Thapsigargin increases cytoplasmic free Ca2+ without influencing steroidogenesis in chicken granulosa cells.

The effects of thapsigargin on intracellular Ca2+ concentration ([Ca2+]i) and progesterone production were determined in granulosa cells from the two largest preovulatory follicles of laying hens. [Ca2+]i was measured in cells loaded with the Ca(2+)-responsive fluorescent dye Fura-2. Thapsigargin stimulated a 4.6 +/- 0.2-fold increase in [Ca2+]i from a resting level of 55 +/- 6 nM up to 233 +/- 23 nM (n = 8) in 100% of the cells tested (n = 86). However, two different response patterns were observed. Dependent on the cell populations, a maximally effective concentration of thapsigargin (100 nM) stimulated either a rapid (within 16 +/- 2 s) transient increase in [Ca2+]i or a slowly (99 +/- 20 s) developing and sustained increase in [Ca2+]i. Both [Ca2+]i responses were concentration (0.001-1 microM)-dependent with an EC50 around 40 nM. The transient [Ca2+]i response occurred in the absence of extracellular Ca2+ and was unaffected by pretreating the cells with the Ca2+ channel blockers methoxyverapamil (50 microM) or lanthanum (1 mM). The plateau phase of the sustained [Ca2+]i response returned to resting level in the absence of extracellular Ca2+, but remained elevated in the presence of methoxyverapamil (50 microM) or lanthanum (1 mM). Despite its ability to cause transient or prolonged increases in [Ca2+]i, thapsigargin (0.001-1 microM) did not affect basal or luteinizing hormone-stimulated progesterone production by chicken granulosa cells.     

Calcium influx mediated by nicotinic receptors and voltage sensitive calcium channels in SK-N-SH human neuroblastoma cells

When SK-N-SH human neuroblastoma cells were exposed to nicotine (NIC) or KCl they showed a dose-dependent transient increase (2- to 4-fold) in intracellular Ca2+ concentration ([Ca2+])i as detected by quin-2 fluorescence, with half maximal effects (EC50) observed at 13 microM and 26 mM, respectively. Tubocurarine and 1-isodihydrohistrionicotoxin potently blocked the NIC-evoked (IC50 congruent to 1 microM and 0.3 microM, respectively), but not the high [K+]o-evoked [Ca2+]i accumulation. The KCl-induced response was inhibited by verapamil and diltiazem (IC50 = 1.4 and 10.9 microM, respectively). Tetrodotoxin (3 microM) and tetraethylammonium (10 microM) had no effect on [Ca2+]i accumulation induced by either agent. Increases in [Ca2+]i could be evoked sequentially by NIC and KCl in the same cells suggesting independent mechanisms of Ca2+ entry. In a Ca2+-free medium, no response to either KCl or NIC was observed. However, when Ca2+ ions were restored, [Ca2+]i accumulation was enhanced to the same extent as cells suspended in a Ca2+-containing buffer. Long-term (18 hr) pretreatment of SK-N-SH cells with pertussis (100 ng/ml) or cholera toxins (10 nM) had no effect on NIC or KCl-induced [Ca2+]i accumulation. Together, these data demonstrate the presence of NIC receptors and voltage-sensitive Ca2+ channels on SK-N-SH neuroblastoma cells, through which [Ca2+]i may be modulated.     

Arteriolar smooth muscle Ca2+ dynamics during blood flow control in hamster cheek pouch.

Intracellular calcium concentration ([Ca2+]i) governs the contractile status of arteriolar smooth muscle cells (SMC). Although studied in vitro, little is known of SMC [Ca2+]i dynamics during the local control of blood flow. We tested the hypothesis that the rise and fall of SMC [Ca2+]i underlies arteriolar constriction and dilation in vivo. Aparenchymal segments of second-order arterioles (diameter 35 +/- 2 microm) were prepared in the superfused cheek pouch of anesthetized hamsters (n = 18) and perifused with the ratiometric dye fura PE-3 (AM) to load SMC (1 microM, 20 min). Resting SMC [Ca2+]i was 406 +/- 37 nM. Elevating superfusate O2 from 0 to 21% produced constriction (11 +/- 2 microm) that was unaffected by dye loading; [Ca2+]i increased by 108 +/- 53 nM (n = 6, P < 0.05). Cycling of [Ca2+]i during vasomotion (amplitude, 150 +/- 53 nM; n = 4) preceded corresponding diameter changes (7 +/- 1 microm) by approximately 2 s. Microiontophoresis (1 microm pipette tip; 1 microA, 1 s) of phenylephrine (PE) transiently increased [Ca2+]i by 479 +/- 64 nM (n = 8, P < 0.05) with constriction (26 +/- 3 microm). Flushing blood from the lumen with saline increased fluorescence at 510 nm by approximately 45% during excitation at both 340 and 380 nm with no difference in resting [Ca2+]i, diameter or respective responses to PE (n = 7). Acetylcholine microiontophoresis (1 microA, 1 s) transiently reduced resting SMC [Ca2+]i by 131 +/- 21 nM (n = 6, P < 0.05) with vasodilation (17 +/- 1 microm). Superfusion of sodium nitroprusside (10 microM) transiently reduced SMC [Ca2+]i by 124 +/- 18 nM (n = 6, P < 0.05), whereas dilation (23 +/- 5 microm) was sustained. Resolution of arteriolar SMC [Ca2+]i in vivo discriminates key signaling events that govern the local control of tissue blood flow.     

Large changes in intracellular pH and calcium observed during heat shock are not responsible for the induction of heat shock proteins in Drosophila melanogaster.

Heat shock caused significant changes in intracellular pH (pHi) and intracellular free calcium concentration [( Ca2+]i) which occurred rapidly after temperature elevation. pHi fell from a resting level value at 25 degrees C of 7.38 +/- 0.02 (mean +/- standard error of the mean, n = 15) to 6.91 +/- 0.11 (n = 7) at 35 degrees C. The resting level value of [Ca2+]i in single Drosophila melanogaster larval salivary gland cells was 198 +/- 31 nM (n = 4). It increased approximately 10-fold, to 1,870 +/- 770 nM (n = 4), during a heat shock. When salivary glands were incubated in calcium-free, ethylene glycol-bis(beta-aminoethyl ether)-N,N',N'-tetraacetic acid (EGTA)-buffered medium, the resting level value of [Ca2+]i was reduced to 80 +/- 7 nM (n = 3), and heat shock resulted in a fourfold increase in [Ca2+]i to 353 +/- 90 nM (n = 3). The intracellular free-ion concentrations of Na+, K+, Cl-, and Mg2+ were 9.6 +/- 0.8, 101.9 +/- 1.7, 36 +/- 1.5, and 2.4 +/- 0.2 mM, respectively, and remained essentially unchanged during a heat shock. Procedures were devised to mimic or block the effects of heat shock on pHi and [Ca2+]i and to assess their role in the induction of heat shock proteins. We report here that the changes in [Ca2+]i and pHi which occur during heat shock are not sufficient, nor are they required, for a complete induction of the heat shock response.     

Alamethicin channel permeation by Ca2+, Mn2+ and Ni2+ in bovine chromaffin cells.

Alamethicin causes a concentration-dependent increase of [Ca2+]i in suspensions of bovine adrenal chromaffin cells loaded with fura-2. The basal levels of Cai2+ (234 +/- 37 nM; n = 4) increased to a maximum of 2347 +/- 791 nM (n = 3) with 100 micrograms/ml alamethicin. In the presence of 1 mM Cae2+ the increase reached a plateau within about 2-5 s. This increase was due to Ca2+ entry into chromaffin cells, since in the absence of Cae2+ alamethicin did not modify [Ca2+]i. This contrasts with ionomycin (1 microM) which produced a Cai2+ transient even in the absence of Cae2+. Mn2+ ions also entered chromaffin cells in the presence of alamethicin, as measured by the quenching of fura-2 fluorescence following excitation at 360 nm. Resting chromaffin cells had a measurable permeability to Mn2+ which was drastically increased by cell depolarization by K+ (50 mM) addition. This suggests that Mn2+ is able to permeate voltage-dependent Ca2+ channels. Ni2+ uptake into either resting or K(+)-stimulated chromaffin cells was undetectable, but addition of alamethicin induced rapid uptake of this cation. The alamethicin-induced entry of Ni2+ was decreased by 50 mM K+. Overall, the results are compatible with the formation by alamethicin of ion channels in chromaffin cell plasma membranes.     

Thienopyridines: effects on cultured endothelial cells.

In cultured endothelial cells harvested from human umbilical vein (HUVEC) or bovine aorta (BAEC) the 30 min incubation with calcium ionophore A 23187 (1 microM) or ticlopidine (100 microM) caused an increase in nitrite generation in HUVEC from basal 227 +/- 37 to 372 +/- 60 or to 325 +/- 33 pmoles per 10(6) cells, respectively, and in BAEC from basal 182 +/- 17 to 378 +/- 18 or to 423 +/- 66 pmoles per 106 cells (n = 6), respectively. Calcium ionophore A 23187 (1 microM) or ticlopidine (100 microM) next to 30 min incubation with BAEC increased release of 6-keto-PGF 1alpha from basal level of 9.4 +/- 1.8 to 96.2 +/- 5.1 or to 99.5 +/- 10.2 pmoles per 10(6) cells, respectively. The pretreatment with aspirin (300 microM) cut down this rise to 4.2 +/- 0.1 pmoles per 10(6) cells (n = 8). Basal cytoplasmic calcium levels, [Ca2+]i, in immortalised HUVEC cell line - ECV304, HUVEC and BAEC were 47.7 +/- 3.3 nM (n = 53), 68.3 +/- 5.0 nM (n = 30) and 53.1 +/- 3.0 nM (n = 15), respectively. In these cultured endothelial cells calcium ionophore A 23187 (0.1 microM) produced net maximum rise in [Ca2+]i by 157 +/-27 nM (n = 16)[ ECV304], by 107 +/- 58 nM (n=4) [HUVEC], and by 231.0 +/- 41.3 nM (n = 8) [BAEC], respectively, while ticlopidine (30 microM) produced net maximum rise in [Ca2+]i by 30.0 +/- 3.2 nM (n=9)[ECV304], 48.8 +/- 15.6 nM (n = 4)[HUVEC] and 28.4 +/- 5.4 nM (n = 8)[BAEC], respectively. Effect of ticlopidine on [Ca2+]i was not only weaker than that of calcium A 23187 but also its maximum appeared after a lag period that was 2 3 times longer than that for A23187. In ECV304 clopidogrel at concentrations of 10, 30 and 100 microM produced maximum increment of [Ca2+]i by 16.5 +/- 3.8 nM (n = 7), 47.0 +/- 6.9 nM (n = 8) and 67.2 +/- 8.3 nM (n = 8), respectively. Incubation of BAEC with A23187 (microM), ticlopidine or clopidogrel (100 microM) for 2 h did not influence viability of cultured endothelial cells. We claim that thienopyridines, independently of their delayed anti-platelet properties ex vivo do release NO and PGI2 from cultured endothelial cells in vitro. The above endothelial action of thienopyridines might be mediated by a rise in [Ca2+]i, however, this possibility has not been proved.     

Heat shock protein synthesis and cytoskeletal rearrangements occur independently of intracellular free calcium increases in Drosophila cells and tissues

Heat shock causes significant changes in intracellular free calcium ([Ca2+]i) which occur rapidly following temperature elevation. The resting level of free calcium in single Drosophila melanogaster larval salivary gland cells measured with the fluorescent indicator fura-2 is 198 +/- 31 nM (n = 4). It increases approximately 10-fold to 1870 +/- 770 nM (n = 4), during a heat shock. When salivary glands are incubated in calcium-free, EGTA-buffered medium the resting free calcium is reduced to 80 +/- 7 nM (n = 3) and heat shock results in a 4-fold increase in free calcium to 353 +/- 90 nM (n = 3). Drosophila Kc cells show a heat shock-induced increase in [Ca2+]i from 118.4 +/- 2 nM (n = 11) to 323 +/- 18 nM. Procedures were devised to block the effects of heat shock on the increase in intracellular calcium and assess its role in the induction of heat shock proteins and in the stress-induced rearrangement of the vimentin cytoskeleton. We report here the changes in [Ca2+]i are not required for a complete induction of the heat shock response or for the collapse of the vimentin cytoskeleton.     

Inositol 1,4,5-trisphosphate increases myoplasmic [Ca2+] in isolated muscle fibers. Depolarization enhances its effects

Inositol 1,4,5-trisphosphate (InsP3) has been proposed as an intracellular messenger which mobilizes calcium from the sarcoplasmic reticulum, during excitation-contraction coupling in skeletal muscle. We have measured the myoplasmic free calcium concentration ([Ca2+]i) by means of calcium selective microelectrodes in intact fibers isolated from Leptodactylus insularis microinjected with InsP3. In muscle fibers bathed in normal Ringer, the mean resting [Ca2+]i was 0.11 +/- 0.01 microM (M +/- SEM, n = 30). The microinjection of 0.3, 0.5 and 1 microM InsP3 induced transient increments in the [Ca2+]i to 0.35 +/- 0.02 microM (n = 9), to 0.53 +/- 0.03 microM (n = 11) and 0.94 +/- 0.06 microM (n = 10) respectively. Microinjection of 0.3, 0.5 and 1 microM InsP3 in muscle fibers incubated in low Ca2+ solution induced increments in [Ca2+]i similar to those observed in fibers bathed with normal Ringer. The microinjection of 0.3, 0.5 and 1 microM InsP3 in muscle fibers partially depolarized with 10 mM [K+]o induced transient enhancements of the resting [Ca2+]i that were greater than the transients observed in the normally polarized muscle. In partially depolarized fibers microinjected with 0.3, 0.5 and 1 microM InsP3, the [Ca2+]i was changed to 1.45 +/- 0.14 microM (n = 20), to 3.37 +/- 0.34 microM (n = 7) and to 7.43 +/- 0.70 microM (n = 6) respectively. In all partially depolarized fibers these increments in [Ca2+]i were associated with local contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurement of sarcoplasmic reticulum Ca2+ content in intact amphibian skeletal muscle fibres with 4-chloro-m-cresol.

Single skeletal muscle fibres were isolated from the toad (Bufo marinus) and isometric force and myoplasmic free calcium concentration ([Ca2+]i) were measured. Brief applications of 4-chloro- m-cresol (4-CmC, 0.2-5 mM) elevated [Ca2+]i reversibly in a dose-dependent manner. The lowest concentration of 4-CmC which reliably gave maximal [Ca2+]i was 2 mM and it was, therefore, used for measurement of sarcoplasmic reticulum (SR) Ca2+ content. Tetanic stimulations (100 Hz) increased [Ca2+]i from a resting level of 105 +/- 47 nM (n = 10) to 1370 +/- 220 nM (n = 6). Application of 2 mM 4-CmC produced a contracture that was 54 +/- 16% (n = 6) of the tetanic force and elevated [Ca2+]i to a peak of 3520 +/- 540 nM (n = 8). Both force and [Ca2+]i levels (resting and tetanic) were restored after 10 min of washout of 4-CmC. In skinned muscle fibres, the myofibrillar Ca(2+)-sensitivity was not changed by 4-CmC, but maximal force was reduced to 74 +/- 10% (n = 4). The magnitude of the peak of the 4-CmC-induced Ca2+ transient was not significantly changed by removal of extracellular Ca2+ nor by inhibiting the SR Ca2+ pump with 2,5-di-tert-butylhydroquinone. Treatment of intact fibres with 30 mM caffeine produced a peak Ca2+ level that was indistinguishable from 2 mM 4-CmC. These results indicate that it is possible to measure the SR Ca2+ content in the same fibre with 4-CmC without loss of normal muscle function.     

Control of cytosolic free calcium in rat and chicken osteoclasts. The role of extracellular calcium and calcitonin

Single cell [Ca2+], studies were performed in chicken and rat osteoclasts loaded with fura-2 and exposed to a variety of treatments. Under resting conditions, basal [Ca2+]i, was 79.2 +/- 47.3 and 84.3 +/- 65.7 nM (averages +/- S.D.; n = 141 and 126) in the osteoclasts of the two species, respectively. Basal [Ca2+]i was stable in all rat and in approximately 80% of chicken osteoclasts. In the remaining 20%, spontaneous, irregular [Ca2+], fluctuations were observed (amplitude range: 50-200 nm over basal values). Increase of [Ca2+]o over the concentration of the Krebs-Ringer incubation medium (2 mM) induced rises of [Ca2+] in almost all cells investigated. [Ca2+] rises were already appreciable with 0.5 mM [Ca2+]o additions and reached high values with 4 mM additions: 390 +/- 113 and 364 +/- 214 nM [Ca2+], in rat and chicken osteoclasts, respectively (n = 122 and 101). Qualitatively, the responses to [Ca2+]o additions consisted of discrete [Ca2+]i transients, biphasic (an initial spike followed by a plateau), or monophasic (either the spike or the plateau). In a few chicken osteoclasts, the [Ca2+]i increase occurring after [Ca2+]o addition consisted of multiple, irregular fluctuations, similar to those observed in 20% of these cells under resting conditions. In individual osteoclasts subsequently exposed to multiple [Ca2+]o increase pulses, the type of the [Ca2+]i transient (mono- or biphasic) was maintained, and the size was dependent on the magnitude of the [Ca2+]o additions. Effects similar to those of [Ca2+]o were induced by the addition of Cd2+ or Ba2+ (but not La3+ or Mg2+) into the medium. The Cd2+ effect was maintained in part even in a Ca2+-free medium. Of various hormones and factors, parathormone, 1,25-dihydroxyvitamin D3, and prostaglandin E2 were inactive. In contrast, calcitonin was active in rat osteoclasts (which express numerous receptors). [Ca2+]i increases were small (19 +/- 17.9 nM; n = 21) when the hormone was administered alone; they were synergistic (severalfold potentiation) when the hormone was administered before or after [Ca2+]o. The [Ca2+]i effects of calcitonin were mimicked by 8Br-cAMP (31 +/- 26 nM; n = 12) when the nucleotide was administered alone; marked synergism when it was administered in combination with [Ca2+]o. This paper demonstrates for the first time that changes of [Ca2+]i are induced in osteoclasts by treatments with [Ca2+]o and calcitonin and can therefore be involved in intracellular mediation of the physiological effects of these two extracellular signals.     

Inhibition of endothelium-dependent vasorelaxation by extracellular K(+): a novel controlling signal for vascular contractility

The effects of extracellular K+ on endothelium-dependent relaxation (EDR) and on intracellular Ca2+ concentration ([Ca2+]i) were examined in mouse aorta, mouse aorta endothelial cells (MAEC), and human umbilical vein endothelial cells (HUVEC). In mouse aortic rings precontracted with prostaglandin F2alpha or norepinephrine, an increase in extracellular K+ concentration ([K+]o) from 6 to 12 mM inhibited EDR concentration dependently. In endothelial cells, an increase in [K+]o inhibited the agonist-induced [Ca2+]i increase concentration dependently. Similar to K+, Cs+ also inhibited EDR and the increase in [Ca2+]i concentration dependently. In current-clamped HUVEC, increasing [K+]o from 6 to 12 mM depolarized membrane potential from -32.8 +/- 2.7 to -8.6 +/- 4.9 mV (n = 8). In voltage-clamped HUVEC, depolarizing the holding potential from -50 to -25 mV decreased [Ca2+]i significantly from 0.95 +/- 0.03 to 0.88 +/- 0.03 microM (n = 11, P < 0.01) and further decreased [Ca2+]i to 0.47 +/- 0.04 microM by depolarizing the holding potential from -25 to 0 mV (n = 11, P < 0.001). Tetraethylammonium (1 mM) inhibited EDR and the ATP-induced [Ca2+]i increase in voltage-clamped MAEC. The intermediate-conductance Ca2+-activated K+ channel openers 1-ethyl-2-benzimidazolinone, chlorozoxazone, and zoxazolamine reversed the K+-induced inhibition of EDR and increase in [Ca2+]i. The K+-induced inhibition of EDR and increase in [Ca2+]i was abolished by the Na+-K+ pump inhibitor ouabain (10 microM). These results indicate that an increase of [K+]o in the physiological range (6-12 mM) inhibits [Ca2+]i increase in endothelial cells and diminishes EDR by depolarizing the membrane potential, decreasing K+ efflux, and activating the Na+-K+ pump, thereby modulating the release of endothelium-derived vasoactive factors from endothelial cells and vasomotor tone.     

Calcium influx in internally dialyzed squid giant axons

A method has been developed to measure Ca influx in internally dialyzed squid axons. This was achieved by controlling the dialyzed segment of the axon exposed to the external radioactive medium. The capacity of EGTA to buffer all the Ca entering the fiber was explored by changing the free EGTA at constant [Ca++]i. At a free [EGTA]i greater than 200 microM, the measured resting Ca influx and the expected increment in Ca entry during electrical stimulation were independent of the axoplasmic free [EGTA]. To avoid Ca uptake by the mitochondrial system, cyanide, oligomycin, and FCCP were included in the perfusate. Axons dialyzed with a standard medium containing: [ATP] = 2 mM, [Ca++]i = 0.06 microM, [Ca++]o = 10 mM, [Na+]i = 70 mM, and [Na+]o = 465 mM, gave a mean Ca influx of 0.14 +/- 0.012 pmol.cm-2.s-1 (n = 12. Removal of ATP drops the Ca influx to 0.085 +/- 0.007 pmol.cm-2.s-1 (n = 12). Ca influx increased to 0.35 pmol.cm-2,s-1 when Nao was removed. The increment was completely abolished by removing Nai+ and (or) ATP from the dialysis medium. At nominal zero [Ca++]i, no Nai-dependent Ca influx was observed. In the presence of ATP and Nai [Ca++]i activates the Ca influx along a sigmoid curve without saturation up to 1 microM [Ca++]i. Removal of Nai+ always reduced the Ca influx to a value similar to that observed in the absence of [Ca++]i (0.087 +/- 0.008 pmol.cm-2.s-1; n = 11). Under the above standard conditions, 50-60% of the total Ca influx was found to be insensitive to Nai+, Cai++, and ATP, sensitive to membrane potential, and partially inhibited by external Co++.     

Increased cytosolic calcium. A signal for sulfonylurea-stimulated insulin release from beta cells

The mechanisms by which glyburide and tolbutamide signal insulin secretion were examined using a beta cell line (Hamster insulin-secreting tumor (HIT) cells). Insulin secretion was measured in static incubations, free cytosolic Ca2+ concentration ([Ca2+]i) was monitored in quin 2-loaded cells, and cAMP quantitated by radioimmunoassay. Insulin secretory dose-response curves utilizing static incubations fit a single binding site model and established that glyburide (ED50 = 112 +/- 18 nM) is a more potent secretagogue than tolbutamide (ED50 = 15 +/- 3 microM). Basal HIT cell [Ca2+]i was 76 +/- 7 nM (mean +/- S.E., n = 141) and increased in a dose-dependent manner with both glyburide and tolbutamide with ED50 values of 525 +/- 75 nM and 67 +/- 9 microM, respectively. The less active tolbutamide metabolite, carboxytolbutamide, had no effect on [Ca2+]i or insulin secretion. Chelation of extracellular Ca2+ with 4 mM EGTA completely inhibited the sulfonylurea-induced changes in [Ca2+]i and insulin release and established that the rise in [Ca2+]i came from an extracellular Ca2+ pool. The Ca2+ channel blocker, verapamil, inhibited glyburide- or tolbutamide-stimulated insulin release and the rise in [Ca2+]i at similar concentrations with IC50 values of 3 and 2.5 microM, respectively. At all concentrations tested, the sulfonylureas did not alter HIT cell cAMP content. These findings provide direct experimental evidence that glyburide and tolbutamide allow extracellular Ca2+ to enter the beta cell through verapamil-sensitive, voltage-dependent Ca2+ channels, causing a rise in [Ca2+]i which is the second messenger that stimulates insulin release.     

Secretagogue-induced mobilization of an intracellular Mg2+ pool in rat sublingual mucous acini.

The regulation of the intracellular free Mg2+ concentration ([Mg2+]i) was monitored in rat sublingual mucous acini using dual wavelength microfluorometry of the Mg(2+)-sensitive dye mag-fura-2. Acini attached to coverslips and superfused continuously with a Mg(2+)-containing medium (0.8 mM) have a steady-state [Mg2+]i of 0.35 +/- 0.01 mM. Adjusting the extracellular Mg2+ concentration to 0 and 10 mM or removing extracellular Na+ did not alter the resting [Mg2+]i. Stimulation with the Ca(2+)-mobilizing, muscarinic agonist, carbachol, induced a sustained increase in [Mg2+]i (approximately 50%; t1/2 < 20 s; Kd approximately 1.5 microM), the magnitude and the duration of which were unchanged in Mg(2+)-depleted medium indicating that the rise in [Mg2+]i was generated by Mg2+ release from an intracellular Mg2+ pool. Forskolin, which increases the intracellular cAMP content, produced a small, transient increase in the [Mg2+]i (< 10%). Muscarinic stimulation in a Ca(2+)-free medium blunted the initial increase in [Mg2+]i by approximately 50%, whereas the sustained increase in [Mg2+]i was lost. When the muscarinic-induced increase in [Ca2+]i was blocked by 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate, an inhibitor of the agonist-sensitive intracellular Ca2+ release pathway, both the initial and the sustained phases of the increase in [Mg2+]i were virtually eliminated. Thapsigargin and 2,5-di-(terbutyl)-1,4-benzohydroquinone, which increase [Ca2+]i by inhibiting microsomal Ca(2+)-ATPase, caused a dramatic increase in [Mg2+]i. Stimulation in a Na(+)-free medium or in the presence of bumetanide, an inhibitor of Na+/K+/2Cl- cotransport, blunted the agonist-induced rise in [Mg2+]i (approximately 50%), whereas ouabain, a Na+,K(+)-ATPase inhibitor, had no significant effect. FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), a mitochondrial uncoupler, mobilized an intracellular Mg2+ pool as well. The carbachol-induced increase in [Mg2+]i was markedly inhibited by FCCP (approximately 80%), suggesting that the same pool(s) of Mg2+ were primarily involved. The above results provide strong evidence that Ca(2+)-mobilizing agonists increase cytoplasmic free [Mg2+] by releasing an intracellular pool of Mg2+ that is associated with a rise in the [Na+]i.     

The contributions of plasma membrane Na+-Ca2+-exchange and the Ca2+-ATPase to the regulation of cytosolic calcium ([Ca2+]i) in a clonal pituitary cell line (AtT-20) of mouse corticotropes

Single cell calcium microfluorimetry was used to examine the regulation of [Ca2+]i homeostasis in a clonal cell line of corticotropes (AtT-20 cells). Single cells, loaded with fura-2/AM, were exposed briefly to elevated potassium chloride (KCI, 40 mM, 5 sec). The time constant of decay of the [Ca2+]i signal was used as an index of [Ca2+]i extrusion and/or sequestration. Substitution of extracellular sodium with lithium, N-methyl-D-glucamine (NMDG), or Tris, increased resting levels of [Ca2+]i and significantly increased the time constant of [Ca2+]i decay by 40% compared to control indicating the participation of Na+-Ca2+-exchange. Prior exposure of single cells to thapsigargin (1 microM) or BuBHQ (10 microM). inhibitors of the SERCA Ca2+-ATPases, and/or the mitochondrial uncoupler FCCP (1 microM) did not significantly change the time constant of [Ca2+]i decay following KCl. Lanthanum ions (La3+), applied during the decay of the KCI-induced increase in [Ca2+]i, significantly increased the time constant of the return of [Ca2+]i to resting levels by 70% compared to control. Brief exposure of cells to sodium orthovanadate, an inhibitor of ATP-dependent pump activity, slowed and longer exposures prevented, the return of [Ca2+]i to resting levels. We conclude that neither intracellular SERCA pumps nor mitochondrial uptake contribute significantly to [Ca2+]i sequestration following a [Ca2+]i load and that the plasma membrane Ca2+-ATPase contributes to a greater extent than the Na+-Ca2+-exchanger to the return of [Ca2+]i to resting levels following a [Ca2+]i load under these experimental conditions.     

Voltage-regulated calcium channels involved in the regulation of enkephalin synthesis are blocked by phorbol ester treatment

Treatment of bovine chromaffin cells with 40 mM KCl stimulates a 3-fold increase in total methionine enkephalin immunoreactivity (medium plus cells) and a 4-fold increase in proenkephalin mRNA (mRNAenk). These effects of KCl, which are dependent on extracellular calcium, can be blocked by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), although release of methionine enkephalin appears less affected. Using fura-2-loaded chromaffin cells and a dual-excitation wavelength spectrofluorometer, we have examined whether the actions of KCl and TPA on methionine enkephalin synthesis and release can be explained by changes in intracellular free calcium ([Ca2+]i). KCl produced a rapid 600 nM increase in [Ca2+]i from resting levels of approximately 170 nM. Subsequently, [Ca2+]i declined to a new steady-state plateau which was approximately 275 nM higher than the original resting levels. The postdepolarization plateau of [Ca2+]i was reduced by TPA, (-)-(R)-202,791 (a dihydropyridine calcium channel antagonist), and LaCl3 (a nonselective calcium channel blocker). TPA also inhibited potentiation of the KCl-stimulated plateau of [Ca2+]i due to (+)-(S)-202,791, a calcium channel agonist. In contrast, TPA had no effect on resting [Ca2+]i and only slightly inhibited the initial rapid KCl-stimulated increase in [Ca2+]i. The inhibitory effects were maintained for 24 h in the continuous presence of TPA. We conclude 1) that TPA inhibits enkephalin synthesis by inactivating dihydropyridine-sensitive voltage-dependent calcium channels, 2) that these channels alone maintain elevated [Ca2+]i following KCl depolarization, and 3) that sustained elevation in [Ca2+]i is necessary in order to increase enkephalin synthesis in KCl-treated chromaffin cells.     

Cytosolic free magnesium in cardiac myocytes: identification of a Mg2+ influx pathway

Regulation of intracellular Mg2+ activity in the heart is not well characterized. Cardiac myocytes were prepared as primary cultures from 7 day old chick embryo hearts and intracellular Mg2+ concentration [( Mg2+]i) was determined in single ventricular cells with mag-fura-2. Basal [Mg2+]i was 0.48 +/- 0.03 mM in normal culture medium. There was no correlation of basal [Mg2+]i with cellular contraction or intracellular [Ca2+]i (determined with fura-2). Cardiocytes cultured (16 hr) in low Mg (0.16 mM) media contained 0.21 +/- 0.05 mM Mg2+ which returned to normal levels when placed in Mg media with a refill time of 20 min. Basal [Ca2+]i (121 +/- 11 nM) and stimulated [Ca2+]i (231 +/- 41 nM) was similar to control cells. Verapamil, 25 microM, reversibly blocked Mg2+ refill. In conclusion, the basal [Mg2+]i of isolated cardiomyocytes is considerably below the Mg2+ electrochemical equilibrium allowing passive Mg2+ influx. The influx pathway for Mg2+ is inhibited by verapamil and appears to be independent of Ca2+ as assessed by fura-2.     

Stimulus-secretion coupling in bovine parathyroid cells. Dissociation between secretion and net changes in cytosolic Ca2+

The relationship between the concentration of cytosolic free Ca2+ ([Ca2+]i) and secretion of parathyroid hormone (PTH) was investigated in isolated bovine parathyroid cells using the fluorescent Ca2+ indicator, quin 2. Increasing the concentration of extracellular Ca2+ from 0.5 to 2.0 mM caused a 3-fold increase in [Ca2+]i (from 183 +/- 4 to 568 +/- 21 nM) which was associated with a 2-4-fold decrease in secretion of PTH. Decreasing extracellular Ca2+ to about 1 microM caused a corresponding fall in [Ca2+]i to 60-90 nM. Extracellular Ca2+-induced changes in [Ca2+]i were not affected by omission of extracellular Na+. Depolarizing concentrations of K+ (30 mM) depressed [Ca2+]i at all concentrations of extracellular Ca examined, and this was associated with increased secretion of PTH. Ionomycin (0.1 or 1 microM) increased [Ca2+]i at extracellular Ca2+ concentrations of 0.5, 1.0, and 2.0 mM, but inhibited secretion of PTH only at Ca concentrations near the "Ca2+ set point" (1.25 microM). In contrast, dopamine, norepinephrine (10 microM each), and Li+ (20 mM) potentiated secretion of PTH without causing any detectable change in [Ca2+]i. The results obtained with these latter secretagogues provide evidence for a mechanism of secretion which is independent of net changes in [Ca2+]i. The phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) did not alter [Ca2+]i or secretion of PTH at low (0.5 mM) extracellular Ca2+ concentrations. At 2.0 mM extracellular Ca2+, however, TPA (20 nM or 1 microM) depressed [Ca2+]i and potentiated secretion of PTH. The addition of TPA prior to raising the extracellular Ca2+ concentration reduced the subsequent increase in [Ca2+]i. The results show that the effects of TPA on secretion in the parathyroid cell are not readily dissociated from changes in [Ca2+]i and suggest that some TPA-sensitive process, perhaps involving protein kinase C, may be involved in those mechanisms that regulate [Ca2+]i in response to changes in extracellular Ca2+.     

Diverse effects of hydrogen peroxide on cytosolic Ca2+ homeostasis in rat pancreatic beta-cells

Oxygen-free radicals are thought to be a major cause of beta-cell dysfunction in diabetic animals induced by alloxan or streptozotocin. We evaluated the effect of H2O2 on cytosolic Ca2+ concentration ([Ca2+]i) and the activity of ATP-sensitive potassium (K+ATP) channels in isolated rat pancreatic beta-cells using microfluorometry and patch clamp techniques. Exposure to 0.1 mM H2O2 in the presence of 2.8 mM glucose increased [Ca2+]i from 114.3+/-15.4 nM to 531.1+/-71.9 nM (n=6) and also increased frequency of K+ATP channel openings. The intensity of NAD(P)H autofluorescence was conversely reduced, suggesting that H2O2 inhibited the cellular metabolism. These three types of cellular parameters were reversed to the control level on washout of H2O2, followed by a transient increase in [Ca2+]i, the transient inhibition of K+ATP channels associated with action currents and increase of the NAD(P)H intensity with an overshoot. In the absence of external Ca2+, 0.1 mM H2O2 increased [Ca2+]i from 88.8+/-7.2 nM to 134.6+/-8.3 nM. Magnitude of [Ca2+]i increase induced by 0.1 mM H2O2 was decreased after treatment of cells with 0.5 mM thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ pump (45.8+/-4.9 nM vs 15.0+/-4.8 nM). Small increase in [Ca2+]i in response to an increase of external Ca2+ from zero to 2 mM was further facilitated by 0.1 mM H2O2 (330.5+/-122.7 nM). We concluded that H2O2 not only activates K+ATP channels in association with metabolic inhibition, but also increases partly the Ca2+ permeability of the thapsigargin-sensitive intracellular stores and of the plasma membrane in pancreatic beta-cells.     

N(omega)-nitro-L-arginine decreases resting cytosolic [Ca2+] and enhances heat stress-induced increase in cytosolic [Ca2+] in human colon carcinoma T84 cells

N(omega)-nitro-L-arginine (LNNA) inhibits the synthesis of heat shock proteins in animals and cultured cells exposed to heat stress. Heat shock protein synthesis is known to be Ca2+-dependent. In this study, we have characterized the effect of LNNA on [Ca2+]i before and after heat stress in human colon carcinoma T84 cells. In untreated cells incubated in the presence of external Ca2+, the resting [Ca2+]i was 201+/-3 nM. If these cells were exposed to 45 degrees C for 10 min, [Ca2+]i increased by 50+/-2%. Preincubation with LNNA (100 microM) without subsequent heating led to a decrease in [Ca2+]i in a LNNA concentration-dependent manner. Preincubation with LNNA followed by heating increased [Ca2+]i to levels 88+/-5% greater than cells heated without LNNA pretreatment. Incubating cells in medium without external Ca2+ (no heating, no LNNA treatment) lowered resting [Ca2+]i to 115+/-2 nM and greatly reduced the increase in [Ca2+]i observed if cells were heated in the presence of Ca2+, indicating that external Ca2+ plays an important role in the maintenance of [Ca2+]i in T84 cells. With external Ca2+ absent, LNNA pretreatment further reduced [Ca2+]i in unheated cells, and heating failed to enhance [Ca2+]i. We determined (with external Ca2+ present) that the heat-stress induced increase in [Ca2+]i in T84 cells was blocked by dichlorobenzamil, a Na+/Ca2+ exchanger inhibitor, suggesting that the exchanger mediates Ca2+ entry. The median inhibitory concentration (IC50) in cells not treated with LNNA was 0.970+/-0.028 microM. With LNNA pretreatment, the IC50 was 5.099+/-0.107 microM. Heat stress of T84 cells did not affect the binding affinity of the Na+/Ca2+ exchanger for external Ca2+, but it increased the maximal velocity of the exchanger. In unheated cells, preincubation with LNNA decreased the binding affinity of the exchanger for Ca2+, but after heat treatment, both the binding affinity and maximal velocity of the exchanger increased. Our data are consistent with the idea that LNNA affects the activity of the Na+/Ca2+ exchanger. We also determined there are intracellular Ca2+ pools in T84 cells sensitive to thapsigargin, monensin, and ionomycin. Treatment with TMB-8, a blocker of Ca2+ sequestration and mobilization, or ionomycin inhibited the LNNA-induced decrease in [Ca2+]i observed in the absence of external Ca2+, suggesting that LNNA promotes Ca2+ sequestration.