The role of cytoplasmic free calcium in the responses of quin2-loaded human platelets to vasopressin.

Responses to vasopressin were studied in human platelets loaded with the fluorescent Ca2+ indicator, quin2. In the presence of 1 mM external Ca2+, vasopressin caused a transient rise in [Ca2+]i from the basal level near 100nM to about 700 nM; peak [Ca2+]i was reached in a few seconds and the level then declined towards resting over several minutes. In the absence of external Ca2+ there was a much smaller rise of similar time-course, suggesting that vasopressin increases [Ca2+]i mainly by stimulated-influx across the plasma membrane but also by partly releasing internal Ca2+. Inhibition of thromboxane A2 formation somewhat reduced the peak [Ca2+]i in the presence of external Ca2+, but had no effect on the response attributed to release of internal Ca2+. With external Ca2+, vasopressin stimulated shape-change, secretion and aggregation. Secretion and aggregation were decreased by about half following blockage of thromboxane production. The ability of vasopressin to induce shape-change and secretion even at near basal [Ca2+]i suggests that activators other than Ca2+ are involved.     

Synchronized oscillations in cytoplasmic free calcium concentration in confluent bradykinin-stimulated bovine pulmonary artery endothelial cell monolayers

Bradykinin-evoked rises in [Ca2+]i were measured in fura-2-loaded bovine pulmonary artery endothelial cell monolayers by dual wavelength excitation fluorimetry. In monolayers seeded thinly and grown to confluence, bradykinin, in the presence of external Ca2+, evoked a rise in [Ca2+]i composed of an initial peak and subsequent oscillating plateau. In the absence of external Ca2+, bradykinin evoked a rise in [Ca2+]i which then returned to the basal value without oscillating. In monolayers seeded near confluent density, the bradykinin-evoked peak in [Ca2+]i was followed by a steady plateau which showed no oscillation. The addition of the phorbol ester, phorbol 12,13-dibutyrate, to a monolayer during bradykinin-evoked oscillations abolished the oscillations and lowered [Ca2+]i partway back toward the basal level. The addition of the protein kinase C inhibitor, H7, did not abolish oscillatory activity, although the frequency of oscillation was reduced. These results indicate that synchronized oscillatory activity can occur in endothelial cell monolayers. It is suggested that these oscillations are dependent on intercellular coupling developed when the cells are grown to confluence and that the mechanism responsible for generating oscillations in [Ca2+]i requires extracellular Ca2+ and involves protein kinase C.     

Agonist-dependent patterns of cytosolic Ca2+ changes in single bovine adrenal chromaffin cells: relationship to catecholamine release.

The patterns of agonist-induced elevations of cytosolic free Ca2+ ([Ca2+]i) were characterized and compared by the use of single adrenal chromaffin cells. Initial histamine- or angiotensin II (AII)-induced elevations of [Ca2+]i were equal in magnitude (peaks 329 +/- 20 [SE] and 338 +/- 46 nM, respectively). These initial increases of [Ca2+]i were transient, insensitive to either Gd3+ or removing external Ca2+, and were primarily the result of Ca2+ release from intracellular stores. After the initial peak(s) of [Ca2+]i, a second phase of moderately elevated [Ca2+]i was observed, and this response was sensitive to either Gd3+ or removing external Ca2+, supporting a role for Ca2+ entry. In most cases, the second phase of elevated [Ca2+]i was sustained during histamine stimulation but transient during AII stimulation. Maintenance of the second phase was a property of the agonist rather than of the particular cell being stimulated. Thus, individual cells exposed sequentially to histamine and AII displayed distinct patterns of [Ca2+]i changes to each agonist, regardless of the order of addition. Histamine also stimulated twice as much [3H]catecholamine release as AII, and release was completely dependent on external Ca2+. Therefore, the ability of histamine and AII to sustain (or promote) Ca2+ entry appears to underlie their efficacy as secretagogues. These data provide evidence linking agonist-dependent patterns of [Ca2+]i changes in single cells with agonist-dependent functional responses.     

Regulation of cytosolic free calcium in fura-2-loaded rat parotid acinar cells

In order to analyze the factors regulating agonist-stimulated Ca2+ mobilization, cytosolic free [Ca2+] ([Ca2+]i) was measured directly in fura-2-loaded rat parotid acinar cells. Stimulation of muscarinic receptors by carbachol produced a dose-dependent rise in [Ca2+]i. In the presence of external Ca2+, the initial transient rise was followed by a maintained elevation. The maintained elevation is dependent on the presence of external Ca2+. Removal of Ca2+ by addition of EGTA caused a rapid decline in [Ca2+]i back to base line. In the absence of external Ca2+, only an initial transient peak in [Ca2+]i was seen which then declined to base line; the maintained elevation in [Ca2+]i could then be evoked by addition of Ca2+ in the continued presence of carbachol. Muscarinic receptor occupation by carbachol is required to maintain the elevated level of [Ca2+]i; addition of the muscarinic antagonist, atropine, caused [Ca2+]i to decline back to the basal level. The maintained elevation in [Ca2+]i, but not the initial transient peak, can also be blocked by Ni2+ but was unaffected by the organic Ca2+ antagonists. Total substitution of external Na+ with the impermeant cation, N-methyl-D-glucamine, had no effect on either the initial or the maintained response to carbachol; however, total substitution of Na+ with K+ attenuated the maintained response while not affecting the initial peak. Refilling of the intracellular Ca2+ store was also studied and found to take place in the absence of agonist and with no substantial elevation in [Ca2+]i. These experiments also showed that not all of the intracellular vesicular Ca2+ stores can be released by agonists. From these results, we propose a model for the regulation of [Ca2+]i.     

Slow changes in cytosolic free Ca2+ in Escherichia coli highlight two putative influx mechanisms in response to changes in extracellular calcium.

Free intracellular Ca2+ ([Ca2+]i) in Escherichia coli was measured using the bioluminescent protein aequorin. Overall, the bacteria maintained a tight control on their free [Ca2+]i. The results indicated a slow Ca2+ influx, the magnitude of the initial rise in free [Ca2+]i being dependent upon the concentrations of external Ca2+. This was followed by the slow removal of free Ca2+ until normal levels were restored. Specifically, addition of external Ca2+ (0.25-10 mM) resulted in a gradual rise in intracellular free Ca2+ from a basal level of approximately 272 nM, maximally reaching a peak of 0.85-5.4 microM within 30-40 min. This was followed by a slow fall over the next 30 min, culminating in an oscillatory pattern of free [Ca2+]i (range 0.3-0.7 microM for 0.25 mM external Ca2+). In the presence of EGTA, free [Ca2+]i was dramatically reduced. Neither the influx of Ca2+ nor restoration of intracellular free Ca2+ required protein synthesis. Moreover, preincubation with Ca2+ increased the rising phase of intracellular Ca2+ in response to further exposure to external Ca2+. This was further evidence against a specific adaptation process such as the synthesis of calcium exporters. A putative Ca2+ influx channel was demonstrated in stationary phase cells in particular, which could be blocked by La3+. This channel was consistent with the voltage-activated poly-3-hydroxybutyrate/polyphosphate Ca2+ channels previously detailed by Reusch et al. [23] Even in the presence of La3+, however, the free [Ca2+]i of log phase and stationary phase bacteria still increased two-fold over resting values in response to external Ca2+. This suggested the presence of at least two Ca2+ influx processes, one inhibited by La3+ and the other not.     

The effects of substance P and carbachol on inositol tris- and tetrakisphosphate formation and cytosolic free calcium in rat parotid acinar cells. A correlation between inositol phosphate levels and calcium entry

Both substance P and carbachol produced increases in inositol tris- and tetrakisphosphate and increased cytosolic free [Ca2+] in dispersed parotid acinar cells loaded with fura-2. The increase in [Ca2+]i in response to each agonist was due to a combination of mobilization of internal Ca2+ and entry of extracellular Ca2+. Kinetic studies of the initial response to substance P, and measurement of peak [Ca2+]i, demonstrated that the initial rapid rise in [Ca2+]i was due to both internal release and entry of Ca2+. Substance P could evoke a greater initial increase in [Ca2+]i and inositol trisphosphate than could carbachol. However, after 1 min in the presence of external Ca2+, the maintained [Ca2+]i level in response to substance P was considerably smaller than that seen with carbachol, an effect apparently due to homologous desensitization of the substance P receptor. The two agonists each produced a similar 4-5-fold increase in inositol tetrakisphosphate levels within 30 s; this level was maintained in the presence of carbachol, but decreased with substance P. Similarly, the level of inositol (1,4,5)-trisphosphate decreased after prolonged incubation with substance P. Thus, the maintained level of [Ca2+]i, and by deduction Ca2+ entry, correlated with the levels of inositol (1,4,5)-trisphosphate and inositol tetrakisphosphate; a result consistent with a possible role for these inositol phosphates in the control of receptor-mediated Ca2+ channels.     

Use of manganese to discriminate between calcium influx and mobilization from internal stores in stimulated human neutrophils

Stimulation of human neutrophils with f-met-leu-phe, platelet-activating factor, or leukotriene B4 resulted in an increase in [Ca2+]i. The [Ca2+]i rise was greater in the presence than absence of external Ca2+; the component that was dependent on external Ca2+ was blocked by Ni2+, or could be reconstituted by addition of external Ca2+ following discharge of the internal Ca2+ store. These measurements of [Ca2+]i responses provide only indirect evidence for agonist-stimulated Ca2+ entry, and here we have used an alternative approach to demonstrate directly agonist-stimulated divalent cation entry. In the presence of extracellular Mn2+, f-met-leu-phe, leukotriene B4, and platelet-activating factor stimulate a quench in fluorescence of fura-2-loaded human neutrophils. This quench was due to stimulated Mn2+ influx and was blocked by Ni2+. When Mn2+ was added in the continued presence of agonist, after discharge of the internal store of Ca2+, a stimulated quench was seen; this result shows that an elevated [Ca2+]i is not needed for the stimulation of Mn2+ entry. Depolarization by high [K+] or addition of the L-type Ca2+ channel agonist, BAY-R-5417, had little or no effect on either [Ca2+]i or Mn2+ entry. These results show that agonists stimulate divalent cation entry (Ca2+ or Mn2+) by a mechanism independent of changes in [Ca2+]i and unrelated to voltage-dependent Ca2+ channels.     

The kinetics of changes in intracellular calcium concentration in fura-2-loaded human platelets

We have investigated the sub-second kinetics of changes in cytosolic free calcium, [Ca2+]i, in fura-2-loaded human platelets by stopped-flow fluorimetry. Thrombin, vasopressin, platelet-activating factor, and the thromboxane A2 analogue U46619 all evoked a rise in [Ca2+]i which was delayed in onset by 200-400 ms in the presence of 1 mM external Ca2+. The responses to these agonists in media containing 1 mM EGTA or 1 mM Ni2+, to prevent Ca2+ influx, were delayed by an additional 60-100 ms. These results indicate that agonist-evoked Ca2+ influx precedes the release of Ca2+ from internal stores. The delays in onset of both responses are sufficient for one or more biochemical steps to lie between ligand-receptor binding and Ca2+ flux generation. ADP responses in media containing EGTA or Ni2+ were similar to those evoked by other agonists, but the response in the presence of external Ca2+ was markedly shorter, occurring without measurable delay at optimal ligand concentration. Analysis of this response showed some delay in ADP-evoked influx at lower concentrations, but this delay was markedly less than that observed with thrombin at doses giving the same elevation in [Ca2+]i. These results suggest that ADP evokes influx using a different transduction system, more closely coupled to the Ca2+ entry system than that used by other agonists. Differences between thrombin- and ADP-evoked influx were further demonstrated by the inhibitory actions of cAMP, which reduced and substantially increased the delay in onset of thrombin-evoked influx but did not measurably delay the influx evoked by an optimal concentration of ADP.     

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.     

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.     

Overshooting cytosolic Ca2+ signals evoked by capacitative Ca2+ entry result from delayed stimulation of a plasma membrane Ca2+ pump

The effect of capacitative Ca2+ entry on cytosolic free Ca2+ concentration ([Ca2+]c) was examined in calf pulmonary artery endothelial cells treated with thapsigargin. Restoration of extracellular Ca2+ evoked an overshoot in [Ca2+]c: the initial rate of Ca2+ influx was 12.4 +/- 0.5 nM/s as [Ca2+]c rose monoexponentially (time constant, tau = 36 +/- 2 s) to a peak (322 +/- 16 nM) before declining to 109 +/- 14 nM after 2000 s. Rates of Ca2+ removal from the cytosol were measured throughout the overshoot by recording the monoexponential decrease in [Ca2+]c after rapid removal of extracellular Ca2+. The time constant for recovery (tau rec decreased from 54 +/- 4 s when Ca2+ was removed after 10 s to its limiting value of 8.8 +/- 1.0 s when it was removed after 2000 s. The time dependence of the changes in tau rec indicate that an increase in [Ca2+]c is followed by a delayed (tau = 408 s) stimulation of Ca2+ removal, which fully reverses (tau approximately 185 s) after Ca2+ entry ceases. Numerical simulation indicated that the changes in Ca2+ removal were largely responsible for the overshooting pattern of [Ca2+]c. Because prolonged (30 min) Ca2+ entry did not increase the total 45Ca2+ content of the cells, an increased rate of Ca2+ extrusion across the plasma membrane most likely mediates the Ca2+ removal, and since it persists in the absence of extracellular Na+, it probably results from stimulation of a plasma membrane Ca2+ pump. We conclude that delayed stimulation of a plasma membrane Ca2+ pump by capacitative Ca2+ entry may protect cells from excessive increases in [Ca2+]c and contribute to oscillatory changes in [Ca2+]c.     

ADP evokes biphasic Ca2+ influx in fura-2-loaded human platelets. Evidence for Ca2+ entry regulated by the intracellular Ca2+ store.

Stopped-flow fluorimetric studies at 37 degrees C have shown that ADP, at optimal concentrations, can evoke Ca2+ or Mn2+ influx in fura-2-loaded human platelets without measurable delay. In contrast, the release of Ca2+ from intracellular stores is delayed in onset by about 200 ms. By working at a lower temperature, 17 degrees C, we have now shown that the rise in cytosolic calcium concentration ([Ca2+]i) evoked by ADP in the presence of external Ca2+ is biphasic. The use of Mn2+ as a tracer for bivalent-cation entry indicates that both phases of the ADP-evoked response are associated with influx. The fast phase of the ADP-evoked rise in [Ca2+]i, which occurs without measurable delay at both 17 degrees C and 37 degrees C, is consistent with Ca2+ entry mediated by receptor-operated channels in the plasma membrane. The delayed phase, indicated by Mn2+ quench, is coincident with the discharge of the intracellular Ca2+ stores. Forskolin did not inhibit the fast phases of ADP-evoked rise in [Ca2+]i or Mn2+ quench, but completely abolished ADP-evoked discharge of the intracellular stores, the delayed phase of the rise in [Ca2+]i observed in the presence of external Ca2+ and the second phase of Mn2+ quench. The timing of the delayed event appears to be modulated by [Ca2+]i: the delayed phase of Mn2+ quench coincides with discharge of the intracellular stores in the absence of added Ca2+, but with the second phase of the ADP-evoked rise in [Ca2+]i in the presence of extracellular Ca2+. Similarly, blockade of the early phase of Ca2+ entry by SK&F 96365 further delays the second phase. It is suggested that a pathway for Ca2+ entry which is regulated by the intracellular Ca2+ store exists in platelets. This pathway operates alongside, and appears to be modulated by the activity of other routes for Ca2+ entry into the cytosol.     

Cytoplasmic calcium transients due to single action potentials and voltage-clamp depolarizations in mouse pancreatic B-cells.

Changes in the cytoplasmic free calcium concentration ([Ca2+]i) in pancreatic B-cells play an important role in the regulation of insulin secretion. We have recorded [Ca2+]i transients evoked by single action potentials and voltage-clamp Ca2+ currents in isolated B-cells by the combination of dual wavelength emission spectrofluorimetry and the patch-clamp technique. A 500-1000 ms depolarization of the B-cell from -70 to -10 mV evoked a transient rise in [Ca2+]i from a resting value of approximately 100 nM to a peak concentration of 550 nM. Similar [Ca2+]i changes were associated with individual action potentials. The depolarization-induced [Ca2+]i transients were abolished by application of nifedipine, a blocker of L-type Ca2+ channels, indicating their dependence on influx of extracellular Ca2+. Following the voltage-clamp step, [Ca2+]i decayed with a time constant of approximately 2.5 s and summation of [Ca2+]i occurred whenever depolarizations were applied with an interval of less than 2 s. The importance of the Na(+)-Ca2+ exchange for B-cell [Ca2+]i maintenance was evidenced by the demonstration that basal [Ca2+]i rose to 200 nM and the magnitude of the depolarization-evoked [Ca2+]i transients was markedly increased after omission of extracellular Na+. However, the rate by which [Ca2+]i returned to basal was not affected, suggesting the existence of additional [Ca2+]i buffering processes.     

Glucagon and vasopressin interactions on Ca2+ movements in isolated hepatocytes.

The effects of glucagon and vasopressin, singly or together, on cytosolic free Ca2+ concentration [( Ca2+]i) and on the 45Ca2+ efflux were studied in isolated rat liver cells. In the presence of 1 mM external Ca2+, glucagon and vasopressin added singly induced sustained increases in [Ca2+]i. The rate of the initial fast phase of the [Ca2+]i increase and the magnitude of the final plateau were dependent on the concentrations (50 pm-0.1 microM) of glucagon and vasopressin. Preincubating the cells with a low concentration of glucagon (0.1 nM) for 2 min markedly accelerated the fast phase and elevated the plateau of the [Ca2+]i increase caused by vasopressin. In the absence of external free Ca2+, glucagon and vasopressin transiently increased [Ca2+]i and stimulated the 45Ca2+ efflux from the cells, indicating mobilization of Ca2+ from internal store(s). Preincubating the cells with 0.1 nM-glucagon accelerated the rate of the fast phase of the [Ca2+]i rise caused by the subsequent addition of vasopressin. However, unlike what was observed in the presence of 1 mM-Ca2+, glucagon no longer enhanced the maximal [Ca2+]i response to vasopressin. In the absence of external free Ca2+, higher concentrations (1 nM-0.1 microM) of glucagon, which initiated larger increases in [Ca2+]i, drastically decreased the subsequent Ca2+ response to vasopressin (10 nM). At these concentrations, glucagon also decreased the vasopressin-stimulated 45Ca2+ efflux from the cells. It is suggested that, in the liver, glucagon accelerates the fast phase and elevates the plateau of the vasopressin-mediated [Ca2+]i increase respectively by releasing Ca2+ from the same internal store as that permeabilized by vasopressin, probably the endoplasmic reticulum, and potentiating the influx of extracellular Ca2+ caused by this hormone.     

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.     

Histamine type I receptor occupancy increases endothelial cytosolic calcium, reduces F-actin, and promotes albumin diffusion across cultured endothelial monolayers

Considerable evidence suggests that Ca2+ modulates endothelial cell metabolic and morphologic responses to mediators of inflammation. We have used the fluorescent Ca2+ indicator, quin2, to monitor endothelial cell cytosolic free Ca2+, [Ca2+]i, in cultured human umbilical vein endothelial cells. Histamine stimulated an increase in [Ca2+]i from a resting level of 111 +/- 4 nM (mean +/- SEM, n = 10) to micromolar levels; maximal and half-maximal responses were elicited by 10(-4) M and 5 X 10(-6) M histamine, respectively. The rise in [Ca2+]i occurred with no detectable latency, attained peak values 15-30 s after addition of stimulus, and decayed to a sustained elevation of [Ca2+]i two- to threefold resting. H1 receptor specificity was demonstrated for the histamine-stimulated changes in [Ca2+]i. Experiments in Ca2+-free medium and in the presence of pyrilamine or the Ca2+ entry blockers Co2+ or Mn2+, indicated that Ca2+ mobilization from intracellular pools accounts for the initial rise, whereas influx of extracellular Ca2+ and continued H1 receptor occupancy are required for sustained elevation of [Ca2+]i. Ionomycin-sensitive intracellular Ca2+ stores were completely depleted by 4 min of exposure to 5 X 10(-6) M histamine. Verapamil or depolarization of endothelial cells in 120 mM K+ did not alter resting or histamine-stimulated [Ca2+]i, suggesting that histamine-elicited changes are not mediated by Ca2+ influx through voltage-gated channels. Endothelial cells grown on polycarbonate filters restricted the diffusion of a trypan blue-albumin complex; histamine (through an H1- selective effect) promoted trypan blue-albumin diffusion with a concentration dependency similar to that for the histamine-elicited rise in [Ca2+]i. Exposure of endothelial cells to histamine (10(-5) M) or ionomycin (10(-7) M) was associated with a decline in endothelial F- actin (relative F-actin content, 0.76 +/- 0.07 vs. 1.00 +/- 0.05; histamine vs. control, P less than 0.05; relative F-actin content, 0.72 +/- 0.06 vs. 1.00 +/- 0.05; ionomycin vs. control, P less than 0.01). The data support a role for cytosolic calcium in the regulation of endothelial shape change and vessel wall permeability in response to histamine.     

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.     

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.     

Rapid kinetics of agonist-evoked changes in cytosolic free Ca2+ concentration in fura-2-loaded human neutrophils.

The initial kinetics of agonist-evoked rises in the cytosolic Ca2+ concentration [Ca2+]i were investigated in fura-2-loaded human neutrophils by stopped-flow fluorimetry. The rises in [Ca2+]i evoked by chemotactic peptide (fMet-Leu-Phe), platelet-activating factor and ADP all lagged behind agonist addition by 1-1.3 s. Lag times were not significantly different in the presence and in the absence of external Ca2+. Stimulation of the cells in the presence of extracellular Mn2+ resulted in a quench of fluorescence with a similar lag time to [Ca2+]i rise. The delay in onset of the rise in [Ca2+]i evoked by fMet-Leu-Phe was dependent on concentration, becoming longer at lower concentrations of agonist. These results indicate that both the agonist-evoked discharge of the intracellular Ca2+ stores and the generation of bivalent-cation influx lag behind agonist-receptor binding in neutrophils. Both pathways thus appear to be mediated by indirect mechanisms, rather than by a directly coupled process such as a receptor-operated channel. The temporal coincidence of the onset of store discharge with the commencement of bivalent-cation influx suggests that the two events may be causally linked.     

Modulation of the epithelial calcium channel, ECaC, by intracellular Ca2+

We have studied the modulation by intracellular Ca2+ of the epithelial Ca2+ channel, ECaC, heterologously expressed in HEK 293 cells. Whole-cell and inside-out patch clamp current recordings were combined with FuraII-Ca2+ measurements:1. Currents through ECaC were dramatically inhibited if Ca2+ was the charge carrier. This inhibition was dependent on the extracellular Ca2+ concentration and occurred also in cells buffered intracellularly with 10 mM BAPTA.2. Application of 30 mM [Ca(2)]e induced in non-Ca2+] buffered HEK 293 cells at -80 m V an increase in intracellular Ca2+([Ca2]i) with a maximum rate of rise of 241 +/-15nM/s (n= 18 cells) and a peak value of 891 +/- 106 nM. The peak of the concomitant current with a density of 12.3 +/- 2.6 pA/pF was closely correlated with the peak of the first-time derivative of the Ca2+ transient, as expected if the Ca2+ transient is due to influx of Ca2+. Consequently, no Ca2+] signal was observed in cells transfected with the Ca2+ impermeable ECaC mutant, D542A, in which an aspartate in the pore region was neutralized.3. Increasing [Ca2+]i by dialyzing the cell with pipette solutions containing various Ca2+] concentrations, all buffered with 10 mM BAPTA, inhibited currents through ECaC carried by either Na+ or Ca2+] ions. Half maximal inhibition of Ca(2+)currents in the absence of monovalent cations occurred at 67 nM (n between 6 and 8), whereas Na+ currents in the absence of Ca2+] and Mg2+ were inhibited with an IC50 of 89 nM (n between 6 and 10). Currents through ECaC in the presence of 1 mM Ca2+ and Na+, which are mainly carried by Ca2+, are inhibited by [Ca2]i with an IC50of 82 nM (n between 6 and 8). Monovalent cation currents through the Ca2+impermeable D542A ECaC mutant were also inhibited by an elevation of [Ca2]i (IC50 = 123 nM, n between 7 and 18). 4. The sensitivity of ECaC currents in inside-out patches for [Ca2]i was slightly shifted to higher concentrations as compared with whole cell measurements. Half-maximal inhibition occurred at 169 nM if Na+ was the charge carrier (n between 4 and 11) and 228 nM at 1 mM [Ca2]e (n between 4 and 8).5. Recovery from inhibition upon washout of extracellular Ca2+ (whole-cell configuration) or removal of Ca2+ from the inner side of the channel (inside-out patches) was slow in both conditions. Half-maximal recovery was reached after 96 +/- 34 s (n= 15) in whole-cell mode and after 135 +/- 23 s (n = 17) in inside-out patches.6. We conclude that influx of Ca2+ through ECaC and [Ca2]i induce feedback inhibition of ECaC currents, which is controlled by the concentration of Ca2+ in a micro domain near the inner mouth of the channel. Slow recovery seems to depend on dissociation of Ca( 2+ from an internal Ca2+ binding site at ECaC.