Voltage-Dependent, Pertussis Toxin Insensitive Inhibition of Calcium Currents by Histamine in Bovine Adrenal Chromaffin Cells

Kevin P. M. Currie and Aaron P. Fox

Department of Neurobiology, Pharmacology and Physiology, The University of Chicago, Chicago, Illinois 60637


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Currie, Kevin P. M. and Aaron P. Fox. Voltage-Dependent, Pertussis Toxin Insensitive Inhibition of Calcium Currents by Histamine in Bovine Adrenal Chromaffin Cells. J. Neurophysiol. 83: 1435-1442, 2000. Histamine is a known secretagogue in adrenal chromaffin cells. Activation of G-protein linked H1 receptors stimulates phospholipase C, which generates inositol trisphosphate leading to release of intracellular calcium stores and stimulation of calcium influx through store operated and other channels. This calcium leads to the release of catecholamines. In chromaffin cells, the main physiological trigger for catecholamine release is calcium influx through voltage-gated calcium channels (ICa). Therefore, these channels are important targets for the regulation of secretion. In particular N- and P/Q-type ICa are subject to inhibition by transmitter/hormone receptor activation of heterotrimeric G-proteins. However, the direct effect of histamine on ICa in chromaffin cells is unknown. This paper reports that histamine inhibited ICa in cultured bovine adrenal chromaffin cells and this response was blocked by the H1 antagonist mepyramine. With high levels of calcium buffering in the patch pipette solution (10 mM EGTA), histamine slowed the activation kinetics and inhibited the amplitude of ICa. A conditioning prepulse to +100 mV reversed the kinetic slowing and partially relieved the inhibition. These features are characteristic of a membrane delimited, voltage-dependent pathway which is thought to involve direct binding of G-protein beta gamma subunits to the Ca channels. However, unlike virtually every other example of this type of inhibition, the response to histamine was not blocked by pretreating the cells with pertussis toxin (PTX). The voltage-dependent, PTX insensitive inhibition produced by histamine was modest compared with the PTX sensitive inhibition produced by ATP (28% vs. 53%). When histamine and ATP were applied concomitantly there was no additivity of the inhibition beyond that produced by ATP alone (even though the agonists appear to activate distinct G-proteins) suggesting that the inhibition produced by ATP is maximal. When experiments were carried out under conditions of low levels of calcium buffering in the patch pipette solution (0.1 mM EGTA), histamine inhibited ICa in some cells using an entirely voltage insensitive pathway. We demonstrate that activation of PTX insensitive G-proteins (most likely Gq) by H1 receptors inhibits ICa. This may represent a mechanism by which histamine exerts inhibitory (in addition to previously identified stimulatory) effects on catecholamine release.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The role of histamine as a secretagogue in adrenal chromaffin cells is well documented. Chromaffin cells express high affinity H1 histaminergic receptors (Chang et al. 1979) which generally couple to the pertussis toxin (PTX) insensitive Gq type G-protein (Hill et al. 1997). In chromaffin cells H1 receptor activation leads to the generation of inositol trisphosphate (IP3), release of intracellular calcium stores, and calcium influx through store operated and other channels (Bunn and Boyd 1992; Cheek et al. 1993, 1994; Noble et al. 1988; Zerbes et al. 1998). Sustained elevation of intracellular calcium ([Ca2+]i) and catecholamine release is dependent on this influx of extracellular calcium. It has been suggested that voltage-gated calcium channels (ICa) contribute to the calcium influx (O'Farrell and Marley 1999) but this may be a secondary consequence caused by depolarization of the cells. The direct effects (if any) of histamine on ICa in chromaffin cells have not been studied.

Physiologically, calcium influx through ICa is the main trigger for catecholamine release from chromaffin cells (Boarder et al. 1987). On stimulation, the preganglionic sympathetic neurons of the splanchnic nerve release acetylcholine (ACh), which activates nicotinic ACh receptors, depolarizes the cell and thereby activates ICa (Douglas 1968). Regulation of ICa is an important mechanism for the control of catecholamine secretion.

One major example of ICa regulation is the inhibition of N-type channels via activation of heterotrimeric G-proteins by seven transmembrane receptors. There are several distinct pathways by which the channels are inhibited. One of these pathways, which is only apparent under conditions of low intracellular calcium buffering and often utilizes PTX insensitive G-proteins, is thought to involve an unidentified diffusible second messenger (for review see Hille 1994). A different inhibitory pathway is membrane delimited and thought to involve direct coupling of the G-protein with the channel (for reviews see Dolphin 1995; Hille 1994; Ikeda and Dunlap 1999; Zamponi and Snutch 1998). In addition to suppressing the peak amplitude of the current, this type of inhibition is characterized by a slowing of the activation kinetics of ICa. The kinetic slowing and most of the current suppression are relieved at very depolarized potentials (Bean 1989; Elmslie et al. 1990; Penington et al. 1991). These features have been incorporated into models in which the channels exist in two functional gating states, one in the absence (willing) and one in the presence (reluctant) of inhibition (Bean 1989; Boland and Bean 1993; Elmslie et al. 1990; Golard and Siegelbaum 1993). P/Q-type ICa are inhibited by the same mechanisms but to a lesser extent in chromaffin cells, neurons, and recombinant systems (Bourinet et al. 1996; Currie and Fox 1997; Mintz and Bean 1993; Roche and Treistman 1998; Zhang et al. 1996).

At present, the voltage-dependent inhibition of ICa is thought to result from direct binding of the G-protein beta gamma subunit to the channel (De Waard et al. 1997; Herlitze et al. 1996; Ikeda 1996; Zamponi et al. 1997). In virtually every case this inhibition can be prevented or attenuated by pretreatment of the cells with PTX which ADP ribosylates the alpha  subunit of Gi/Go type G-proteins and disrupts the coupling to neurotransmitter/hormone receptors (Gierschik 1992). This suggests that there is a preferential coupling between PTX sensitive G-proteins and N-type Ca channels, although the reasons why this should be the case are not clear. There are a few exceptions found in the literature where voltage-dependent inhibition is mediated by PTX insensitive G-proteins including LHRH and ATP inhibition of ICa in frog sympathetic ganglion neurons (Elmslie 1992) and VIP inhibition of ICa in rat sympathetic ganglion neurons (Zhu and Ikeda 1994).

In chromaffin cells ICa is inhibited in a voltage-dependent manner by several transmitters including ATP, opioids, and GABA (Albillos et al. 1996; Currie and Fox 1996; Doroshenko and Neher 1991; Gandia et al. 1993; Kleppisch et al. 1992). The inhibition produced by all of these transmitters is blocked by pretreatment of the cells with PTX. However, we recently reported that PGE2 also inhibits ICa in chromaffin cells but that this inhibition is only partially blocked by PTX (Currie and Fox 1998). In this paper we report that histamine, which likely couples to the PTX insensitive G-protein Gq, inhibits ICa in chromaffin cells in a PTX-insensitive fashion. This outlines a previously unidentified mechanism by which histamine may participate in the autocrine/paracrine regulation of catecholamine release.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Culture of cells

Chromaffin cells were prepared by digestion of bovine adrenal glands with collagenase and purified by density gradient centrifugation as previously described (Artalejo et al. 1992a). The cells were plated on collagen-coated glass coverslips in 35 mm tissue culture dishes (2 ml of cell suspension; 0.15-0.3 × 106 cells/ml) and maintained in an incubator at 37°C in an atmosphere of 92.5% air and 7.5% CO2 with a relative humidity of 90%. Fibroblasts were effectively suppressed with cytosine-arabinoside (10 µM), leaving relatively pure chromaffin cell cultures. Although mixed, the cultures were somewhat enriched for epinephrine containing over norepinephrine containing cells. One-half of the incubation medium was exchanged every day. This medium consisted of DMEM/F12 (1:1, Gibco) supplemented with fetal bovine serum (10%), glutamine (2 mM), penicillin/streptomycin (100 unit ml-1/100 µg ml-1), cytosine arabinoside (10 µM), and 5-fluorodeoxyuridine (10 µM).

Electrophysiology

Chromaffin cells were voltage clamped in the whole cell configuration of the patch clamp technique (Hamill et al. 1981) using an Axopatch 1C amplifier (Axon Instruments) at a holding potential of -80 mV and ICa were activated by step depolarizations. Current voltage curves were generated by voltage ramps of 100 ms duration from the holding potential (-80 mV) to +100 mV. Peak inward calcium channel currents were activated at approximately +20 mV. Leak currents were generated by averaging 16 hyperpolarizing sweeps (steps or ramps). All the data reported in this paper were capacitance and leak subtracted. The data were filtered at 2 kHz and then digitized at 100 µs/point. Series resistance was partially compensated (approx 80%) using the series resistance compensation circuit of the Axopatch-1C amplifier. Electrodes were pulled from microhaematocrit capillary tubes (Drummond) and coated with sylgard (Dow Corning). After fire polishing, final electrode resistance when filled with the CsCl based patch pipette solution (see Solutions) was ~1.5-2.5 MOmega . Voltage protocols and data analysis were carried out in AxoBasic. Data are reported as mean ± SE and statistical significance was determined using paired or independent Student's t-test. All recording was performed at room temperature (~23°C)

Solutions

Electrodes were filled with solution composed of (in mM) 110 CsCl, 4 MgCl2, 20 HEPES, 10 EGTA, 0.35 GTP, 4 ATP, and 14 creatine phosphate (pH = 7.3 adjusted by CsOH; osmolarity ~310 mOsm). Note that in some experiments (Fig. 4) the patch pipette solution contained 0.1 mM EGTA. The NaCl based extracellular recording medium contained 140 mM NaCl, 2 mM KCl, 10 mM glucose, 10 mM HEPES, 10 mM CaCl2, and 0.3-1.0 µM tetrodotoxin (TTX) (pH = 7.3 adjusted with NaOH, osmolarity ~310 mOsm). In a few experiments the TTX was omitted. Nisoldipine was prepared as a stock solution (10 mM) in ethanol and stored, protected from light, at -20°C. It was added to all extracellular solutions (1 µM) to block any facilitation ICa (L-type) present. PGE2 (Calbiochem) was prepared as a stock solution in DMSO and aliquots frozen. Final dilutions yielded DMSO concentrations of <0.03% which had no effect on the currents by itself. omega -Conotoxin GVIA (Alomone Labs) was diluted in sterile H2O and aliquots (300 µM) frozen until use. ATP (Boehringer Mannheim), histamine, and reactive blue-2 (RBI) were prepared as stock solutions in sterile H2O daily and diluted to final concentrations in recording medium immediately before use. Pertussis toxin (RBI) was prepared as a stock solution in sterile H2O and stored at 4°C until dilution in culture medium before use.

The recording bath was <1 cm in diameter with a volume of ~250-350 µl. The bath solution was gravity fed from reservoirs at a flow rate of 3-4 ml/min which ensured efficient perfusion of the recording chamber. Agonists and antagonists were applied to the cells by including them in the recording solution and washing them into the bath. There was a latency between switching solutions at the reservoirs and the drugs reaching the cell as a result of "dead space" in the tubing leading to the bath. This accounted for most of the delay seen between agonist application and inhibition of ICa. Cgtx (35-50 µL) was added directly to the bath, with the flow of extracellular solution stopped, at 10 times the desired final concentration. Thus Cgtx was added at 10 µM to give a final concentration of ~1 µM.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Histamine inhibited ICa in a voltage-dependent manner

In these experiments, histamine was applied by continuous perfusion through the recording chamber. In most cells tested (59 of 64) histamine produced an inhibition of ICa like that shown in Fig. 1. In this experiment, about one quarter of the whole cell ICa was inhibited by a supramaximal dose of histamine (300 µM). Histamine (300 µM) suppressed the peak amplitude of ICa by 28 ± 1.5% (n = 31, see Fig. 1) and slowed the activation kinetics. A 50 ms prepulse to +100 mV relieved 71 ± 4% (n = 9) of the inhibition of ICa amplitude and reversed the kinetic slowing (Fig. 1B). The slowing of activation kinetics by histamine and the voltage-dependent reversal of its inhibition are characteristic of direct G-protein mediated inhibition of ICa (Bean 1989; Elmslie et al. 1990; Penington et al. 1991). In some cells, prolonged application of histamine (>30 s) resulted in a desensitization of the response which was also seen with repeated applications in some cells.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1. Histamine inhibits ICa in a voltage-dependent manner. A: 3 superimposed current records are shown. Currents were activated by step depolarization from -80 mV to +20 mV before application of histamine (control), during application of 300 µM histamine (histamine), and in the continued presence of histamine preceded by a 50-ms prepulse to +100 mV (histamine + prepulse). B: plots mean data from 9 cells like the one shown above. Peak current amplitude was normalized to controls (before histamine application).

Pharmacology of histamine response

Because prolonged application of histamine produced a desensitization of the histamine response, cells were first exposed to a brief application of histamine (100 nM-100 µM) and then exposed a second time to 300 µM histamine to determine the dose response curve for inhibition of ICa. Exposing cells to two brief histamine applications reduced possible complications from desensitization. The dose response curve (Fig. 2A) yielded an EC50 of 585 nM and a maximal inhibition of 28%.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2. Inhibition by histamine is mediated by H1 histaminergic receptors. A: percentage inhibition of ICa is plotted against the log10 of the histamine concentration. Mean inhibition produced by each concentration is shown with number of cells and SE bars. Also shown is a sigmoidal fit to data which yielded an EC50 of 585 nM and a maximal inhibition of 28%. B: mean percentage inhibition of ICa produced by 10 µM histamine in control conditions or in the presence of 1 µM mepyramine, a H1 receptor antagonist. Mepyramine blocked the action of histamine.

Activation of H1 histaminergic receptors is known to elicit release of intracellular calcium stores and to trigger catecholamine release in chromaffin cells (Bunn and Boyd 1992; Noble et al. 1988). To determine if these receptors were responsible for coupling to ICa, histamine was applied to cells either in the absence or presence of mepyramine (1 µM), a selective H1 receptor antagonist (Fig. 2B). In the absence of mepyramine 300 µM histamine produced an inhibition of 26 ± 3.5% (n = 4) and in the presence of mepyramine the response to histamine was abolished (1 ± 0.7%, n = 4; P < 0.001).

ATP, which is stored at high concentrations in chromaffin cell secretory granules, is coreleased with catecholamines and causes a negative feedback inhibition of ICa through activation of P2Y purinergic receptors (Currie and Fox 1996). Thus exposing cells to histamine, a known secretagogue, likely results in the release of ATP from surrounding cells in the recording chamber which may lead to the inhibition of ICa. This was unlikely because the recording chamber was continuously perfused with fresh medium which should have washed away any ATP released from the surrounding chromaffin cells before it could concentrate sufficiently to inhibit ICa (see Currie and Fox 1996). However, to rule out involvement of ATP in the inhibition, histamine was applied to chromaffin cells in the presence of reactive blue-2 (RB-2), a purinergic receptor antagonist, which completely inhibits the ATP-mediated inhibition of ICa. Before application of RB-2, histamine inhibited ICa by 23 ± 0.9% and in the presence of RB-2 the inhibition was not significantly different at 29 ± 3.3% (n = 5). Hence the inhibition produced by histamine was not caused by the release of ATP from surrounding cells in the recording bath.

Inhibition was not blocked by pertussis toxin treatment

Neurotransmitters and hormones inhibit ICa in neurons (and other cells) using several pathways (Hille 1994). One of these pathways is thought to involve direct binding of the G-protein beta gamma subunits to the channel producing a voltage-dependent inhibition and kinetic slowing. Virtually every example of this type of voltage-dependent inhibition can be blocked by pretreating cells with pertussis toxin (PTX) which selectively ADP-ribosylates Gi/Go type G-proteins and prevents their coupling to and activation by neurotransmitter/hormone receptors.

To determine the type of G-proteins involved in the inhibition of ICa produced by histamine, cells were incubated for 18-24 h with 300 ngml-1 PTX. Control cells were from the same cultures and were recorded from on the same days as PTX treated cells. The inhibition produced by 300 µM histamine was not significantly different in PTX treated cells compared with control cells (26 ± 2.7%, n = 12 and 20 ± 3%, n = 11, respectively; Fig. 3). As a positive control for the action of PTX, the cells were also tested with ATP (100 µM) which we have shown previously to inhibit ICa in a PTX sensitive fashion (Currie and Fox 1996). In control cells ATP inhibited ICa by 50 ± 1.9% (n = 12) and in PTX treated cells by 3 ± 0.7% (n = 14; Fig. 3). Therefore the H1 receptors use a PTX insensitive G-protein as part of the signaling pathway that inhibits calcium channels in chromaffin cells.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 3. Pertussis toxin pretreatment does not block the inhibition produced by histamine. Control cells are shown with open bars and cells pretreated for 18-24 h with 300 ngml-1 pertussis toxin (PTX) are shown with filled bars. ATP (100 µM) is known to inhibit ICa in a PTX sensitive manner and was therefore used as a control for the action of PTX. The inhibition produced by ATP was virtually abolished in PTX treated cells. In contrast the inhibition produced by histamine (300 µM) was not significantly different in PTX treated cells compared with control cells.

Voltage-independent inhibition of ICa in cells recorded using low intracellular EGTA

Hille and colleagues (1994) have demonstrated the existence of at least 5 pathways by which neurotransmitters and hormones can modulate ICa in rat sympathetic ganglion neurons. In addition to the fast, membrane delimited, voltage-dependent pathway like the inhibition described so far in this paper, there is a pathway which involves an unidentified diffusible messenger (Beech et al. 1991, 1992; Bernheim et al. 1991); this pathway is voltage-independent and involves PTX insensitive G-proteins. It is only seen under conditions of low intracellular calcium buffering. All the data presented above were recorded with high intracellular calcium buffering (10 mM EGTA) in the patch pipette solution. To determine whether this second pathway could be observed in chromaffin cells some experiments were performed using a patch pipette solution in which the concentration of EGTA was reduced to 0.1 mM.

Under these recording conditions histamine (300 µM) inhibited ICa in all 11 cells tested (average inhibition was 35 ± 6%, n = 11). Although this was not significantly greater than the inhibition reported in cells recorded using 10 mM EGTA, the voltage dependence of the inhibition was significantly different. Of the 11 cells tested only 7 exhibited voltage-dependent inhibition as illustrated in the example shown in Fig. 4A. In the presence of histamine the current amplitude was suppressed and the activation kinetics were slowed. A 50-ms prepulse to +100 mV relieved ~30% of the inhibition and reversed the kinetic slowing. In these seven cells the mean inhibition produced by histamine (300 µM) was 27 ± 5% and a prepulse relieved 34 ± 5% of this inhibition. In the remaining four cells ICa was inhibited by histamine in a voltage-independent fashion as illustrated in the example shown in Fig. 4B. In this cell the current amplitude was inhibited by ~70% but there was no slowing of activation kinetics and a prepulse produced no relief of the inhibition. The mean inhibition in these four cells was 49 ± 13%. The onset of this voltage independent inhibition and its washout was rather slow (Fig. 4C), which is consistent with the involvement of a diffusible messenger as suggested previously in sympathetic ganglion neurons (Beech et al. 1991, 1992; Bernheim et al. 1991).



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 4. Other inhibitory pathways are apparent under conditions of low intracellular Ca2+ buffering. In these experiments alone the patch pipette solution contained lowered levels of Ca2+ chelator (0.1 mM EGTA). A: representative traces from a cell which exhibited voltage-dependent relief from inhibition. Three superimposed current traces recorded in the absence (control) or in the presence of 300 µM histamine or in the presence of histamine but with a depolarizing prepulse which preceeded the test pulse. Note that kinetic slowing and prepulse relief of inhibition were evident. B: representative traces from another cell which exhibited no voltage-dependent relief from inhibition. Three superimposed currents recorded in the absence (control) or in the presence of histamine or in the presence of histamine but with a depolarizing prepulse which preceeded the test pulse. Note in this cell there was no kinetic slowing and the prepulse did not relieve the inhibition. C: plots peak current amplitude, for step depolarizations to +20 mV, as a function of time from the same cell as shown in B. Application of test-pulses preceded by prepulses are labeled with "p". Note the slow onset of inhibition and slow, incomplete washout.

Both N- and P/Q-type ICa are inhibited

The remaining experiments were all performed using 10 mM EGTA in the patch pipette solution. As illustrated above (Fig. 3) the maximal inhibition produced by histamine is about one-half that of ATP. The calcium current in chromaffin cells is carried by three types of channels. The L-type channels, also called facilitation Ca channels, can be recruited by depolarizing prepulses similar to those used to relieve the G-protein inhibition of the N- and P/Q-type channels (Artalejo et al. 1992b; Fenwick et al. 1982; Hoshi et al. 1984) and so have been blocked by the inclusion of nisoldipine in all recording solutions. In the presence of nisoldipine the remaining current is approximately one-half N-type (blocked by 1 µM omega -conotoxin GVIA) and one-half P/Q type (blocked by 400 nM omega -Agatoxin IVA) (Currie and Fox 1996, 1997). Both the N- and P/Q type channels are inhibited by ATP in a PTX sensitive, voltage-dependent manner. One explanation for the difference in the magnitude of the inhibition produced by histamine could be that one or other of the channel types was selectively inhibited by the PTX insensitive G-proteins. Therefore we tested the ability of histamine to inhibit ICa before and after block of N-type channels by omega -conotoxin GVIA (omega -CgTx).

Consistent with our previous studies omega -CgTx (1-2 µM) blocked about one-half of the whole cell current (Fig. 5A). On average omega -CgTx blocked 49 ± 3.6% (n = 7) of ICa. After block with omega -CgTx, 300 µM histamine was still able to inhibit part of the remaining P/Q-type ICa (Fig. 5A). On average histamine inhibited the remaining (P/Q-type) current by 26 ± 3.3% (n = 7) (Fig. 5). In the presence of histamine activation kinetics of ICa were slowed; a conditioning prepulse relieved 44 ± 6% (n = 5) of the inhibition and reversed the kinetic slowing (not shown). Thus both channel types are inhibited by histamine.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 5. Both N- and P/Q-type currents are inhibited by histamine. A: peak amplitude of ICa is plotted against time. Open bar indicates the application of omega -conotoxin GVIA (1 µM)(CgTx) used to block the N-type current. Current remaining after block with CgTx (P/Q-type) was reversibly inhibited when histamine was washed through the bath as indicated by filled bar. B: 2 currents from cell in A are shown, one after application of CgTx (CgTx) and one after application of CgTx and in the presence of histamine. C: mean amplitude of ICa is normalized to control before application of CgTx. After block with CgTx about one-half the current remains (CgTx). This current was inhibited by ~26% by application of 300 µM histamine (CgTx + Hist). The dashed line is for illustrative purposes and represents the current amplitude before block with CgTx.

Additivity of the inhibition produced by histamine with that produced by ATP or PGE2

As shown in Fig. 3, ATP inhibits ICa by using a PTX sensitive G-protein whereas histamine uses a PTX insensitive G-protein. Activation of both receptor types at the same time could lead to a higher concentration of activated G-proteins in the vicinity of the channels. To see if the inhibition produced by the two pathways was additive, cells were exposed to a supramaximal dose of histamine (300 µM) followed immediately by histamine plus a supramaximal dose of ATP (100 µM). Histamine caused an inhibition of 21 ± 1.9% whereas histamine + ATP inhibited the current by 50 ± 2.4% (n = 6; P < 0.001; Fig. 6, A and C). The cells were washed and subsequently exposed to ATP alone which inhibited the current by 47 ± 2.6%. In a different group of cells the same experiment was repeated except the order of agonist application was reversed (Fig. 6, B and D). The mean inhibition produced by ATP was 53 ± 3.8% (n = 4) and by ATP + histamine was not significantly different at 54 ± 4.3% (n = 4). These results suggest the channels are maximally inhibited by ATP and are saturated by the PTX sensitive G-proteins activated by the P2Y receptors.



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 6. Inhibition produced by histamine is not additive with that produced by ATP. Mean percentage inhibition of ICa amplitude produced by histamine (300 µM), ATP (100 µM), or both agonists in combination is shown. A: cells were exposed to histamine alone and then histamine + ATP and the mean inhibition plotted. B: mean inhibition produced in cells exposed to ATP alone and then ATP + histamine. C: 3 superimposed current records from a cell shown in A are plotted before application of histamine (control), during application of histamine, and during application of histamine + ATP. D: 3 superimposed current records from a cell shown in B are plotted before application of ATP (control), during application of ATP, and during application of ATP + histamine. Inhibition produced by ATP was maximal because histamine produced no further suppression of ICa.

Prostaglandin E2 (PGE2) also inhibits ICa in chromaffin cells using both PTX sensitive and insensitive G-proteins (Currie and Fox 1998). Cells treated for 18-24 h with 300 ngml-1 PTX were exposed to histamine (300 µM) or histamine + PGE2 (300 nM; Fig. 7). The inhibition produced by histamine alone was 17 ± 4.6% and by histamine + PGE2 was significantly greater at 30 ± 5.1% (n = 5; P < 0.0001). The same was true if the order of agonist application was reversed so that PGE2 alone produced an inhibition of 22 ± 1.9% and PGE2 + histamine was 33 ± 2.9% (n = 6; P < 0.0001). This partial additivity suggests that the submaximal inhibition produced by PTX insensitive G-proteins activated either by histamine or by PGE2 is due, at least in part, to insufficient levels of activated G-protein in the vicinity of the channel.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 7. Inhibition of ICa produced by histamine and PGE2 in pertussis toxin treated cells is additive. All cells were pretreated for 18-24 h with 300 ngml-1 PTX. A: mean percentage inhibition of ICa produced by PGE2 (300 nM) and histamine (300 µM) alone or in combination is shown. In the 6 cells illustrated on the left, PGE2 was applied alone and then in combination with histamine. Bars on the right are from a different 5 cells in which the order of application was reversed (histamine alone then histamine + PGE2). Inhibition produced by the 2 agonists was additive regardless of order of application. B: 3 superimposed current records from the same cell illustrating additivity of inhibition produced by PGE2 and histamine. Currents were recorded before application of PGE2 (control), in the presence of PGE2 alone, or in the presence of PGE2 + histamine.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In chromaffin cells, activation of H1 histaminergic receptors leads to the generation of IP3, release of intracellular calcium stores, and calcium influx through both store operated and other less well defined channels which triggers catecholamine secretion (Bunn and Boyd 1992; Cheek et al. 1993, 1994; Noble et al. 1988; Zerbes et al. 1998). However, as with neurotransmission, the main physiological trigger for catecholamine release is calcium influx through ICa (Boarder et al. 1987). Ca2+ channels are important sites for the regulation of catecholamine release. In this paper we demonstrate that histamine inhibits ICa in adrenal chromaffin cells. In experiments with 10 mM EGTA in the patch pipette solution inhibition of ICa occurred in >90% of the cells tested and was blocked by mepyramine, indicating the involvement of H1 receptors. Histamine not only suppressed ICa amplitude but also slowed its activation. The kinetic slowing and most of the current suppression could be reversed by depolarizing prepulses, features which are characteristic of N- and P/Q-type ICa inhibition (Dolphin 1995; Hille 1994; Ikeda and Dunlap 1999; Zamponi and Snutch 1998). Although multiple pathways are known to cause inhibition of ICa, the voltage-dependent pathway is thought to result from a direct interaction between the activated G-protein beta gamma subunit complex and the alpha 1 subunit of the calcium channel (De Waard et al. 1997; Herlitze et al. 1996; Ikeda 1996; Zamponi et al. 1997).

Modulation of ICa by transmitters is a widespread phenomenon. In adrenal chromaffin cells a variety of transmitters inhibit ICa including ATP, dopamine, opioids, and GABA (Bigornia et al. 1990; Doroshenko and Neher 1991; Gandia et al. 1993; Kleppisch et al. 1992). However, the inhibition produced by histamine reported in this paper is unique in that it is not mediated by PTX sensitive G-proteins. In virtually every case, voltage-dependent inhibition is blocked or substantially diminished by pretreatment of the cells with PTX. There are few instances in the literature where the voltage-dependent inhibition has been reported to be PTX insensitive; these include VIP inhibition in rat sympathetic ganglion neurons (Zhu and Ikeda 1994) and LHRH and ATP inhibition of ICa in frog sympathetic ganglion neurons (Elmslie 1992). As a positive control for the action of PTX we showed the inhibition of ICa produced by ATP, which is known to be PTX sensitive in chromaffin cells (Currie and Fox 1996; Gandia et al. 1993), was abolished in the same cells that responded to histamine. Typically H1 receptors couple to the PTX-insensitive Gq type G-protein (Hill et al. 1997) and in chromaffin cells the elevation of intracellular calcium and catecholamine release are not blocked by PTX (Bunn and Boyd 1992). It seems likely that the same PTX insensitive G-protein (most likely Gq) couples to ICa.

Because histamine can initiate secretion from chromaffin cells we were concerned that the inhibition of ICa could be secondary to the release of other transmitters such as ATP or catecholamines from the chromaffin cells. In a previous study (Currie and Fox 1996) we showed that when chromaffin cells in the bath were stimulated (using tetraethylammonium chloride to block K+ channels and depolarize the cells) they released sufficient ATP to suppress ICa. However, the flow of solution through the recording chamber had to be stopped to allow ATP to accumulate. If the solution through the bath was kept flowing, as in the experiments reported in this paper, then the inhibitor never accumulated sufficiently to suppress ICa. Furthermore, RB2, a purinergic receptor antagonist, blocked the "endogenous inhibition" produced by ATP. In this study RB2 had no significant effect on the inhibition produced by histamine. The PTX data also rule out an involvement of locally released ATP in producing the inhibition because this inhibition is mediated by PTX-sensitive G-proteins.

Hille and colleagues have identified a number of different pathways by which neurotransmitters inhibit ICa in rat sympathetic ganglion neurons (reviewed by Hille 1994). In addition to the membrane delimited, voltage-dependent pathway that is typically sensitive to PTX there is another pathway that typically uses PTX insensitive G-proteins. This pathway is not voltage-dependent, is blocked by higher concentrations of intracellular calcium buffers, and involves an unidentified diffusible second messenger (Beech et al. 1991, 1992; Bernheim et al. 1991). Our results indicate the presence of a second inhibitory pathway in chromaffin cells. With 0.1 mM EGTA in the patch pipette solution, histamine inhibited ICa in all 11 cells tested but only 7 of these cells showed kinetic slowing and prepulse relief of inhibition. In addition the proportion of the inhibition that was relieved by a prepulse was only about one-half of that in cells recorded with 10 mM EGTA. The other four cells were strongly inhibited by histamine but there was no kinetic slowing evident and a prepulse failed to relieve any of the inhibition.

Both N- and P/Q-type channels were inhibited to the same extent by histamine. The mean inhibition of the total current (both N- and P/Q-type) by histamine was 28%. After block of N-type ICa with omega -CgTx about one-half the current remained. Histamine inhibited this P/Q-type current by 26%.

Assuming that the voltage-dependent inhibition is produced by direct binding of the G-protein beta gamma subunit to the channel, the smaller inhibition produced by histamine (compared with ATP) could be explained by there being fewer channels occupied by beta gamma subunits after H1 receptor activation than by P2Y receptor activation. It has been shown that some beta gamma subunits are less effective at producing inhibition than others and that this correlated with weaker interactions with a sequence encoding the I-II linker of the channel where the beta gamma subunits bind (Garcia et al. 1998). Another explanation could be that the species of beta gamma activated by the two agonists bind to the channels equally well but elicit the inhibition with different efficacies once bound. To begin to probe these questions, the additivity of the inhibition produced by ATP and histamine was investigated. Because ATP and histamine utilize different G-proteins in their respective signaling pathways (PTX sensitive and insensitive respectively), when both agonists are applied simultaneously one would expect there to be a higher concentration of activated G-proteins in the vicinity of the channels. Our results show that when ATP was applied in the continued presence of histamine there was an increased inhibition of ICa. However, the inhibition produced by both agonists together was not significantly different from that produced by ATP alone. Similarly if ATP was applied first and then histamine added, there was no further inhibition of ICa. This suggests that the channels are maximally inhibited and are saturated by the beta gamma subunits released by P2Y receptor activation. It also suggests the reason for the smaller inhibition produced by histamine is partially caused by nonsaturating amounts of beta gamma subunits being activated near the channels. If the beta gamma subunits activated by histamine saturated the channels but were less effective at producing the inhibition once bound one might expect a competitive block of the ATP inhibition (because the beta gamma binding sites would already be occupied). Furthermore, we show that in PTX treated cells PGE2 inhibits ICa by about 20%. This inhibition and that produced by histamine were partially additive no matter what the order of agonist application. This again suggests that the amounts of beta gamma subunits generated by activation of PTX insensitive G-proteins is nonsaturating and that this accounts at least in part for the sub-maximal inhibition of ICa.

Physiologically it is not clear what role the inhibition of ICa by histamine may have. Histamine is produced by adrenal chromaffin cells and may also be produced locally by mast cells or reach the adrenal gland through the bloodstream. Inhibition of ICa will almost certainly reduce catecholamine secretion. However, it is clear that histamine also has the ability to induce secretion through elevation of [Ca2+]i by release of intracellular calcium stores and calcium influx through channels other than ICa. There may be a balance between these seemingly opposing mechanisms whereby acute histamine exposure could inhibit catecholamine release by suppressing ICa. Although our results suggest that desensitization is possible in some cells with exposures >30 s, it is still possible that prolonged exposure may augment release due to summation of the calcium influx pathways stimulated by histamine with the calcium influx through the noninhibited ICa. These complicated interactions will require further investigation, but this study identifies multiple pathways that result in the inhibition of ICa and which may modulate the functioning of adrenal chromaffin cells.


    ACKNOWLEDGMENTS

This work was supported by National Institutes of Health grants to A. P. Fox.


    FOOTNOTES

Address for reprint requests: K. Currie, Dept. of Neurobiology, Pharmacology and Physiology, The University of Chicago, 947 E. 58th St., Chicago, IL 60637.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 16 September 1999; accepted in final form 30 November 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/00 $5.00 Copyright © 2000 The American Physiological Society