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.     

Ca2+-sensitivity and cGMP-independent effects of NO in vascular smooth muscle.

The effects of NO on Ca2+-sensitivity of vascular smooth muscle (VSM) myofilaments have been the focus of this study. Simultaneous measurements of [Ca2+]i and force were carried out in rat tail artery segments. NO, 10(-7) M, evoked a transient decrease in [Ca2+]i accompanied by sustained relaxation (45.3+/-6.3 vs. 69.45+/-7.2%, P<0.05, respectively) of VSM precontracted with K+ (70 mM), suggesting a decrease in Ca2+-sensitivity of VSM. This decrease in Ca2+-sensitivity was completely abolished by preincubation of VSM with ODQ (10(-6) M) (63.9+/-7.8% for [Ca2+]i vs. 20.5+/-8.4% for relaxation, P<0.05). Ca2+-presensitization of VSM myofilaments with PE (10(-6) M) decreased the efficacy of NO to relax VSM (44.25+/-6.9% vs. 69.45+/-7.2%, P<0.05), but increased its ability to lower [Ca2+]i (70.5+/-6.8% vs. 45.3+/-6.3%, P<0.05). Application of DTT (10(-3) M) together with ODQ (10(-6) M) to subtract possible cGMP-independent effects revealed the total suppression of both the relaxant responses and [Ca2+]i of VSM under high-K+ preactivation of VSM. The data indicate that NO not only relaxes VSM and lowers [Ca2+]i in K+-preactivated VSM, but also decreases Ca2+-sensitivity of VSM myofilaments and these effects are strongly cGMP-dependent. In PE-induced contractions of VSM, NO relaxed VSM of rat tail artery and lowered [Ca2+]i, but failed to reverse Ca2+-presensitized myofilaments. We suggest that alternative cGMP-independent effects of NO are primarily manifested via activation of K+-channels and inhibition of Ca2+ current rather than to affect relaxation. An importance of reduced SH-groups within VSM myoplasm for both relaxation and [Ca2+]i disposal evoked by NO is evident whatever Ca2+-mobilization pathways are involved.     

Parathyroid hormone inhibition of Na+/phosphate cotransport in OK cells: intracellular [Ca2+] as a second messenger

Parathyroid hormone increases cellular cAMP, 1,2-diacylglycerol, inositol 1,4,5-trisphosphate and cytosolic Ca2+ concentration ([Ca2+]i) in OK cells. In the present study, we determined the importance of the PTH-dependent increase in [Ca2+]i in the control of sodium-dependent phosphate (Na+/Pi) cotransport. PTH (10(-7) M) results in a transient increase in [Ca2+]i from basal levels of 67 +/- 4 nM to maximal concentrations of 190 +/- 9 nM. The increase in [Ca2+]i was dose-dependent with half-maximal increases at about 5.10(-8) M PTH. These hormone levels were 10(3)-fold higher than that required for half-maximal inhibition of Na+/Pi cotransport. Clamping [Ca2+]i with either intracellular Ca2+ chelators or by ionomycin in the presence of high concentrations of extracellular Ca2+ did not alter PTH-dependent inhibition of Na/Pi cotransport. Nor did indomethacin, an inhibitor of the cyclooxygenase pathway, influence the hormonal inhibition of cotransport. Accordingly, these data suggest that changes in [Ca2+]i and/or activation of the phospholipase A2 and the cyclooxygenase pathways are not involved in signal induction of the PTH-mediated control of Na+/Pi cotransport.     

Membrane potential and Na(+)-K+ pump activity modulate resting and bradykinin-stimulated changes in cytosolic free calcium in cultured endothelial cells from bovine atria

The effects of membrane potential on resting and bradykinin-stimulated changes in [Ca2+]i were measured in fura-2 loaded cultured endothelial cells from bovine atria by spectrofluorimetry. The basal and bradykinin-stimulated release of endothelium-derived relaxing factor, monitored by bioassay methods, were dependent on extracellular Ca2+. Similarly, the plateau phase of the biphasic [Ca2+]i response to bradykinin stimulation exhibited a dependence on extracellular Ca2+, whereas the initial transient [Ca2+]i peak was refractory to the removal of extracellular Ca2+. The effect of membrane depolarization on the plateau phase of the bradykinin-induced change in [Ca2+]i was determined by varying [K+]o. The resting membrane potential measured under current clamp conditions was positively correlated with the extracellular [K+] (52 mV change/10-fold change in [K+]o). The observed decrease in resting and bradykinin-stimulated changes in [Ca2+]i upon depolarization is consistent with an ion transport mechanism where the influx is linearly related to the electrochemical gradient for Ca2+ entry (Em - ECa). The inhibition of bradykinin-stimulated Ca2+ entry by isotonic K+ was not due to the absence of extracellular Na+ since Li+ substitution did not inhibit the agonist-induced Ca2+ entry. In K(+)-free solutions and in the presence of ouabain, bradykinin evoked synchronized oscillations in [Ca2+]i in confluent endothelial cell monolayers. These [Ca2+]i oscillations between the plateau and resting [Ca2+]i levels were dependent on extracellular Ca2+ and K+ concentrations. Although the mechanism(s) underlying [Ca2+]i oscillations in vascular endothelial cells is unclear, these results suggest a role of the membrane conductance.     

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.     

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.     

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.     

Regulation of intracellular pH in human platelets. Effects of thrombin, A23187, and ionomycin and evidence for activation of Na+/H+ exchange and its inhibition by amiloride analogs

Intracellular pH (pHi) of human platelets was measured with the fluorescent dye 2',7'-bis(carboxyethyl)5,6-carboxyfluorescein under various conditions. Stimulation by thrombin at 23 degrees C caused a biphasic change in pHi (initial pHi 7.09); a rapid fall of 0.01-0.04 units (correlated with the rise of [Ca2+]i measured with quin2) followed after 10-15 s by a sustained rise of 0.1-0.15 units pHi. The fall of pHi and [Ca2+]i mobilization was reduced by early (5 s) addition of hirudin, but the later elevated pHi was not reversed by hirudin added after 30 s, although this strips thrombin from receptors and rapidly returns [Ca2+]i to basal levels. In Na+-free medium, or in presence of the Na+/H+ antiport inhibitors, 5-(N,N-dimethyl)amiloride (DMA) or 5-(N-ethyl-N-isopropyl)amiloride (EIPA), thrombin caused a greater fall of pHi (0.22-0.26 units) that was sustained. DMA or EIPA could also reverse the alkalinization response to thrombin. Ca2+ ionophores (ionomycin, A23187) decreased platelet pHi by 0.02-0.15 units, but without an increase of pHi comparable to that following thrombin; DMA and EIPA enhanced the fall of pHi (0.14-0.33 units). Cytoplasmic acidification produced by nigericin (K+/H+ ionophore) was followed by return towards normal that was abolished by Na+/H+ antiport inhibitors. The phorbol diester phorbol 12-myristate 13-acetate had little effect on resting pHi but increased the rate of recovery 2-3-fold after cytoplasmic acidification by nigericin, ionomycin, or sodium propionate. These results indicate that elevation of [Ca2+]i by thrombin enhances H+ production, but the subsequent alkalinization is independent of receptor occupancy or elevated [Ca2+]i and stimulation of the Na+/H+ antiporter by thrombin probably involves some mechanism apart from regulation by H+ and protein kinase C.     

Effect of diethylstilbestrol on Ca2+ handling and cell viability in human breast cancer cells

In human breast cancer cells, the effect of the widely prescribed estrogen diethylstilbestrol (DES) on intracellular Ca2+ concentrations ([Ca2+]i) and cell viability was explored by using fura-2 and trypan blue exclusion, respectively. DES caused a rise in [Ca2+]i in a concentration-dependent manner (EC50 = 15 microM). DES-induced [Ca2+]i rise was reduced by 80 % by removal of extracellular Ca2+. DES-induced Mn(2+)-associated quench of intracellular fura-2 fluorescence also suggests that DES induced extracellular Ca2+ influx. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca2+]i rise, after which the increasing effect of DES on [Ca2+]i was greatly inhibited. Conversely, pretreatment with DES to deplete intracellular Ca2+ stores totally prevented thapsigargin from releasing more Ca2+, whereas ionomycin added afterward still released some Ca2+. These findings suggest that in human breast cancer cells, DES increases [Ca2+]i by stimulating extracellular Ca2+ influx and also by causing intracellular Ca2+ release from the endoplasmic reticulum. Acute trypan blue exclusion studies suggest that 10-20 NM DES killed cells in a time-dependent manner.     

Alpha 1-adrenoceptor activation elevates cytosolic calcium in rat pinealocytes by increasing net influx

The regulation of [Ca2+]i in rat pinealocytes was studied using the fluorescent indicator quin2. Pinealocyte resting [Ca2+]i was approximately 100 nM; this rapidly decreased in low Ca2+ medium (approximately 10 microM), indicating there was a high turnover of [Ca2+]i in these cells. Norepinephrine (NE, 10(-6) M) increased [Ca2+]i to approximately 350 nM within 1 min; [Ca2+]i then remained elevated for 30 min. The relative potency of adrenergic agonists was NE greater than phenylephrine much greater than isoproterenol. Phentolamine (10(-6) M) and prazosin (10(-8) M) blocked the effects of adrenergic agonists; in contrast, propranolol (10(-6) M) or yohimbine (10(-6) M) had little or no effect. These observations indicate NE acts via alpha 1-adrenoceptors to elevate [Ca2+]i. The [Ca2+]i response to NE did not occur when [Ca2+]e was reduced to approximately 10 microM by adding EGTA 5s before NE, indicating an increase in net Ca2+ influx is involved rather than mobilization of Ca2+ from intracellular stores. The effect of NE was not blocked by nifedipine (10(-6) M), which did block a K+-induced increase in [Ca2+]i, presumably involving voltage-sensitive channels. Ouabain (10(-5) M) caused a gradual increase in [Ca2+]i; this increase was not blocked by nifedipine. Together these data indicate that pinealocyte [Ca2+]i may be influenced by mechanisms regulated by alpha 1-adrenoceptors, voltage-dependent Ca2+ channels, and perhaps a Na+/Ca2+ exchange mechanism stimulated by ouabain. These studies indicate that the pinealocyte is an interesting model to use to study the adrenergic regulation of [Ca2+]i because of the rapid and prolonged changes in [Ca2+]i produced by alpha 1-adrenoceptor activation.     

Changes in luminal flow rate modulate basal and bradykinin-stimulated cell [Ca2+] in aortic endothelium.

Hemodynamic forces influence many endothelial cell functions. The coupling between hemodynamic forces and cell function could be mediated by mechano-sensitive ion channels present in the plasma membrane of endothelial cells. Because one of these channels is permeable to Ca2+, we tested whether hemodynamic forces influence endothelial cell Ca2+ ([Ca2+]i). Bovine aortic endothelial cells were grown inside cylindrical glass tubes, loaded with fura-2, and perfused at different pressures and flow rates on the stage of a fluorescence microscope. Decreasing flow from 110 to 2 ml.min-1 raised [Ca2+]i from 57 +/- 11 to 186 +/- 29 nM (mean +/- SEM, p less than 0.01) by increasing the entry of extracellular Ca2+ into the cytoplasm. Increasing flow from 2 to 110 ml.min-1 transiently decreased [Ca2+]i from 62 +/- 3 to 33 +/- 5 nM (p less than 0.01) apparently due to reduced Ca2+ entry and concomitant extrusion by the plasma membrane Ca(2+)-ATPase. The rise in [Ca2+]i induced by bradykinin was magnified during a decrease in flow; in control cells, 10(-7) M bradykinin increased [Ca2+]i by 162 +/- 26 nM, whereas [Ca2+]i increased 350 +/- 67 nM (p less than 0.05) in cells previously exposed to 110 ml.min-1. These observations suggest that flow-induced changes in [Ca2+]i might be a signal-transduction mechanism for endothelial functions responsive to hemodynamic forces and may also modulate the magnitude of hormonally mediated increases in [Ca2+]i.     

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.     

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.     

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.     

The thiol reagent, thimerosal, evokes Ca2+ spikes in HeLa cells by sensitizing the inositol 1,4,5-trisphosphate receptor.

The thiol reagent, thimerosal, has been shown to cause an increase in intracellular Ca2+ concentration ([Ca2+]i) in several cell types, and to cause Ca2+ spikes in unfertilized hamster eggs. Using single cell video-imaging we have shown that thimerosal evokes repetitive Ca2+ spikes in intact Fura-2-loaded HeLa cells that were similar in shape to those stimulated by histamine. Both thimerosal- and histamine-stimulated Ca2+ spikes occurred in the absence of extracellular (Ca2+ o), suggesting that they result from mobilization of Ca2+ from intracellular stores. Whereas histamine stimulated formation of inositol phosphates, thimerosal, at concentrations that caused sustained Ca2+ spiking, inhibited basal and histamine-stimulated formation of inositol phosphates. Thimerosal-evoked Ca2+ spikes are therefore not due to the stimulated production of inositol 1,4,5-trisphosphate (InsP3). The effects of thimerosal on Ca2+ spiking were probably due to alkylation of thiol groups on intracellular proteins because the spiking was reversed by the thiol-reducing compound dithiothreitol, and the latency between addition of thimerosal and a rise in [Ca2+]i was greatly shortened in cells where the intracellular reduced glutathione concentration had been decreased by preincubation with DL-buthionine (S,R)-sulfoximine. In permeabilized cells, thimerosal caused a concentration-dependent inhibition of Ca2+ accumulation, which was entirely due to inhibition of Ca2+ uptake into stores because thimerosal did not affect unidirectional 45Ca2+ efflux from stores preloaded with 45Ca2+. Thimerosal also caused a concentration-dependent sensitization of InsP3-induced Ca2+ mobilization: half-maximal mobilization of Ca2+ stores occurred with 161 +/- 20 nM InsP3 in control cells and with 62 +/- 5 nM InsP3 after treatment with 10 microM thimerosal. We conclude that thimerosal can mimic the effects of histamine on intracellular Ca2+ spiking without stimulating the formation of InsP3 and, in light of our results with permeabilized cells, suggest that thimerosal stimulates spiking by sensitizing cells to basal InsP3 levels.     

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.     

Calmodulin mediates Ca2+-dependent modulation of M-type K+ channels

To quantify the modulation of KCNQ2/3 current by [Ca2+]i and to test if calmodulin (CaM) mediates this action, simultaneous whole-cell recording and Ca2+ imaging was performed on CHO cells expressing KCNQ2/3 channels, either alone, or together with wild-type (wt) CaM, or dominant-negative (DN) CaM. We varied [Ca2+]i from <10 to >400 nM with ionomycin (5 microM) added to either a 2 mM Ca2+, or EGTA-buffered Ca2+-free, solution. Coexpression of wt CaM made KCNQ2/3 currents highly sensitive to [Ca2+]i (IC50 70 +/- 20 nM, max inhibition 73%, n = 10). However, coexpression of DN CaM rendered KCNQ2/3 currents largely [Ca2+]i insensitive (max inhibition 8 +/- 3%, n = 10). In cells without cotransfected CaM, the Ca2+ sensitivity was variable but generally weak. [Ca2+]i modulation of M current in superior cervical ganglion (SCG) neurons followed the same pattern as in CHO cells expressed with KCNQ2/3 and wt CaM, suggesting that endogenous M current is also highly sensitive to [Ca2+]i. Coimmunoprecipitations showed binding of CaM to KCNQ2-5 that was similar in the presence of 5 mM Ca2+ or 5 mM EGTA. Gel-shift analyses suggested Ca2+-dependent CaM binding to an "IQ-like" motif present in the carboxy terminus of KCNQ2-5. We tested whether bradykinin modulation of M current in SCG neurons uses CaM. Wt or DN CaM was exogenously expressed in SCG cells using pseudovirions or the biolistic "gene gun." Using both methods, expression of both wt CaM and DN CaM strongly reduced bradykinin inhibition of M current, but for all groups muscarinic inhibition was unaffected. Cells expressed with wt CaM had strongly reduced tonic current amplitudes as well. We observed similar [Ca2+]i rises by bradykinin in all the groups of cells, indicating that CaM did not affect Ca2+ release from stores. We conclude that M-type currents are highly sensitive to [Ca2+]i and that calmodulin acts as their Ca2+ sensor.     

Stimulus-response coupling in monocytes infected with Leishmania. Attenuation of calcium transients is related to defective agonist-induced accumulation of inositol phosphates.

Mononuclear phagocytes infected with Leishmania have been shown to have defective responses to extracellular stimuli. To investigate the potential relationship of these findings to alterations in calcium-dependent signaling pathways, the regulation of [Ca2+]i concentrations was examined in human peripheral blood monocytes infected with amastigotes of Leishmania donovani. Measurements of [Ca2+]i in fura-2-loaded monocytes were made at the single cell level by microfluorimetry. In normal monocytes, resting [Ca2+]i was 56 +/- 2 nM (mean +/- SEM). In contrast, in monocytes infected with Leishmania there was an approximately twofold increase in basal [Ca2+]i (122 +/- 5 nM, p less than 0.01 vs control). Treatment of cells with pertussis toxin before infection did not abrogate infection-induced increases in basal [Ca2+]i, suggesting that this effect was not mediated via the activation of a G protein coupled to phospholipase C. However, elevated resting [Ca2+]i did correlate with increased rates of 45Ca2+ uptake by infected monocytes. As expected, in response to treatment with 10(-7) M FMLP, control monocytes showed rapid net increases in [Ca2+]i of 303 +/- 19 nM. In contrast, net transients of [Ca2+]i in infected monocytes in response to FMLP were attenuated to only 137 +/- 9 nM (p less than 0.01 vs control). This result was not related to excess buffering of [Ca2+]i in infected cells as both control and infected monocytes showed equivalent transients of [Ca2+]i in response to the calcium ionophore A23187. Rather, inhibition of agonist-induced calcium release in infected cells appeared related to defective generation of second messenger because compared to control cells labeled with myo-[2-3H]inositol, little accumulation of inositol 1,4,5-trisphosphate was detected in infected monocytes. Attenuation of inositol phosphate accumulation and calcium release in response to chemotactic peptide correlated with decreased FMLP-induced superoxide and hydrogen peroxide production by infected monocytes. These results provide direct evidence for defective regulation of [Ca2+]i and calcium-dependent signaling in Leishmania-infected monocytes and provide a basis for understanding abnormalities in activation-related responses that involve signaling through Ca(2+)-regulated pathways.     

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.     

Effect of vasopressin on Na+ kinetics in cultured rat vascular smooth muscle cells

The effect of arginine vasopressin (AVP) on Na+ kinetics was examined in cultured rat vascular smooth muscle cells (VSMC) and rat renal papillary collecting tubule cells (RPCT) by the direct measurement of intracellular sodium concentration [(Na+]i) using fluorescence dye; SBFI. AVP increased [Na+]i in a dose-dependent manner at a concentration of 10(-9) M or higher in rat VSMC but did not affect [Na+]i in rat RPCT. The calcium (Ca2+)-free solution completely blocked the increasing effect of AVP on [Na+]i in rat VSMC. A Ca2+ ionophore, ionomycin (1-2 x 10(-6) M) increased [Na+]i both in rat VSMC and RPCT. The Ca2(+)-free solution abolished the ionomycin-increased [Na+]i both in rat VSMC and RPCT. These results therefore indicate that after binding the V1 receptor AVP increases [Na+]i mediated through an increase in cellular Ca2+ uptake in VSMC.