Adreno-muscarinic synergy in the bladder trigone: calcium-dependent and -independent mechanisms

We have recently demonstrated a strong synergy between both excitatory pathways in the trigone which--as part of the bladder base--is believed to play a key role in outflow control. The aim of this study was to reveal whether modulation of intracellular Ca2+, [Ca2+]i, is mainly responsible for this synergistic effect. Intact muscle strips from the superficial trigone of male guinea-pigs were used for tension experiments. In isolated trigonal cells, [Ca2+]i was measured by epifluorescence microscopy using the fluorescent Ca2+-indicator Fura-2. Phenylephrine (PE, 10 microM) augmented contractions induced by carbachol (1 microM) to 4.0+/-0.8-fold of control, while corresponding [Ca2+]i levels did not exceed 1.3+/-0.2-fold of control. Furthermore, PE generated significantly greater contractions for a given rise of [Ca2+]i, compared to depolarising KCl solutions. The protein kinase C inhibitor GF 109203X (5 microM) and the Rho-kinase inhibitor Y-27632 (5microM) reduced the PE contracture to 37.3+/-9.4 and 60.1+/-12.4% of control, respectively, without significantly altering the [Ca2+]i transients. GF 109203X reduced the augmentation of 1microM carbachol by PE to 1.5+/-0.1-fold. Muscarinic and adrenergic receptor activation exerts a strong synergistic effect in the bladder trigone without similar changes to the [Ca2+]i transient. Ca2+-sensitisation of contractile proteins is likely to play a key role in this synergism, particularly for adrenergic activation.     

Intracellular Ca2+ requirement for activation of the Na+/H+ exchanger in vascular smooth muscle cells

The role for intracellular Ca2+ in modulating activity of the Na+/H+ exchanger was studied in cultured vascular smooth muscle cells. Na+/H+ exchange was activated by four distinct stimuli: 1) phorbol 12-myristate 13-acetate, 2) thrombin, 3) cell shrinkage, and 4) intracellular acid loading. [Ca2+]i was independently varied between 40 and 200 nM by varying the bathing Ca2+ from 10 nM to 5.0 mM. Thrombin-induced intracellular Ca2+ transients were blocked with bis(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (MAPTAM). In the absence of stimulators of Na+/H+ exchange, varying [Ca2+]i above or below the basal level of 140 nM did not activate Na+/H+ exchange spontaneously. However, varying [Ca2+]i did affect stimulus-induced activation of Na+/H+ exchange. Activation of the exchanger by phorbol 12-myristate 13-acetate was blunted by reduced intracellular Ca2+ (half-maximal activity at 50-90 nM [Ca2+]i), consistent with a Ca2+ requirement for protein kinase C (Ca2+/phospholipid-dependent enzyme). Activation of the exchanger by thrombin in protein kinase C-depleted cells was also sensitive to reduced intracellular Ca2+ (half-maximal activity at 90-140 nM [Ca2+]i) and was increased 40% by raising [Ca2+]i to 200 nM. Activation of the exchanger by cell shrinkage or intracellular acid loads was not significantly affected over the range of [Ca2+]i tested. Thus, altered [Ca2+]i does not itself affect Na+/H+ exchange activity in vascular smooth muscle but instead modulates activation of the transporter by particular stimuli.     

Effects of endothelin on sodium transport mechanisms: potential role in cellular Ca2+ mobilization

The effects of endothelin on cellular Ca2+ mobilization were examined in cultured rat vascular smooth muscle cells (VSMC). Endothelin (10(-8)M) induced a rapid transient increase of [Ca2+]i from 77 +/- 3 to 104 +/- 5 nM (p less than .05) in VSMC. Preincubation (60 min) with endothelin (2 x 10(-6)M) increased basal [Ca2+]i from 77 +/- 3 to 105 +/- 8 nM (p less than .05). Preincubation with endothelin also enhanced vasopressin (10(-7)M)-stimulated peak levels of [Ca2+]i (528 +/- 20 nM vs 969 +/- 21 nM, p less than .01). Endothelin (10(-7)M) induced an intracellular alkalinization (7.18 +/- 0.03 vs 7.37 +/- 0.04, p less than .01) which was blocked by pretreatment with amiloride. The biphasic effects of endothelin on [Ca2+]i were similar to those of an endogenous inhibitor of Na-K-ATPase that we examined in a previous study. Therefore, we examined the effects of endothelin on Na-K-ATPase in an enzyme preparation from hog cerebral cortex. At high concentrations, endothelin (10(-5)M) inhibited Na-K-ATPase in vitro. Thus, endothelin may exert its vasoconstrictor effects at least in part via alterations of cellular Ca2+ mobilization in VSMC. While the rapid transient increase of [Ca2+]i appears to reflect intracellular Ca2+ mobilization, the sustained effect on [Ca2+]i may be related to an increase of intracellular sodium mediated by inhibition of Na-K-ATPase and/or more likely by stimulation of the Na+/H+-antiport.     

Magnesium regulates intracellular free ionized calcium concentration and cell geometry in vascular smooth muscle cells.

Regulatory effects of extracellular magnesium ions ([Mg2+]o) on intracellular free ionized calcium ([Ca2+]i) were studied in cultured vascular smooth muscle cells (VSMCs) from rat aorta by use of the fluorescent indicator fura-2 and digital imaging microscopy. With normal Mg2+ (1.2 mM)-containing incubation media, [Ca2+]i in VSMCs was 93.6 +/- 7.93 nM with a heterogeneous cellular distribution. Lowering [Mg2+]o to 0 mM or 0.3 mM (the lowest physiological range) resulted in 5.8-fold (579.5 +/- 39.99 nM) and 3.5-fold (348.0 +/- 31.52 nM) increments of [Ca2+]i, respectively, without influencing the cellular distribution of [Ca2+]i. Surprisingly, [Mg2+]o withdrawal induced changes of cell geometry in many VSMCs, i.e., the cells rounded up. However, elevation of [Mg2+]o up to 4.8 mM only induced slight decrements of [Ca2+]i (mean = 72.0 +/- 4.55 nM). The large increment of [Ca2+]i induced by [Mg2+]o withdrawal was totally inhibited when [Ca2+]o was removed. The data suggest that: (1) [Mg2+]o regulates the level of [Ca2+]i in rat aortic smooth muscle cells, and (2) [Mg2+] acts as an important regulatory ion by modulating cell shapes in cultured VSMc and their metabolism to control vascular contractile activities.     

Carbachol and KCl-induced changes in intracellular free calcium concentration in isolated, fura-2 loaded smooth-muscle cells from the anterior byssus retractor muscle of Mytilus edulis

Intracellular free calcium concentration [( Ca2+]1) was measured in suspensions of fura-2 loaded smooth-muscle cells isolated from the anterior byssus retractor muscle of Mytilus edulis. Successive application of 5mM carbachol (CCh) and 100mM KCl to the cells transiently elevated [Ca2+]1 from the resting value of 124 +/- 4.5nM (mean +/- S.E., n = 14) to 295 +/- 15.3 and 383 +/- 20.5 nM, respectively. The response to CCh was concentration-dependent with an ED50 of 10(-5) M. Under the microscope, 67 +/- 3.0 and 83 +/- 1.3 % of fura-2 loaded cells contracted on the addition of 5mM CCh and 100mM KCl, respectively. In Ca2+ -free sea water, the CCh induced change in [Ca2+]1 was partially suppressed whereas that induced by KCl was completely abolished, suggesting an agonist-evoked release of stored Ca2+.     

Effect of lead on parathyroid hormone-induced responses in rat osteoblastic osteosarcoma cells (ROS 17/2.8) using 19F-NMR

Using 19F-NMR and the intracellular divalent cation indicator, 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid, we have recently demonstrated that Pb2+ treatment elevates the intracellular free calcium ion concentration ([Ca2+]i) of rat osteoblastic osteosarcoma cells (ROS 17/2.8) (Proc. Natl. Acad. Sci. USA (1989) 86, 5133-5135). In this study, we have examined the effects of Pb2+ on the basal and parathyroid hormone (PTH)-stimulated levels of [Ca2+]i and cAMP in cultured ROS 17/2.8 cells. PTH treatment (400 ng/ml) stimulated a 150% elevation in [Ca2+]i from a control level of 105 +/- 25 nM to a concentration of 260 +/- 24 nM. Treatment of ROS 17/2.8 cells with Pb2+ (5 microM) alone produced a 50% elevation in the [Ca2+]i to 155 +/- 23 nM. Pb2+ treatment diminished subsequent elevation in [Ca2+]i in response to PTH administration thereby limiting the peak increase in [Ca2+]i to only 25% or 193 +/- 22 nM. In contrast to the dampening effect of Pb2+ on the peak rise in [Ca2+]i produced by PTH, Pb2+ (1 to 25 microM) had no effect on PTH-induced increments in intracellular cAMP levels. Hence, Pb2+ dissociated the PTH stimulation of adenylate cyclase from PTH effects on [Ca2+]i and shifted the regulation of [Ca2+]i beyond the control of PTH modulation. These observations further extend the hypothesis that an early toxic effect of Pb2+ at the cellular level is perturbation of [Ca2+]i homeostasis.     

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.     

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.     

Intracellular calcium of longitudinal muscles isolated from pregnant rat myometrium.

Longitudinal muscle cells were successfully isolated from pregnant rat myometrium (21 days of gestation) with more than a 95% survival rate. The approximate size of relaxed cells was 232.2 +/- 74 microns in length and 16.2 +/- 7.0 microns in width. Using the fluorescent indicator Fura-2, the concentration of intracellular free calcium ([Ca2+]i) in resting state cells was calculated to be 116 +/- 18.5 nM. The isolated cells responded well to K+, acetylcholine and oxytocin in terms of contraction as well as the increase in [Ca2+]i. The increase in [Ca2+]i induced by acetylcholine and K+ appeared to be mainly due to an influx of extracellular Ca2+. On the other hand, the oxytocin-induced increase in [Ca2+]i was mainly due to a release of Ca2+ from intracellular storage sites in the isolated cells. Isolated longitudinal muscle cells can serve as a useful tool in establishing the relationship between [Ca2+]i and regulation of the uterine contraction at the final stage of pregnancy.     

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.     

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.     

Unusual sensitivity of cytosolic free Ca2+ to changes in extracellular Ca2+ in rat C-cells

An essential function of C-cells is to monitor extracellular Ca2+ concentration ([Ca2+]e) and to respond to changes in [Ca2+]e by regulating hormone secretion. Using the calcitonin-secreting rat C-cell line rMTC 44-2, we have investigated a possible tight linkage between [Ca2+]e and cytosolic free Ca2+ ([Ca/+]i). We have demonstrated, using the Ca2+ indicator Quin 2, that the [Ca2+]i is particularly sensitive to changes in [Ca2+]e. Sequential increases in [Ca2+]e as small as 0.1 mM evoke clear elevations in [Ca2+]i. In contrast, other cell types tested did not alter their [Ca2+]i in response to increasing [Ca2+]e even to levels as high as 4.0 mM. Sequential 1.0 mM increments in [Ca2+]e caused the [Ca2+]i to rise from a base line of 357 +/- 20 nM Ca2+i at 1.0 mM Ca2+e to a maximum of 1066 +/- 149 nM Ca2+i at 5.0 mM Ca2+e. [Ca2+]e above 2.0 mM produced a biphasic response in [Ca2+]i consisting of an immediate (less than 5 s) spike followed by a decay to a new plateau. Treatment of rMTC 44-2 cells with either 50 mM K+ or 100 nM ionomycin at 1.0 mM Ca2+e caused an immediate spike in [Ca2+]i to micromolar levels. Pretreatment with EGTA or verapamil inhibited completely the increase in [Ca2+]i induced by 50 mM K+. However, pretreatment with EGTA only slightly attenuated the spike phase in [Ca2+]i produced by ionomycin, demonstrating that ionomycin released intracellular stores of calcium. We conclude that rMTC 44-2 cells regulate [Ca2+]i by monitoring small physiological changes in [Ca2+]e, the primary secretagogue for C-cells.     

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.     

Evidence that binding of Indo-1 to cardiac myocyte protein does not markedly change Kd for Ca2+

Quantitative measurement of [Ca2+]i with the fluorescent Ca(2+)-indicators Indo-1 and Fura-2 is complicated by the possibility that the value of the dissociation constant (Kd) may be influenced by binding to intracellular proteins. We investigated this question in cultured chick ventricular myocytes by use of two different Indo-1 calibration methods. First, the Indo-1 fluorescence ratio (R) (400/500 nm) was measured in beating myocytes loaded by exposure to Indo-1/AM. Then, cells were exposed to the Ca2+ ionophore Br A-23187 and fluorescence ratio was measured in the presence of 500 nM Ca2+ (EGTA-Ca2+ buffer). Subsequently cells were permeabilized to Ca2+ by a 1 min exposure to 25 microM digitonin in the presence of 'zero' Ca2+ (10 mM EGTA) and saturating 1 mM Ca2+ to obtain Rmin, Rmax and beta. We then calculated [Ca2+]i from the formula ([Ca2+]i = Kd [( R - Rmin)/(Rmax - R)]beta). With Kd = 250 nM, calculated systolic [Ca2+]i was 750 +/- 44 nM and diastolic 269 +/- 19 nM (means +/- SEM, n = 16). The R value calculated for an assumed [Ca2+]i = 500 nM using the above formula and digitonin derived constants was very similar to the value measured using Br A-23187 (digitonin, 0.67 +/- 0.03: Br A-23187, 0.66 +/- 0.03, ns). As the Br A-23187 method is independent of the value chosen for Kd, we conclude that the Kd of 250 nM for Indo-1 measured in free solutions closely approximates the Kd for intracellular Indo-1 in these cells, and that therefore the Kd of Indo-1 for Ca2+ does not appear to be markedly affected by binding to proteins or other intracellular molecules.     

Production of NAADP and its role in Ca2+ mobilization associated with lysosomes in coronary arterial myocytes

The present study was designed to determine the production of nicotinic acid adenine dinucleotide phosphate (NAADP) and its role associated with lysosomes in mediating endothelin-1 (ET-1)-induced vasoconstriction in coronary arteries. HPLC assay showed that NAADP was produced in coronary arterial smooth muscle cells (CASMCs) via endogenous ADP-ribosyl cyclase. Fluorescence microscopic analysis of intracellular Ca2+ concentration ([Ca2+]i) in CASMCs revealed that exogenous 100 nM NAADP increased [Ca2+]i by 711 +/- 47 nM. Lipid bilayer experiments, however, demonstrated that NAADP did not directly activate ryanodine (Rya) receptor Ca2+ release channels on the sarcoplasmic reticulum. In CASMCs pretreated with 100 nM bafilomycin A1 (Baf), an inhibitor of lysosomal Ca2+ release and vacuolar proton pump function, NAADP-induced [Ca2+]i increase was significantly abolished. Moreover, ET-1 significantly increased NAADP formation in CASMCs and resulted in the rise of [Ca2+]i in these cells with a large increase in global Ca2+ level of 1,815 +/- 84 nM. Interestingly, before this large Ca2+ increase, a small Ca2+ spike with an increase in [Ca2+]i of 529 +/- 32 nM was observed. In the presence of Baf (100 nM), this ET-1-induced two-phase [Ca2+]i response was completely abolished, whereas Rya (50 microM) only markedly blocked the ET-1-induced large global Ca2+ increase. Functional studies showed that 100 nM Baf significantly attenuated ET-1-induced maximal constriction from 82.26 +/- 4.42% to 51.80 +/- 4.36%. Our results suggest that a lysosome-mediated Ca2+ regulatory mechanism via NAADP contributes to ET-1-induced Ca2+ mobilization in CASMCs and consequent vasoconstriction of coronary arteries.     

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.     

Effect of BAY K 8644 on cytosolic free calcium in isolated rabbit gall-bladder epithelial cells

Rabbit gall-bladder epithelial cells were isolated by a combination of Ca2+ omission, enzymatic treatment, and mechanical detachment and had a viability of 96-98% and well preserved morphology. Measurements of cytosolic free Ca2+ concentration ([Ca2+]i) in these cells with the Ca2+-fluorescent indicator fura-2 demonstrated a resting [Ca2+]i level of 115 +/- 12 nM. When used in concentrations which inhibit rabbit gall-bladder isosmotic NaCl absorption (1-100 microM), the Ca2+-channel activator BAY K 8644 caused a dose-dependent increase in the epithelial [Ca2+]i to a maximal value of 850 nM. The effect was dependent on extracellular Ca2+, and was not altered by 1 microM L-verapamil. Depolarization of the epithelial cells with KCl had no effect on [Ca2+]i. The results suggest that BAY K 8644 activates a Ca2+ influx which is not dependent on voltage-gated channels. Cytosolic Ca2+ may be involved in the regulation of isosmotic NaCl absorption in the mammalian gall-bladder.     

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.     

Thyrotropin-releasing hormone-induced changes in intracellular [Ca2+] measured by microspectrofluorometry on individual quin2-loaded cells

We have developed an accurate and practical method for measuring intracellular Ca2+ concentration [( Ca2+]i) in single cells in monolayer culture using the fluorescent Ca2+-binding dye quin2. Quin2 was loaded into cells as a membrane-permeant ester which is hydrolyzed in the cytoplasm to the impermeant free acid, which is the indicator form (Tsien, R.Y., T. Pozzan, and T.J. Rink, 1982, J. Cell Biol., 94:325-334). The method involves the measurement of fluorescence at 340- nm excitation (I340), where dye fluorescence is dependent on Ca2+, and at 360-nm excitation (I360), where dye fluorescence is independent of Ca2+. The ratio of these two values (I340/I360) is thus related to the concentration of Ca2+ but independent of dye concentration and can be used as a measure of [Ca2+]. To test the ratio method in the microscope, we measured [Ca2+]i in GH3 cells in monolayer culture. We found a resting [Ca2+]i of 44 +/- 28 nM (mean +/- SD, n = 34), as compared with a suspension value (Gershengorn, M., and C. Thaw, 1983, Endocrinology, 113:1522-1524) of 118 +/- 18 nM. We also measured [Ca2+]i during stimulation of the cells with thyrotropin-releasing hormone (TRH) and found a 2.4-fold increase above resting levels within 20 s, a trough at 73% of resting at 90-100 s, and a peak slightly above resting at 3 min. Depolarization of the plasma membrane with KCl produced a sustained increase in [Ca2+]i. All of these data are in good agreement with the results of Gershengorn and Thaw on suspension cultures. When measuring both resting [Ca2+]i and the effects of TRH and KCl on small groups of cells, we found some variation among experiments. Using an image intensifier-video camera, we videotaped cells during TRH stimulation. Digital image analysis of these pictures demonstrated that there was a large variation in responsiveness from cell to cell. The microscope ratio method offers the possibility of resolving regions of differing [Ca2+] within the cytoplasm.     

Simultaneous analysis of cell Ca2+ and Ca2(+)-stimulated chloride conductance in colonic epithelial cells (HT-29).

We used perforated patch, whole-cell current recordings and video-based fluorescence ratio imaging to monitor the relation of plasma membrane ionic conductances to intracellular free Ca2+ within individual colonic epithelial cells (HT-29). The Ca2(+)-mediated agonist, neurotensin, activated K+ and Cl- conductances that showed different sensitivities to [Ca2+]i. The Cl- conductance was sensitive to increases or decreases in [Ca2+]i around the resting value of 76 +/- 32 (mean +/- SD) nM (n = 46), whereas activation of the K+ conductance required at least a 10-fold rise in [Ca2+]i. Neurotensin increased [Ca2+]i by stimulating a transient intracellular Ca2+ release, which was followed by a sustained rise in [Ca2+]i due to Ca2+ influx from the bath. The onset of the initial [Ca2+]i transient, monitored at a measurement window over the cell interior, lagged behind the rise in Cl- current during agonist stimulation. This lag was not present when the [Ca2+]i rise was due to Ca2+ entry from the bath, induced either by the agonist or by the Ca2+ ionophore ionomycin. The temporal differences in [Ca2+]i and Cl- current during the agonist-induced [Ca2+]i transient can be explained by a localized Ca2+ release from intracellular stores in the vicinity of the plasma membrane Cl- channel. Chloride currents recover toward basal values more rapidly than [Ca2+]i after the agonist-induced [Ca2+]i transient, and, during a sustained neurotensin-induced [Ca2+]i rise, Cl- currents inactivate. These findings suggest that an inhibitory pathway limits the increase in Cl- conductance that can be evoked by agonist. Because this Cl- current inhibition is not observed during a sustained [Ca2+]i rise induced by ionomycin, the inhibitory pathway may be mediated by another agonist-induced messenger, such as diacylglycerol.