Intercellular cAMP

Some of the main examples of biological rhythms of nonelectrical nature are discussed below, among which are glycolytic oscillations (Section III), oscillations and waves of cytosolic Ca + (Section IV), cAMP oscillations that underlie pulsatile intercellular communication in Dictyostelium amoebae (Section V), circadian rhythms (Section VI), and the cell cycle clock (Section VII). Section VIII is devoted to some recently discovered cellular rhythms. The transition from simple periodic behavior to complex oscillations including bursting and chaos is briefly dealt with in Section IX. Concluding remarks are presented in Section X. 

The three best-known examples of biochemical oscillations were found during the decade 1965-1975 [40,41]. These include the peroxidase reaction, glycolytic oscillations in yeast and muscle, and the pulsatile release of cAMP signals in Dictyostelium amoebae (see Section V). Another decade passed before the development of Ca " " fluorescent probes led to the discovery of oscillations in intracellular Ca +. Oscillations in cytosolic Ca " " have since been found in a variety of cells where they can arise spontaneously, or after stimulation by hormones or neurotransmitters. Their period can range from seconds to minutes, depending on the cell type [56]. The oscillations are often accompanied by propagation of intracellular or intercellular Ca " " waves. The importance of Ca + oscillations and waves stems from the major role played by this ion in the control of many key cellular processes—for example, gene expression or neurotransmitter secretion. 

Figure 1.10 Model of a G protein-coupled receptor with 7 membrane-spanning domains. Binding of an agonist to the receptor causes GDP to exchange with GTP. The a-GTP complex then dissociates from the receptor and the py complex and interacts with intercellular en mes or ion channels. The Py complex can activate an ion channel or possibly also interact with intercellular enzymes. GDP, guanine diphosphate GTP, guanine triphosphate cAMP, cyclic adenosine monophosphate PKC, protein kinase C PLC, phospholipase C DAG, diacylglycerol.

In contrast to cAMP, a stimulation of PKC with phorbol esters (TPA) has been shown to play an important role in the downregulation of gap junctional coupling [Yancey et al., 1982]. Since reestablishment of intercellular coupling was not seen after wash out of TPA in the presence of the protein synthesis inhibitor, puromycin [Fitzgerald et al., 1983], the phorbol ester probably induces the elimination of junctional channels under these conditions. Thus, it might be possible that PKC is involved in the regulation of channel degradation. 

Obviously, there is some kind of cross-talk between PKC and cAMP in the modulation of intercellular coupling, since cAMP can inhibit the uncoupling effect of phorbol esters if cells are exposed to both agents from the start of the experiment [Kanno et al., 1984], but this protective effect can be abolished by the protein synthesis inhibitor cycloheximide [Enomoto et al., 1984] in Balb/ cells. 

Similar to a P-adrenoceptor stimulation intracellular cAMP can be increased by inhibition of phosphodiesterase. Thus, in turtle retina cells, cAMP leads to uncoupling and this can be mimicked by stimulation of adenylate cyclase with forskolin and concomitant inhibition of phosphodiesterase by IBMX [Piccolino et al., 1984]. In cardiac cells inhibition of phosphodiesterase has been investigated using methylxanthine derivates [De Mello, 1989], resulting in an enhancement of intercellular coupling. 

Darrow J, Fast VG, Kleber AG, Beyer EC, Saffitz JE Functional and structural assessment of intercellular communication. Increased conduction velocity and enhanced connexin expression in dibutyryl cAMP-treated cultured cardiac myocytes. Circ Res 1996 79 174-183. 

De Mello WC Influence of the sodium pump on intercellular communication in heart fibers Effect of intracellular injection of sodium ion on electrical coupling. J Physiol (Lond) 1976 263 171-197. De Mello WC Effect of intracellular injection of cAMP on the electrical coupling of mammalian cardiac cells. Biochem Biophys Res Commun 1984 119 1001-1007. 

Somatostatin. Somatostatin is present in numerous tissues including pancreatic D-cells and pituitary cells. Its secretion appears to be mediated via increases in cAMP (Patel et al., 1991). The peptide inhibits insulin and glucagon release in a paracrine or intercellular fashion involving inhibition of cAMP formation (Pipeleers, 1987). Somatostatin, like a2-agonists such as clonidine, stimulates inhibitory Gj-protein in B-cells. Moreover, somatostatin induces repolarization and decreases [Ca2+]j in the B-cell (for a review see Berggren et al., 1992). Previously, Hsu et al. (1991) reported that somatostatin inhibits insulin secretion by a G-protein-mediated decrease in Ca2+ entry via a voltage-dependent Ca2+ channel in the B-cell. 

Part IV is devoted to the function of pulsatile signalling in intercellular communication. The function of cAMP pulses in Dictyostelium is first addressed in chapter 8. Experiments have shown that cAMP signals... 

The first model proposed for the mechanism of intercellular communication in D. discoideum (fig. 5.10) successfully predicted the existence of sustained oscillations of cAMP (Goldbeter, 1975). The experimental data on which that model was based, however, turned out to be partly inexact due to an experimental artefact. Although the theoretical prediction of cAMP oscillations was soon corroborated by the observations of Gerisch Wick (1975), the source of the oscillations lay in another regulatory mechanism that remained to be determined. 

Experimentally, the mechanism of intercellular communication by cAMP pulses in the course of D. discoideum aggregation is characterized by its periodicity. The latter is reflected by the wavelike movement of amoebae towards the aggregation centres, as a result of the periodic pulses of cAMP that the latter emit at regular intervals (Durston, 1974a). The periodic behaviour of the model based on receptor desensitization accounts for the periodic secretion of cAMP by aggregation centres, whereas excitable behaviour accounts for the relay of cAMP pulses by cells that amplify the suprathreshold signals emitted by the centres. 

Fig. 7.1. Evolution of the system of intercellular communication by cAMP signals in D. discoideum in the course of development. Time is measured in hours after the beginning of starvation. The black zone at the bottom of the figure shows the evolution of the basal activity of adenylate cyclase. Until 2 h after the beginning of starvation, the enzyme fails to be activated by cAMP signals the transient activation, corresponding to the phenomenon of relay, is observed thereafter, followed by the onset of autonomous oscillations of cAMP. The latter spontaneously appear about 4 h after the beginning of starvation and continue for about 3 h, before the system recovers its excitable behaviour (Gerisch et a/.,1979).

Fig. 7.3. Evolution of the biochemical parameters of the mechanism of intercellular communication during the hours that follow starvation in D. discoideum. The variation in the activity of (a) adenylate cyclase and (b) intra- and extracellular phosphodiesterase and (c) the evolution of the quantity of cAMP receptor measured in experiments performed with two different levels of cAMP. In (b), circles and triangles refer to total phosphodiesterase and to a protein inhibitor of the enzyme, respectively closed symbols relate to cells treated by pulses of cAMP resulting in 10" M cAMP every 5 min, whereas open symbols relate to untreated cells (data collected from various authors by Loomis, 1979).

Fig. 7.4. Developmental path for the mechanism of intercellular communication by cAMP signals in D. discoideum, in agreement with the variations observed for the activity of adenylate cyclase and phosphodiesterase after starvation. The diagram is constructed as indicated in fig. 7.2, for system (5.1) to which the term (-k a) has been added in the evolution equation for variable a, to take into account the utilization of ATP to ends other than cAMP synthesis. In these conditions, the developmental path accounting for the sequential transitions of fig. 7.1 corresponds to the increase in the two enzyme activities that is observed in the hours that follow starvation (fig. 7.3). Domains A, B and C have the same meaning as in fig. 7.2 domain D corresponds to a stable steady state characterized by an elevated level of cAMP, while two stable steady states can coexist in E. The signal considered for amplification in B is y = 10. Parameter values are V = 0.04 s, k, - 0.4 s k = 10" s", = 10, L = lO", q = 100 (Goldbeter Segel, 1980).

Fig. 7.5. Incorporating the variation of biochemical parameters into the description of the evolution of the mechanism of intercellular communication in D. dis-coideum. The sigmoidal increase observed during the 6h that follow the beginning of starvation for adenylate cyclase (

These results are of general significance for the study of biological rhythms as they show how the continuous variation of certain control parameters can lead to the emergence of a rhythm in the course of development of an organism. Here, the level of certain proteins augments once the amoebae begin to synthesize the components of their intercellular communication system after starvation. As soon as the concentration of the cAMP receptor and the activity of enzymes such as adenylate cyclase and phosphodiesterase reach a critical value, oscillations appear spontaneously. Rinzel Baer (1988) have shown, however, that a certain delay separates the time at which the parameters cross their bifurcation values and the moment at which oscillations... 

Similar experiments carried out with the wild type (Gerisch et al, 1975) showed that periodic signals of cAMP, in contrast to constant stimuli, accelerate cell differentiation by inducing the precocious synthesis of proteins involved in the aggregation process. Initial experiments demonstrated the induction of proteins necessary for the establishment of intercellular contacts similar experiments (Klein Darmon, 1977 Juliani Klein, 1978 Chisholm, Hopkinson Lodish, 1987 Kimmel, 1987 Mann Firtel, 1987 Mann, Pinko Firtel, 1988) established that the periodic signals of cAMP induce a differentiation programme in which other proteins such as adenylate cyclase, the cAMP receptor, and phosphodiesterase are synthesized. These developmental responses depend on the periodic nature of the stimulation and do not occur when the cAMP stimulus remains constant. 

When applying these results to intercellular communication in cellular slime moulds, we need to remember that the amoebae relaying cAMP signals during aggregation are excitable. These cells therefore possess an absolute and a relative refractory period, but only the latter period characterizes the response of the system when self-amplification in cAMP synthesis is suppressed under conditions where the level of... 

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