The Role of Calcium in Stimulus-Secretion Coupling in Excitable and Non-Excitable Cells

There is hardly any cellular function that is not influenced directly or indirectly by the universal second messenger calcium. Particularly, the release of vesicular contents by exocytosis has become the most extensively studied process for establishing the crucial role of calcium as the triggering and controlling event. Historically, this view emerged from studies on quantal transmitter release at the neuromuscular junction (Katz, 1969). It was extended to other excitable cells, such as adrenal chromaffin cells and pancreatic beta cells, and soon became the universal mechanism for control of secretion (Douglas, 1968). The following main observations corroborated the ‘calcium hypothesis’ of exocytosis: (1) extracellular calcium is required to evoke transmitter release, (2) voltage-activated Ca²⁺ channels allow influx of Ca²⁺, the magnitude of which determines the amount of transmitter release, (3) procedures that elevate intracellular calcium concentration ([Ca²⁺]_i) induce exocytosis, and blocking Ca²⁺ influx abolishes secretion.

The concept of Ca²⁺ control of exocytosis also appeared to apply to nonexcitable cells which seem to be devoid of voltage-activated Ca²⁺ channels. This was primarily based on observations that closely resemble those mentioned above: (1) antigenic stimulation of mast cells or rat basophil leukaemic cells depends on extracellular [Ca²⁺] and is paralleled by Ca²⁺ uptake (Foreman, Hallett & Mongar, 1977), (2) injection of Ca²⁺ into mast cells has been reported to induce secretion (Kanno, Cochrane & Douglas, 1973), (3) Ca²⁺ ionophores, which translocate external Ca²⁺ into the cytosol, produce secretory responses in mast cells (Cochrane & Douglas, 1974; Penner & Neher, 1988), RBL cells (Beaven et al. 1987), neutrophils (Rubin, Sink & Freer, 1981) and platelets (Feinman & Detwiler, 1974). Furthermore, studies with fluorescent Ca²⁺ indicator dyes have revealed increases in [Ca²⁺]_i following stimulation with secretagogues in virtually every type of non-excitable cell investigated (Tsien, Pozzan & Rink, 1984). It soon became clear that in many of these cells the source of calcium was not extracellular Ca²⁺, but was storage organelles that sequester Ca²⁺ and release it upon stimulation by the recently discovered phosphatidylinositol pathway (Berridge & Irvine, 1984). This did not temper, but instead corroborated, the overwhelming evidence in support of the dominant role of [Ca²⁺]_i in the secretory process, although now Ca²⁺ released from internal stores by a second messenger had to be considered as a ‘third messenger’.

Some findings, however, disturb the simplistic view that an increase in [Ca²⁺]_i induces secretion in a straightforward manner. (1) In neutrophils, platelets, RINm5F cells and mast cells secretion can be induced at the cells’ resting [Ca²⁺]_i level or even in the almost complete absence of [Ca²⁺]_i (Sha ‘afi et al. 1983; Rink, Sanchez & Hallam, 1983; Di Virgilio, Lew & Pozzan, 1984; Barrowman, Cockroft & Gomperts, 1986; Wollheim, Ullrich, Meda & Vallar, 1987; Neher & Aimers, 1986; Neher, 1988) and (2) in Ca²⁺-ionophore-treated RBL cells (Beaven et al. 1987) or patch-clamped mast cells (Penner & Neher, 1988) a rise of [Ca²⁺]_i into the range produced by physiological stimulation does not elicit secretion. This would suggest that in some non-excitable cells [Ca²⁺]_i is neither a necessary nor sufficient stimulus for secretion.

This article aims to review the role of [Ca²⁺]_i as a second messenger in the secretory responses of excitable and non-excitable cells. Special attention will be paid to the mechanisms by which these cell types bring about changes in [Ca²⁺]_i and the modulations exerted by other second messenger systems. It is not intended, and in fact not possible, to give a full account of the overwhelming literature being published on this topic. Instead, this is an attempt to sketch roughly what we think are the most important and most intriguing features in the regulation of [Ca²⁺]_i.

Although the formulation of the ‘calcium hypothesis’ dates back almost 20 years, only recently developed methodological approaches provide a proper quantitative evaluation of cell calcium function and regulation, namely: (1) permeabilization techniques which have enabled control of the composition of the cell’s intracellular milieu (Gomperts & Fernandez, 1985; Knight & Scrutton, 1986) and the introduction of defined Ca²⁺ concentrations, (2) fluorescent Ca²⁺ indicator dyes that have been extensively used to monitor changes in [Ca²⁺]_i following stimulation with secretagogues (Tsien et al. 1984) and (3) the patchclamp technique which enables the effective control of the composition of the cytosol and at the same time measures cell membrane parameters such as membrane current or capacitance (Neher, 1988). Although the current gives an indication of transmembrane ion fluxes, the capacitance reflects cell membrane area. The fusion of vesicles with the plasma membrane brings about an increase in membrane area, thus reflecting the exocytotic activity of a single cell (Neher & Marty, 1982). The combined application of the patch-clamp technique and fluorescence measurement provides the most effective methodological assay to monitor simultaneously [Ca²⁺]_i, membrane currents and secretion.

The relationship between [Ca²⁺]_i and secretory responses in three different cell types is illustrated in Fig. 1. To compare these responses, nearly identical compositions of extracellular and intracellular solutions were used. The cells were challenged by dialysing the cytosol through the orifice of a patch pipette containing free Ca²⁺ (buffered to about 1 μmoll⁻¹ with the aid of EGTA). Secretory responses were monitored by measuring the cell membrane capacitance. With excitable cells, such as bovine adrenal chromaffin cells (Fig. 1A) and mouse pancreatic beta cells (Fig. IB), the elevated Ca²⁺ level is sufficient stimulus to produce secretion, as witnessed by an increase in cell membrane capacitance. The different magnitudes of these capacitance changes presumably reflect differences in the number of vesicles contained in chromaffin and beta cells or different ratios of exocytosis to endocytosis, both of which determine the measured capacitance changes. With non-excitable mast cells (Fig. 1C), a similar increase in Ca²⁺ concentration is not sufficient to induce sizeable secretory responses in patch clamp experiments.

Ca²⁺-induced secretion in mast cells can be achieved by clamping [Ca²⁺]_i, in the high micromolar range, or by adding Ca²⁺ ionophores to produce large increases in [Ca²⁺]_i (Penner & Neher, 1988). However, the apparent requirement for [Ca²⁺]_i in mast cells (Penner & Neher, 1988) and RBL cells (Beaven et al. 1987) is about an order of magnitude higher than that for Ca²⁺-induced exocytosis in permeabilized chromaffin cells (Knight & Baker, 1982). It is also much higher than [Ca²⁺]_i typically observed during degranulation in physiologically stimulated cells.

Thus in chromaffin cells, secretion may be induced by elevating [Ca²⁺]_i into the physiological range, 0·4–1·5μmoll⁻¹, whereas unphysiologically high [Ca²⁺]_i levels of several micromolar are required to ‘force’ secretion in mast cells. These findings raise the question whether non-excitable cells have developed alternative means either to bring about secretion by Ca²⁺-independent mechanisms or to vary the Ca²⁺ dependence of secretion in terms of lowering the Ca²⁺ requirement of the secretory process in response to the adequate stimulus. In fact, in permeabilized mast cells, a variety of [Ca²⁺]_i concentration-response relationships can be obtained which cover the range of physiologically observed [Ca²⁺]_i levels, depending on the additional provision of different nucleotides (Howell, Cockcroft & Gomperts, 1987). Similarly, there is a shift of the concentration-response curve for Ca²⁺-induced secretion in the presence of phorbol esters which mediate activation of protein kinaseC (Heiman & Crews, 1985). These findings indicate that the dual pathway may act synergistically to promote secretion in non-excitable cells.

In non-excitable cells, the transduction cascade is initiated by agonist-induced receptor stimulation, followed normally by activation of G-proteins which mediate activation of membrane-associated enzymes and eventual execution of cellular functions (Berridge, 1987). It is, therefore, not surprising that one may bypass and mimic receptor stimulation by directly activating G-proteins with non-hydro-lysable GTP-analogues such as GTP-γ-S. Dramatic secretory responses leading to complete degranulation of mast cells (Fig. 2A) can be elicited by internally perfusing the cells with GTP-γ-S (Fernandez, Neher & Gomperts, 1984). The action of GTP-γ-S, which irreversibly activates GTP-binding proteins, results in activation of phospholipase C leading to the generation of inositol trisphosphate (InsP₃) and diacylglycerol (DAG). The transient rise of [Ca²⁺]_i seen in Fig. 2A presumably reflects the InsP₃-mediated release of Ca²⁺ from internal stores. Similar Ca²⁺ transients can also be induced by intracellular perfusion of mast cells with InsP₃ alone (Neher, 1986) or by externally applied secretagogues which are known to cause polyphosphoinositide breakdown (Neher & Penner, 1988). They can readily be elicited in the absence of extracellular calcium (Neher & Aimers, 1986). Ironically, similar Ca²⁺ transients are very often observed when removing divalent ions from the external medium in the absence of any additional stimulus (R. Penner & E. Neher, unpublished observations).

Whether the secretory response is a consequence of DAG-mediated activation of protein kinase C is not clear. However, secretion does not seem to be mediated by the rise in [Ca²⁺]_i since little capacitance increase is associated with the Ca²⁺ transient, and degranulation proceeds at near-resting levels of [Ca²⁺]_i. In fact, degranulation of mast cells does not require Ca²⁺ transients at all when stimulating with GTP-γ-S or compound 48/80, as demonstrated by experiments in which cells were dialysed with Ca²⁺ buffers that kept [Ca²⁺]_i at near-resting levels (Neher & Penner, 1988; Neher, 1988). Apart from mast cells, the ability of non-excitable cells to secrete at basal [Ca²⁺]_i levels following stimulation has also been shown to exist in RBL cells (Sagi-Eisenberg, Lieman & Pecht, 1985), platelets (Rink et al. 1983; Haslam & Davidson, 1984) and parotid cells (Takemura, 1985). In the case of GTP-γ-S-stimulation, secretion can be induced even in the presence of 10mmol1⁻¹ internal EGTA, albeit delayed and at a slow rate (Neher, 1988). Apparently, there is a signal being provided by the stimulus to enable the cell to secrete at resting or even reduced levels of [Ca²⁺]_i. In neutrophils a novel GTP-binding protein G_E has been postulated to mediate such Ca²⁺-independent secretion (Barrowman et al. 1986).

It is clear, however, that elevated levels of [Ca²⁺]_i raise the rate of secretion following stimulation. Mast cells become gradually more responsive to Ca²⁺ after stimulation (Neher, 1988), suggesting synergistic actions of [Ca²⁺]_i and another signal delivered by GTP-γ-S. Another hint of such a hidden signal is the observation that 48/80-induced secretion is rapidly lost in a whole-cell patch clamp recording, whereas Ca²⁺ transients are immune to such washout (Penner, Pusch & Neher, 1987). It should be noted that permeabilized mast cells have been reported to show an essential synergy between Ca²⁺ and guanine nucleotides (Howell et al. 1987), whereas in patch-clamped mast cells this synergy is not essential. GTP-γ-S-induced secretion in the almost complete absence of [Ca²⁺]_i has also been shown to occur in insulin-secreting RINm5F cells (Wollheim et al. 1987) and neutrophils (Barrowman et al. 1986).

Neuronal tissue is known to possess the phospholipid composition, the G-proteins and the enzymatic machinery to employ the polyphosphoinositide protein kinaseC pathway in the regulation of cellular functions (Fisher & Agranoff, 1986). Furthermore, a growing number of receptors that are known to activate phospholipid turnover is found in excitable cells. However, little information is available as to what purpose the dual-signal pathway serves in neuronal cells. Activation of protein kinaseC through phorbol esters has been reported to augment transmitter release at the neuromuscular junction (Shapira, Silberberg, Ginsburg & Rahamimoff, 1987), to induce secretion in permeabilized adrenal chromaffin cells (Knight & Baker, 1983; Pocotte et al. 1985) and to mimic cellular responses normally associated with long-term potentiation in brain slices (Hu et al. 1987).

If this signal transduction pathway included a G-protein it should be possible to mimic receptor-mediated responses by introducing GTP-γ-S into the cell. Conse quently, it would be anticipated that secretion could be induced by GTP-γ-S through activation of protein kinaseC, since this is the case following direct activation of this enzyme by phorbol esters. However, GTP-γ-S does not induce secretion in permeabilized chromaffin cells, but instead causes a rightward inhibitory shift of the concentration-response relationship of Ca²⁺-dependent secretion (Knight, 1987). Other reports, demonstrating that phorbol esters caused a moderate leftward shift of the curve (Knight & Baker, 1983), have made it difficult to interpret secretion in terms of a G-protein-mediated activation of protein kinase C in these cells.

In Fig. 2 the responses of two excitable cell types are compared with a typical mast cell response following stimulation by internally administered GTP-γ-S. In adrenal chromaffin cells, repetitive Ca²⁺ transients can be elicited by GTP-γ-S (Fig. 2B). These Ca²⁺ transients are likely to reflect the generation of InsP₃ due to the G-protein-mediated activation of phospholipaseC. This response closely resembles the actions of GTP-γ-S in mast cells (Fig. 2A). However, there is no secretory response induced by the nucleotide apart from a small capacitance increase during the transient increases in [Ca²⁺]_i This clearly contrasts with the case of non-excitable mast cells where during the initial Ca²⁺ transients no appreciable secretion occurs, but instead degranulation proceeds as [Ca²⁺]_i returns to resting levels.

Pancreatic beta cells also show Ca²⁺ transients when stimulated by GTP-γ-S. The pattern of [Ca²⁺]_i changes depends on the ATP concentration provided by the pipette solution with which the cells are dialysed. At low ATP concentrations (0·2 mmol 1⁻¹) in the intracellular pipette solution, GTP-γ-S usually evokes a single long-lasting Ca²⁺ transient (Fig. 2C). Nevertheless, capacitance increases can be seen after [Ca²⁺]_i has returned to pre-stimulus levels. In the presence of high levels of ATP (3 mmol 1⁻¹), a series of Ca²⁺ transients can be elicited which would suggest a major role for ATP in the cycling of uptake and release of intracellular Ca²⁺. Interestingly enough, the pattern of intracellular Ca²⁺ transients induced by GTP-γ-S under hyperpolarizing voltage-clamp conditions is quite similar to the well-known repetitive bursting activity recorded under current clamp (Matthews & Sakamoto, 1975), in spite of the fact that voltage-activated Ca²⁺ channels remain closed. It will be interesting to learn how excitable cells, and particularly beta cells, coordinate release from internal stores with Ca²⁺ influx driven by voltage-dependent Ca²⁺ channels. In the case of beta cells, it seems that secretion may be under dual control: elevated physiological levels of [Ca²⁺]_i are as effective as G-protein-mediated stimulation in inducing secretion. Parallel and synergistic actions of these two pathways provide an effective means for controlling insulin release through neurotransmitters as well as hormones or glucose.

It is evident, from what has been discussed above, that both excitable and nonexcitable cells utilize Ca²⁺ in control of secretion. Whereas in excitable cells Ca²⁺ alone is sufficient to trigger secretion, it appears likely that non-excitable cells use Ca²⁺ as a modulator of secretion in addition to a synergistic signal provided by another second messenger system (Fig. 3). So far, the discussion has centred around the role of [Ca²⁺]_i in secretion. But what are the mechanisms by which excitable and non-excitable cells bring about changes in intracellular calcium concentration?

In excitable cells, the predominant mechanism by which an increase in [Ca²⁺]_i is accomplished is the pathway provided by voltage-activated Ca²⁺ channels that allow extracellular Ca²⁺ to permeate the plasma membrane down its electro chemical gradient (Hagiwara, 1983). At least three different types of voltage-activated Ca²⁺ channels may be distinguished based upon their electrophysiological and pharmacological properties (Nowycky, Fox & Tsien, 1985). The trigger for the opening of these channels is provided by the depolarization occurring during the action potential.

In Fig. 4A the effect of membrane depolarization on [Ca²⁺]_i is exemplified for adrenal chromaffin cells. In this experiment the cell was clamped at a holding potential of −70 mV and subjected to depolarizing voltage pulses for periods of 4 s. Three different potential levels were chosen to illustrate the basic properties of voltage-activated Ca²⁺ currents. The first sequence of voltage pulses clamped the membrane potential from −70 mV to −30 mV which is just above the threshold for Ca²⁺ channel activation. In the [Ca²⁺]_i trace this is accompanied by small increases in [Ca²⁺]_i during the depolarizing episodes, and decreases in [Ca²⁺]_i as the cell is repolarized to −70 mV. A sequence of stronger depolarizations to 0 mV, which fully activate Ca²⁺ channels, elicits larger Ca²⁺ transients during the depolarization. Stronger depolarizations into the range of +50 mV, which also fully activate Ca²⁺ channels, are not as effective in increasing [Ca²⁺]_i, because approaching the reversal potential for Ca²⁺ reduces the driving force for Ca²⁺. Thus, the observed changes in [Ca²⁺]_i reflect the current-voltage relationship of voltage-activated calcium currents. A similar parallelism has been observed for currents and capacitance changes (Clapham & Neher, 1984).

Fluxes through Ca²⁺ channels are regulated by several mechanisms. Hyper polarization, through voltage changes and/or by Ca²⁺-activated K⁺ currents, deactivates Ca²⁺ channels, whereas increased [Ca²⁺]_i provides negative feedback control by inactivating Ca²⁺ channels (Eckert & Chad, 1984). These mechanisms constitute an effective and fast safety device to prevent Ca²⁺ overload. In addition, long-term modulatory mechanisms regulate the amount of Ca²⁺ influx by affecting the open probability of Ca²⁺ channels. Most prominent is the capability of cyclic nucleotides (cyclic AMP and cyclic GMP) or catalytic subunits of cyclic-AMP-dependent and cyclic-GMP-dependent protein kinase to increase Ca²⁺ currents via phosphorylation (Hescheler, Kameyama & Trautwein, 1986; Paupardin-Tritsch et al. 1986). Similarly, injection of protein kinase C into mollusc neurones causes an increase of Ca²⁺ current (DeRiemer et al. 1985), whereas injection into chick dorsal root ganglion neurones causes a decrease in Ca²⁺ current (Rane & Dunlap, 1986). Interestingly, purified exogenous G-proteins added to a recording patch pipette in the whole-cell configuration can substitute for endogenous G-proteins in the modulation of Ca²⁺ channels by opioids (Hescheler et al. 1986). This suggests that G-proteins may interact directly with Ca²⁺ channels, in analogy with the case of K⁺ channels in heart cells, as discussed by Dunlap, Holz & Rane (1987).

Since, by definition, non-excitable cells lack voltage-activated Ca²⁺ channels, other mechanisms for [Ca²⁺]_i control must be present. In recent years a new second messenger system has gained much attention as it provides a mechanism to increase [Ca²⁺]_i by releasing Ca²⁺ from internal stores. No electrical activity and no external source for Ca²⁺ is required for this process. Following receptor occupancy, a G-protein mediates the activation of phospholipaseC which hydrolyses membrane-integral polyphosphoinositides yielding two second messengers: inositol-(l,4,5)-trisphosphate (Ins(l,4,5)P₃) and diacylglycerol (DAG) (see Ber-ridge & Irvine, 1984 for a review). InsP₃ is well established as a cause of release of Ca²⁺ from the endoplasmic reticulum while DAG activates protein kinase C such that its affinity towards Ca²⁺ is increased. This dual signal is predestined to promote cellular responses synergistically.

Due to the limited accessibility of intracellular organelles, little information and much speculation is available about how release of Ca²⁺ from internal stores is regulated. It is believed that InsP₃ mediates the opening of some channel-like structure through which Ca²⁺ flows down its concentration gradient. The InsP₃-induced Ca²⁺ release may be caused by putative Ca²⁺ channels in the endoplasmic reticulum membrane, either directly gated by InsP₃ or possibly indirectly activated through a GTP-binding protein (Dawson, 1985). In mast cells, however, agonist-induced release of Ca²⁺ from internal stores occurs even at GTP levels that fail to support secretion (Penner et al. 1987).

InsP₃-induced release of Ca²⁺ from internal stores is typically transient in nature (see Fig. 2). Several mechanisms could curtail Ca²⁺ release induced by InsP₃. These include Ca²⁺-dependent or time-dependent inactivation of putative endoplasmic reticulum Ca²⁺ channels in analogy to plasma membrane Ca²⁺ channels, and desensitization of the InsP₃ receptor. Modulation of polyphosphoinositide breakdown may also occur at the level of phospholipaseC. So far, evidence for inhibition of phosphoinositide (PI) breakdown by cyclic AMP and cyclic GMP has been presented (Knight & Scrutton, 1984). Furthermore, the phospholipase A₂ pathway through arachidonic acid and its metabolites may interact with the phospholipaseC pathway by enhancing or reducing PI break down (see Rubin, 1986, and references cited therein). The removal of the agonist InsP₃ may serve as an additional mechanism for terminating Ca²⁺ release. Fast degradation of InsP₃ due to phosphatases as well as the generation of a number of different inositol phosphates, including other forms of InsP₃, lnsP₄ and cyclic derivatives, are known (see Irvine, 1986 for a review). Finally, by analogy with desensitization of the β-adrenergic receptor (Sibley & Lefkowitz, 1985), receptor desensitization has been reported to occur following activation of protein kinase C by phorbol esters (Kelleher, Pessin, Ruoho & Johnson, 1984). As a result, receptor stimulation is ineffective in releasing inositol phosphates (Orellana, Solski & Brown, 1985; Watson & Lapetina, 1985). Neither degradation nor negative feedback can be responsible for the decline in [Ca²⁺]_i seen in experiments where InsP₃ was directly injected into mast cells (Neher, 1986), since InsP₃ is constantly supplied from the recording pipette in the absence of receptor stimulation.

Another modulatory component of the Ca²⁺ release process has emerged from the finding that replenishment of intracellular stores may involve complicated regulation. Apparently, the stores cannot be refilled in the sustained presence of receptor agonists (Berridge & Fain, 1979). Furthermore, the emptying and refilling of the internal stores, as proposed by the capacitative model of Ca²⁺ release and uptake (Putney, 1986), has been suggested to be under the control of InsP₃ or InsP₄ or a combination of both (Putney, 1987). InsP₃ and InsP₄ are also candidates as agonists for the gating of plasma membrane Ca²⁺ channels (see Houslay, 1987).

As emphasized above, excitable cells mainly utilize voltage-activated Ca²⁺ channels to bring about changes in [Ca²⁺]_i. In addition, they are able to release Ca²⁺ from internal stores by using the ubiquitous second messenger system of PI turnover. Yet another conceptually distinct way of allowing changes in [Ca²⁺]_i is provided by so-called receptor-operated channels in excitable cells and may possibly be realized also in non-excitable cells. Some classical receptor-operated channels like the acetylcholine or glutamate receptor are known to be permeant also to Ca²⁺ (Lewis, 1979; MacDermott et al. 1986). Fluxes through the acetylcholine receptor channel are modulated by [Ca²⁺]_i (Nastuk, 1977; Miledi, 1980), cyclic AMP (presumably via phosphorylation through cyclic-AMP-dependent kinase) (Huganir, Delcour, Greengard & Hess, 1986) and GTP-γ-S (presumably via phosphorylation through DAG-activated protein kinaseC) (Eusebi,. Grassi, Molinaro & Zani, 1987). In addition to their Ca²⁺ permeability these channels determine the general excitability of cells, such that their regulation also affects the Ca²⁺ influx through voltage-operated Ca²⁺ channels. More recently, a more Ca²⁺-specific receptor-operated channel has been described in smooth muscle cells (Benham & Tsien, 1987). This channel is gated by the putative sympathetic neurotransmitter ATP, apparently without the involvement of second messengers. Presumably, more receptor-operated Ca²⁺ channels in excitable and non-excitable cells (where they have been postulated based on tracer flux studies) await discovery. In analogy to these receptor-operated channels which are gated by external ligands, there are channels that are gated by internal ligands and may be termed second messenger-activated channels. Well-known examples of such channels are the cyclic GMP-dependent conductance in photoreceptors (Fesenko, Kolesnikov & Lyubarski, 1985) and the cyclic AMP-dependent currents in olfactory receptor cells (Nakamura & Gold, 1987).

In non-excitable cells, Ca²⁺ release from internal stores through second messengers appears to be the primary mechanism by which [Ca²⁺]_i is regulated. However, as originally proposed (Michell, 1975), and only recently overshadowed by the excitement caused by the discovery of internal Ca²⁺ release, increasing evidence suggests that Ca²⁺ influx from the extracellular space may be just as important. In many cell types [Ca²⁺]_i shows a characteristic diphasic behaviour (Rasmussen & Barrett, 1984). An initial transient increase in [Ca²⁺]_i that is independent of extracellular Ca²⁺ is followed by a more sustained phase which depends on external [Ca²⁺] and is believed to result from changes in plasma membrane permeability for Ca²⁺ induced by second messengers. There is only little but promising information available about the gating of these so-called second messenger-activated channels in the plasma membrane. One mechanism of Ca²⁺ influx present in neutrophils may depend on [Ca²⁺]_i itself (presumably brought about by initial receptor-stimulated release of Ca²⁺ from intracellular stores) in such a way that [Ca²⁺]_i-gated cation channels permeant to Ca²⁺ are activated (von Tscharner, Prod ‘hom, Baggiolini & Reuter, 1986). In other cells, InsP₃ may open not only Ca²⁺ channels in the endoplasmic reticulum but also Ca²⁺-permeant channels in the plasma membrane, as reported in lymphocytes (Kuno & Gardner, 1987). A membrane current with similar properties has been observed in mast cells (Matthews, Neher & Penner, 1988). Modulation of a class of Ca²⁺ channels by InsP₃ has also been suggested to occur in Xenopus oocytes (Parker & Miledi, 1987a). In the same cells, injection of InsP₄ (physiologically derived from InsP₃ and subsequent phosphorylation by the InsP₃-kinase) is believed to activate voltage-sensitive Ca²⁺ channels via a process that may require ‘priming’ by InsP₃ (Parker & Miledi, 1987b). This is reminiscent of the findings in sea urchin eggs, where InsP₄ was suggested to control Ca²⁺ entry across the plasma membrane (Irvine & Moor, 1986). Similarly, synergistic actions of InsP₃ and InsP₄ have been reported in lacrimal gland cells (Morris, Gallacher, Irvine & Petersen, 1987). In these, InsP₄ greatly augments and prolongs InsP₃-induced [Ca²⁺]_i increases (as measured by increased activity of Ca²⁺-activated K⁺ channels). In another study, however, injection of GTP-γ-S or InsP₃ alone produced notable [Ca²⁺]_i responses that were dependent on extracellular Ca²⁺ and membrane voltage (Llano, Marty & Tanguy, 1987).

In conclusion, it may appear surprising how many diverse and elaborate mechanisms have evolved in controlling and modulating the intracellular calcium concentration. However, given the central role of [Ca²⁺]_i in so many cellular processes, we should be prepared to learn in the not too distant future that our current understanding of the mechanisms involved in Ca²⁺ regulation is far too simplistic.

We would like to thank Dr G. Trube for providing pancreatic beta cells and Dr G. G. Matthews for reading the manuscript.