Mediators of Ca2(+)-dependent secretion.

Ca2+, an obligatory mediator of the secretory process, acts in concert with other second messengers that further amplify or inhibit the secretory response. In this overview, we will consider the relative roles of diacylglycerol (DAG), arachidonic acid, and cyclic AMP (cAMP) in modulating Ca2(+)-dependent secretion in nonexcitable cells. DAG, a product of phospholipase C (PLC)-catalyzed breakdown of phosphoinositides, stimulates protein kinase C. Ca2+ ionophores and phorbol esters (or DAG analogues) elicit a synergistic secretory response in the exocrine pancreas and parotid gland. These findings suggest that the complete activation of secretion requires stimulation of both Ca2(+)-dependent and protein kinase C-dependent pathways. Hydrolysis of phospholipids can also lead to the liberation of arachidonic acid in secretory cells. Endogenously generated arachidonic acid inhibits polyphosphoinositide synthesis in exocrine pancreas, leading to inhibition of agonist-induced IP3 formation, Ca2(+)-mobilization and amylase secretion. By contrast, arachidonic acid and its metabolites stimulate PLC in the rabbit peritoneal neutrophil, causing Ca2(+)-mobilization and lysosomal enzyme secretion. Arachidonic acid can thus serve as a positive or negative feedback regulator of secretion induced by Ca2(+)-mobilizing agonists. Finally, in the parotid gland, stimulation of amylase secretion by norepinephrine, the physiological mediator, which stimulates both the alpha and beta adrenoceptors, requires the interaction of both Ca2+ and cAMP pathways to produce a full secretory response. These studies, taken together, indicate that phosphoinositide and cAMP-dependent pathways play coordinate roles in signal transduction, leading to the Ca2(+)-mediated secretion.

Mediators of Ca2 -Dependent Secretion by Archana Chaudhry* and Ronald P. Rubin* Ca", an obligatory mediator of the secretory process, acts in concert with other second messengers that further amplify or inhibit the secretory response. In this overview, we will consider the relative roles of diacylglycerol (DAG), arachidonic acid, and cyclic AMP (cAMP) in modulating Ca2,-dependent secretion in nonexcitable cells. DAG, a product of phospholipase C (PLC)-catalyzed breakdown of phosphoinositides, stimulates protein kinase C. Ca2" ionophores and phorbol esters (or DAG analogues) elicit a synergistic secretory response in the exocrine pancreas and parotid gland. These findings suggest that the complete activation of secretion requires stimulation of both Ca2+-dependent and protein kinase C-dependent pathways. Hydrolysis of phospholipids can also lead to the liberation of arachidonic acid in secretory cells. Endogenously generated arachidonic acid inhibits polyphosphoinositide synthesis in exocrine pancreas, leading to inhibition of agonist-induced IP3 formation, Ca2+-mobilization and amylase secretion. By contrast, arachidonic acid and its metabolites stimulate PLC in the rabbit peritoneal neutrophil, causing Ca2+-mobilization and lysosomal enzyme secretion. Arachidonic acid can thus serve as a positive or negative feedback regulator of secretion induced by Ca21-mobilizing agonists. Finally, in the parotid gland, stimulation of amylase secretion by norepinephrine, the physiological mediator, which stimulates both the a and P adrenoceptors, requires the interaction of both Ca + and cAMP pathways to produce a full secretory response. These studies, taken together, indicate that phosphoinositide and cAMP-dependent pathways play coordinate roles in signal transduction, leading to the Ca2+-mediated secretion.
The role of calcium (Ca2") in stimulus-secretion coupling has been unequivocally established. In electrically excitable cells such as the neuron, adrenal medullary chromaffin cell, the P cell of the endocrine pancreas, and cells of the adeno-and neurohypophysis, the rise in cellular Ca2" following stimulation is derived to a large extent from influx of cation through voltage-sensitive channels (1). In nonexcitable secretory cells, such as those of exocrine glands and neutrophils, cellular stores of Ca2' play a more predominant role in regulating secretion, although Ca2" influx through receptor-operated channels also increases cellular Ca2' availability ( Fig. 1). The initial secretory response seems dependent on Ca2' released from intracellular stores, but prolonged secretion requires the presence of extracellular Ca2' (2).

The concept that increases in intracellular ionic
Ca2"stimulate secretion in nonexcitable cells is supported by the following pieces of evidence: a) secretagogues evoke increases in cytosolic Ca2" (3,4) and cause a rapid efflux of 45Ca from cells (2); b) Ca2" ionophores which bypass receptors to raise cytoplasmic Ca2' stimulate enzyme secretion (4); c) depletion Ca2"-mobilizing agonists stimulate phospholipase C (PLC), which catalyzes the phosphodiesteratic cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) (Fig. 1). This leads to the formation of 1,4,5-inositol trisphosphate (1P3) and diacylglycerol (DAG), both of which have important second messenger roles. 1,4,5-IP3 releases cellular Ca2' by interacting with a specific receptor site on the endoplasmic reticulum (6), and DAG activates protein kinase C, a key regulatory enzyme (7). Arachidonic acid is also liberated from phosphoinositides during stimulation by secretagogues, and free arachidonic acid and/or its metabolites may also serve as cellular messengers to modulate the secretory response (8). Also, some secretory cells possess a signaling system that uses cyclic AMP (cAMP) as a second messenger. In such systems Ca2' and cAMP may act either sequentially or in concert to regulate secretion. Simultaneous changes in the intracellular concentrations of cytosolic Ca2' and cAMP have been reported after stimulation of secretory cells by a variety of secretagogues (9,10). However, in contrast to Ca2 , cAMP has not been characterized as a direct mediator of exocytosis.
In this brief overview, we will consider the concept that the second messengers DAG, arachidonic acid, and cAMP interact with Ca2" to modulate the secretory response (Fig. 1). We will employ the parotid and pancreatic acinar cells, as well as the rabbit neutrophil, to offer evidence to support this thesis. A large body of evidence suggests that in many tissues optimal secretion requires both Ca2' and DAG ( Fig. 1). Nishizuka and his colleagues (7) first demonstrated that DAG activates a phospholipid-dependent kinase (protein kinase C) by increasing the affinity of the kinase for Ca2". Thus, in the presence of DAG, protein kinase C can be maximally stimulated at submicromolar concentrations of Ca2+. The interactions between Ca2+ and protein kinase C in cellular secretion have been probed by using calcium ionophores (which bypass receptors to raise cytoplasmic Ca2') and phorbol esters (which substitute for DAG) to activate the Ca2+-dependent and protein kinase Cdependent pathways separately. In isolated pancreatic acini, phorbol 12,13-dibutyrate (PDBu) when added together with a threshold concentration of the Ca2+ ionophore, ionomycin, causes a synergistic potentation of amylase secretion, with no further elevation in cytoplasmic Ca2+ than the one elicited by ionomycin alone (Fig. 2). Diacylglycerols containing unsaturated fatty acids also stimulate amylase secretion and exhibit synergistic effects on secretion in combination with ionomycin (4). These results suggest that complete activation of amylase secretion by the pancreas requires stimulation of both Ca2+-dependent and protein kinase C-dependent pathways. Similar synergistic effects of ionophores and phorbol esters have been reported in other model secretory systems (11,12). Apart from interacting with the protein kinase C pathway, Ca2" may interact with the arachidonic acid messenger system to modulate secretion (13). Mammalian phospholipids are enriched in arachidonic acid, and Ca2"-mobilizing agonists liberate free arachidonic acid either through activation of phospholipase A2, the sequential activation of PLC and DAG lipase, or phosphatidate-specific phospholipase A2 (13). For example, the mucarinic agonist carbachol elevates free arachidonate levels in pancreatic acinar cells (14). The time course of this event parallels that of other cellular responses to carbachol, including IP3 accumulation and amylase 600-1    (14) (14). These findings indicate that in exocrine pancreas, arachidonic acid inhibits the synthesis of the polypholphoinositide pool used by Ca2'-mobilizing agonists.
To confirm the inhibitory role of endogenously released arachidonic acid on phosphoinositide turnover and, consequently, on agonist-mediated Ca2" mobilization as well as amylase secretion, we utilized tetrahydrocannabinol (THC) to increase endogenous T* levels of unesterified arachidonate. THC is an inhibitor of acyl-CoA transferase and stimulates arachidonic acid release from cells by activating 25 50 phospholipase A2 (17)(18)(19) (Fig. 4). The effects of THC on arachidonate release l-inedxued ocacrubmulal and [3P]Pi levels are dose-related over the concentrag the radioactivity in tion range of 1 to 20 jM (unpublished observations). mean ± SE (n = 5).
Pretreatment with THC causes a dose-related inhibiferent from samples tion of [3H]IP3 accumulation (unpublished observations), as well as cytoplasmic Ca2' and amylase secretion (Table 1) elicited by cerulein in the exocrine nous arachidonic pancreas. These results indicate that endogenously lent reduction in generated arachidonic acid and/or its metabolites can 14). The decrease serve as a negative feedback regulator of phosphoi-,-stimulated PIP2 nositide turnover and, thus, inhibit agonist-induced one does not pro-rises in cytosolic [Ca2'] and amylase secretion.
Basal values for [Ca2+]i and amylase secretion have been subtracted from the data that is shown here as mean ± SE (n = 3-5).
*Significantly different from samples treated with cerulein alone (p < 0.05). mechanism also appears operative in the blood platelet (22) and rat corpus luteum (23). Thus it appears that agonist-induced liberation of arachidonic acid can either amplify Ca2"-induced secretion or inhibit it, depending upon the secretory system under scrutiny. Recent evidence suggests that Ca2+ can also interact with the cAMP-dependent pathway to enhance the secretory responses of secretagogues. Cyclic AMP generated through beta adrenoceptor action appears to be a critical modulator of amylase secretion by the rat parotid gland. By contrast, Ca2+-mobilizing agonists which express their actions through muscarinic and a-adrenergic receptors cause a predominance of water and electrolyte release (24). Norepinephrine (NE), the physiological neurotransmitter for salivary amylase secretion, which stimulates both f and a adrenoceptors, requires participation of both the cAMP and Ca2+ pathways to produce a full secretory response. Figure 5 shows that amylase secretion induced by NE is greater than the sum of the release Further support for an interaction between the Ca2' and cAMP pathways comes from our observation that a subthreshold concentration of carbachol causes a significant enhancement of isoproterenol-induced secretion (Fig. 6). The site of interaction between the two transduction systems is distal to the catalytic site of adenylate cyclase because carbachol failed to elevate isoproterenol-stimulated cAMP levels (unpublished observations). One possible explanation for the above findings is that there is an enhancement of Ca2" availability produced by the coordinate interactions of the two pathways. Parotid acinar cells exposed to Time (min) FIGURE 7. Effect of carbachol (CCh) and isoproterenol (ISO), alone and in combination, on the steady-state accumulation of 45Ca in rat parotid acinar cells. Cells were incubated with 45Ca (8 gCi/ mL) for 60 min and then exposed to either CCh (100 gM) or ISO (200 nM) for various times. A third group of cells was pretreated for 2 min with 200 nM ISO prior to exposure to CCh. The incubation was terminated by filtering the cells through Millipore filters. The filters were counted, and the cellular 45Ca content of each sample was calculated as a percent of its corresponding control value. Each value is mean ± SE (n = 3-8). 45Ca under steady-state conditions show an enhanced 45Ca accumulation in cells exposed to carbachol, but not to isoproterenol (Fig. 7). However, the combination of carbachol plus isoproterenol produces a further elevation in cellular 45Ca content. The above findings, therefore, suggest that the Ca2'-mediated pathway interacts with the cAMP-mediated pathway to regulate amylase secretion by increasing Ca2' availability.
In conclusion, this brief account has dealt with the concept that phosphoinositideand cAMP-dependent pathways play coordinate roles in signal transduction leading to the activation of Ca2'-mediated exocytotic secretion. While Ca2+ appears to be a sine qua non for activation of the secretory apparatus, there is convincing evidence that DAG, arachidonic acid, and cAMP serve to modulate this pivotal action of Ca2'. In secretory cells it appears as though controls are arranged in an integrative system in which information from several levels or sources may influence the final Ca2'-dependent message that is transmitted to the secretory apparatus. Continued intensive experimentation will be required to define the full scope of the close association, if not tight coupling, between Ca2+ and these messengers systems. The current intensity of attention to this subject seems likely to generate additional paradigms for this aspect of cell activation.