Effect of buffer systems and pHi on the measurement of [Ca2+]i with fura 2

The fluorescent probe, fura 2, is widely used to measure agonist-induced changes in intracellular calcium concentration ([Ca2+]i) in cultured cells. However, in many instances, the results obtained in the same cell type have differed from one study to the next. The possibility that such differences might be due to experimental conditions was examined by using fura 2 in four different cell types responding to appropriate agonists when the cells were incubated in either CO2/HCO3-- or HEPES-buffered media. Examined were: 1) the response of rat glomerular mesangial cells to arginine vasopressin, 2) the response of vascular smooth muscle cells to angiotensin II, 3) the response of adrenal glomerulosa cells to angiotensin II, and 4) the response of hypothalamic cells to insulin-like growth factor-1. In each cell type there was a significant difference in the pattern of agonist-induced change in [Ca2+]i when HEPES vs. CO2/HCO3- was used as the buffer system: in HEPES buffer, agonist addition led to a transient rise in [Ca2+]i followed by a fall to a sustained plateau 27 to 34 nM higher than the original basal value, whereas in CO2/HCO3- buffer, agonist addition led to an identical transient increase in [Ca2+]i followed by a fall to a value within 10 nM or less of the preagonist level. The plateau value of [Ca2+]i in the different buffers was examined in relationship to known differences in intracellular pH (pHi). It was found that measurements of [Ca2+]i with fura 2 were influenced by shifts in pHi that occur when cells are incubated in either HEPES-buffered or CO2/HCO3- media of differing pHo values. However, at any given value of pHi, the apparent [Ca2+]i measured in cells incubated in HEPES-buffered media was slightly higher than in cells incubated in CO2/HCO3- buffered media.     

Angiotensin II type 1 receptor activation modulates L- and T-type calcium channel activity through distinct mechanisms in bovine adrenal glomerulosa cells

In adrenal zona glomerulosa cells, calcium entry is crucial for aldosterone production and secretion. This influx is stimulated by increases of extracellular potassium in the physiological range of concentrations and by angiotensin II (Ang II). The high threshold voltage-activated (L-type) calcium channels have been shown to be the major mediators for the rise in cytosolic free calcium concentration, [Ca2+]c, observed in response to a depolarisation by physiological potassium concentrations. Paradoxically, both T- and L-type calcium channels have been shown to be negatively modulated by Ang II after activation by a sustained depolarisation. While the modulation of T-type channels involves protein kinase C (PKC) activation, L-type channel inhibition requires a pertussis toxin-sensitive G protein. In order to investigate the possibility of additional modulatory mechanisms elicited by Ang II on L-type channels, we have studied the effect of PKC activation or tyrosine kinase inhibition. Neither genistein or MDHC, two strong inhibitors of tyrosine kinases, nor the phorbol ester PMA, a specific activator of PKC, affected the Ang II effect on the [Ca2+]c response and on the Ba2+ currents elicited by cell depolarisation with the patch-clamp method. We propose a model describing the mechanisms of the [Ca2+]c modulation by Ang II and potassium in bovine adrenal glomerulosa cells.     

Clearance of large Ca2+ loads in a single smooth muscle cell: examination of the role of mitochondrial Ca2+ uptake and intracellular pH

Redistribution of cytosolic free Ca2+ following Ca2+ influx into the cytoplasm was studied in single smooth muscle cells isolated from guinea-pig urinary bladder. Voltage-clamped cells were loaded with a low-affinity fluorophore Indo-1FF. A decay of free intracellular Ca2+ ([Ca2+]i) after the termination of the depolarizing pulse (1 s from -50 mV to +20 mV) was fitted with a single exponential and the effect of various substances on the time constant was compared. At a holding potential of +80 mV the [Ca2+]i decay was 1.56 times slower compared to that at -50 mV suggesting the presence of a voltage-dependent process redistributing Ca2+. In the presence of cyclopiazonic acid (CPA, 10 microM), an inhibitor of sarco(endo)plasmatic Ca2+ pump (SERCa), the [Ca2+]i decay was 3.93 times slower than that in the absence of the inhibitor. Introduction of a polycation Ruthenium Red (RR) (20 microM), an inhibitor of the mitochondrial Ca2+ uniporter, into a cell or collapsing a transmitochondrial H+ gradient with the protonophore CCCP (2 microM) slowed down the [Ca2+]i decay 6.05-fold and 9.78-fold, respectively. The apparent amplitude of [Ca2+]i increments was also increased by CCCP. Increasing H+ buffering power in the intracellular solution from 10 mM to 40 mM of HEPES greatly reduced the effect of CCCP on [Ca2+]i decay. A further increase in HEPES concentration to 100 mM eliminated the effects of CCCP both on the time course of [Ca2+]i decay and on the amplitude of [Ca2+]i increment. Perfusion of RR together with 100 mM HEPES into the cytoplasm was without effect on the decay time course of [Ca2+]i. The effect of CPA on [Ca2+]i decay was also reduced in cells loaded with 100 mM HEPES; the time constant in the presence of CPA was slowed down by a factor of 2.18. Application of 10 mM Na(+)-butyrate to the cells loaded with 10 mM HEPES resulted in a slowing down of [Ca2+]i decay: the time constant was increased by a factor of 5.84. Measurement of intracellular pH with SNARF-1 confirmed cytoplasmic acidification during application of Na(+)-butyrate and CCCP. It is concluded that the contribution of mitochondrial Ca2+ uptake to the rapid [Ca2+]i decay is much less than could be extrapolated from action of protonophores in these smooth muscle cells. The results also demonstrate the importance of intracellular pH for Ca2+ handling in the cytoplasm of smooth muscle cells.     

Control of cytosolic free calcium by intracellular organelles in bovine adrenal glomerulosa cells. Effects of sodium and inositol 1,4,5-trisphosphate

The regulation of cytosolic free Ca2+ concentration ([Ca2+]c) by intracellular organelles was studied in permeabilized bovine adrenal glomerulosa cells. Two compartments, with distinct characteristics, were able to pump Ca2+. A first pool, sensitive to ruthenium red and presumably mitochondrial, required respiratory chain substrates to maintain [Ca2+]c around 700 nM. Ca2+ efflux from this compartment was activated by Na+ (ED50 = 5 mM). Inositol 1,4,5-trisphosphate (IP3) had no effect on this pool. A second nonmitochondrial pool required ATP to lower [Ca2+]c to about 200 nM and released Ca2+ transiently upon addition of IP3. When the two systems were allowed to work simultaneously, the nonmitochondrial pool regulated [Ca2+]c and IP3 released Ca2+ in a concentration-dependent manner (EC50 = 0.6 microM). Under these conditions the mitochondria seemed Ca2+ depleted. Upon repeated stimulations with IP3, a marked attenuation of the response was observed. This phenomenon was due to Ca2+ sequestration by a nonmitochondrial IP3-insensitive pool. Neither dantrolene (200 microM) nor 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (10 microM) were able to abolish IP3-induced Ca2+ release, though both compounds efficiently inhibited aldosterone production in intact cells stimulated with angiotensin II (10 nM) or K+ (12 mM). These results suggest that in permeabilized adrenal glomerulosa cells: the nonmitochondrial pool is responsible for buffering [Ca2+]c and for releasing Ca2+ in response to IP3; at resting [Ca2+]c levels, the mitochondria appear Ca2+ depleted; when [Ca2+]c rises above their set point, the mitochondria accumulate Ca2+ as a function of [Na+]c; 4) the mitochondria are not involved in the desensitization mechanism of the response to IP3.     

Intracellular alkalinization leads to Ca2+ mobilization from agonist-sensitive pools in bovine aortic endothelial cells

Receptor-stimulated phosphoinositide turnover leads to activation of Na+/H+ exchange and subsequent intracellular alkalinization. To probe the effect of increased intracellular pH (pHi) on Ca2+ homeostasis in cultured bovine aortic endothelial cells (BAEC), we studied the effect of weak bases, ammonium chloride (NH4Cl) and methylamine (agents which increase pHi by direct passive diffusion), on resting and ATP (purinergic receptor agonist)-induced Ca2+ fluxes. Changes in cytosolic free Ca2+ ([Ca2+]i) or pHi were monitored in BAEC monolayers using the fluorescent dyes, fura-2 or 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, respectively. NH4Cl-induced, dose-dependent (5-20 mM) increases in [Ca2+]i (maximum change = 195 +/- 26 nM) which were temporally similar to the NH4Cl-induced pHi increases. Methylamine (20 mM) induced a more sustained pHi increase and also stimulated a prolonged [Ca2+]i increase. When BAEC were bathed in HCO3- buffer, removal of extracellular CO2/bicarbonate caused pHi to increase and also induced [Ca2+]i to increase transiently. Extracellular Ca2+ removal did not abolish the rapid NH4Cl-induced rise in [Ca2+]i, although the response was blunted and more transient. NH4Cl addition to BAEC cultures resulted in an increase in 45Ca efflux and decrease in total cell 45Ca content. BAEC treatment with ATP (100 microM) to deplete inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pools completely blocked the NH4Cl (20 mM)-induced rise in [Ca2+]i. Likewise, prior NH4Cl addition partially inhibited ATP-induced increases in [Ca2+]i, as well as slowed the frequency of repetitive [Ca2+]i spikes in single endothelial cells due to agonist. NH4Cl augmented the rate of [Ca2+]i increase that occurs in response to the depletion of agonist-sensitive intracellular Ca2+ pools. However, the internal Ca2+ store remained depleted during the continued presence of NH4Cl, as indicated by a decreased [Ca2+]i response to ATP in Ca2(+)-free medium. Finally, NH4Cl exerted these actions without affecting basal or ATP-stimulated IP3 formation. These observations provide direct evidence that increased pHi leads to Ca2+ mobilization from an agonist-sensitive pool and impairs Ca2+ pool(s) refilling mechanisms without altering cellular IP3 levels.     

Direct demonstration of adrenocorticotropin-induced changes in cytoplasmic free calcium with aequorin in adrenal glomerulosa cell

Effects of adrenocorticotropin (ACTH) on cytoplasmic free calcium concentration, [Ca2+]c, have been measured in adrenal glomerulosa cells using a calcium-sensitive photoprotein, aequorin. ACTH causes a rapid and transient increase in [Ca2+]c. Dose response study demonstrates that 1 pM ACTH induces an elevation of [Ca2+]c and that effect of ACTH appears to be saturated at 100 pM. ACTH action is greatly inhibited but not abolished by removal of extracellular calcium and is completely blocked in medium containing no added calcium and 1 mM EGTA. Under similar conditions, angiotensin II induces a remarkable rise in [Ca2+]c. ACTH action is not affected by pretreatment with dantrolene, which considerably decreases angiotensin II action on [Ca2+]c. One micromolar forskolin, which mimics 1 nM ACTH-mediated elevation of intracellular cAMP, does not increase [Ca2+]c nor modulates changes in [Ca2+] induced by a low dose of ACTH. One hundred micromolar forskolin or 1 mM 8-bromo-cAMP, however, increases [Ca2+]c even in calcium-free medium containing 1 mM EGTA. When glomerulosa cells are co-loaded with aequorin and quin2, angiotensin II-induced change in aequorin signal is greatly reduced, and ACTH-induced change is abolished. Quin2 loading results in accumulation of calcium in the cell under both unstimulated and stimulated conditions. These results indicate that ACTH increases [Ca2+]c by cAMP-independent mechanism, that ACTH action on [Ca2+]c is exclusively dependent on extracellular calcium, and that quin2 is unable to detect the rapid change in [Ca2+]c because of its calcium chelating activity.     

Characterization of oscillations in cytosolic free Ca2+ concentration and measurement of cytosolic Na+ concentration changes evoked by angiotensin II and vasopressin in individual rat aortic smooth muscle cells. Use of microfluorometry and digital imaging

Dual wavelength microfluorometry was used to characterize the changes in cytosolic free Ca2+ concentration [( Ca2+]i) in individual cultured rat aortic vascular smooth muscle cells (VSMC). Angiotensin II (ANG II) at 10(-8) M induced a transient rise in [Ca2+]i from 43 +/- 2 to 245 +/- 23 nM, lasting for approximately 60 s (n = 42). In half of the population, discrete oscillations in [Ca2+]i of smaller amplitude occurred after the initial [Ca2+]i peak, with a period of 58 +/- 8 s and a maximum height of 132 +/- 24 nM. A similar oscillatory pattern was observed with arginine vasopressin (AVP). The oscillations depended upon the presence of extracellular Ca2+. Cytosolic free Na+ concentration ([Na+]i) in VSMC was also measured using the fluorescent Na+ probe sodium-binding benzofuran isophthalate. ANG II induced a gradual and sustained elevation of [Na+]i, from 24.0 +/- 6.2 to 36 +/- 9.7 mM. In response to AVP, [Na+]i rose to 41.0 +/- 11.6 mM. Video imaging of individual VSMC, with on-line ratio calibration of [Ca2+]i, revealed an inhomogeneous distribution of Ca2+ within the cell. [Ca2+] in the nucleus was invariably lower than in the cytoplasm in resting cells. In the cytoplasm, there were small regions in which [Ca2+] was elevated, or "hot spots." In Ca(2+)-containing medium, the initial rise in [Ca2+]i triggered by ANG II and AVP appeared to emanate from the hot spots and to spread evenly throughout the cytoplasm. Between [Ca2+]i oscillations, Ca2+ retreated back to the original hot spots. This study demonstrates the cellular and subcellular heterogeneity of [Ca2+]i both in resting VSMC and during stimulation by ANG II and AVP and reports the direct measurement of [Na+]i in VSMC. The results suggest an action of Ca2+ in both the initial and sustained phases of the response in VSMC and a link between changes in [Ca2+]i and [Na+]i.     

AT1 angiotensin II receptor mediates intracellular calcium mobilization in normal and cancerous breast cells in primary culture

Angiotensin II (Ang II) increases intracellular calcium concentration ([Ca2+]i) in both normal and cancerous human breast cells in primary culture. Maximal [Ca2+]i increase is obtained using 100nM Ang II in both cell types; in cancerous breast cells, [Ca2+]i increase (delta[Ca2+]i) is 135+/-10nM, while in normal breast cells it reaches 65+/-5 nM (P<0.0001). In both cell types, Ang II evokes a Ca2+ transient peak mediated by thapsigargin (TG) sensitive stores; neither Ca2+ entry through L-type membrane channels or capacitative Ca2+ entry are involved. Type I Ang II receptor subtype (AT1) mediates Ang II-dependent [Ca2+]i increase, since losartan, an AT1 inhibitor, blunted [Ca2+]i increase induced by Ang II in a dose-dependent manner, while CGP 4221A, an AT2 inhibitor, does not. Phospholipase C (PLC) is involved in this signaling mechanism, as U73122, a PLC inhibitor, decreases Ang II-dependent [Ca2+]i transient peak in a dose-dependent mode.Thus, the present study provides new information about Ca2+ signaling pathways mediated through AT1 in breast cells in which no data were yet available.     

Histamine-Induced increases in intracellular free Ca2+ levels in hepatoma cells

The effect of histamine on intracellular free Ca2+ levels ([Ca2+]i) in HA22/VGH human hepatoma cells were evaluated using fura-2 as a fluorescent Ca2+ dye. Histamine (0.2-5 microM) increased [Ca2+]i in a concentration-dependent manner with an EC50 value of about 1 microM. The [Ca2+]i response comprised an initial rise, a slow decay, and a sustained phase. Extracellular Ca2+ removal inhibited 50% of the [Ca2+]i signal. In Ca2+-free medium, after cells were treated with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 5 microM histamine failed to increase [Ca2+]i. After pretreatment with 5 microM histamine in Ca2+-free medium for 4 min, addition of 3 mM Ca2+ induced a [Ca2+]i increase of a magnitude 7-fold greater than control. Histamine (5 microM)-induced intracellular Ca2+ release was abolished by inhibiting phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), and by 5 microM pyrilamine but was not altered by 50 microM cimetidine. Together, this study shows that histamine induced [Ca2+]i increases in human hepatoma cells by stimulating H1, but not H2, histamine receptors. The [Ca2+]i signal was caused by Ca2+ release from thapsigargin-sensitive endoplasmic reticulum in an inositol 1,4,5-trisphosphate-dependent manner, accompanied by Ca2+ entry.     

Characterization of histamine-induced increases in intracellular free Ca2+ concentrations in Chang liver cells.

The effect of histamine on intracellular free Ca2+ levels ([Ca2+]i) in Chang liver cells were investigated by using fura-2 as a Ca2+ dye. Histamine (0.2-50 microM) increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 0.8 microM. The [Ca2+]i response comprised an initial rise, a slow decay, and a sustained phase. Extracellular Ca2+ removal inhibited 50% of the maximum [Ca2+]i signal and abolished the sustained phase. After pretreatment with 5 microM histamine in Ca2+-free medium for 4 min, addition of 3 mM Ca2+ induced a [Ca2+]i increase with a magnitude 7-fold greater than control. In Ca2+-free medium, after treatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 5 microM histamine failed to increase [Ca2+]i. Histamine (5 microM)-induced intracellular Ca2+ release was abolished     

IgE receptor-mediated phosphatidylinositol hydrolysis and exocytosis from rat basophilic leukemia cells are independent of extracellular Ca2+ in a hypotonic buffer containing a high concentration of K+

In isotonic buffer, IgE receptor-mediated exocytosis from rat basophilic leukemia cells is dependent on extracellular Ca2+, with half-maximal degranulation requiring 0.4 mM Ca2+. No significant exocytosis occurs in the absence of extracellular Ca2+. This absolute requirement for Ca2+ is eliminated by suspending the cells in a hypotonic buffer containing 60 to 80 mM K+; Na+ cannot substitute for K+. Optimal Ca2(+)-independent exocytosis occurs in a buffer containing 20 mM dipotassium Pipes, pH 7.1, 40 mM KCl, 5 mM glucose, 7 mM Mg acetate, 0.1% BSA, and 1 mM EGTA. The cells maintain this Ca2(+)-independent exocytosis even if they are preincubated with 1 mM EGTA for 40 min at 37 degrees C before triggering. Exocytosis is eliminated as isotonicity is approached by adding sucrose, NaCl, KCl, or potassium glutamate to the buffer. Quin 2 fluorescence measurements reveal only a very small rise in [Ca2+]i when the cells are triggered in hypotonic buffer in the absence of extracellular Ca2+ and the presence of 1 mM EGTA. In isotonic buffer, degranulation does not occur under conditions that lead to such a small rise in [Ca2+]i. Sustained IgE receptor-mediated phosphatidylinositol hydrolysis, which is also Ca2+ dependent in isotonic buffer, becomes independent of Ca2+ in the hypotonic buffer. In fact, the rate of phosphatidylinositol hydrolysis in hypotonic buffer in the absence of Ca2+ (and presence of 1 mM EGTA) is twice that observed in isotonic buffer in the presence of 1 mM Ca2+. These data show that in hypotonic buffer, the requirement of IgE receptor-mediated PI hydrolysis for extracellular Ca2+ is eliminated, and degranulation proceeds with a [Ca2+]i of 0.1 microM, the baseline level of [Ca2+]i found in resting cells. These results are consistent with the hypothesis that, in isotonic buffer, the Ca2+ requirement for mast cell degranulation is for the generation of second messengers via hydrolysis of membrane phosphatidylinositols.     

Cells from human bone giant cell tumors show a [Ca2+]o-sensing.

Giant cells from a human giant cell tumor of bone, showing several osteoclast features were tested for their capability of detecting the [Ca2+]o by a receptor like [Ca2+]o sensing. We found that cultured cells responded to elevation of [Ca2+]o, obtained adding 4 mM Ca2+ to the 2 mM Ca2+ containing buffer, by a transient increase of [Ca2+]i. Proliferative cells induced to differentiate by treatment with 10(-8) M 1,25 dihydroxyvitamin D3, were upregulated in their capability of responding to elevated [Ca2+]o. In fact, in this circumstance, the peak of [Ca2+]o-induced [Ca2+]i rise was increased compared to untreated cells. This suggests that 1,25 dihydroxyvitamin D3 induces a more efficient regulation of osteoclast activity.     

Angiotensin II and its receptors in human endocardial endothelial cells: role in modulating intracellular calcium

The aims of the present study are to investigate the presence and distribution of angiotensin II (Ang II), as well as AT1 and AT2 receptors, in endocardial endothelial cells (EECs) and to determine if the effect of Ang II on intracellular calcium in these cells is mediated via the AT1 or the AT2 receptor. Immunofluorescence and 3D confocal microscopy techniques were used on 20-week-old fetal human EECs. Our results showed that Ang II and its receptors, the AT1 and the AT2 types, are present and exhibit a different distribution in human EECs. Ang II labelling is found throughout the cell with a fluorescence signal higher in the cytosol when compared with the nucleus. Like Ang II, the AT1 receptor fluorescence signal is also homogeneously distributed in human EECs but with a preferential labelling at the level of the nucleus, while the AT2 receptor labelling is solely present in the nucleus. Using fluo-3 and 3D confocal microscopy technique, superfusion of human EECs with increasing concentration of Ang II induced a dose-dependent sustained increase in free cytosolic and nuclear Ca2+ levels. This effect of Ang II on human EEC's intracellular Ca2+ ([Ca2+]) was completely prevented by losartan, an AT1 receptor antagonist. Our results suggest that Ang II, as well as AT1 and AT2 receptors, is present but differentially distributed in EECs of 20-week-old fetal human hearts, and that the AT1 receptor mediates the effects of Ang II on [Ca2+]i in these cells.     

Endoplasmic reticulum Ca2+ store: regulation of Ca2+ release and reuptake by intracellular and extracellular Ca2+ in pancreatic acinar cells

We investigated the effect of cytosolic and extracellular Ca2+ on Ca2+ signals in pancreatic acinar cells by measuring Ca2+ concentration in the cytosol([Ca2+]c) and in the lumen of the ER([Ca2+]Lu). To control buffers and dye in the cytosol, a patch-clamp microelectrode was employed. Acetylcholine released Ca2+ mainly from the basolateral ER-rich part of the cell. The rate of Ca2+ release from the ER was highly sensitive to the buffering of [Ca2+]c whereas ER Ca2+ refilling was enhanced by supplying free Ca2+ to the cytosol with [Ca2+]c clamped at resting levels with a patch pipette containing 10 mM BAPTA and 2 mM Ca2+. Elevation of extracellular Ca2+ to 10 mM from 1 mM raised resting [Ca2+]c slightly and often generated [Ca2+]c oscillations in single or clustered cells. Although pancreatic acinar cells are reported to have extracellular Ca2+-sensing receptors linked to phospholipase C that mobilize Ca2+ from the ER, exposure of cells to 10 mM Ca2+ did not decrease [Ca2+]Lu but rather raised it. From these findings we conclude that 1) ER Ca2+ release is strictly regulated by feedback inhibition of [Ca2+]c, 2) ER Ca2+ refilling is determined by the rate of Ca2+ influx and occurs mainly in the tiny subplasmalemmal spaces, 3) extracellular Ca2+-induced [Ca2+]c oscillations appear to be triggered not by activation of extracellular Ca2+-sensing receptors but by the ER sensitised by elevated [Ca2+]c and [Ca2+]Lu.     

Spatially resolved cytosolic calcium response to angiotensin II and potassium in rat glomerulosa cells measured by digital imaging techniques

The response of cytosolic calcium [Ca2+]i to angiotensin II (AII) and potassium (K+) in individual rat glomerulosa cells was determined using the calcium-sensitive fluorescent dye, fura-2 and digital imaging. Control (4 mM K+) cytosolic calcium levels were generally in the 80-120 nM range and increased monotonically as [K+] was increased from 4 to 12 mM. There was no delay in the onset of the response. In most cells the [Ca2+]i decreased from its peak after 3-4 min, even in the presence of superfusate containing elevated K+. The time course of the change in [Ca2+]i in response to AII stimulation, on the other hand, was more variable. It was most often characterized by an early decrease followed by a large delayed increase. The response also was observed to decline during sustained AII stimulation. The majority of the cells showed some response to one or the other secretagogue with a sizeable minority (25%) having an increase in [Ca2+]i in excess of 200%. While the majority showed a response, the cell to cell variation was substantial. Finally, the pattern of cytosolic calcium increase sometimes showed a marked dependence on the secretagogue used, with different regions of the same cell being more strongly affected by one agent or the other. A few cells (10%) responded to AII only at one pole, establishing a large concentration gradient of calcium across the cell. Because of differences in time course, pattern, and degree of responsiveness, it is likely that the mechanisms underlying the Ca2+ elevation with K+ and AII are different.     

Dissociation of Ca2+ entry and Ca2+ mobilization responses to angiotensin II in bovine adrenal chromaffin cells

In fura-2-loaded bovine adrenal chromaffin cells, 0.5 microM angiotensin II (AII) stimulated a 185 +/- 19 nM increase of intracellular-free calcium [( Ca2+]i) approximately 3 s after addition. The time from the onset of the response until achieving 50% recovery (t 1/2) was 67 +/- 10 s. Concomitantly, AII stimulated both the release of 45Ca2+ from prelabeled cells, and a 4-5-fold increase of [3H]inositol 1,4,5-trisphosphate [( 3H]Ins(1,4,5)P3) levels. In the presence of 50 microM LaCl3, or when extracellular-free Ca2+ [( Ca2+]o) was less than 100 nM, AII still rapidly increased [Ca2+]i by 95-135 nM, but the t 1/2 for recovery was then only 23-27 s. In medium with 1 mM MnCl2 present, AII also stimulated a small amount of Mn2+ influx, as judged by quenching of the fura-2 signal. When [Ca2+]o was normal (1.1 mM) or low (less than 60 nM), 1-2 microM ionomycin caused [Ca2+]i to increase 204 +/- 26 nM, while also releasing 45-55% of bound 45Ca2+. With low [Ca2+]o, ionomycin pretreatment abolished both the [Ca2+]i increase and 45Ca2+ release stimulated by AII. However, after ionomycin pretreatment in normal medium, AII produced a La3+-inhibitable increase of [Ca2+]i (103 +/- 13 nM) with a t 1/2 of 89 +/- 8 s, but no 45Ca2+ release. No pretreatment condition altered AII-induced formation of [3H]Ins(1,4,5)P3. We conclude that AII increased [Ca2+]i via rapid and transient Ca2+ mobilization from Ins(1,4,5)P3- and ionomycin-sensitive stores, accompanied (and/or followed) by Ca2+ entry through a La3+-inhibitable divalent cation pathway. Furthermore, the ability of AII to activate Ca2+ entry in the absence of Ca2+ mobilization (i.e. after ionomycin pretreatment) suggests a receptor-linked stimulus other than Ca2+ mobilization initiates Ca2+ entry.     

Evidence for TRH-induced influx of extracellular Ca2+ in pituitary GH4C1 cells

The aim of the study was to investigate the relationship between thyrotropin-releasing hormone (TRH)-induced changes in intracellular free Ca2+ ([Ca2+]i), and influx of extracellular Ca2+ in Fura 2 loaded pituitary GH4C1 cells. Stimulating the cells with TRH in a Ca(2+)-containing buffer induced a biphasic change in [Ca2+]i. First, a transient increase in [Ca2+]i, followed by a sustained phase. In cells stimulated with TRH in a Ca(2+)-free buffer, the transient increase in [Ca(2+)]i was decreased (p less than 0.05), and the sustained phase was totally abolished. Addition of Ni2+ prior to TRH blunted the component of the TRH-induced transient increase in [Ca2+]i dependent on influx of Ca2+. In the presence of extracellular Mn2+, TRH stimulated quenching of Fura 2 fluorescence. This quenching was blocked by Ni2+. The results indicate that both the TRH-induced transient increase in [Ca2+]i as well as the sustained phase in [Ca2+]i in GH4C1 cells is dependent on influx of extracellular Ca2+.     

Inhibitory effects of digoxin and ouabain on aldosterone synthesis in human adrenocortical NCI-H295 cells

The present study was to investigate the effects and action mechanisms of digoxin and ouabain on steroidogenesis in human adrenocortical NCI-H295 cells. Administration of digoxin or ouabain for 24 h decreased the basal and angiotensin II (Ang II)-stimulated release of aldosterone by NCI-H295 cells. The conversions of corticosterone (substrate of cytochrome P450 aldosterone synthase, P450c11AS) to aldosterone or deoxycortisol (substrate of cytochrome P450 11beta-hydroxylase, P450c11beta) to cortisol were reduced by digoxin or ouabain. The basal and 22-hydroxy-cholesterol (a membrane-permeable cholesterol, substrate of cytochrome P450 side-chain cleavage enzyme, P450scc)-stimulated pregnenolone release in mitochondria was inhibited by digoxin or ouabain. Digoxin or ouabain suppressed the basal and Ang II-stimulated protein expression of steroidogenic acute regulatory (StAR) protein and P450scc. Incubation of digoxin or ouabain for 24 h reduced P450c11AS mRNA expression in NCI-H295 cells. Digoxin or ouabain (10(-6) M, 24 h)-treated cells showed a lower resting intracellular Ca2+ concentration ([Ca2+]i) and an attenuated response of [Ca2+]i to Ang II. Since no significant cytotoxicity was observed at 10(-6) M digoxin or ouabain, the digoxin- or ouabain-induced decrease of aldosterone or cortisol release was not associated with cytotoxicity. These results demonstrate that digoxin or ouabain inhibits the aldosterone or cortisol release via reduction of P450c11AS or P450c11beta and P450scc activities, inhibition of StAR and P450scc protein expression, suppression of P450c11AS mRNA expression, and attenuation of Ca2+ mobilization in NCI-H295 cells.     

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.     

Phosphoinositide and calcium signalling responses in smooth muscle cells: comparison between lipoproteins, Ang II, and PDGF.

The effects of low density lipoprotein (LDL) and high density lipoprotein (HDL3) on second messenger systems were investigated in cultured human vascular smooth muscle cells (VSMC) and compared with those of angiotensin II (Ang II) and platelet-derived growth factor (PDGF-BB). Phosphoinositide metabolism was studied in myo-[2-3H]-inositol prelabelled VSMC using high performance liquid anion-exchange chromatography. The spectra of inositol phosphate isomers increased after stimulation with either Ang II, LDL, HDL3 or PDGF-BB were qualitatively identical. Major increases occurred in 4-IP1, 1,4-IP2, 1,3,4-IP3 and 1,3,4,5-IP4. These are metabolic conversion products of 1,4,5-IP3 for which only a minor increase was found. Thus lipoproteins, like Ang II and PDGF-BB, activate polyphosphatidylinositol-specific phospholipase C. Intracellular Ca2+ concentrations ([Ca2+]i) were studied in fura-2 loaded VSMC. In monolayer cultures LDL and HDL3 increased [Ca2+]i with kinetics comparable to those for Ang II. Relative to the effects of these agonists, the PDGF-BB-induced increase in [Ca2+]i was slower in onset and the decay from peak [Ca2+]i levels more gradual. Fluorescence recordings from single cells exposed to LDL and HDL3 revealed a prolonged series of transient oscillations of [Ca2+]i, a phenomenon typical for calcium-mobilizing hormones. Additionally, as found for Ang II, preincubation of VSMC with either phorbol 12-myristate, 13-acetate, forskolin or 8-bromo-cyclic GMP inhibited LDL- and HDL-induced accumulation of [3H]-inositol monophosphate. We propose that LDL and HDL3 stimulate signal transduction in VSMC via mechanisms analogous to those of Ca(2+)-mobilizing hormones.