55

Journal of Physiology (1990), 421, pp. 55-77 With 12figures Printed in Great Britain

AGONIST-STIMULATED DIVALENT CATION ENTRY INTO SINGLE CULTURED HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS BY RON JACOB From Smith Kline & French Research Ltd, The Frythe, Welwyn AL6 9AR, Herts

(Received 16 June 1989) SUMMARY

1. The free cytoplasmic Ca2+ concentration ([Ca2+]1) can be measured using Fura-2 in superfused single human umbilical vein endothelial cells. When an endothelial cell is stimulated by a maximal dose of histamine (100 /tM), [Ca2+]i rises to a peak and then falls back to a maintained plateau which is due to a stimulated Ca2+ influx. 2. If extracellular Ca2+ is replaced by 50 ,uM-Mn2+ then 100 ,tM-histamine causes a rise in [Ca2+]i accompanied by a fluorescence quench that signals the stimulated entry of Mn2+ into the cytoplasm. 3. If in Ca2+-free solution a cell is stimulated by 100 JtM-histamine for 120 s to discharge the internal Ca2+ store, and then exposed to 50 /tM-Mn2+ after removal of the histamine, a similar stimulated Mn2+ entry is seen. This quench is unaffected by readdition of histamine and is not seen if the store is refilled by exposure to 1 mM-extracellular Ca2+ for 180 s before exposure to the Mn2+. 4. The refilling of the internal store by exposure to 1 mM-Ca2+ and the stimulated entry of Mn2+ are both blocked by 2 mM-Ni2+. 5. If [Ca2+], is stimulated to produce repetitive spikes by a low dose of histamine (0-3-1 /IM) in nominally Ca2 -free solution containing Mn2 , then the stimulated quench is uniform and is not modulated by the [Ca2+], spiking. 6. If the internal store is discharged by exposure to histamine in Ca2+-free solution and then refilled for a short period then the cell is in a state where the internal store is partly full to an extent that depends on the duration of the refilling. In such an experiment, the rate of Mn2+ influx may be estimated by measuring the rate of quench during a short exposure to 50 /tM-Mn2+. The rate of Mn2+ entry varies inversely with the degree of fullness of the internal Ca2+ store. 7. If a similar experiment is repeated but with the fullness of the internal store being varied by varying the period of the initial exposure to 100 /tM-histamine, with no refilling, the same inverse relationship between Mn2+ influx and fullness of the internal store is obtained. 8. These experiments show that Mn2+ enters human umbilical vein endothelial cells following agonist stimulation by a pathway that is controlled by the degree of fullness of the internal store; it does not, however, enter the cytoplasm by exactly the same route as Ca2±. It is proposed that Mn2± enters by a pathway that is in part the same as the pathway by which Ca2+ refills into the internal store. The common MS 7770

56

R. JACOB

element of these two pathways contains the point at which divalent cation entry is controlled by the degree of fullness of the internal store. INTRODUCTION

The typical intracellular free Ca2+ concentration ([Ca2+]i) response to agonist stimulation of an electrically non-excitable cell is that [Ca2+]i rises rapidly to a peak and then falls to a maintained elevated plateau. Many studies have shown that the initial peak is due to a release of Ca2+ from an internal store whereas the plateau is due to a stimulated entry of Ca2+ from outside the cell into the cytoplasm. The stimulated influx may serve two functions. Firstly it prolongs the [Ca2+]i response past the point when the internal store is depleted and so no longer able to maintain an elevated [Ca2+]i by itself. Secondly, the influx may be linked to the mechanism for refilling the internal store after removal of the agonist. Several mechanisms have been proposed for the stimulated entry of Ca2+ and these vary in the degree of coupling between the binding of agonist at the receptor and the entry of Ca2+ through a channel. Starting with the tightest coupling, Ca2+ could enter via a receptor-operated calcium channel, i.e. a channel which is tightly coupled to a receptor, perhaps via a G-protein. This appears to be the mechanism for ATPstimulated Ca2+ entry into rabbit ear artery smooth muscle cells (Benham & Tsien, 1987; Benham, 1988). A less tightly coupled mechanism is the entry of Ca2+ through a channel that is controlled by a second messenger generated at the membrane following receptor-agonist binding. This has been proposed as the mode of action of inositol-1,3,4,5-tetrakisphosphate (1P4) in lacrimal glands (Morris, Gallacher, Irvine & Petersen, 1987). The most loosely coupled Ca2+ entry mechanism is that proposed for rat parotid acinar cells where the stimulated Ca2+ entry appears to be a consequence of the agonist-stimulated discharge of the internal Ca2+ store (Putney, 1986; Merritt & Rink, 1987) rather than the presence of agonist per se. Two Ca2+ entry mechanisms which are prevalent in other cell types, namely sodium-calcium exchange and voltage-operated Ca2+ channels, do not appear to play a role in determining the agonist-stimulated changes of [Ca2+], in human umbilical vein endothelial cells (Rotrosen & Gallin, 1986; Jacob, Merritt, Hallam & Rink, 1988). The nature of the stimulated Ca2+ entry in populations of cultured human umbilical vein endothelial cells has been investigated by using Mn2+ as an indicator of divalent cation entry (Hallam, Jacob & Merritt, 1988a, 1989a). Mn2+ has three desirable properties. Firstly, it quenches Fura-2 fluorescence so that its entry into the cytoplasm is readily detected. Secondly, Mn2+ entry appears to be activated under the same circumstances as that of Ca2+, suggesting a common pathway. Thirdly, since there is no endogenous agonist-releasable Mn2+ store, a fluorescence quench unambiguously signals that the Mn2+ entering the cytoplasm originates from outside the cell and not from an internal store. Hallam et al. (1989a) showed that after the releasable Ca2+ store had been depleted by stimulating endothelial cells with histamine or thrombin for a short period (~ 120 s) in Ca2+-free medium, the divalent cation entry system was activated even after an antagonist had been added to cancel the direct action of the agonist. Thus the divalent cation entry could not be through a receptor-operated calcium channel or through a second messengeroperated channel with the second messenger being generated as a direct consequence

DIVALENT CATION ENTRY INTO ENDOTHELIAL CELLS 57 of agonist binding. The results suggested that the divalent cation entry was a consequence of the agonist-induced store depletion. An unresolved question was the nature of the coupling between the Ca2" store and the channel (Rink & Hallam, 1989); the coupling could be a physical one based on the proximity of the store to the channel (Putney, 1986; Merritt & Rink, 1987) or Ca21 could enter through a second messenger-operated channel with the second messenger being generated as a consequence of the agonist-induced store depletion. In this article I extend the analysis of agonist-stimulated divalent cation entry in populations of human umbilical vein endothelial cells by making measurements on single cells. The importance of experimenting with single cells rather than with populations lies in the fact that qualitatively different results may be obtained since population measurements average out heterogeneous responses. An example of this is that the response of a population of human umbilical vein endothelial cells to low doses of histamine is a scaled-down version of the maximal response (Rotrosen & Gallin, 1986) whereas single-cell measurements reveal the appearance of [Ca2+]± oscillations (Jacob et al. 1988). The results presented here strengthen the analogy between Ca2+ and Mn2± movements and clarify the relationship between store depletion and Mn2+ entry. Some of the results have been reported previously in abstracts (Hallam, Jacob & Merritt, 1989b; Jacob, Merritt, Rink & Hallam, 1989). METHODS

Endothelial cells were prepared according to Hallam, Pearson & Needham (1988b). Briefly, cells obtained from the vein of human umbilical cords, and were cultured for 7 days in 25 cm2 flasks in culture medium comprising 80% medium 199, 20% fetal calf serum, 50,uU ml-' penicillin and 50 jug ml-' streptomycin. Cells were detached by exposure to 0-05 % trypsin in Ca2+and Mg2+-free phosphate-buffered saline for 120 s, reseeded in culture medium on No. 1 glass cover-slips and kept in culture for 3 or 4 days before use. Cells were loaded with Fura-2 by incubation in HEPES (20 mM)-buffered Dulbecco's modified Eagle's medium with 20% fetal calf serum and 1 uM-Fura-2-AM (Molecular Probes) for 30 min at room temperature and then kept before use at room temperature, for at least 30 min, in the superfusion medium supplemented with 1 % bovine serum albumin. To measure [Ca2+]1, the cover-slip was placed on the thermostatted stage of an inverted microscope (Zeiss Axiomat) equipped with a glycerin-immersion x 40 Nikon fluorite objective and superfused at 0 3 ml min-' with a medium containing 145 mM-NaCl, 5 mM-KCl, 1 mM-CaCl2, 1 mM-MgCl2, 10 mM-glucose, 01 % bovine serum albumin and 10 mM-HEPES (pH 7-4) at 37 'C. CaCl2 was omitted from Ca2+-free solution which also contained 100 ,uM-EGTA where indicated. Mn2± and Ni2+ were added as chloride salts. The stage was shaped in such a way that the coverslip formed the bottom of a trough which was 15 mm long and 3 mm wide. In order to obtain a stable upper surface and laminar flow through this trough, a cover-slip was fixed, by means of ledges, 1 mm above the bottom cover-slip so that the trough was actually a channel of rectangular cross-section. Superfusion media were switched using miniature solenoid valves (Lee Valve Co.) with a dead volume of 100 ,ul. After operating the valves, there was a delay of several seconds before the new solution started to appear over the cell and the solution change was 90 % complete within a further 7 s. Cells were illuminated via the epifluorescence port of the microscope. In some experiments the illumination was taken from a Spex fluorimeter providing alternating excitation either at 350 or at 360 nm, and at 380 nm. In these experiments, fluorescence was detected by a photon-counting photomultiplier connected to the Spex controller and counted in 1 s periods. In other experiments, excitation light was obtained from a 150 W Xe arc lamp (Ealing Electro-Optics Ltd) and a filter wheel (Cairn Research Ltd) rotating at 50 Hz and containing 350, 360 and 380 nm interference filters. The fluoresced light was detected by a Hammamatsu R928 photomultiplier run at were

-

-

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58

approximately -900 V and the signal was fed into the virtual ground current input preamplifier of a Cairn spectrophotometer system. The demodulated outputs were sampled using a Tandon PCA plus (IBM PC AT compatible) computer equipped with a Metrabyte DAS 16 analog-to-digital conversion board. The data acquisition and display was controlled by custom-written software: incoming data were averaged in blocks of 25 points so that the effective acquisition rate was 2 Hz. Where Ca2+ (rather than Mn2+ quench) was being measured, an autofluorescence correction was made at the end of the run by exposing the cell to 2 mM-Mn2+ and 0 5 4aM-ionomycin to flood the cell with Mn2+ and quench the Fura-2 fluorescence (Hallam et al. 1988b). [Ca21]1 was calculated from the ratio of the fluorescences measured when exciting at 350 and 380 nm (Grynkiewicz, Poenie & Tsien, 1985) using in vitro-determined values for the ratios in zero [Ca2+] and in saturating

(1 mM) [Ca2+]. B

A

_

6~~~30

nm

6.0

3 20 s C

pCa

0

0 Ca2.~~~~~~~~~~~FL

7.5

_________________ 100 uM-histamine

0

Ca2++50IM-Mn2+ 100 gM-histamine

Fig. 1. Histamine-stimulated divalent cation influx. A, the response of [Ca2+], in a single human umbilical vein-endothelial cell to 100 /M-histamine in the presence or nominal absence of extracellular Ca2+. The initial phase (the transient peak) was independent of extracellular Ca2+ unlike the following plateau phase which was only seen in the presence of extracellular Ca2+. B, in the presence of Mn2+ in nominally Ca2+-free solution 100,UMhistamine stimulated not only a rise in [Ca2+], (signalled by a sharp decrease in the 380 nm fluorescence) but also the entry of Mn2+ (signalled by the quench of fluorescence, most clearly seen at 360 nm). Direct comparisons between data presented within one figure are made on the basis that the data were obtained from cells from one culture, unless explicitly stated otherwise (i.e. Figs 2 and 9). Means are quoted + the standard error of the mean. RESULTS

Agonist-stimulated divalent cation entry The fundamental observation implying an agonist-stimulated Ca2+ entry into human umbilical vein endothelial cells is that when a cell is stimulated by agonist (100 gM-histamine) the [Ca2+], shows a transient peak followed by a maintained elevated plateau (800 + 140 nM, n = 16) in the presence of extracellular Ca2+ whereas in the absence of extracellular Ca2+ there is no significant elevated plateau phase ([Ca2+]i at the end of exposure to histamine: 81 + 17 nm, n = 16) although the initial transient is still present (Fig. 1A); these averages were obtained from measurements on cells from eight cultures. This pattern of response has been reported in many different non-excitable cells. The initial [Ca2+], peak is attributed to a release of an internal Ca2+ store whereas the plateau is attributed to some form of stimulated Ca2+ entry. The dose of histamine used in these experiments is a maximal one, as assessed

DIVALENT CATION ENTRY INTO ENDOTHELIAL CELLS

59

either by stimulation of inositol phosphate production (Lo & Fan, 1987; Resink, Grigorian, Moldabaeva, Danilov & Biihler, 1987) or by the increase in [Ca2+], (Rotrosen & Gallin, 1986). Mn2+ is well suited for investigating the nature of the stimulated Ca2+ entry because it quenches the Fura-2 fluorescence on entering the cell. This is best observed when exciting at the isoemissive wavelength (nominally 360 nm) where the fluorescence is independent of [Ca2+]. In the following experiments where the quench was being monitored and only two excitation wavelengths were available, measurements were made at 360 and 380 nm. Although 350 nm measurements would have provided simpler traces (at this wavelength fluorescence increases with increasing [Ca2+]i) the signal at this wavelength is weaker than at 380 nm (where fluorescence decreases with increasing [Ca2+]i) because of attenuation by the microscope optics. The ability of agonist to stimulate Mn2+ entry is shown in Fig. l B. At the start of the experiment there was a basal rate of fluorescence decay, partly due to photobleaching and partly due to Fura-2 leakage from the cell. The removal of extracellular Ca2+ and addition of 50,tm-Mn2+ slightly augmented the rate of fluorescence decay although in many cells addition of Mn2+ at this stage had no detectable effect (e.g. Figs 2A, 7 and 10). On adding 100 /LM-histamine, the 380 nm fluorescence decreased sharply without any accompanying change in the 360 nm fluorescence. This indicates that [Ca2+]i rose sharply, consistent with the result in Fig. 1 A. Following this rise in [Ca2+]i there was a marked increase in the rate of decay of fluorescence at 360 nm that was not seen in the absence of extracellular Mn2+ (see Figs 2-4) and was due to a stimulated entry of Mn2+. The 380 nm fluorescence was also quenched although this is less clear because of the superimposed effect of the changes in [Ca2+]i. Because the decay in fluorescence is pseudo-exponential, the rate of decay for any particular segment of a quench experiment is best quantified as a rate constant; this was estimated by dividing the absolute rate of decay of the 360 nm fluorescence trace by the magnitude of the fluorescence at the instant at which the rate of decay was measured. In eleven cells from four cultures subjected to the manoeuvre shown in Fig. 1B, the basal decay rate constant was 0-0026+0-0007 s-' and the rate constant after exposure to histamine was 0-017 + 0-003 s-1; this increase was significant at P < 0-00025 (paired one-tailed t test). The exact temporal relationship between the rise in [Ca2+]i and the stimulated Mn2+ entry is not clear because of the rounded shoulder on the 360 nm trace although the Mn2+ influx did not precede the Ca2+ release.

Stimulated divalent cation entry following agonist removal Hallam et al. (1989a) showed that the stimulated Mn2+ influx was not a direct consequence of the presence of histamine but rather a consequence of the discharge of the internal store. The equivalent experiment for a single cell is shown in Fig. 2A; the rate constants quoted in parentheses were obtained by averaging data from four cells. An initial short exposure to 50 ,uM-Mn2+ had no effect on the basal quench rate (0-0008+0-0004 s-'). The cell was then exposed to 100 gM-histamine for 120 s causing a [Ca2+], transient which returned almost to basal during the exposure to histamine. This implies that the histamine exposure was sufficiently long to discharge the internal store completely. After a further 300 s in nominally Ca2+-free solution the

R. JACOB

60

cell was exposed to 50 /tM-Mn2+ and, despite the absence of histamine, it caused a large stimulated quench (0-014 + 0006 s-1) of a similar magnitude to that seen in Fig. IB; the increase in the quench rate constant was significant at P < 0-015 (paired one-tailed t test). This shows that the stimulated Mn2+ entry is not a direct A

1 mn

360 nm 0

0 0 0 0

ce

0

Histamine

.

Ca2+

Mn2+

Mn2+

B 4

am 0

360 nm

o -

Ca2+

Histamine -

Histamine Ca2+ Mn2+

Fig. 2. Stimulated Mn2+ influx in the absence of agonist after depletion of the internal store. A, after removing Ca2+ from the superfusate at the start of the experiment, the internal store was depleted by exposure to 100 ,SM-histamine. Following this, exposure to 50 AuM-Mn2+ caused a rapid quench, not seen during the exposure to Mn2+ before the store was depleted. B, if the internal store was refilled by exposure to 1 mM-extracellular Ca2+ for 180 s before applying 50 ,sM-Mn2 , the quench was marginal. The ability of this cell to sustain a stimulated Mn2+ entry was verified by reapplying 100 /SM-histamine at the end of the experiment to obtain a rapid quench.

consequence of the presence of histamine but perhaps a consequence of the Ca2+ store depletion. If this were the case, refilling the internal store in the period between its discharge by histamine and the exposure to Mn2+ should block the stimulated Mn2+ entry. The experiment in Fig. 2B (repeated on four cells) shows that this is indeed so because exposure to Mn2+ after refilling the internal store by exposure to 1 mmCa2+ for 180 s caused a small but insignificant increase (P > 01, paired one-tailed t test) in the rate constant from a basal value of 0-0008 + 0-0002 s- to a value of 000 16 + 00013 s-1; a refilling period of 180 s is sufficient to refill completely the

DIVALENT CATION ENTRY INTO ENDOTHELIAL CELLS

61

internal store (Jacob et al. 1988; also see below). To ensure that the cell was capable of supporting a stimulated Mn2+ entry, it was exposed at the end of the experiment to 100 /uM-histamine in the continued presence of 50 fuM-Mn2+ and a rapid quench was observed. The data for Fig. 2 were taken from cells from three cultures with two of the cells for the experiment without refilling (Fig. 2A) and three of the cells for the experiment with refilling (Fig. 2B) coming from the same culture; the traces for Fig. 2 were obtained from two cells from the same culture. The rise in [Ca2+]i during refilling of the internal store is variable (Jacob et al. 1988; Rink & Hallam, 1989) being pronounced in some cells as in Fig. 2B but quite small in other cells, e.g. Figs 5A and 7. The presence of agonist does not directly affect Mn2+ entry These results show that the Mn2+ influx detected in the presence of histamine may also be observed after the removal of histamine, provided the internal store remains empty; they do not rule out the possibility that the entry of Mn2+ might be faster in the presence of histamine than in its absence which would indicate, perhaps, an additional divalent cation entry mechanism that is more tightly coupled to the binding of histamine to its receptor. To test this, the internal store was discharged by exposure to 100 /tM-histamine for 120 s and then exposed to 50 4tM-Mn2+ to establish a stimulated quench during which 100 /um-histamine was readded (Fig. 3.) The stimulated quench rate constant measured after the readdition of histamine (0-0049+0±0010 s-1) was not significantly different (P > 0.1, paired two-tailed t test) from that measured beforehand (0-0055 + 0-0012 s-1) suggesting that there is only one route for Mn2+ entry. I have shown so far that a stimulated Mn2+ entry can be observed either in the presence of histamine or after the internal store has been discharged by a prior exposure to histamine. In both circumstances one can argue that if extracellular Ca2+ had been present instead of Mn2+ then there would have been a stimulated Ca2+ entry. In the presence of histamine a maintained elevated level of [Ca2+]i is observed provided extracellular Ca2+ is present, implying that the plateau is due to a stimulated Ca2+ entry. In the absence of histamine, exposure to extracellular Ca2+ results in a rapid filling of the internal store (see below), again implying some form of stimulated Ca2+ entry.

Ni2+ blocks both Mn2+ entry and store refilling by Ca2+ Another correlation between Mn2+ and Ca2+ entry is that both are blocked by 2 mM-Ni2+. Hallam et al. (1988a) showed that 2 mM-Ni2+ could block thrombinstimulated Mn2+ entry either when the Mn2+ entry was measured during the application of thrombin or after discharge of the internal store by thrombin followed by its inhibition by hirudin. The effect of Ni2+ cannot be tested in the presence of histamine because Ni2+ binds avidly to histamine (Dawson, Elliott, Elliott & Jones, 1986) and inactivates it (R. Jacob, unpublished observation). However, 2 mM-Ni2+ can be shown to block reversibly the Mn2+ entry that follows a histamine-induced depletion of the internal store (Fig. 4). In three experiments, addition of 50 /tM-Mn2+ in the presence of 2 mM-Ni2+ resulted in a quench rate constant (0-0017 + 0-0004 s-1) which was not significantly different (P > 0 4, paired two-tailed t test) from the basal

62

R. JACOB A

380

01

380

C

0

0~~~~~~5 Ca2+ B

-

Hitain

Ca 2+ o ,E Ca 8

~~~Histamine

=

360 nm~~60

Hstmie550n 380 nm 360 nm 30n Histamine

Mn2+

o L_CDI9 0

2.0-

0

Fig. 3. The lack of effect of histamine on the stimulated Mn2+ entry. A, the raw data obtained with the spinning filter wheel. On applying 100 /SM-histamine the 350 and 380 nm fluorescence traces changed in a reciprocal fashion, as expected. The 360 nm trace showed a small dip, indicating that the filter was not providing excitation light precisely at the isoemissive point for Fura-2. B, the processed data. The top trace is the 360 nm trace with a small correction applied (7 % of the 380 nm trace) to remove the small dip mentioned above. The bottom trace is the [Ca2+], calculated from the 350/380 nm ratio. After removal of histamine, application of 50 /sM-Mn2+ caused a quench which continued at the same rate after the reapplication of 100 /tM-histamine. The presence of the reapplied histamine is verified by the small transient on the [Ca2+]i trace.

quench rate constant (00015 +0 0001 s-1). On removal of Ni2+ in the continued presence of Mn2+, the quench rate constant increased significantly (P < 0 003, paired one-tailed t test) to 00082 +0-0006 s-1. The control experiment in Fig. 4A, also repeated on three cells, shows that addition of Mn2+ with the same timing as in Fig. 4B but in the absence of Ni2+ caused a rapid increase in the quench rate constant from 00017 + 0-0003 to 0-015 + 0O005 s-1. This increase was significant at P < 0-08 (paired one-tailed t test), the relatively poor value of this significance being a consequence of the stimulated quench rate being unusually large for one of the three cells, leading to a large standard error of the mean for the stimulated quench rate constant. The Ca2+ corollary of the experiment in Fig. 4 is shown in Fig. 5. Figure 5A shows that a 180 s exposure to 1 mM-Ca2+ after discharge of the internal store by a 120 s exposure to 100 /LM-histamine in nominally Ca2+-free solution allows an almost normal [Ca2+]i transient to be stimulated by a subsequent exposure to 100 /Mhistamine. In these experiments, the basal [Ca2+], was 54 + 7 nM (n = 10), the initial peak [Ca2+], was 645+114 nM (n = 7) and the peak [Ca2+]i after refilling was

DIVALENT CATION ENTRY INTO ENDOTHELIAL CELLS

63

A 1 min 0 0 a 0 G)

0 ir

M n2+ B

C)8

c) 0

0)

Histamine Mn2+

Nj2+

Fig. 4. Ni2+ (2 mM) blocks the stimulated entry of Mn2+. The experiments were carried out in nominally Ca2+-free solution. A, the control showing the stimulated quench on exposure to 50 #M-Mn2+ after the internal store was discharged by exposure to 100 /SM-histamine for 120 s. B, in the presence of 2 mM-Ni2+ no quench was seen; the effect was reversible.

528+85 nM (n = 3). But, if 2 mM-Ni2+

was present during the 180 s exposure to mM-Ca2+ (Fig. 5B) then there was no detectable rise in [Ca2+]i during the exposure to extracellular Ca2+ and the peak response was very attenuated (118 + 33 nM, n = 3) and similar to that seen when no attempt was made to refill the internal stores before the second exposure to histamine (91 + 7 nm, n = 3). The block by Ni2+ of the second peak could conceivably be due to residual amounts of Ni2+ binding to, and inactivating, the histamine. As a control, therefore, cells in the first part of the experiment (Fig. 5A) were exposed to 2 mM-Ni2+ up to 60 s before the first exposure to 100 #zM-histamine. This did not affect the peak response (602 + 95, n = 3), indicating that any residual Ni2+ would have been insufficient to inactivate the histamine. Although Ni2+ quenches Fura-2 fluorescence (J. E. Merritt, personal communication), no quench was seen in these experiments indicating that Ni2+ did not enter the cytoplasm. 1

Mn2+ entry during [Ca2+]1 spiking Stimulation of single endothelial cells by low doses of histamine causes a repetitive spiking of [Ca2+], (Jacob et al. 1988). It is reasonable to expect that if such spiking

R. JACOB

64 A

1 min

800

[Ca2+],A (nm)

Histamine

Histamine

Ca2+

Ca2+

Ni2+ B 800

-

[Ca2+]i

I

(nM)

Histamine

Histamine

Ca2+

Ca2+

Ni 2+ Fig. 5. Ni2+ (2 mM) blocks refilling of the internal store. A, if the cell was exposed to 1 mMl,M-histamine for Ca2+ for 180 s after discharging the internal store by exposure to 100 120 s, then a normal histamine response could subsequently be obtained. B, if 2 mM-Ni2+ was present during the exposure to 1 mM-Ca2+ then the refilling was blocked because a normal histamine response could not be subsequently obtained.

were induced in the presence of Mn2+ then a stepwise quench of the Fura-2 fluorescence would be seen due to the release of discrete packets of Mn2+ into the cytoplasm alongside the Ca2+. However, as shown in Fig. 6, the major part of the quench by Mn2+ was a smooth one and not phasic despite the spiking of [Ca2+],. In all, twenty-three cells were examined for their response to low doses of histamine (0-3-1 gm) in the presence of Mn2+ (50-200 gM). In six cases the traces were not stable enough to draw a firm conclusion about the presence or absence of a phasic Mn2+ entry. In sixteen cases there was no clear evidence of phasic entry. In one case there appeared to be a phasic entry starting just before the initiation of the [Ca2+], spike and ending before the end of the spike. I cannot say whether this was an uncommon cell or an uncommon artifact.

Dependence of Mn2+ entry on fullness of the internal store Because Mn2+ influx is modulated by the fullness of the internal store, it is reasonable to propose that this modulation of divalent cation entry is the mechanism that controls the refilling of the internal store with Ca2+ following its agonist-induced depletion. However, the similarity between the observed Mn2+ influx and the supposed Ca2+ influx could be coincidental. It might be, for example, that Mn2+ entry

DIVALENT CATION ENTRY INTO ENDOTHELIAL CELLS

65

60 s 380 nm

0 C.,

0 Ca2++50

jiM-Mn2+

0.5 gIM-histamine Fig. 6. Exposure to 50 /M-Mn2+ causes a smooth quench when [Ca2+], is stimulated to spike repetitively by exposure to 0-5 ,tM-histamine.

is stimulated only when the store is less than half full, in which case the mechanism could not regulate the filling of the store up to its capacity. Another possibility is that agonist exposure in nominally Ca2+-free solution renders the cell permeable to Mn2+ and that this permeability is resealed by exposure to extracellular Ca2±, in which case the observations would result from the direct action of Ca2+ outside the cell rather than from store refilling. To address these issues I measured the rate of Mn2+ entry with the internal store filled to various degrees. In the first series of experiments, the store was fully depleted by exposure to 100 /tM-histamine for 120 s in Ca2+-free solution and then refilled by exposure to 1 mM-Ca2+ for periods of 5-120 s (Fig. 7). In these experiments, Ca2+-free solution that did not contain Mn2+ did contain 100 ,tM-EGTA. In Fig. 7, ignoring for the moment the exposures to 50 /M-Mn2+, the internal store was discharged by exposure to 100 /uM-histamine for 120 s, partly refilled by exposure to 1 mM-Ca2+ for 30 s and then re-exposed to 100 /tM-histamine for 120 s approximately 500 s after the first exposure. Because of the short refilling period, the internal store was only partly full at the time of the second exposure to histamine so that the height of the second peak was considerably smaller than the height of the first one. A measure of the degree of fullness of the internal store after refilling is the ratio of the height of the second peak to the height of the first peak. If this normalized peak height is plotted as a function of the duration of the exposure to 1 mM-Ca2+, a refilling curve is obtained (Fig. 8). In addition to the exposures to histamine and Ca2+, there were also three exposures to 50 /tM-Mn2+. The first exposure was a control to show that there was no significant basal rate of Mn2+ entry. The second exposure was just before the second exposure to histamine, i.e. at a time when the internal store was partly full; the duration of this exposure was sufficient to establish the rate of quench and was usually between 30 and 60 s. The third exposure was after the second exposure to histamine, i.e. when the internal store was fully depleted, so that the rate of quench measured during this 3

PH Y 421

R. JACOB

66

E

120 s

co 00

o

2-2 0

(0 0.4,

Ca2+

Ca2

Histamine

Histamine Mn2+

Mn2+

Mn2+

Fig. 7. An experiment to investigate the relationship between the state of the internal store and the rate of Mn2+ entry. The experiment was performed using the spinning filter wheel. The top trace is the 360 nm fluorescence, corrected as in Fig. 3, and the bottom trace is the 350/380 nm fluorescence ratio (proportional to [Ca2+]j). This experiment is one of a series in which the refilling time in between the two exposures to 100 /uM-histamine was varied; in the above experiment the refilling time was 30 s. The data obtained from this series of experiments are tabulated in Table 1. See text for further explanation.

1.2 r 0

._

0)

0.8

[

0

Q -1 a)

co N

0

0*4 [

z

0

0.0

L 0

50

100

150

200

Refilling time (s) Fig. 8. The effect of refilling time on store repletion plotted from the data in Table 1, these data having been measured from experiments similar to that shown in Fig. 7. The normalized peak height is a normalized measure of the state of the internal store and is referred to in Table 1 as peak 2/peak 1. The curve was fitted by eye.

DIVALENT CATION ENTRY INTO ENDOTHELIAL CELLS

67

period represented a maximal rate of Mn2+ entry. A normalized rate of quench when the internal store was partly full could then be obtained by taking a ratio of the quench rate constants measured during the second and third exposures to Mn2+. A correction was made for the basal rate of quench by subtracting the basal rate 0.8

0@8.

-

c

0.4 ~ 04-

0\

E 0*

°0

04

Z 0-2

-

0-2

1-2 1.0 0-8 0.6 Normalized peak height Fig. 9. The dependence of the rate of Mn2+ entry on the state of the internal store. 0, data from Table 1; *, data from Table 2. The normalized peak height is a normalized measure of the state of the internal store and is referred to in Tables 1 and 2 as peak 2/peak 1. The normalized quench rate is a normalized measure of the rate of Mn2+ entry; it is referred to in Tables 1 and 2 as rate 1/rate 2. The line was fitted by linear regression. The two sets of points were obtained from experiments on different cultures.

0.0

0.4

constant from both values before taking their ratio. The normalized rate of quench could then be plotted against the normalized peak height (Fig. 9, 0). This showed a smooth monotonic relationship indicating that the rate of Mn2+ entry could reflect the control of store refilling. The data used to generate Figs 8 and 9 are shown in Table 1. The second issue raised above was whether Mn2+ entry is modulated by the presence of extracellular Ca2+ per se or whether it really is a consequence of store refilling. To address this issue, the above series of experiments was repeated in a slightly different way. Instead of fully depleting the internal store and then partly refilling it, the store was partly depleted by exposure to 100 ,tM-histamine for periods between 5 and 120 s and there was no refilling (Fig. 10). (For the shortest histamine exposure of 5 s, the [Ca2+]i transient did not reach its peak value before the histamine was removed so the peak response was determined by inserting an additional 15 s exposure to 100lM-histamine followed by complete refilling of the internal store before the other manipulations.) Using the same procedure as in the previous series of experiments, the normalized rate of quench was plotted against the normalized peak height (Fig. 9, @); the data are shown in Table 2. The points from the two 3-2

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TABLE 1. Effect on the rate of Mn2' entry of varying the fullness of the internal Ca2+ store by variable refilling Basal Refill Rate 1 Rate 2 Rate 1/ Peak 2/ rate time Peak 1 Peak 2 peak 1 rate 2 (s-') (s-') (s-') (s) 1-02 070 0 0007 0 0094 034 0-0262 30 1-46 0-56 00077 090 15 162 040 0-0193 0-0001 0 0000 0-22 0-11 0-0248 0-0170 0 203 0-69 5 1 78 044 040 0-0383 0-0002 0-0155 0-78 1-09 201 009 0-0315 0-0029 180 1 85 0-0001 1-10 0-06 0 0005 1-75 1 60 0 0509 0-0036 90 0-27 0 0036 1-32 1 66 079 0-0129 0-0001 50 0-58 044 0 0445 0-0259 070 5 1-61 0-0001 2 14 0-56 0-28 0-0711 0 0400 0 0000 0-61 0 Data obtained from experiments similar to that shown in Fig. 7. Rates 1 and 2 are the quench rate constants measured during the second and third exposures to Mn2+. The basal rate is the quench rate constant measured between the second and third exposures to Mn2 . Peaks 1 and 2 are the heights of the fluorescence ratio (350/380 nm) peaks elicited by the two exposures to histamine, measured in dimensionless ratio units. The ratio peak 2/peak 1 is referred to as the normalized peak height in Figs 8 and 9. The ratio rate 1/rate 2 is referred to as the normalized quench rate in Fig. 9.

A

'T

C,) -CDC

E

o 0)0

60 s

U-

1-8r a 00

CY) 0

C') 0

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0 4-

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Histamine Mn2+

Mn2+

Mn2+

Fig. 10. An experiment to investigate the relationship between the state of the internal store and the rate of Mn2+ entry. The experiment was performed using the spinning filter wheel. The top trace is the 360 nm fluorescence, corrected as in Fig. 3, and the bottom trace is the 350/380 nm fluorescence ratio (proportional to [Ca2+]M). This experiment is one of a series in which the duration of the first exposure to 100 /tM-histamine was varied. In the above experiment this duration was 20 s. The data obtained from this series of experiments are tabulated in Table 2. See text for further explanation.

DIVALENT CATION ENTRY INTO ENDOTHELIAL CELLS

69

TABLE 2. Effect on the rate of Mn2' entry of varying the fullness of the internal Ca2+ store by variable discharge Histamine Basal Rate 1/ duration rate Rate 1 Rate 2 Peak 2/ (s) Peak 1 Peak 2 (s-1) (s-1) (s-1) peak 1 rate 2 20 1-20 0-78 0 0007 0.0151 0-0412 0-65 0-36 0 0010 50 0.51 0-76 0-0072 0-0256 0-67 0-25 0-24 120 0-83 0-0019 0-0259 0-0506 0-29 0 49 0 95 0 94 5 0 90 0 0009 0-0058 0-14 0-0368 120 1-46 0-47 0-0004 0-0188 0-0247 0-32 0-76 120 1-46 0-31 -0 0001 0-0288 0-0383 0-22 0-75 0-42 0 0344 30 1-30 0 0001 0 54 0-0632 0 33 10 1-40 0-60 0 0005 0-0116 0-0203 0 43 0-56 Data tained from experiments similar to that shown in Fig. 10. Rates 1 and 2 are the quench rate coIiztants measured during the second and third exposures to Mn2+. The basal rate is the qi nch rate constant measured between the second and third exposures to Mn2 . Peaks 1 and 2 are i;f heights of the fluorescence ratio (350/380 nm) peaks elicited by the two exposures to histamine, measured in dimensionless ratio units. Histamine duration is the duration for the first application of histamine. The ratio peak 2/peak 1 is referred to as the normalized peak height and the ratio rate 1/rate 2 is referred to as the normalized quench rate in Fig. 9.

experiments can be fitted by the same line even though in one case the degree of store fullness was varied by a varying exposure to extracellular Ca2+ whereas in the other case it was not. This suggests that it is not the presence of extracellular Ca2+ per se which controls Mn2+ entry but rather the degree of store fullness. DISCUSSION

In a wide variety of electrically non-excitable cells agonist stimulation causes a Ca2+ influx into the cytoplasm from outside the cell. This stimulated influx has been directly measured by 45Ca uptake in, for example, parotid acinar cells (Poggioli & Putney, 1982). A stimulated Ca2+ influx in the presence of agonist is also indicated by the [Ca2+]i response since in endothelial cells (Fig. 1A; Hallam et al. 1988a) and in most other cases (e.g. gastric parietal cells, Negulescu & Machen, 1988; parotid acinar cells, Merritt & Rink, 1987; neutrophils, Merritt, Jacob & Hallam, 1989; platelets, Merritt & Hallam, 1988) the initial [Ca2+], peak due to agonist-induced Ca2+ release from the internal store is followed by a maintained [Ca2+]i plateau which is absolutely dependent on the presence of extracellular Ca2+. Although the widespread nature of this observation strongly suggests the simple explanation that there is a receptor-mediated entry of Ca2+, it is possible that the observation could be due to a modulation by extracellular Ca2+ of the intracellular response. Use of Mn2+ An alternative to 45Ca uptake or [Ca2+]i measurements is to make use of the ability of Mn2+ to enter the cell by the agonist-stimulated Ca2+ entry pathway (Hallam &

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Rink, 1985). The ability of Mn2+ to pass through Ca2+ channels is wide-spread. Not only does it pass through the better known voltage-operated Ca2+ channels (Nelson, 1986; Tsien, Hess, McClesky & Rosenberg, 1987) but it is also able to pass through the less well-defined channels that effect receptor-mediated Ca2+ entry. These channels are not voltage gated and, although cation selective, show varying degrees of selectivity for divalent cations over monovalent cations. Neutrophils (von Tscharner, Prod'hom, Baggiolini & Reuter, 1986), mast cells (Penner, Matthews & Neher, 1988) and BALB/c 3T3 cells (Matsunaga, Nishimoto, Kojima, Yamashita, Kurokawa & Ogata, 1988) possess non-selective cation channels which promote receptor-mediated Ca2+ entry. On the other hand, the channels isolated from thrombin-activated platelets are very selective for divalent over monovalent cations (Zschauer, van Breeman, Biihler & Nelson, 1988) as are the NMDA receptoroperated channels in neurones (Mayer & Westbrook, 1987). The ability of these channels to conduct Mn2+ has only been directly tested (i.e. by patch clamp measurements) for the NMDA receptor-operated channels where the permeability to Mn2+ is about one-eighth of the permeability to Ca2+. But, entry of Mn2+ via the receptor-mediated Ca2+ entry pathway has also been detected in other cells by its ability to quench a fluorescent Ca2+ indicator. Thus, stimulated Mn2+ entry has been reported in platelets (Hallam & Rink, 1985; Sage, Merritt, Hallam & Rink, 1989), neutrophils (Merritt et al. 1989), parathyroid cells (Johansson, Larsson, Wallfelt, Rastad, Akerstr6m & Gylfe, 1988) and smooth muscle cells (Benham, 1988) and is seen in single endothelial cells (Fig. 1B). The receptor-mediated Ca2+ entry pathway is not permeable to Ni2+ which acts as a blocker. This has been directly determined from electrical measurements of channel activity (Mayer & Westbrook, 1987; Zschauer et al. 1988). The ability of Ni2+ to block Ca2+ influx without entering the cell may also be inferred from the combination of two facts observed in endothelial cells (Hallam et al. 1988 a), platelets (Merritt & Hallam, 1988) or neutrophils (Merritt et al. 1989). (1) Agonist stimulation of these cells in the presence of Ni2+ does not cause a quench of Fura-2 even though Ni2+ is capable of quenching Fura-2. (2) In the presence of Ni2+ and Ca2+, the [Ca2+]i response to agonist stimulation is the same as that observed in the absence of Ca2+ (and Ni2+). There are two important reservations about the use of Mn2+ to detect Ca2+ entry. The first reservation is that there may be receptor-mediated Ca2+ entry pathways that are impermeable to Mn2+. A clear example of this is that agonist-stimulated parotid cells do not take up Mn2+ into their cytoplasm even though they have a welldefined receptor-mediated Ca2+ entry (Merritt & Hallam, 1988). Also, rapid ,measurements of the Mn2+ quench and the rise of [Ca2+]i in thrombin-stimulated platelets (Sage et al. 1989) hint at the possibility of two Ca2+ entry pathways, only one of which is permeable to Mn2+. The second reservation is that Mn2+ may not follow an identical route to that of receptor-mediated Ca2+ entry. This is clearly a possibility in endothelial cells given the observation that stimulation by a low dose of histamine stimulates Mn2+ to enter the cytoplasm in an apparently non-pulsatile manner whilst [Ca2+], is repetitively spiking.

Correlation between Ca2+ and Mn2+ entry Bearing in mind these reservations about the use of Mn2+, there are four points of correlation between Mn2+ entry into the cytoplasm and Ca2+ entry into the internal

DIVALENT CATION ENTRY INTO ENDOTHELIAL CELLS

71

store. (1) In the presence of Mn2+ and the nominal absence of Ca2+, histamine induces a quench at a time when under normal circumstances (no Mn2+, 1 mM-Ca2+) it would induce a maintained elevation of [Ca2+], which is dependent on the presence of extracellular Ca2+. (2) If the internal store is discharged by prior exposure to histamine in Ca2+-free medium then the cell is in a state where Mn2+ can rapidly enter the cytoplasm (Fig. 2A). This correlates with the ability to refill the internal store by exposing the cell to extracellular Ca2 . (3) If the internal store is refilled after discharge then there is no stimulated Mn2+ entry (Fig. 2B); since the cell is back at its resting steady state after refilling, there is also no net Ca2+ uptake. It is conceivable that in the steady state there is still a large Ca2+ influx which is balanced by a large efflux. This is unlikely because it is energetically very wasteful. In pancreatic acinar cells, 45Ca uptake measurements show that this is not the case (Muallem, Schoeffield, Fimmel & Pandol, 1988). (4) Ni2+ blocks both the refilling and the stimulated Mn2+ entry (Figs 4 and 5). The fact that after discharge of the internal store, the same rate of Mn2+ entry is seen in the presence or absence of histamine (Fig. 3) suggests that there is only one route for Mn2+ entry that is controlled by the state of the internal store, and that there is no pathway that is directly coupled to the histamine receptor. The block by Ni2+ of the (albeit sometimes small) rise in [Ca2+]i during refilling is consistent with its proposed action of blocking Ca2+ entry. A similar result has been reported for the action of La3+ on pancreatic acinar cells (Pandol, Schoeffield, Fimmel & Muallem, 1987) and parotid acinar cells (Takemura & Putney, 1989). The small transient in Fig. 3 seen on the second exposure to histamine is a consistent feature (see also Fig. 5A) and may be related to the small fall in [Ca2+], that is often seen when histamine is removed after discharging the internal store in Ca2+-free solution (Figs 2 and 4). Perhaps the internal store Ca2+-ATPase has a slightly higher affinity for Ca2+ than does the plasmalemmal Ca2+-ATPase so that, on removal of histamine, the internal store takes up a small amount of Ca2+ from the cytoplasm thereby slightly lowering [Ca2+],.

Mn2+ entry during [Ca2+]i spiking Careful examination of the quench records obtained in presence of spiking [Ca2+],

does show some phasic variation in the 360 nm fluorescence that is synchronous with the spiking of [Ca2+]1. Two patterns of variation would correlate very nicely with the other experimental data. One pattern would be caused by a pulsatile entry of Mn2+ into the cytoplasm with each spike of [Ca2+],; this would give a stepwise fluorescence quench as shown in Fig. II A. Alternately, following the idea that the rate of Mn2+ entry is determined by the fullness of the internal store, the rate of quench should increase with each [Ca2+], spike (Fig. 1 lB) since in Ca2+-free solution each spike is associated with a further depletion of the store. A third pattern of variation (Fig. 11 C) is an artificial one caused by having the longer wavelength monochromator set to a wavelength that is slightly longer than the in vivo isoemissive wavelength of Fura-2. The small phasic variations in the 360 nm fluorescence seen in Fig. 6 and many of the other records closely resemble those illustrated in Fig. 11 C and cannot be identified as the patterns shown in Fig. llA and B. It is possible that there are small undetected phasic variations in quench rate of the type illustrated in Fig. I1B. In theory, these might be detected by comparing the patterns of quenching induced

R. JACOB

72 A

a)

_

--

L_

B

Time

B

Time

N

2c E

Q

Co

OE

o~ c-

\

iv

Time

Fig. I11. Possible patterns of quenching when stimulating [Ca2+], spikes by applying a low dose of histamine to a cell in Ca2+ -free solution containing Mn2+. These diagrams assume the absence of a basal rate of quench and do not show the pseudo-exponential nature of the Mn2+ -induced quench. A, a stepwise quench due to a phasic influx of Mn2+ into the cytoplasm concomitant with the phasic influx of Ca2+ that generates the [Ca2+], spikes. B, a phasic increase in the rate of quench; the rate of Mn 2+ influx is inversely related to the fullness of the internal Ca2+ store and, with each spike, the rate of quench increases because the store becomes progressively more depleted. C, a pattern of quench due to the wavelength (nominally 360 nm) being slightly longer than the in vivo isoemissive wavelength.

DIVALENT CATION ENTRY INTO ENDOTHELIAL CELLS

73

by high and low doses of histamine; in practice the variability of the rates of quench in different cells precludes comparison between cells. The correlation between Mn2+ and Ca2+ entry appears to be contradicted by the uniform entry of Mn2+ seen when [Ca2+], is spiking in response to a low dose of histamine (Fig. 6). However, this observation only implies that the pathway for the entry of the two ions into the cytoplasm is different. It does not contradict the hypothesis that in part the two ions share a common entry pathway and that this common part includes the point at which the state of the internal store controls its refilling by Ca2+.

Refilling of the internal store The refilling of the internal store by exposure to extracellular Ca2+ is rapid, being complete within 90 s (Fig. 8). Similarly rapid refilling is seen in parotid acinar cells, which can be refilled by a 2 min exposure to Ca21 (Aub, McKinney & Putney, 1982; Merritt & Rink, 1987), and in gastric parietal cells, which are half-filled within 90 s (Negulescu & Machen, 1988). If the control of Mn2+ entry is to reflect a control mechanism for regulating the filling of the internal store then the influx of Mn2+ must approach zero only when the store is full. This is borne out by the result in Fig. 9. The rate of quench is not necessarily a linear measure of Mn2+ entry since the degree of quench is determined by competitive binding of Mn2+ and Ca2+ to Fura-2. Also, the normalized peak height elicited by exposure to histamine is unlikely to be a linear measure of the fullness of the internal store. Thus, no conclusions can be drawn from the fortuitous linear relationship between the two parameters. The fact that the same relationship is obtained irrespective of whether it is determined by fully depleting the store and then partly refilling it (Fig. 7) or by partly depleting the store with no repletion (Fig. 10) implies that it is the fullness of the internal store which determines the rate of Mn2+ entry; the relationship is not a consequence of some slow inhibition of a divalent cation entry pathway by exposure to millimolar concentrations of extracellular Ca2+. Figure 9 could have been plotted using normalized peak [Ca2+]i or normalized peak changes in pCa. Such measures would not be any more likely to be linearly related to the fullness of the internal store than the one that I have used. But, using changes in [Ca2+]i or pCa as a measure would have the disadvantage of introducing a nonlinear transformation of the experimentally determined variable which would distort the scatter of the points. Control of Ca2+ entry In many types of cell, the unresolved issue relating to agonist-stimulated Ca2+ entry is the nature of its control. Intimately connected with this issue is the route by which Ca2+ enters the cytoplasm; does it cross the plasma membrane directly into the cytoplasm or does it cross directly into an internal store, thence to be released into the cytoplasm? For some cells the issue has been resolved electrophysiologically. For example, both the ATP-sensitive channel in smooth muscle cells (Benham & Tsien, 1987) and the insulin-like growth factor II-sensitive channels in BALB/c 3T3 cells (Matsunaga et al. 1988) are only stimulated in a whole-cell-attached patch if the

R. JACOB

74

agonist is present in the patch pipette and not if the agonist is outside the pipette; this demonstrates a very close coupling of the receptor to the channel. This cannot be the case with endothelial cells since the stimulated Mn2+ influx is seen after removal of the agonist. B Ca2+ Mn2+ A

P3 'P3~~~~~~~~~~~~~~~~~~~~~~~Y Ca2+ Mn2+

Ca2a Mn2+ \

Ca2+ Fig. 12. Three minimal models of the control of divalent cation entry into endothelial cells. The dashed lines indicate modulation pathways and a circle with a tangential arrow indicates a Ca2+-ATPase. A, Ca2+ and Mn2+ enter the cytoplasm directly from outside the cell. The store exerts its control of divalent cation entry by a soluble second messenger. B, an extension of the model of Merritt & Rink (1987) in which Ca2+ enters directly into the internal store from outside the cell. Ca2+ can leave the store either by the IP3modulated channel or by a leak pathway. Mn2+ can only leave by the leak pathway. A rise in [Ca2+] within the store inactivates the Ca2+ (and Mn2+) entry. C, the 'capacitative' model of Putney (1986). Ca2+ and Mn2+ enter into a restricted space between the plasmalemma and the internal store. Ca2+ can be pumped from this space into the internal store. A rise in [Ca2+] within the restricted space between the plasma membrane and the store inactivates the Ca2+ entry.

One unlikely possibility for endothelial cells is that the divalent cation entry is controlled by a second messenger whose production is directly stimulated by agonist. This second messenger would have to decay very slowly on removal of the agonist but then rapidly on refilling of the internal store. In some cases inositol 1,4,5trisphosphate (JP3) itself may directly stimulate Ca2+ entry (e.g. Kuno & Gardner, 1987) and in some cases IP4 may be involved in the control of Ca2+ entry (e.g. Irvine & Moor, 1986; Morris et al. 1987) but the metabolism of these inositol phosphates is unlikely to be slow and, in pancreatic acinar cells, IP3 and 1P4 levels decay with halftimes of 8 and 43 s respectively (Hughes, Takemura & Putney, 1988). The other possibility for endothelial cells is that the influx is directly controlled by the fullness of the internal store. Three classes of model for this are illustrated in Fig. 12. In Fig. 12A, the internal store is remote from the plasma membrane so that it must refill from the cytoplasm and it must exert its control on divalent cation entry via a soluble second messenger. This messenger cannot be Ca2+ because Mn2+ entry

DIVALENT CATION ENTRY INTO ENDOTHELIAL CELLS

75

is seen when [Ca2+], is at its basal value (e.g. Fig. 3). The ability of some cells to refill with only a marginal concomitant increase in [Ca2+], would be explained by a steep activation curve for the store Ca2+-ATPase coupled with a high capacity of the ATPase to pump Ca2+ relative to the capacity of the second messenger-operated channels to conduct Ca2+. This model might also require a Ca2+ leak pathway from the internal store to account for the fact that after removal of agonist in the absence of extracellular Ca2+, the [Ca2+], is not pumped down to a very low level. The model in Fig. 12B is an extension of that proposed by Merritt & Rink (1987) for parotid acinar cells. Ca2+ can enter the internal store directly from outside the cell through a gap junction-like structure which is inactivated by high [Ca2+] within the store. Ca2+ can leave the store either by the IP3-sensitive channel or by a leak pathway that is also permeable to Mn2+. This model clearly accounts for the smallness of the refilling transient seen in some cells but would need a modification, such as the modulation of one of the channels out of the store by the [Ca2+] within the store, to account for the significant refilling transients seen in other cells. A model which is intermediate to these two models is illustrated in Fig. 12C; it is the 'capacitative' model proposed by Putney (1986), also for parotid cells. Ca2+ enters a restricted space lying between the internal store and the plasma membrane through a channel that is inactivated by high [Ca2+] and that is permeable to Mn2+. From this space Ca2+ is taken up into the internal store by a Ca2+-ATPase. The store indirectly controls the channel by its ability to take up Ca2+ from the restricted space; when the store is empty it maintains a low [Ca2+] in the restricted space, but when it is full, the [Ca2+] in this space rises to inactivate the channel. According to this model, the appearance of a refilling transient would depend on the relative capacities of the channels and the ATPase. In each of these three models, Mn2+ passes through the point at which the state of the internal store would exert its control on refilling by Ca2+. Each of these three models could account for cells in which a refilling transient (either small or large) is seen because in each case there is a leak pathway, either direct or indirect, for Ca2+ into the cytoplasm. Consequently, each model could account for the block of the refilling transient by di- or trivalent cations (e.g. Ni2+ or La3+) so that, contrary to the suggestion of Pandol et al. (1987), the block does not imply that the internal store must refill from the cytoplasm. One possible problem with these models is that if Mn2+ influx depends on the fullness of the internal store, why does the rate of quench seen in the presence of [Ca2+], spiking not increase with each [Ca2+], spike (Fig. 11B) ? It could be that only a small fraction of the store is released during the production of a single spike, as suggested by the results of Jacob et al. (1988). Alternatively, the effect of the store fullness may be moderated by a delay between a change in the fullness of the store and a change in the activation of the channel that passes the Mn2+. Such a delay could be introduced in the models in Fig. 12A and C by the time required to effect a change in the second messenger concentration or a change in [Ca2+] in the restricted space between store and plasma membrane. These models are minimal models presented to clarify possible relationships between Ca2+ and Mn2+ entry. The actual situation may be more complex, perhaps, for example, involving more than one internal store. In conclusion, studying the Mn2+ quench is a powerful tool in investigating Ca2+

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entry in endothelial cells. Although Mn2+ enters the cytoplasm by a different route to Ca2+, it appears to pass through the point at which Ca2+ entry into the internal store is modulated and has provided information on the manner in which the internal store controls Ca2+ entry. However, it is not possible to use the data to resolve the route by which Ca2+ (or Mn2+) enters the cell, although a study of the kinetics of entry of these two ions may supply the answer. I would like to thank Drs Chris Benham, Janet Merritt, Stuart Sage and Tim Rink and Professor Tom Bolton for much helpful discussion and, in particular, Dr Trevor Hallam for extensive discussion and advice. I would also like to thank Messrs Colin Burns, David Harrison and Allan Long for technical support, and Dr Jeremy Pearson and Mr Tom Carter for supplying endothelial cells. REFERENCES

AUB, D. L., McKINNEY, J. S. & PUTNEY, J. W. JR. (1982). Nature of the receptor-regulated calcium pool in the rat parotid gland. Journal of Physiology 331, 557-565. BENHAM, C. D. (1988). Ca entry through ATP-activated channels elevates cytoplasmic Ca in single smooth muscle cells from rabbit ear artery. Journal of Physiology 407, 92P. BENHAM, C. D. & TSIEN, R. W. (1987). A novel receptor-operated Ca2+-permeable channel activated by ATP in smooth muscle. Nature 328, 275-278. DAWSON, R. M. C., ELLIOTT, D. C., ELLIOTT, W. H. & JONES, K. M. (1986). Data for Biochemical Research, chap. 17, third edn, pp. 399-415. Clarendon Press, Oxford. GRYNKIEWICZ, G., POENIE, M. & TSIEN, R. Y. (1985). A new generation of Ca2+ indicators with greatly improved fluorescence properties. Journal of Biological Chemistry 260, 3440-3450. HALLAM, T. J., JACOB, R. & MERRITT, J. E. (1988a). Evidence that agonists stimulate bivalentcation influx into human endothelial cells. Biochemical Journal 255, 179-184. HALLAM, T. J., JACOB, R. & MERRITT, J. E. (1989a). Influx of bivalent cations can be independent of receptor stimulation in human endothelial cells. Biochemical Journal 259, 125-129. HALLAM, T. J., JACOB, R. & MERRITT, J. E. (1989b). Nickel blocks store refilling and stimulated divalent cation entry in single cultured human umbilical vein endothelial cells. Journal of Physiology 410, 49P. HALLAM, T. J., PEARSON, J. D. & NEEDHAM, L. A. (1988b). Thrombin-stimulated elevation of human endothelial-cell cytoplasmic free calcium concentration causes prostacyclin production. Biochemical Journal 251, 243-249. HALLAM, T. J. & RINK, T. J. (1985). Agonists stimulate divalent cation channels in the plasma membrane of human platelets. FEBS Letters 186, 175-179. HUGHES, A. R., TAKEMURA, H. & PUTNEY, J. W. JR. (1988). Kinetics of inositol 1,4,5-trisphosphate and inositol cyclic 1: 2,4,5-trisphosphate metabolism in intact rat parotid acinar cells. Journal of Biological Chemistry 263, 10314-10319. IRVINE, R. F. & MOOR, R. M. (1986). Micro-injection of inositol 1,3,4,5-tetrakisphosphate activates sea urchin eggs by a mechanism dependent on external Ca2+. Biochemical Journal 240, 917-920. JACOB, R., MERRITT, J. E., HALLAM, T. J. & RINK, T. J. (1988). Repetitive spikes in cytoplasmic calcium evoked by histamine in human endothelial cells. Nature 335, 4045. JACOB, R., MERRITT, J. E., RINK, T. J. & HALLAM, T. J. (1989). Repetitive spiking of cytoplasmic calcium in human endothelial cells. Biochemical Society Transactions 17, 92. JOHANSSON, H., LARSSON, R., WALLFELT, C., RASTAD, J., AKERSTROM, G. & GYLFE, E. (1988). Calcium-agonistic action of Mn2+ in the parathyroid cell. Molecular and Cellular Endocrinology 59, 77-82. KUNO, M. & GARDNER, P. (1987). Ion channels activated by inositol 1,4,5-trisphosphate in plasma membrane of human T-lymphocytes. Nature 326, 301-304. Lo, W. W. Y. & FAN, T.-P. D. (1987). Histamine stimulates inositol phosphate accumulation via the H1-receptor in cultured human endothelial cells. Biochemical and Biophysical Research Communications 148, 47-53. MATSUNAGA, H., NISHIMOTO, I., KOJIMA, I., YAMASHITA, N., KUROKAWA, K. & OGATA, E. (1988).

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