Role Of cAMP Signaling Research Articles

The role of hexose transport and phosphorylation in cAMP signalling in the yeast Saccharomyces cerevisiae.

Glucose-induced cAMP signalling in Saccharomyces cerevisiae requires extracellular glucose detection via the Gpr1-Gpa2 G-protein coupled receptor system and intracellular glucose-sensing that depends on glucose uptake and phosphorylation. The glucose uptake requirement can be fulfilled by any glucose carrier including the Gal2 permease or by intracellular hydrolysis of maltose. Hence, the glucose carriers do not seem to play a regulatory role in cAMP signalling. Also the glucose carrier homologues, Snf3 and Rgt2, are not required for glucose-induced cAMP synthesis. Although no further metabolism beyond glucose phosphorylation is required, neither Glu6P nor ATP appears to act as metabolic trigger for cAMP signalling. This indicates that a regulatory function may be associated with the hexose kinases. Consistently, intracellular acidification, another known trigger of cAMP synthesis, can bypass the glucose uptake requirement but not the absence of a functional hexose kinase. This may indicate that intracellular acidification can boost a downstream effect that amplifies the residual signal transmitted via the hexose kinases when glucose uptake is too low.

The role of hexose transport and phosphorylation in cAMP signalling in the yeast

Glucose-induced cAMP signalling in Saccharomyces cerevisiae requires extracellular glucose detection via the Gpr1-Gpa2 G-protein coupled receptor system and intracellular glucose-sensing that depends on glucose uptake and phosphorylation. The glucose uptake requirement can be fulfilled by any glucose carrier including the Gal2 permease or by intracellular hydrolysis of maltose. Hence, the glucose carriers do not seem to play a regulatory role in cAMP signalling. Also the glucose carrier homologues, Snf3 and Rgt2, are not required for glucose-induced cAMP synthesis. Although no further metabolism beyond glucose phosphorylation is required, neither Glu6P nor ATP appears to act as metabolic trigger for cAMP signalling. This indicates that a regulatory function may be associated with the hexose kinases. Consistently, intracellular acidification, another known trigger of cAMP synthesis, can bypass the glucose uptake requirement but not the absence of a functional hexose kinase. This may indicate that intracellular acidification can boost a downstream effect that amplifies the residual signal transmitted via the hexose kinases when glucose uptake is too low.

Modification of representational difference analysis applied to the isolation of forskolin-regulated genes from Schwann cells.

Many aspects of the response of Schwann cells to axonal cues can be induced in vitro by the adenylyl cyclase activator forskolin, yet the role of cAMP signaling in regulating Schwann cell differentiation remains unclear. To define better the relationship between cAMP signaling and Schwann cell differentiation, we used a modification of cDNA representational difference analysis (RDA) that permits the analysis of small amounts of mRNA and identified additional genes that are differentially expressed by forskolin-treated and untreated Schwann cells. The genes that we have identified, including MKP3, a regulator of ERK signaling, and the sphingosine-1-phosphate receptor edg3/lp(B3), may play important roles in mediating Schwann cell differentiation.

Role of the cAMP Signaling Pathway in the Regulation of Gonadotropin-Releasing Hormone Secretion in GT1 Cells

Development of a line of highly differentiated neuronal cells (GT1 cells) that secrete gonadotropin-releasing hormone (GnRH) has permitted studies on the signaling mechanisms involved in regulating secretion of GnRH. This study focused on the pathways coupling GnRH secretion to elevated levels of cyclic adenosine monophosphate (cAMP) in the GT1 cell line. Two mechanisms were examined: a direct action of increased cAMP by means of cyclic nucleotide-gated (CNG) cation channels and an indirect action through activation of cAMP-dependent protein kinase A (PKA). Patch-clamp experiments were carried out on two lines of GT1 cells in both the on-cell and excised patch configurations. Treatment of perifused GT1 cells with dopamine led to a substantial rise in cAMP levels, which resembled accumulation of intracellular cAMP in GT1 cells exposed to dopamine. GT1 cells were found to express the three CNG subunits present in olfactory neurons: CN2, CNG4.3, and CNG5. The cells possessed functional cAMP-gated cation channels. Activation of PKA did not seem to be required for increased cAMP to stimulate the release of GnRH. Indeed, inhibiting PKA activity was associated with increased basal GnRH secretion. PKA inhibited adenylyl cyclase activity, probably by suppressing the cellular expression of adenylyl cyclase V. Stimulation of GnRH release by increased cAMP seems to result from neuronal depolarization triggered by increased conductance of cations by cAMP-gated cation channels. Activation of PKA may be a negative-feedback means of lowering cAMP levels. The resultant oscillations in cAMP could provide a basis for timing the pulsatile release of GnRH. These findings can be tested in vivo by using transgenic methods to examine the role of cAMP signaling in regulating pulsatile GnRH release in the rat. Proc Natl Acad Sci U S A 2000;97:1861–1866

Impaired growth and fertility of cAMP-specific phosphodiesterase PDE4D-deficient mice.

In eukaryotic cells, the inactivation of the cyclic nucleotide signal depends on a complex array of cyclic nucleotide phosphodiesterases (PDEs). Although it has been established that multiple PDE isoenzymes with distinct catalytic properties and regulations coexist in the same cell, the physiological significance of this remarkable complexity is poorly understood. To examine the role of a PDE in cAMP signaling in vivo, we have inactivated the type 4 cAMP-specific PDE (PDE4D) gene, a mammalian homologue of the Drosophila dunce. This isoenzyme is involved in feedback regulation of cAMP levels. Mice deficient in PDE4D exhibit delayed growth as well as reduced viability and female fertility. The decrease in fertility of the null female is caused by impaired ovulation and diminished sensitivity of the granulosa cells to gonadotropins. These pleiotropic phenotypes demonstrate that PDE4D plays a critical role in cAMP signaling and that the activity of this isoenzyme is required for the regulation of growth and fertility.

Protein kinase C in cultured human placental trophoblasts: identification of isoforms and role in cAMP signalling.

The protein kinase C (PKC) superfamily is a family of serine/threonine kinases involved in the regulation of many cell functions. The objective of the present work was to identify the PKC isoenzymes present in human placental trophoblasts and to compare their relative responses to acute and chronic phorbol 12-myristate 13-acetate (PMA) treatment. In addition, the effect of PMA treatment on ligand-stimulated cAMP production was determined. In total extracts prepared from cultured or freshly purified trophoblasts, PKC isoforms alpha, epsilon and zeta were detected. Following acute treatment with PMA, PKC alpha and PKC epsilon were translocated from the cytosol to the particulate fraction. Prolonged treatment (up to 36 h) with PMA resulted in the temporal down-regulation of PKC alpha and PKC epsilon. PKC zeta did not respond to either acute or chronic treatment with PMA. An acute 10 min treatment of the cells with PMA (10(-10)-10(-6) M) enhanced isoprenaline- and adrenaline-stimulated cAMP production. An enhanced response was observed at all concentrations of isoprenaline tested (10(-9)-10(-4) M), suggesting an increased capacity to respond. The acute PMA effect was evident within 2 min and near maximal by 5 min. The acute response to PMA was lost in cells where PKC was down-regulated by prior PMA treatment. Epidermal growth factor, a potential ligand for the activation of PKC in trophoblasts, enhanced isoprenaline-stimulated cAMP production. In summary, activation of PMA-responsive PKCs (PKC alpha or PKC epsilon) appears to enhance ligand-stimulated cAMP production in trophoblasts. This may be a physiologically important example of 'cross-talk' between various signalling pathways in human placental trophoblasts.

Altered circadian pacemaker functions and cyclic AMP rhythms in the drosophila learning mutant dunce

Neural circadian pacemakers can be reset by light, and the resetting mechanism may involve cyclic nucleotide second messengers. We have examined pacemaker resetting and free-running activity rhythms in Drosophila dunce (dnc) and DC0 mutants, which identify a cAMP-specific phosphodiesterase and the catalytic subunit of cAMP-dependent protein kinase, respectively. dnc mutants exhibit augmented light-induced phase delays and shortened circadian periods, which indicate altered pacemaker function. Interestingly, however, light-induced phase advances are normal in dnc, suggesting a selective effect on one component of the pacemaker resetting response. Furthermore, we demonstrate the presence of circadian rhythms in cAMP content in head tissues and show that dnc mutations increase the amplitude of daily cAMP peaks. These results show that cAMP levels are not chronically elevated in the dnc mutant. A role for cAMP signaling in circadian processes is also suggested by an analysis of DC0 mutants, which have severe kinase deficits and display arrhythmic locomotor activity.

In vitro fertilization of partially zona dissected or partially zona removed equine oocytes

We previously demonstrated that male mice deficient in the soluble adenylyl cyclase (sAC) are sterile and produce spermatozoa with deficits in progressive motility and are unable to fertilize zona-intact eggs. Here, analyses of sAC−/− spermatozoa provide additional insights into the functions linked to cAMP signaling. Adenylyl cyclase activity and cAMP content are greatly diminished in crude preparations of sAC−/− spermatozoa and are undetectable after sperm purification. HCO3− is unable to rapidly accelerate the flagellar beat or facilitate evoked Ca2+ entry into sAC−/− spermatozoa. Moreover, the delayed HCO3−-dependent increases in protein tyrosine phosphorylation and hyperactivated motility, which occur late in capacitation of wild-type spermatozoa, do not develop in sAC−/− spermatozoa. However, sAC−/− sperm fertilize zona-free oocytes, indicating that gamete fusion does not require sAC. Although ATP levels are significantly reduced in sAC−/− sperm, cAMP-AM ester increases flagellar beat frequency, progressive motility, and alters the pattern of tyrosine phosphorylated proteins. These results indicate that sAC and cAMP coordinate cellular energy balance in wild-type sperm and that the ATP generating machinery is not operating normally in sAC−/− spermatozoa. These findings demonstrate that sAC plays a critical role in cAMP signaling in spermatozoa and that defective cAMP production prevents engagement of multiple components of capacitation resulting in male infertility.