Abstract [Arg8]-vasopressin (AVP) is both a potent vasoconstrictor and a mitogen for vascular smooth muscle cells. AVP binds to a single class of receptors (V1a) in the A7r5 rat aortic smooth muscle cell line (Kd ≈2 nmol/L). Stimulation of these cells with AVP results in an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) by releasing intracellular Ca2+ stores and increasing Ca2+ influx; the EC50 for these effects is ≈5 nmol/L. AVP has recently been reported to stimulate arachidonic acid release in primary cultures of rat aortic smooth muscle over a much lower concentration range (EC50 ≈0.05 nmol/L). The present study examined the effects of varying concentrations of AVP on spontaneous Ca2+ spiking activity in fura 2–loaded A7r5 cells. Frequency of Ca2+ spiking increased with increasing [AVP] in the range of 10 to 500 pmol/L. Higher concentrations of AVP inhibited spiking but elicited the characteristic [Ca2+]i changes ascribed to the release of Ca2+ stores and increased Ca2+ entry. The effects of both low and high concentrations of AVP were inhibited by [1-(β-mercapto-β,β,-pentamethylenepropionic acid),2-O-methyltyrosine]arginine vasopressin, a selective V1a vasopressin antagonist. Nimodipine (50 nmol/L), a blocker of L-type voltage-sensitive Ca2+ channels, abolished the Ca2+-spiking activity without inhibiting a maximal [Ca2+]i response to AVP (1 μmol/L). AVP-stimulated Ca2+ spiking, but not release of intracellular Ca2+ stores, was also abolished by ONO-RS-082 (1 μmol/L), an inhibitor of phospholipase A2. These results suggest that occupation of a small fraction of V1a vasopressin receptors by AVP results in stimulation of phospholipase A2 and leads to increased Ca2+-spiking activity. This effect may be important for fine tuning of vascular tone, whereas maximal stimulation by AVP (full receptor occupancy) may be required for more vigorous or sustained vasoconstriction or mitogenesis.
Vascular smooth muscle (VSM) forms a layer of contractile cells in blood vessel walls. Coordinated contraction or relaxation of these cells determines the vessel diameter and thereby influences the flow of blood through the lumen of the vessel. The contractile state of VSM is regulated primarily by the cytosolic Ca2+ concentration ([Ca2+]i). An increase in [Ca2+]i leads to the activation of myosin light chain kinase and myosin phosphorylation and ultimately produces contraction. Physiologically, an increase in [Ca2+]i may occur in response to circulating or locally released hormones or neurotransmitters that bind to cell surface receptors; binding is then transduced into an increase in Ca2+ influx across the plasma membrane and/or a release of intracellular Ca2+ stores. The release of intracellular Ca2+ occurs by the action of InsP3, a soluble second messenger that binds to its receptor in the membrane of the sarcoplasmic reticulum and activates an intrinsic Ca2+ channel. Another mechanism for increasing [Ca2+]i in VSM is through the activation of L-type voltage-sensitive Ca2+ channels. These channels are involved in spontaneous increases in [Ca2+]i (Ca2+ spikes), which occur as a result of the inherent ability of VSM to generate action potentials. It has been suggested that moment-to-moment regulation of blood flow and pressure is determined by the spontaneous electrical activity of the VSM in resistance vessels.1 Hormone-induced Ca2+ signals are important both for vasoconstriction and initiation of cell proliferation.2
AVP is a peptide hormone that is produced in the hypothalamus and released from the posterior pituitary into the systemic circulation. AVP has also very recently been shown to be produced by rat aortic smooth muscle.3 Plasma [AVP] may increase from a few picomoles per liter to several hundred picomoles per liter in response to a large decrease in blood volume. AVP, which is a potent vasoconstrictor, binds to a single class of receptors (V1a) in the A7r5 rat aortic smooth muscle cell line.4 As in other VSM preparations, stimulation of A7r5 cells with AVP results in an increase in [Ca2+]i by releasing intracellular Ca2+ stores and increasing Ca2+ influx, but the EC50 for these effects is ≈5 nmol/L,4 5 much too high to account for the vasoconstrictor effects of concentrations of AVP in the picomolar range found in the systemic circulation. The release of intracellular Ca2+ is attributed to production of InsP3 resulting from activation of PLC. AVP has recently been reported to stimulate arachidonic acid release in primary cultures of rat aortic smooth muscle over a much lower concentration range (EC50 ≈0.05 nmol/L) than was required for InsP3 formation (EC50 ≈2 nmol/L; see Reference 6).
A7r5 cells exhibit spontaneous Ca2+ spikes in the absence of AVP. The mechanism involved in generating this Ca2+-spiking activity involves activation of L-type voltage-sensitive Ca2+ channels and is independent of the release of intracellular Ca2+ stores.7 Ca2+ spiking in VSM may be of fundamental importance in the physiological control of blood flow and pressure. The present study reveals that the frequency of Ca2+ spiking in A7r5 cells is exquisitely sensitive to concentrations of AVP found in the systemic circulation and that this effect apparently requires activation of PLA2.
Materials and Methods
Cell culture media were from GIBCO BRL. Fura 2-AM, fura 2 pentapotassium salt, and Pluronic F127 were from Molecular Probes, Inc. Nimodipine was from Research Biochemicals International. ONO-RS-082 and HELSS were purchased from Biomol. d(CH2)5[Tyr(CH3)2,Ala-NH29]AVP was a gift from Dr M. Manning (Medical College of Ohio, Toledo). [3H]Arachidonic acid (180 Ci/mmol) was from New England Nuclear. All other chemicals, including AVP and ionomycin, were from Sigma Chemical Co.
Measurement of [Ca2+]i
Detailed methods for the culture of A7r5 cells and measurement of [Ca2+]i have been published previously.7 Briefly, A7r5 cells were subcultured on glass coverslips and grown to confluence. The cells were loaded with the fluorescent Ca2+ indicator fura 2 by incubating with fura 2-AM (2 μmol/L) for 2.5 hours at room temperature. Fura 2 fluorescence was then measured either with a videomicroscopy system for monitoring [Ca2+]i changes in single cells or with a fluorescence spectrophotometer for population studies. This combination of population and single-cell measurements is important for characterizing responses in terms of global changes that may reflect the behavior of VSM as a tissue while retaining information about the proportion of cells and heterogeneity of the individual cells that contribute to these global changes.
The videomicroscopy system (Universal Imaging Corp) was composed of a Nikon Diaphot inverted epifluorescence microscope, an eight-position rotating filter wheel for alternating interference filters between the light source (75 W xenon lamp) and the microscope, and an intensified CCD video camera (model XC77/C2400, Hamamatsu) for capturing low light level images at up to video rate (60 Hz). Universal Imaging software allows the user to select regions of the field of view for analysis (typically individual cells). The coverslip was mounted in a chamber (Warner Instrument Corp) on the stage of the microscope and superfused with media at a rate of 5 to 10 mL/min; a four-way valve mounted adjacent to the chamber allowed rapid switching of solutions from four gravity-fed reservoirs. For experiments with fura 2, the field of cells was excited alternately with 340- and 380-nm light, and the average brightness of the pixels within each selected region of the field was recorded to disk. Background fluorescence was determined at the end of the experiment by quenching the fura 2 fluorescence for 15 minutes in the presence of 1 μmol/L ionomycin and 6 mmol/L MnCl2 in Ca2+-free medium. After background fluorescence was subtracted, the 340- and 380-nm fluorescence ratio was calculated and calibrated in terms of [Ca2+]i. This procedure allows simultaneous measurement of [Ca2+]i in up to 20 individual cells, with a temporal resolution of ≈1 second.
The control medium used for all experiments was a modified Krebs’ solution containing (mmol/L) NaCl 135, KCl 5.9, CaCl2 1.5, MgCl2 1.2, glucose 11.5, and HEPES 11.6, pH 7.3. Ca2+-free medium lacked CaCl2 and contained 0.1 mmol/L EGTA. All [Ca2+]i measurements shown are from experiments conducted at 25°C.
A Perkin-Elmer LS50B fluorescence spectrophotometer was used to measure fura 2 fluorescence from populations of A7r5 cells. This instrument was equipped with a rotating filter wheel for alternating 340- and 380-nm excitation wavelengths. A coverslip was mounted vertically on a 30° angle to the light path in a cuvette, which was continuously perfused with media. A four-way valve mounted just above the cuvette allowed rapid switching of solutions; the half-time for replacement of the medium bathing the cells was ≈20 seconds. The excitation light illuminates an area of ≈30 mm2 on the coverslip for recording of fluorescence from several thousand cells. Background fluorescence was determined as for single-cell experiments and subtracted from individual wavelength measurements before ratio calculation and calibration.
Calibration of fura 2 fluorescence in terms of [Ca2+]i, as described previously,8 routinely used solutions of known [Ca2+] to construct a standard curve; a lookup table was then prepared for analysis of fluorescence ratios recorded from cells. The [Ca2+] was calculated using software (MaxChelator, version 6.60), which accounts for binding of Ca2+ to each constituent of the solution. In situ calibration of fura 2 fluorescence by determination of maximum and minimum ratios9 from within cells yields similar calibrated values (not shown).
To measure arachidonic acid release, A7r5 cells were grown to confluence in 60-mm Petri dishes and incubated in low-serum medium (0.1% fetal bovine serum) for 48 hours. The cells were then labeled for 3 hours with [3H]arachidonic acid (1.2 μCi per dish in control medium at 37°C), washed with control medium containing 5 mg/mL fatty acid–free BSA, and pretreated for 30 minutes in this same medium in the presence or absence of the PLA2 inhibitor ONO-RS-082 (1 μmol/L, 37°C). This medium was removed, and the cells were then treated for 30 minutes in control medium containing 5 mg/mL BSA alone (control) or with ONO-RS-082 (1 μmol/L) and varying concentrations of AVP at 37°C (cells pretreated with 1 μmol/L ONO-RS-082 were also incubated with this agent during this final treatment). After 30 minutes, the medium was collected, centrifuged to remove cell debris, and counted for 10 minutes per sample in an LKB 1209 RackBeta liquid scintillation counter.
The concentration dependence for release of intracellular Ca2+ stores by AVP was assessed by treating cells with varying concentrations of AVP in Ca2+-free medium (Fig 1⇓). An increase in [Ca2+]i was detected in individual cells with exposure to AVP at concentrations ≥100 pmol/L (Fig 1⇓, left). The EC50 for the peak [Ca2+]i response to AVP, reflecting release of intracellular Ca2+ stores, was 9.8±2.1 nmol/L (n=4; Fig 1⇓, right). A previous study10 reported that the concentration dependence for the peak [Ca2+]i response to AVP was similar in the presence or absence of extracellular Ca2+.
In Ca2+-containing medium, A7r5 cells display spontaneous Ca2+ spikes, which are dependent on activation of L-type voltage-sensitive Ca2+ channels.7 In the absence of AVP, the spontaneous Ca2+ spiking activity was sporadic and occurred at a very low frequency in most of the coverslips examined (0.02±0.01 min−1, n=47). Treatment of A7r5 cells with increasing concentrations of AVP between 10 and 100 pmol/L (in 10-pmol/L increments) resulted in a marked increase in Ca2+ spike frequency, with little or no change in spike amplitude (Fig 2⇓, top). Ca2+ spike frequency usually increased abruptly when [AVP] was increased above a threshold of 20 to 60 pmol/L and then increased further with each step in [AVP] (Fig 2⇓). The increase in frequency was reversible by lowering [AVP] (not shown). A near-maximal [AVP] (100 nmol/L) still elicited the characteristic Ca2+ transient attributed to the release of intracellular Ca2+ stores but inhibited Ca2+ spiking, although in some experiments the inhibition was transient and spiking resumed at high frequency after ≈10 minutes (Fig 2⇓, top). An increase in spike frequency with increasing [AVP] in the 10- to 150-pmol/L range was observed in every case in >35 experiments.1 In additional experiments, increasing [AVP] in larger increments (100 to 200 pmol/L) revealed Ca2+ spiking activity, which increased up to a frequency of ≈17 min−1 at 500 pmol/L AVP. In experiments using both single cells and cell populations, Ca2+ spiking usually ceased abruptly when [AVP] was increased to 400 or 500 pmol/L (Fig 3⇓, top). Spiking continued at [AVP] up to 500 pmol/L in only two of six experiments on cell populations. In one of these experiments, spike frequency continued to increase up to 19 min−1 at 700 pmol/L AVP, with no further increase when [AVP] was raised to 800 or 900 pmol/L. The relationship between frequency of Ca2+ spiking and [AVP] is shown in Fig 3⇓, bottom. One-way repeated-measures ANOVA followed by pairwise multiple comparison of spike frequency values with control spike frequency (Dunnett’s method, SigmaStat) revealed that frequency of Ca2+ spiking was significantly increased above control values (P<.05) for all AVP concentrations tested that were ≥30 pmol/L.
The different mechanism involved in generating the Ca2+-spiking activity versus the release of intracellular Ca2+ stores was demonstrated by treating cells with nimodipine (50 nmol/L) to block L-type voltage-sensitive Ca2+ channels. Increasing [AVP] up to 150 pmol/L in the presence of nimodipine produced no Ca2+-spiking activity, but a near-maximal [AVP] of 100 nmol/L elicited a typical peak [Ca2+]i response (Fig 4⇓). Peak [Ca2+]i responses following acute exposure to 1 μmol/L AVP in Ca2+-free medium were similar in the presence or absence of 50 nmol/L nimodipine (387±34 nmol/L [n=5] and 411±82 nmol/L [n=4], respectively). Both the Ca2+-spiking response (Fig 5⇓) and the peak [Ca2+]i response to AVP (not shown, but see Reference 10) were inhibited by d(CH2)5[Tyr(CH3)2,Ala-NH29]AVP, a V1a vasopressin receptor antagonist. These results suggest that although both the Ca2+-spiking effects and the peak [Ca2+]i response to AVP are mediated by V1a vasopressin receptors, only the Ca2+-spiking activity requires L-type voltage-sensitive Ca2+ channels; the peak [Ca2+]i response is independent of these channels.
The different concentration dependencies for AVP-stimulated Ca2+ spiking and release of intracellular Ca2+ stores also suggest that different signaling pathways may be involved in these two effects. AVP has recently been shown to be more potent in releasing arachidonic acid than in formation of InsP3 in rat aortic smooth muscle cells,6 suggesting that activation of PLA2 may occur in the [AVP] range that stimulates Ca2+ spiking. To examine the involvement of PLA2 in AVP-stimulated Ca2+ spiking, ONO-RS-082, a PLA2 inhibitor, was tested. ONO-RS-082 (1 μmol/L) had no effect on resting [Ca2+]i levels but completely abolished AVP-stimulated Ca2+ spiking (Fig 6⇓, top and middle); a lower concentration of ONO-RS-082 (100 nmol/L) had no discernible effect (not shown). ONO-RS-082 (1 μmol/L) did not block the peak [Ca2+]i response to 100 nmol/L AVP (Fig 6⇓, middle). Peak [Ca2+]i responses following acute exposure to 1 μmol/L AVP in Ca2+-free medium were similar in the presence or absence of 1 μmol/L ONO-RS-082 (712±128 nmol/L [n=3] and 762±77 nmol/L [n=3], respectively). ONO-RS-082 had no effect on the [Ca2+]i response to a depolarizing solution of high extracellular K+ (59 mmol/L extracellular KCl; Fig 6⇓, bottom), indicating that the inhibition of Ca2+ spiking by ONO-RS-082 was not due to inhibition of voltage-sensitive Ca2+ channels. Peak [Ca2+]i responses following acute exposure to high extracellular K+ were the same in the presence or absence of 1 μmol/L ONO-RS-082 (212±3 nmol/L [n=3] and 212±20 nmol/L [n=3], respectively).
Inhibition of PLA2 by ONO-RS-082 was evaluated by measuring arachidonic acid released from A7r5 cells during a 30-minute incubation with AVP in cells pretreated for 30 minutes in the presence or absence of ONO-RS-082 (1 μmol/L, Fig 7⇓). Arachidonic acid release was significantly increased in cells treated with 500 pmol/L AVP, and this increase was prevented by pretreatment with ONO-RS-082. 1 μmol/L AVP produced a slightly larger increase in arachidonic acid release, which was also prevented by pretreatment with ONO-RS-082 (Fig 7⇓).
Another PLA2 inhibitor, HELSS, a specific inhibitor of Ca2+-independent PLA2, has been found to inhibit AVP-stimulated arachidonic acid release without inhibiting PLC activation in A10 VSM cells.11 HELSS (5 μmol/L) also prevented AVP-stimulated Ca2+ spiking in A7r5 cells (at [AVP] up to 100 pmol/L) without inhibiting the peak [Ca2+]i response to 100 nmol/L AVP but was found to produce a slight elevation of [Ca2+]i in the absence of AVP (not shown), suggesting that it may have other effects in addition to its inhibition of PLA2.
Proliferation of VSM cells is a key event in wound healing and, pathologically, in the development of atherosclerotic lesions and vascularization of tumors. Spontaneous increases in cytosolic [Ca2+] (Ca2+ spikes) in VSM are believed to be important for physiological regulation of vascular tone and may also play a role in hypertension and vasospastic disorders. AVP regulates both proliferation and spontaneous Ca2+ spiking in VSM cells, but probably via distinct mechanisms. Proliferation of human aortic smooth muscle cells was stimulated by AVP with an EC50 of 3 nmol/L,12 similar to the concentrations that elicit phosphatidylinositol turnover and Ca2+ release.4 5 In contrast, the effects of AVP on spontaneous Ca2+ spiking occur at much lower concentrations (EC50 ≈300 pmol/L; Fig 3⇑). This effect correlates more closely with concentrations of AVP that stimulate arachidonic acid release.6
[AVP] in the plasma of normal healthy adults is ≈1 pmol/L,13 but baroreceptor responses triggered by lowering blood pressure by up to 50% in adult volunteers results in an increase in plasma [AVP] in direct proportion to the change in mean arterial blood pressure.14 Concentrations of AVP in volunteers whose mean arterial pressure was decreased by 16% to 44% corresponds to the levels found to stimulate arachidonic acid release in primary cultures of rat aortic smooth muscle6 and Ca2+ spiking in A7r5 cells (10 to 500 pmol/L, Figs 2⇑ and 3⇑). Similarly, a reduction of blood volume of 30% in rats increased plasma [AVP] to 300 to 500 pmol/L within 5 minutes.15 The higher concentrations of AVP required to stimulate proliferation of cultured cells are probably never attained in the systemic circulation, but the recent discovery that AVP may be synthesized by VSM cells3 raises the possibility that locally released AVP may reach concentrations high enough to stimulate proliferation in an autocrine or paracrine manner.
Activation of PLA2 by AVP will lead to production of arachidonic acid and lysophospholipids. Arachidonic acid and its eicosanoid metabolites are ubiquitous compounds with complex effects on vascular tissues. A recent study using endothelium-denuded strips of rat thoracic aorta revealed that spontaneous rhythmic contractions of this preparation were increased in a concentration-dependent manner by arachidonic acid and prostaglandin F2α and inhibited by cyclooxygenase and lipoxygenase inhibitors.16 Other arterial tissues tested in that study also produced rhythmic contractions, leading the authors to suggest that “rhythmic contractions are physiological characteristics of many and perhaps all blood vessels and may play a role in blood flow and turbulence; the likely cause of these oscillations is the cyclic release of one or more eicosanoids.”16 It remains to be determined which products of PLA2 activation are responsible for the Ca2+-spiking effects in A7r5 cells and what the molecular targets are for these signals.
Previous studies have suggested that AVP stimulates PLA2 independently of PLC, by activation of a distinct G protein–coupled signal transduction pathway.6 11 17 The different concentration dependencies for AVP-stimulated PLA2 and PLC also suggest distinct pathways for activating these enzymes. Activation of PLC has an EC50 similar to the Kd for AVP binding to V1a vasopressin receptors (≈2 nmol/L), suggesting a close correlation between this response and the fraction of occupied receptors. In contrast, activation of PLA2 and Ca2+ spiking occur at much lower [AVP] (EC50 ≈50 to 300 pmol/L), corresponding to a fractional occupancy of only 2.5% to 13%, assuming a Kd of 2 nmol/L. Li et al18 found that a lower concentration of a V1 vasopressin receptor antagonist was required to inhibit AVP binding than was required to inhibit Ca2+ signals or mitogen-activated protein kinase activation in rat aortic smooth muscle cells, suggesting that binding to a fraction of the receptors generates maximal levels of second messengers. The existence of “spare receptors” for AVP-stimulated signaling may be important for VSM cells, enabling these cells to detect very low circulating [AVP] and to respond rapidly to changes in [AVP].19 Circulating [AVP] is closely regulated by baroreceptor and osmosensor mechanisms. AVP-stimulated Ca2+ spiking in VSM may therefore represent an important physiological mechanism for the moment-to-moment regulation of vascular tone in response to changes in blood pressure or osmotic balance. A hypothetical scheme for AVP-stimulated Ca2+ signaling pathways is shown in Fig 8⇓.
Several features distinguish AVP-stimulated Ca2+ spiking from the release of intracellular Ca2+ in A7r5 cells: (1) Ca2+ spiking requires extracellular Ca2+7 and is abolished by nimodipine (Fig 4⇑), whereas AVP-stimulated Ca2+ release occurs in the absence of extracellular Ca2+ (Fig 1⇑) and in the presence of nimodipine (Fig 4⇑). (2) AVP-stimulated Ca2+ spiking is abolished by PLA2 inhibitors ONO-RS-082 (Fig 6⇑) and HELSS (not shown), whereas Ca2+ release is resistant to these agents (Fig 6⇑, middle). (3) The Ca2+-spiking response to AVP is a frequency-modulated (FM) response; spike frequency changes dramatically while the amplitude of the Ca2+ spiking is fairly constant over a wide range of AVP concentrations (Figs 2⇑ and 3⇑). The release of intracellular Ca2+ by AVP is an amplitude-modulated (AM) response, which is graded with increasing [AVP], producing increases in [Ca2+]i that reach much higher levels than occur during Ca2+ spiking (Fig 1⇑). (4) AVP-stimulated Ca2+ spiking occurs at much lower [AVP] (10 to 500 pmol/L, Figs 2⇑ and 3⇑) than is required to elicit the Ca2+-release response (100 pmol/L to 1 μmol/L, Fig 1⇑).
Single-cell imaging experiments also reveal that Ca2+ spiking occurs synchronously among all of the cells in a population, whereas the Ca2+-release response occurs asynchronously in individual cells (not shown, but see References 7 and 20). Synchronization of Ca2+ spiking presumably occurs because the cells are electrically coupled via gap junctions,7 21 22 and the spikes are triggered by an electrical stimulus that rapidly propagates between adjacent cells. The differences in the [Ca2+]i signals may have important functional consequences for contraction of VSM cells. A synchronous [Ca2+]i increase may be expected to produce a coordinated contraction within an artery, which may then propagate electrically away from the initial stimulus. The electrical propagation of the Ca2+-spiking signal may also serve to further sensitize VSM cells to AVP, because even a small localized increase in [AVP] detected by a few cells may increase spike frequency in neighboring cells that did not detect the change in [AVP]. The Ca2+-release response, in contrast, is apparently not communicated between cells via gap junctions and may be considered, therefore, to be a localized response that will occur only in those cells that detect a large increase in [AVP]. The latter response may be appropriate to produce a profound vasoconstriction and initiation of cell proliferation locally, eg, at the site of an injury. The more coordinated Ca2+-spiking response may be better suited to regulation of vascular tone in response to changes in plasma osmolality and blood pressure, both because it is exquisitely sensitive to concentrations of AVP found in the systemic circulation and because the frequency-modulated nature of the signal may more finely tune the contractile response than the amplitude-modulated signal that results from release of intracellular Ca2+.
Selected Abbreviations and Acronyms
|d(CH2)5[Tyr(CH3)2, Ala-NH29]AVP||=||[1-(β-mercapto-β,β,-pentamethylenepropionic acid), 2-O-methyl- tyrosine]arginine vasopressin|
|PLA2, PLC||=||phospholipase A2, phospholipase C|
|VSM||=||vascular smooth muscle|
This study was funded intramurally by the Research Committee of the Council of Loyola University Chicago, Stritch School of Medicine. The author gratefully acknowledges the technical assistance of Gary Maszak and Dan Hemsworth.
↵1 In several experiments, raising the temperature from 25°C to 37°C had no effect on the frequency of Ca2+ spiking, nor did it prevent the increase in spike frequency induced by increasing the AVP concentration from 40 to 50 pmol/L (not shown). Experiments were not routinely performed at 37°C, because the fura 2 signal was rapidly lost from the cells.
- Received August 21, 1995.
- Accepted February 27, 1996.
- © 1996 American Heart Association, Inc.
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