mM (percent inhibition = 59% and 15%, respectively). Neomycin (0.05−2 mM) blocked high K⁺-evoked noradrenaline and adrenaline release in a dose-dependent fashion (IC₅₀ = 0.8−1.1 mM), the blockade efficiency being enhanced in the presence of 1 μM nitrendipine (IC₅₀ = 0.17−0.19 mM). It is concluded that neomycin (≤ 0.3 mM) blocks preferentially the dihydropyridine-insensitive Ca²⁺ influx pathway of the chromaffin cell. Moreover, both the dihydropyridine-sensitive and the dihydropyridine-resistant, neomycin-sensitive Ca²⁺ influx pathways contribute strongly to depolarization-evoked catecholamine secretion.     

Lack of Ca2+‐ and ATP‐dependent priming stage in caffeine‐induced exocytosis in bovine adrenal chromaffin cells: comparison with Ca2+

1 Caffeine (20–40 m m ) secreted catecholamines from β‐escin‐permeabilized bovine adrenal chromaffin cells in the presence or absence of 2 m m MgATP. The caffeine‐induced catecholamine secretion in the presence of MgATP was to the same extent as that in the absence of MgATP.
2 Ca2+ (0.1–10 μm ) induced a significantly greater secretion of catecholamines in the presence of MgATP than in the absence of MgATP.
3 ML‐9 (100 μm ) and ML‐7 (100 μm ), myosin light chain kinase inhibitors, and W‐7 (100 μm ) and trifluoperazine (TFP; 30 μm ), calmodulin antagonists, inhibited the Ca2+‐induced catecholamine secretion in the presence of MgATP but not in the absence of MgATP. They did not inhibit the caffeine‐induced catecholamine secretion in the presence of MgATP.
4 The ATP‐independent phase in Ca2+‐dependent exocytosis is thought to be associated with the final step that ultimately leads to fusion, while the ATP‐dependent phase is thought to be associated with a vesicle priming reaction. Therefore, these results suggest that the ATP‐requiring priming stage is lacking in the process of caffeine‐induced exocytosis in bovine adrenal chromaffin cells.     

Fendiline mobilizes intracellular Ca2+ in Chang liver cells

1. The effects of the antianginal drug fendiline (N-[3,3-diphenylpropyl]-alpha-methyl-benzylamine) on intracellular free Ca2+ levels ([Ca2+](i)) in Chang liver cells were evaluated using fura-2 as a fluorescent Ca2+ indicator. 2. Fendiline (1-100 micromol/L) increased [Ca2+](i) in a concentration-dependent manner, with an EC50 of 25 micromol/L. 3. The [Ca2+](i) response was composed of an initial rise and a slow decay to a sustained phase. Removal of extracellular Ca2+ partly reduced the [Ca2+](i) signals. 4. Fendiline (10 micromol/L)-induced release of intracellular Ca2+ was reduced by 65% following pretreatment with 1 micromol/L thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete Ca2+ stored in the endoplasmic reticulum. 5. After pretreatment with 10 micromol/L fendiline in Ca2+-free medium for several minutes, addition of 3 mmol/L Ca2+ induced an increase in [Ca2+](i) of a magnitude four-fold greater than control. This increase in [Ca2+](i) was not reduced by 10 micromol/L SKF96365, econazole, nifedipine or verapamil. 6. Fendiline (10 micromol/L)-induced release of intracellular Ca2+ was not altered by inhibition of phospholipase C with 2 micromol/L 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino) hexyl)-1H-pyrrole-2,5-dione (U73122). 7. The results of the present study show that fendiline induces an increase in [Ca2+](i) in Chang liver cells by releasing stored Ca2+ in an inositol 1,4,5-trisphosphate-independent manner and by causing extracellular Ca2+ influx.     

(+)-Niguldipine binds with very high affinity to Ca2+ channels and to a subtype of alpha 1-adrenoceptors

The enantiomers of the 1,4-dihydropyridine (DHP) niguldipine (3-methyl-5-[3-(4,4-diphenyl-1-piperidinyl)-propyl]- 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-pyridine-3,5-dicarboxylate- hydrochloride) were investigated with respect to their interaction with 1,4-DHP receptors on L-type Ca2+ channels and alpha-adrenoceptors. The Ki values for niguldipine were dependent on the membrane protein concentrations in the radioligand binding assay. 'True' Ki values (at extrapolated 'zero' membrane protein) were determined with guinea-pig membranes for (+)-niguldipine and were found to be 85 pmol/l for the 1,4-DHP receptor of skeletal muscle, 140 pmol/l for that of brain and 45 pmol/l for that of heart. (-)-Niguldipine was approximately 40 times less potent. (+)-Niguldipine (Ki: 78 nmol/l) and (-)-niguldipine (Ki: 58 nmol/l) bound with approximately equal affinity to the alpha 1-adrenoceptors ('alpha 1B') in liver cell membranes. The (+)-niguldipine alpha 1-adrenoceptor inhibition data for rat brain cortex membranes were better fitted by a two-site model. The high-affinity component ('alpha 1A') had a Ki value of 52 pmol/l in competition experiments with [3H]prazosin. The low-affinity site (alpha 1B) had 200- to 600-fold less affinity. (-)-Niguldipine was greater than 40-fold less potent at alpha 1A- but was nearly equipotent to the (+)enantiomer at alpha 1B-sites. (+)-Niguldipine was the most selective compound for discriminating alpha 1A- from alpha 1B-adrenoceptors and is a novel prototype for 1,4-DHPs which bind with nearly equal affinity to skeletal muscle and brain or heart 1,4-DHP receptors.     

Changes in the transmembrane potential of thymocytes during dexamethasone- and Ca2(+)-induced apoptosis]

By means of the method of potential-sensitive fluorescent probes is established that under dexametazone-induced apoptosis (0.1-10 microM) the reduction of transmembrane potentials (TMP, delta phi) on plasmatic (delta phi p) and mitochondrial (delta phi m) membranes of hormone-sensitive thymocytes is observed. Dexametazone does not changes transmembrane potentials of glucocorticoid-resistant thymocytes. Ca-ionophore A23187 realistically reduces (at the average on 26%) value of plasmatic TMP both in glucocorticoid-sensitive and glucocorticoid-resistant thymocytes. Change of delta phi is one of the mechanisms to realization an apoptotic process in rats' thymocytes. Dynamics and nature of TMP changes depend on the agent-inductor of apoptosis.     

Extracellular Ca2+ influx and endothelin-1-induced intracellular mitogenic cascades in rabbit internal carotid artery vascular smooth muscle cells

Endothelin-1 (ET-1) has been proven to activate two types of Ca2+-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca2+ channel (SOCC) in rabbit internal carotid artery vascular smooth muscle cells (ICA VSMCs). Ca2+ influx through these channels plays an essential role for ET-1-induced mitogenesis in ICA VSMCs. The purpose of the current study was to investigate the effects of Ca2+ influx on intracellular pathways of ET-1-induced mitogenesis in ICA VSMCs using receptor-operated Ca2+ channel blockers, SK&F 96365 and LOE 908. We focused on extracellular-signal regulated kinase 1 and 2 (ERK1/2) in this context. PD 98059, an inhibitor of mitogen-activated protein kinase kinase, abolished the ET-1-induced increase in ERK1/2 activity, but only partially suppressed the mitogenesis. ERK1/2 activation by ET-1 was partially suppressed in the absence of extracellular Ca2+. Moreover, based on the sensitivity to SK&F 96365 and LOE 908, Ca2+ influx through NSCC-1, NSCC-2 and SOCC plays essential roles in the extracellular Ca2+-dependent component of ERK1/2 activity. In addition, Ca2+ influx through these channels was also involved in the PD 98059-resistant component of ET-1-induced mitogenesis. These results indicate that (1) the ET-1-induced mitogenesis involves both ERK1/2-dependent and -independent mechanisms in ICA VSMCs (2), ERK1/2 activation by ET-1 involves a Ca2+ influx-dependent cascade as well as a Ca2+ influx-independent cascade (3), Ca2+ influx through NSCC-1, NSCC-2 and SOCC has important roles in the Ca2+ influx-dependent component of ERK1/2-dependent mitogenesis, and (4) Ca2+ influx through these channels also plays important roles in mitogenic pathways downstream of ERK1/2.     

Evidence for SNARE zippering during Ca2+-triggered exocytosis in PC12 cells

SNAREs (soluble NSF attachment protein receptors) are membrane proteins that catalyze membrane fusion. SNAREs are defined by a characteristic 70 residue sequence called the SNARE motif. During synaptic vesicle fusion, the single SNARE motif of the synaptic vesicle SNARE protein synaptobrevin/VAMP associates into a four-helical bundle with SNARE motifs from the plasma membrane SNARE proteins syntaxin 1 and SNAP-25. The four SNARE motifs (one each from synaptobrevin and syntaxin, and two from SNAP-25) assume a parallel orientation in the complex, suggesting that formation of the complex initiates fusion by forcing the membranes containing the SNAREs into close proximity. It has been proposed that SNARE complexes assemble in an N- to C-terminal progression, a process referred to as zippering, but little direct evidence for zippering exists. Furthermore, the SM protein Munc18-1, which binds to syntaxin 1 and is essential for synaptic fusion, is thought to prepare SNAREs for complex formation by an unknown mechanism, possibly by nucleating zippering. We now show that fragments containing the N- and C-terminal regions of the SNARE motif from syntaxin 1A bind SNAP-25 similarly. However, in permeabilized PC12 cells which are used as a biochemical model system to study synaptic fusion, only fragments containing the N-terminal region are powerful inhibitors of fusion. Furthermore, mutations in the N-terminal part of the Syntaxin SNARE motif have only a moderate effect on SNAP-25 binding but abolish the inhibitory activity of the SNARE motif. Finally, larger fragments of syntaxin 1A that strongly bind to Munc18-1 but do not readily assemble into SNARE complexes had no effect on exocytosis in permeabilized PC12 cells. Together these results suggest that Munc18-1 acts before SNARE complex assembly, and is no longer required at the stage of fusion assayed in permeabilized PC12 cells. The selective effect of the N-terminal half of the syntaxin 1A SNARE motif on PC12 cell exocytosis shows that the SNARE motif is functionally polarized, and supports the notion that SNARE complexes assemble in an N- to C-terminal zippering reaction during fusion without a stable, partially assembled intermediate.