Role of the cAMP-binding protein Epac in cardiovascular physiology and pathophysiology

Exchange proteins directly activated by cyclic AMP (Epac) were discovered 10 years ago as new sensors for the second messenger cyclic AMP (cAMP). Epac family, including Epac1 and Epac2, are guanine nucleotide exchange factors for the Ras-like small GTPases Rap1 and Rap2 and function independently of protein kinase A. Given the importance of cAMP in the cardiovascular system, numerous molecular and cellular studies using specific Epac agonists have analyzed the role and the regulation of Epac proteins in cardiovascular physiology and pathophysiology. The specific functions of Epac proteins may depend upon their microcellular environments as well as their expression and localization. This review discusses recent data showing the involvement of Epac in vascular cell migration, endothelial permeability, and inflammation through specific signaling pathways. In addition, we present evidence that Epac regulates the activity of various cellular compartments of the cardiac myocyte and influences calcium handling and excitation–contraction coupling. The potential role of Epac in cardiovascular disorders such as cardiac hypertrophy and remodeling is also discussed.     

Role of the cAMP sensor Epac as a determinant of KATP channel ATP sensitivity in human pancreatic beta-cells and rat INS-1 cells

Protein kinase A (PKA)-independent actions of adenosine 3',5'-cyclic monophosphate (cAMP) are mediated by Epac, a cAMP sensor expressed in pancreatic beta-cells. Evidence that Epac might mediate the cAMP-dependent inhibition of beta-cell ATP-sensitive K(+) channels (K(ATP)) was provided by one prior study of human beta-cells and a rat insulin-secreting cell line (INS-1 cells) in which it was demonstrated that an Epac-selective cAMP analogue (ESCA) inhibited a sulphonylurea-sensitive K(+) current measured under conditions of whole-cell recording. Using excised patches of plasma membrane derived from human beta-cells and rat INS-1 cells, we now report that 2'-O-Me-cAMP, an ESCA that activates Epac but not PKA, sensitizes single K(ATP) channels to the inhibitory effect of ATP, thereby reducing channel activity. In the presence of 2'-O-Me-cAMP (50 microM), the dose-response relationship describing ATP-dependent inhibition of K(ATP) channel activity (NP(o)) is left-shifted such that the concentration of ATP producing 50% inhibition (IC(50)) is reduced from 22 microM to 1 microM for human beta-cells, and from 14 microM to 4 microM for rat INS-1 cells. Conversely, when patches are exposed to a fixed concentration of ATP (10 microM), the administration of 2'-O-Me-cAMP inhibits channel activity in a dose-dependent and reversible manner (IC(50) 12 microM for both cell types). A cyclic nucleotide phosphodiesterase-resistant ESCA (Sp-8-pCPT-2'-O-Me-cAMPS) also inhibits K(ATP) channel activity, thereby demonstrating that the inhibitory actions of ESCAs reported here are unlikely to arise as a consequence of their hydrolysis to bioactive derivatives of adenosine. On the basis of such findings it is concluded that there exists in human beta-cells and rat INS-1 cells a novel form of ion channel modulation in which the ATP sensitivity of K(ATP) channels is regulated by Epac.     

cAMP protects neutrophils against TNF-alpha-induced apoptosis by activation of cAMP-dependent protein kinase, independently of exchange protein directly activated by cAMP (Epac)

It is unclear by which receptor cyclic adenosine monophosphate (cAMP) acts to promote neutrophil survival. We found that 8-(4-chlorophenylthio)-2'-O-methyl-cAMP, a specific activator of the recently discovered cAMP receptor, cAMP-regulated guanosine 5'-triphosphate exchange protein directly activated by cAMP, failed to protect human neutrophils from cell death. In contrast, specific activators of cAMP-dependent protein kinase type I (cA-PKI) could protect against death receptor [tumor necrosis factor receptor 1 (TNFR-1), Fas]-mediated apoptosis as well as cycloheximide-accelerated "spontaneous" apoptosis. A novel "caged" cA-PK-activating analog, 8-bromo (8-Br)-acetoxymethyl-cAMP, was more than 20-fold more potent than 8-Br-cAMP to protect neutrophils challenged with TNF-alpha against apoptosis. This analog acted more rapidly than forskolin (which increases the endogenous cAMP production) and allowed us to demonstrate that cA-PK must be activated during the first 10 min after TNF-alpha challenge to protect against apoptosis. The protective effect was mediated solely through cA-PK activation, as it was abolished by the cA-PKI-directed inhibitor Rp-8-Br-cAMPS and the general cA-PK inhibitor H-89. Neutrophils not stimulated by cAMP-elevating agents showed increased apoptosis when exposed to the cA-PK inhibitors Rp-8-Br-cAMPS and H-89, suggesting that even moderate activation of cA-PK is sufficient to enhance neutrophil longevity and thereby contribute to neutrophil accumulation in chronic inflammation.     

A critical role of the cAMP sensor Epac in switching protein kinase signalling in prostaglandin E2-induced potentiation of P2X3 receptor currents in inflamed rats

Sensitization of purinergic P2X receptors is one of the mechanisms responsible for exaggerated pain responses to inflammatory injuries. Prostaglandin E2 (PGE2), produced by inflamed tissues, is known to contribute to abnormal pain states. In a previous study, we showed that PGE2 increases fast inactivating ATP currents that are mediated by homomeric P2X3 receptors in dorsal root ganglion (DRG) neurons isolated from normal rats. Protein kinase A (PKA) is the signalling pathway used by PGE2. Little is known about the action of PGE2 on ATP currents after inflammation, although the information is crucial for understanding the mechanisms underlying inflammation-induced sensitization of P2X receptors. We therefore studied the effects of PGE2 on P2X3 receptor-mediated ATP currents in DRG neurons dissociated from complete Freund's adjuvant (CFA)-induced inflamed rats. We found that PGE2 produces a large increase in ATP currents. PKCɛ, in addition to PKA, becomes involved in the modulatory action of PGE2. Thus, PGE2 signalling switches from a solely PKA-dependent pathway under normal conditions to both PKA- and PKC-dependent pathways after inflammation. Studying the mechanisms underlying the switch, we demonstrated that cAMP-responsive guanine nucleotide exchange factor 1 (Epac1) is up-regulated after inflammation. The Epac agonist CPT-OMe mimics the potentiating effect of PGE2 and occludes the PKC-mediated PGE2 action on ATP currents. These results suggest that Epac plays a critical role in P2X3 sensitization by activation of de novo PKC-dependent signalling of PGE2 after inflammation and would be a useful therapeutic target for pain therapies.     

Cell models for studying renal physiology

The development over the past 20 years of a variety of cultured renal tubule cell lines derived from different parts of the renal tubule has provided invaluable powerful cell systems for in vitro analyses of the various tubule segment-specific biochemical functions and ion transport processes. Immortalized cell lines have been established using different hybrid gene constructs, most of them carrying the immortalizing simian virus 40 large T antigen (Tag) gene. The development of transgenic mice carrying unregulated Tag, and of others in which the expression of Tag remains controlled, has made it possible to establish permanent cell lines derived from microdissected or immunoselected renal proximal, distal, and collecting duct tubules. This review summarizes the different strategies of cellular immortalization used and the most frequently used human, rabbit, rat, and mouse tubule cell lines. This review provides an overview of the use of immortalized mouse tubule cell lines for in vitro analyses of various tubule cell-specific functions and the regulation of ion transporters and membranous channels. The advantages of using primary cultures of isolated tubules dissected from physiopathological models of transgenic mice are also discussed.     

Cell volume regulation: physiology and pathophysiology

Cell volume perturbation initiates a wide array of intracellular signalling cascades, leading to protective and adaptive events and, in most cases, activation of volume-regulatory osmolyte transport, water loss, and hence restoration of cell volume and cellular function. Cell volume is challenged not only under physiological conditions, e.g. following accumulation of nutrients, during epithelial absorption/secretion processes, following hormonal/autocrine stimulation, and during induction of apoptosis, but also under pathophysiological conditions, e.g. hypoxia, ischaemia and hyponatremia/hypernatremia. On the other hand, it has recently become clear that an increase or reduction in cell volume can also serve as a specific signal in the regulation of physiological processes such as transepithelial transport, cell migration, proliferation and death. Although the mechanisms by which cell volume perturbations are sensed are still far from clear, significant progress has been made with respect to the nature of the sensors, transducers and effectors that convert a change in cell volume into a physiological response. In the present review, we summarize recent major developments in the field, and emphasize the relationship between cell volume regulation and organism physiology/pathophysiology.     

Effect of cAMP on Peptide Formation in Human Erythrocytes Depending on Cell Age

We found that exogenous and endogenous cAMP induces changes in the spectrum and amount of intraerythrocytic peptides in human erythrocytes of different age. Activation of cAMP formation with epinephrine leads to the appearance of peptides of the other type and to an increase in their total amount in young cells, while blockade of these β-adrenoreceptors with propranolol eliminates these effects. Inhibition of cAMP phosphodiesterase with imidazole in the absence of hormonal signals elevates the content of longer peptides in erythrocytes compared to the effect of exogenous cAMP. The degree of this elevation depends on erythrocyte age.     

Contributions of mitochondria to animal physiology: from homeostatic sensor to calcium signalling and cell death

Over recent years, it has become clear that mitochondria play a central role in many key aspects of animal physiology and pathophysiology. Their central and ubiquitous task is clearly the production of ATP. Nevertheless, they also play subtle roles in glucose homeostasis, acting as the sensor for substrate supply in the transduction pathway that promotes insulin secretion by the pancreatic β-cell and that modulates the excitability of the hypothalamic glucose-sensitive neurons involved in appetite control. Mitochondria may also act as sensors of availability of oxygen, the other major mitochondrial substrate, in the regulation of respiration. Mitochondria take up calcium, and the high capacity mitochondrial calcium uptake pathway provides a mechanism that couples energy demand to increased ATP production through the calcium-dependent upregulation of mitochondrial enzyme activity. Mitochondrial calcium accumulation may also have a substantial impact on the spatiotemporal dynamics of cellular calcium signals, with subtle differences of detail in different cell types. Recent work has also revealed the centrality of mitochondrial dysfunction as an irreversible step in the pathway to both necrotic and apoptotic cell death. This review looks at recent developments in these rapidly evolving areas of cell physiology in an attempt to draw together disparate areas of research into a common theme.     

Estradiol stimulation of pituitary cAMP production and cAMP binding

The role of 17 beta-estradiol (E2) in the modulation of N6,O2'-dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP)-induced hormone release was examined in pituitary monolayer cultures prepared from intact and ovariectomized female rats. Incubations with 5 mM DBcAMP for 4 h significantly (P less than 0.05) stimulated both luteinizing hormone (LH) and prolactin (PRL) release in pituitary cultures prepared from rats at diestrus and from cycling rats at random stages of the estrous cycle. However, DBcAMP failed to stimulate the LH or PRL release in cultures prepared from ovariectomized rats in which the basal LH and PRL release was approximately three-fold and one-tenth of that in cycling rats, respectively. Pretreatment with 1 nM E2 augmented or restored the DBcAMP-induced LH release but not the DBcAMP-induced PRL release in cultures prepared from cycling or ovariectomized rats, respectively. Furthermore, E2 treatment alone of cultures prepared from cycling rats significantly increased intracellular cAMP concentrations and cAMP-binding activities by at least twofold over that of the non-E2-treated controls. The E2-induced rise in cellular cAMP concentration preceded the E2-induced rise in cAMP binding. These results indicate that the priming effect of E2 on pituitary LH responsiveness to DBcAMP is associated with increased cAMP production and cAMP binding.     

Epac1-Rap1 signaling regulates monocyte adhesion and chemotaxis

Extravasation of leukocytes is a crucial process in the immunological defense. In response to a local concentration of chemokines, circulating leukocytes adhere to and migrate across the vascular endothelium toward the inflamed tissue. The small guanosinetriphosphatase Rap1 plays an important role in the regulation of leukocyte adhesion, polarization, and chemotaxis. We investigated the role of a guanine nucleotide exchange protein for Rap1 directly activated by cAMP (Epac1) in adhesion and chemotaxis in a promonocytic cell line and in primary monocytes. We found that Epac1 is expressed in primary leukocytes, platelets, CD34-positive hematopoietic cells, and the leukemic cell lines U937 and HL60. Epac activation with an Epac-specific cAMP analog induced Rap1 activation, beta1-integrin-dependent cell adhesion, and cell polarization. In addition, activated Epac1 enhanced chemotaxis of U937 cells and primary monocytes. Similar to activation of Epac1, stimulation of cells with serotonin to induce cAMP production resulted in Rap1 activation, increased cell adhesion and polarization, and enhanced chemotaxis. The effects of serotonin on U937 cell adhesion were dependent on cAMP production but could not be blocked by a protein kinase A inhibitor, implicating Epac in the regulation of serotonin-induced adhesion. In summary, our work reveals the existence of previously unrecognized cAMP-dependent signaling in leukocytes regulating cell adhesion and chemotaxis through the activation of Epac1.     

转录因子环腺苷酸反应元件结合蛋白介导的细胞凋亡及其调控机制

细胞凋亡是一个精细而复杂的过程,需要一系列的蛋白参与.其中环腺苷酸反应元件结合蛋白(CREB)作为一个核转录因子,通过自身磷酸化实现调节转录功能,改变细胞内的信号转导途径,从而调控细胞凋亡过程.我们结合研究结果对CREB介导的细胞凋亡及其调控机制作一综述.     

Clinical physiology: an accepted branch of physiology

Clinical physiology is a branch of physiology particularly dealing with functional disturbances in disease (pathophysiology) and the integrated function of the human body in disease against the background of normal function in healthy subjects, suitable physiological methods for the study of patients--particularly for diagnostic purposes--as well as for research, and the education of medical students and laboratory assistants in these fields. Departments of clinical physiology in university hospitals form a bridge between basic physiology and many clinical specialties. Independent departments of clinical physiology developed early in Sweden due to the work of Professor Torgny Sj?strand at the Karolinska Hospital in Stockholm, and have been models of research, teaching and hospital organization which have been followed in several other countries. The International Union of Physiological Sciences (IUPS) has recognized clinical physiology as a separate branch of physiology by approving a Commission of Clinical Physiology which has contributed to the programme of this and, we hope, future congresses, as well as promote the development of clinical physiology internationally.