Mechanisms of cellular information processing

HYPOTHESES Mechanisms of Cellular Information Processing Christof Schsfl, Klaus Prank, and Georg Brabant

Living cells in multicellular organisms are in simultaneous contact with many regulatory factors such as hormones or neurotransmitters. Many of these factors vary with time in their local concentrations, owing to pulsatile release or production. Therefore, complex patterns of signaling factors act on each living cell in vivo, stimulating or inhibiting second-messenger pathways with potentially complex dynamics. These intracellular pathways do not operate independently but are extensively interconnected, creating complex networks and patterns of intracellular signals that combine to determine the cell’s response. The potential significance of cross-signaling between second-messenger pathways and of dynamic stimulation of receptors for cellular information processing in physiology and pathophysiology are discussed. (Trends Endocrinol Metab 1994;5:53-59)

Biologic information in multicellular organisms is communicated by neuronal pathways and by humor-al transmission. Spatial targeting of information occurs either via selective innervation pathways to the respective target tissues or via specific receptors for a given hormone present only on its target cells. The target cells themselves possess large numbers of different receptors for a variety of neurotransmitters, hormones, and other signaling molecules. On receptor activation, distinct intracellular signaling cascades are activated, thereby translating the extracellular signal into specific target cell responses such as function, proliferation, or differentiation. Target cells are in simultaneous contact with many regulatory factors. Therefore, complex patterns of stimulator-y and inhibitory factors occur at the site of each living cell, which may activate or inhibit simultaneously several distinct signaling

Christof SchiSfl, Klaus Prank, and Georg Brabant are at the Department of Clinical Endocrinology, Medizinische Hochschule Hannover, 30623 Hannover, Germany.

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selectively bind to receptors that in turn selectively activate intracellular signaling pathways such as the CAMP or Ca2+ systems, a temporal code might exist that may be equally important for the “specificity” of cell regulation through extracellular stimuli (Weigle 1987, Li and Goldbeter 1989, Waxman et al. 199 1, Brabant et al. 1992, Haisenleder et al. 1992). If this is true, information encoded in the temporal pattern of cell stimulation (that is, in the frequency and amplitude of agonist pulses) has to be transferred across the plasma membrane and translated into distinct intracellular signals in order to regulate target cell responses differentially. Data are presented here that corroborate this hypothesis.

??

Cross-Signaling and Temporal Aspects in Intracellular Signaling Cascades

Most hormones and regulatory factors vary with time in their local concentrations, owing to pulsatile secretion (Brabant et al. 1992). The kinetics of processes in the signaling pathways may enable target cells to decode biologic information encoded in those temporal patterns of extracellular stimulation. Each receptor has characteristic desensitization and resensitization kinetics; each G protein has characteristic kinetics for activation by receptors and inactivation by GTPase activity, and so on down the

Because of space restrictions, we focus here on receptor systems that are coupled through guanine-nucleotide-binding regulatory proteins (G proteins) to different intracellular signaling cascades such as the CAMP or Ca2+/phosphatidylinositol-signaling pathway. A paradigmatic scheme for the adrenergic receptors is shown in Figure 1. As outlined previously, thresholds, the dose response, and the kinetics of the reactions involved are key features of signaling in biologic systems (Brabant et al. 1992). The sensitivity of a target cell to a hormone is primarily determined by the sensitivity of its receptor. The needs of a receptor as a sensing device are to respond to a low agonist concentration and to rapid changes in its concentration. These goals are mainly achieved by having spare receptors of a relatively low affinity for the agonist (that is, most receptors remain unbound by agonist). This enables the cell to respond to low

signaling cascades. The pathways as a whole may have characteristic refractory and recovery periods, and therefore be sensitive to the frequency of stimulation. In addition to the “conformational specificity” in biologic communication by hormones and neurotransmitters, which

agonist concentrations and to be sensitive to rapid changes in agonist concentration (Taylor 1990), and provides an elegant way to control the threshold concentration of a hormone or to tune the sensitivity to temporal changes in hormone concentrations by either chang-

cascades inside the cell. These intracellular pathways do not operate independently. In contrast, as has been shown in recent years, they are extensively interconnected at almost every level of the signaling cascades, creating complex networks and patterns of intracellular signals, which finally determine the actual target cell responses.

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53

catecholamines

subunits,

are central

to the coupling

of

individual receptors to second-messengercontrolling enzymes such as adenylyl cyclase and phospholipase C (PLC) or ion channels. There is an ever-growing list of G proteins, which are mainly characterized by their a subunits, although there are also differences in their /3and y subunits, which usually form a tight complex (Taylor 1990). Until recently, only the a subunits were thought to be relevant to signal transduction, whereas the @y subunits were believed to regulate mainly the levels of the active a subunit. Recent reports, however, indicate, that the fly subunits might be of prime importance in cross-signaling beresponses cellular tween different G-protein-activated sigFigure 1. Diagram of intracellular signaling naling cascades (Birnbaumer 1992, pathways by which catecholamines and other Lefkowitz 1992, Tang and Gilman 1992, G-protein-coupled receptors regulate target Federman et al. 1992, Milligan 1993, cell responses. cr,Adrenergic receptors (al ), Clapham and Neer 1993). Furthermore, like other Caz+-mobilizing receptors, couple it appears that the time constants for via G,-type G proteins to phospholipase C degradation of, for example, the active (PLC), which generates inositol-1,4,5-trisphosphate (IP,) and diacylglycerol (DAG) form of G,, through GTPase activity are from phosphatidylinositol-4,5_bisphosphate. regulated by its intracellular effecters IP, elevates cytosolic free Ca*+ through re(Bourne and Stryer 1992). This might be lease of Ca*+ from intracellular pools. The important for the concentrations of Gi, increase in cytosolic Ca*+ activates Ca*+/ required to inhibit intracellular effecters calmodulin-dependent kinase (CaM kinase), activated by G,, (Taussig et al. 1993). If and DAG stimulates protein kinase C (PKC). the activation of GTPase activity were to Both kinases regulate a variety of intracellular depend on the state of activation of the effecters via phosphorylation, thereby determining the cellular responses. g-Adrenergic effector, the possibilities for dynamic receptors (8) and other CAMP-elevating recepcomplexity would be further increased tors couple via C&-typeG proteins to adenylyl (Cuthbertson and Chay 1991). Different cyclases (AC), which stimulate adenosine and distinct kinetics for the degradation 3’,5’-monophosphate (CAMP) synthesis from of active species of G-protein subunits ATP. An elevation in CAMP leads to the might enable differential activation or activation of protein kinase A (PKA), which in turn regulates cellular responses via inhibition of intracellular effecters dephosphorylation. Inhibitory receptors like the pendent on the temporal pattern of their a,-adrenergic receptor (a,) couple to G,-type generation. Therefore, G proteins emerge G proteins and inhibit AC activity. as excellent candidates not only for extensive cross-signaling between different intracellular signaling cascades but ing receptor numbers or affinity. Regulaalso for time-specific information proction of receptor numbers and function essing. through cross-signaling has been reNot only G proteins exist in many ported, for example, from adrenergic forms, but their intracellular effecters receptor subtypes coupled to different such as adenylyl cyclase or PLC also signaling pathways (Leeb-Lundberg et exhibit a number of isoforms, which are al. 1987, Morris et al. 1991, Collins et al. distinguished by their control by recep1992). The kinetics of rapid desensitizators, G-protein subunits, and their crosstion or functional uncoupling of recepregulation by other signaling cascades tors as well as resensitization mecha(Birnbaumer 1992, Cockcroft and Thonisms are most important for our mas 1992, Tang and Gilman 1992, Iyenunderstanding of temporal specificity. gar 1993). Type I and III adenylyl However, cross-regulation and temporal aspects are not restricted to membrane receptors. Heterotrimeric G proteins, which consist of a, l3, and y

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cyclase, for example, can be coactivated by the Ca*+/phosphatidylinositol-signaling cascade via Ca*+/calmodulin, whereas other mammalian types of the enzyme

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cannot (Tang and Gilman 1992, Iyengat 1993). Furthermore, there is typespecific regulation of adenylyl cyclase activity by $y subunits, which are provided from other activated G-proteinmediated signaling pathways. In HEK293 cells transfected with a,-adrenergic receptors, which usually inhibit adenylyl cyclase activity via Gi proteins, coexpression of mutationally active G,, with adenylyl cyclase II converted a,-adrenergic agonists into stimulators of CAMP synthesis (Federman et al. 1992). Like adenylyl cyclases, several isoforms of PLC exist that are differentially regulated by CAMP-dependent kinase and by G-protein subunits (Cockcroft and Thomas 1992, Birnbaumer 1992, Park et al. 1993, Clapham and Neer 1993). There is evidence that PLC-y, but not PLC-l3 or PLCB, is phosphorylated and negatively modulated by CAMP-dependent mechanisms (Kim et al. 1989). PLC-), is activated by tyrosine kinase receptors independent from G proteins, whereas PLC-f$l4 are activated by G,,. Recently, it was demonstrated that PLC-fll-3, but not PLC-l34, are also activated by $y subunits. In contrast to the isoform-specific activation of adenylyl cyclase activity by &J subunits, stimulation of PLC-6 activity by fly does not require the presence of G,. Therefore, @y subunits released from other G proteins like G, and Gi could potentially activate the Ca*+/phosphatidylinositol-signaling pathway by activating PLC-b activity (Figure 2). Adenylyl cyclases generate CAMP, and PLC activation produces two messengers: inositol 1,4,5-trisphosphate (IP,), which triggers release of Ca*+ from intracellular pools, and diacylglycerol, which activates protein kinase C (Figure 1). The intracellular Ca*+ signal is thought to be one of the key signals in the Ca*+/phosphatidylinositol-signaling pathway. Its regulation by cross-signaling and by the temporal pattern of receptor stimulation is described below. All secondmessenger systems ultimately activate protein kinases or phosphatases (Figure 1) that divert the signal to a variety of mostly unknown intracellular effecters and finally stimulate distinct cellspecific responses. Sites for crossregulation and temporal specificity have been reported at almost every level of the intracellular signaling cascades, including secretion and transcription (Law et al. 1989, Amm&et al. 1993, Haisenle-

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Figure

2. Examples

for the potential

role of

cross-signaling for the regulation of target cell functions by hormones. (a) A hypothetical situation is depicted where CAMP-dependent mechanisms activated by hormone B inhibit the activation of phospholipase C (PLC) by hormone A. In the presence of hormone A and hormone B, the cellular response is therefore dominated by the CAMP-signaling pathway, as shown on the left. If a third hormone, C, however, is added that inhibits adenylyl cyclase activity through the inhibitory G protein (Ci), inhibition of PLC activity is removed and the cellular response is then predominantly determined by the Ca2+/ phosphatidylinositol pathway, as shown on the right. In situations where hormones activate receptors that couple to both the CAMP pathway and the Ca*‘/phosphatidylinositol pathway, like the PTH, calcitonin, or glucagon receptor (AbouSamra et al. 1992, Chabre et al. 1992, Jelinek et al. 1993, Milhgan 1993), such a negative feedback as proposed on the lefi allows for shifting between CAMP pathway and CaVphosphatidylinositol pathway-regulated target cell responses, depending on the presence or absence of a second inhibitory hormone like hormone C, which could act like a switch between the two pathways. Abbreviations are the same as in Figure 1. R, receptor. (b) The potential role of isoforms of intracellular effecters such as adenylyl cyclases for cellular signaling. Both hormone A and C stimulate CAMP production through two different stimulatory G proteins (G,), which activate adenylyl cyclase type I (AC I) and type II (AC II), respectively. As shown on the [eft, the combination of hormone A with the “inhibitory” hormone B leads to the expected inhibition of CAMP production via inhibition of AC I by fly subunits of Gi (Federman et al. 1992, Tang and Cilman 1992, Iyengar 1993). If hormone C is combined with hormone B, however, a “paradoxical” potentiation of CAMP production is observed, owing to the isoform specific regulation of adenylyl cyclase activity by gy subunits (Fedex-man et al. 1992, Tang and Gilman 1992, Iyengar 1993), as shown on the right. Abbreviations are the same as in Figure 1. R, receptor. (c) The potential role of activation of PLC-fl by l3y subunits for cellular signaling. On the left, the cell is stimulated with hormone A, which activates the CAMP-signaling pathway. The combination of hormone A and the ‘inhibitory” hormone B causes inhibition of adenylyl cyclase activity through l3y subunits (Fedex-man et al. 1992, Tangand Gilman 1992, Iyengar 1993) and possibly G,, (Taussig et al. 1993) as shown on the right. Besides inhibiting adenylyl cyclase, the gy subunits, which are released from Gi on activation of receptor B, might stimulate PLC-0 (Park et al. 1993), thereby activating the Ca*+/ phosphatidylinositol pathway similar to the situation depicted in a. In contrast to a, however, the presence of a Ca2+-mobilizing hormone is not required, since activation of PLC-h by By subunits is independent from Gqa (Park et al. 1993). Abbreviations are the same as in Figure 1. R, receptor.

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a

El

CAMP

I

I A

b

IAC’I

B

I AC

II

A

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??

Regulation

of [Ca2+li in Single

Hepatocytes by Cross-Signaling and Pulsatile Cell Stimulation We recently described a model system using the receptor-mediated intracellular

Ca2+ signal

in single

hepatocytes,

which illustrates the potential cross-signaling and of temporal of cell stimulation

role of aspects

for target cell regula-

tion by hormones (Schdfl et al. 199 1 and 1993). Ca2+ is one of the key messengers in intracellular signaling. In liver cells, a rise in cytosolic Ca*+ is involved in the regulation of various metabolic pathways, protein synthesis, and liver cell regeneration in response to a variety of hormones, for example, epinephrine, norepinephrine, or vasopressin. Catechol-

* t

t

different

cellulur

responses

?

Figure 3. Schematic illustration of the regulation of the cytosolic free Ca*+ concentration by cross-signaling with CAMP-dependent mechanisms in single hepatocytes. Vasopressin and of similar shape and catecholamines, when given alone, generate repetitive Ca2+ transients quality in single rat hepatocytes (Woods et al. 1987, SchBfl et al. 1991, Sanchez-Bueno et al. 1993). Coactivation of CAMP-dependent mechanisms, for example, by glucagon, result in different and distinct changes in the intracellular CaZ+ response depending on the Ca2+mobilizing agonist. Elevation of CAMP causes broadening of vasopressin-induced Ca2+ transients without major effects on the frequency or amplitude of the Ca2+ transients (Sanchez-Bueno et al. 1993), whereas the frequency and amplitude of a,-adrenergic receptor-induced Ca2+ transients increase dramatically by coactivation of the CAMP-signaling pathway (Schijfl et a1.1991, Sanchez-Bueno et al. 1993). Whether both Ca*+ signals are equal in terms of eliciting the same target cell responses or whether they mean something different to the target cell remains to be shown. Abbreviations are the same as in Figure 1. [Ca2+],, intracellular free Ca2+ concentration: t, time; V,, vasopressin V, receptor; and R, receptor for glucagon.

Schematic

adaptations

der et al. 1992, Schwaninger

are from the cited references.

et al. 1993).

These mechanisms are probably tissue specific and cell specific, since it has been shown that the expression of receptor subtypes, the set of G proteins available, or isoforms of signaling effecters as adenylyl cyclases or PLC are cell type specific. Hence, a hormone might control tissue-specific target cell responses by cross-signaling or temporal specificity due to the set of signaling effecters present in the respective tissue. The

56

potential

functional

consequences

of

cross-signaling are schematically illustrated in Figure 2. Since cross-signaling and temporal aspects potentially occur at almost any level of the signaling cascades, one can appreciate how complex and diverse signaling in biologic

amines act on two different adrenergic receptors present on hepatocytes, the a,-adrenergic receptor and the B2adrenergic receptor. The a,-adrenergic receptor activates the Ca*+/phosphatidylinositol pathway, thereby causing repetitive CaZr transients in single liver cells, whereas the p,-adrenergic receptor stimulates CAMP formation. Epinephrine and, to a lesser degree, norepinephrine activate both receptor types. Interactions between the signaling pathways may be important in the adrenergic regulation of liver cells under physiologic conditions. In single-cell experiments, it was recently shown that CAMP-dependent mechanisms potentiate the a,-adrenergic receptor-induced Ca*+ signal by increasing the frequency and amplitude of each Ca*+ transient (Sch6fl et al. 1991, Sanchez-Bueno et al. 1993) (Figure 3). Furthermore, the threshold for activation of the a,-adrenergic receptor pathway was substantially lowered by CAMPdependent mechanisms to such an extent that physiologic concentrations of epinephrine were able to elicit an a,adrenergic receptor-mediated Ca*+ response. This indicates that cross-signaling between the b2- and a,-adrenergic re-

systems could be and how difficult it is to predict the cellular response to a given hormone when several signaling pathways are activated simultaneously, perhaps with different temporal patterns.

ceptor-activated signaling pathways might be of relevance to the catecholaminergic regulation of liver cell functions under physiologic conditions. Interestingly, this effect of a CAMP-dependent mechanism does not seem to be universal to all Ca*+-mobilizing receptors present on liver cells. Vasopressin given alone causes slightly broader Ca*+ transients of similar amplitude and frequency compared with a,-adrenergic receptor-induced Ca2+

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transients

in single liver cells (Woods et

al. 1987). However, coactivation of the CAMP-dependent signaling pathway, for instance,

by glucagon,

results in a totally

different pattern of the intracellular

Ca2+

I

response, with broadening of the vasopressin-induced Ca2+ transients, as schematically shown in Figure 3 (SanchezBueno et al. 1993). This shows that the intracellular Ca2+ signal elicited by different Ca2+-mobilizing hormones can be distinctly regulated via CAMP-dependent mechanisms. Like the example in Figure 2b, receptor-specific information of receptors acting through otherwise apparently identical signaling pathways is retained ing.

and uncovered

t

catecholamines

1

af

by cross-signal-

In contrast to other cell types (Nishizuka 1986) and to the situation depicted in Figure 2a, CAMP-dependent mechanisms potentiate the Ca2+ signal induced by Ca2+-mobilizing hormones in hepatocytes (Morgan et al. 1984, Burgess et al. 1991, Schijfl et al. 1991, SanchezBueno et al. 1993). In permeabilized hepatocytes, CAMP-dependent kinase shifted the dose-response curve for IF’, to release Ca2+ to the left and increased the total amount of Ca2+ released by 25% (Burgess et al. 1991), suggesting that the IP, receptor is a major site of interaction. A higher affinity or efficiency of the IP, receptor is hardly sufficient, however, to explain the differential effects of elevated CAMP on Ca2+ transients generated by phenylephrine or vasopressin in the same hepatocyte (Figure 3). In experiments with liver cells, using AlF, to stimulate G proteins directly, CAMP-dependent mechanisms have been reported to potentiate A1F3-induced Ca2+ mobilization as well as IP, formation (Blackmore and Exton 1986). Furthermore, a, -adrenergic receptors possess sites for phosphorylation by CAMP-dependent protein kinase A (Leeb-Lundberg et al. 1987), and binding of a,-adrenergic receptor agonists to plasma membranes of liver cells is increased by CAMP-dependent mechanisms (Morgan et al. 1984). This points to additional sites of interaction proximal to the hydrolysis of phosphatidylinositol4,5-bisphosphate at the level of membrane receptors, G proteins, or PLC. Since different agonists activate specific membrane receptors, which may couple through different subtypes of G, to different isozymes of PLC, differential modulation of those sites by CAMP-depend-

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t

different

cellular

responses

?

Figure 4. Schematic illustration of the regulation of the cytosolic free Ca*+ concentration by the temporal pattern of a,-adrenergic receptor stimulation in single rat hepatocytes. Two

situations are depicted where a single hepatocyte is stimulated with short pulses of an a,-adrenergic agonist of two different frequencies. Lowering of the extracellular stimulation frequency almost doubles the amplitude of each Ca*+ transient, whereas the frequency of the Ca*+ transients decreases in parallel to the external frequency of the agonist pulses, as shown on the right (Sch6fl et al. 1993). Since there are multiple intracellular Ca*+-effector proteins with distinct characteristics, changes in the amplitude and frequency of Ca** transients produced by changes in the temporal pattern of extracellular stimulation could differentially regulate target cell responses. Abbreviations are the same as in Figure 3. c, concentration of the agonist. Schematic adaptations are from Schijfl et al. (1993).

ent mechanisms might explain the agonistspecific modulation of Ca2+ transients by elevated CAMP in single hepatocytes (Figure 3). Norepinephrine, which mainly acts via a,-adrenergic receptors, is released from sympathetic nerve endings terminating in the liver parenchyma and so is presumably released in pulses following the action potentials in the nerves. In a recent study, it was found that the temporal pattern of a, -adrenergic receptor stimulation markedly influenced the intracellular Ca2+ response in a single liver cell (Schijfl et al. 1993). Effects on both frequency and amplitude of the Ca2+ transients were observed, depending on the pattern of phenylephrine

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application (this is shown schematically in Figure 4). Also, pulsatile delivery of phenylephrine was more efficacious in terms of eliciting an integrated Ca2+ response of a certain magnitude. According to the hypothesis of temporal specificity, this could mean that intercellular information encoded in the temporal pattern of a,-adrenergic receptor stimulation

is transferred

across

the plasma

membrane and translated into distinct intracellular information encoded in the frequency and amplitude of Ca2+ transients. Experiments with vasopressin pulses suggest that the reqtiired temporal patterns for changes in the intracellular Ca2+ signal might be receptor specific (C. Sch&l and K.S.R. Cuthbertson un-

57

published results). Thus, these data provide direct evidence that besides the well-recognized conformational specificity associated with the fitting of the ligand to the receptor temporal specificity might coexist in intercellular communication There is experimental evidence that the highest frequency of Ca*+ transients obtainable in a single cell does not in any case cause a maximal cellular response. In perfusion experiments, where the release of prolactin (PRL) from lactotrophs was measured in response to repetitive Ca*+ transients induced by pulses of high potassium, Ca*+ transients at a frequency of l Ca*+ transient per minute, secretion became less responsive to the Ca*+ transients because of rapid adaptation of the release process (Law et al. 1989). Therefore, optimal frequencies of Ca2+ transients appear to exist for maximal stimulation of cellular responses that are less than the highest frequency obtainable in a single cell. Since there are multiple intracellular Ca*+-effector proteins and Ca*+-dependent processes, which most likely have different and distinct kinetics and affinities for Ca*+ binding (Davis 1992), changes in the frequency and amplitude of the Ca*+ signal through cross-signaling or the temporal pattern of receptor stimulation could be sufficient to activate distinct Ca*+-dependent cellular responses.

??

Significance for Physiology and Pathophysiology in Endocrine Systems

Cross-signaling between incoming signals enables the cell to increase the diversity of cellular responses. Tissuespecific expression of receptor subtypes and isoforms of components of the individual signaling cascades that have distinct characteristics with respect to crossregulation allow for tissue-specific regulation of target cells in response to a given set of hormones. This reduces the number of circulating hormones required to control a myriad of different and distinct functions in the organism. Furthermore, consideration of mathematical models like parallel distributed process networks, which resemble some aspects of intracellular signaling networks, suggests that there are some

58

shared characteristics

of such intercon-

nected networks that cannot easily be predicted by just knowing the elements themselves (Bray 1990). Such emergent properties of networks like the intracellular signaling network include capacities for generalization, familiarity recognition, and error correction (Hopfield 1982, Rumelhart and McClelland 1987). The networks are also remarkably resistant to small changes. This might be one explanation, among others, why not all mutations found so far, for example, in hormone receptors of otherwise normal patients, necessarily lead to overt pathologic features. Major changes, however, especially if they occur at key sites, could well damage the system and lead to pathologic behavior such as uncontrolled proliferation (Allen et al. 1991, Marx 1993). Because of the properties of the network, it might be possible to correct such an effect by overactivating another system, thereby correcting at least partially the defect in the network. One example is the correction of a defect in the retinoic acid receptor a in promyelocytic cells by high doses of retinoic acid, leading to complete differentiation and maturation of the leukemic cells (Chomienne et al. 199 1, Warrell et al. 1991). On the other hand, in a situation as shown in Figure 2a, apparent failure of hormone B to stimulate a CAMPdependent cellular response and to inhibit the action of hormone A could first of all indicate a defect in its membrane receptor or the CAMP-signaling pathway. Abnormal or constitutive activation of the receptor-C-mediated pathway, however, could have similar effects on cell behavior. Thus, changes in receptor pathways apparently unrelated to a given target cell response could have profound effects via cross-signaling. This could account for situations where no abnormalities were found in the receptor or its associated signaling system, which were believed to be defective. The relevance and significance of encoding biologic information in temporal patterns has been outlined previously (Li and Goldbeter 1989, Brabant et al. 1992). A possible advantage of carrying information in temporal patterns of agonist pulses or patterns of intracellular signals such as Ca*+ transients, apart from the relative insensitivity to noise, is to allow for differential target cell regulation in response to just one hormone or one

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intracellular

signal, thereby reducing the

number of hormones, transmitters, and signaling molecules needed for the control of the organism. Since the kinetics of the individual reactions of a signaling pathway as well as the affinity and the numbers of receptors and effecters would be part of the decoding system of the temporal code, the decoding mechanism could be sensitive to cross-signaling at multiple sites. Cross-signaling and temporal aspects of information processing are intimately interrelated. Dysfunction in temporal information transfer could occur either by a distorted incoming temporal signal or by defects in the decoding mechanisms. Examples of the first situation have been reviewed previously (Brabant et al. 1992). The second possibility implies that changes, for example, in the affinity or numbers of cell membrane receptors or in the kinetics of crucial signaling reactions could cause abnormal target cell behavior despite a correct temporal pattern of stimulation. Future research and interpretation of experimental results has to take these aspects into account, especially if results from highly artificial in vitro systems are to be transferred to the in vivo situation. Furthermore, mathematical models and methods are needed to analyze extracellular patterns of agonists responsible for certain target cell functions as well as models to characterize the intracellular signaling networks for the better understanding of target cell regulation under physiologic and pathophysiologic conditions. The possibility of simulating target cell behavior might enable better prediction of the effects of therapeutic interventions and might provide a better rationale for the treatment of defects in intracellular signaling pathways.

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Acknowledgments

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