Regulation of cardiac fibroblast collagen synthesis by adenosine: roles for Epac and PI3K.

Rat cardiac fibroblasts (CF) express multiple adenosine (ADO) receptors. Pharmacological evidence suggests that activation of A(2) receptors may inhibit collagen synthesis via adenylyl cyclase-induced elevation of cellular cAMP. We have characterized the signaling pathways involved in ADO-mediated inhibition of collagen synthesis in primary cultures of adult rat CF. ANG II stimulates collagen production in these cells. Coincubation with agents that elevate cellular cAMP [the ADO agonist, 5'-N-ethylcarboxamidoadensoine (NECA), and forskolin] inhibited the stimulatory effects of ANG II. However, direct stimulators and inhibitors of protein kinase A (PKA) did not alter ANG II-induced collagen synthesis, indicating that PKA does not mediate the inhibitory effects of NECA. Inhibitors of AMP-kinase (AMPK) and extracellular signal-regulated kinase 1/2 (ERK1/2) do not alter NECA-inhibited collagen synthesis. However, activation of exchange factor directly activated by cAMP (Epac) mimicked the effects of NECA on ANG II-stimulated collagen synthesis. Inhibition of phosphoinositol-3 kinase (PI3K) reduced the inhibitory effects of NECA on ANG II-induced collagen synthesis, suggesting that NECA acts via PI3K. Furthermore, inhibition of PI3K also relieved the inhibitory effect of Epac activation on ANG II-stimulated collagen synthesis. Thus it appears that ADO activates the A(2)R-G(s)-adenylyl cyclase pathway and that the resultant cAMP reduces collagen synthesis via a PKA-independent, Epac-dependent pathway that feeds through PI3K.

Adenosine receptors and second messenger signaling pathways in rat cardiac fibroblasts.

The ability of adenosine (ADO) to inhibit proliferation and protein synthesis (in particular, collagen synthesis) in cardiac fibroblasts (CF) may ameliorate adverse cardiac remodeling and fibrosis seen in heart failure patients. However, little is known about the signaling pathways that ADO may modulate in CF to alter cell phenotype. Accordingly, this study was designed to identify ADO receptors (AR) and the signaling pathways linked to them in primary cultures of adult rat CF. Quantitative RT-PCR data indicate that the mRNAs for all four known ARs (A(1)R, A(2a)R, A(2b)R, and A(3)R) are present in rat CF, with a greater prevalence of A(2) receptor subtypes. No coupling of AR to the G(q)-phospholipase C signaling pathway or to mobilization of calcium is measurable. Studies using subtype specific agents imply that the A(2a)R and A(2b)R couple to G(s)-adenylyl cyclase and A(1)R couple weakly to G(i)-adenylyl cyclase. 2-Chloroadenosine, 5'-N-ethylcarboxamidoadensoine, and other agents that elevate cellular cAMP stimulate extracellular signal-regulated kinase 1/2 activity in a pertussis toxin-insensitive manner. We conclude that a combination of cAMP-dependent signals generated via A(2a) and A(2b) receptors likely mediate ADO signaling in adult rat CF.

Endothelin downregulates SERCA2 gene and protein expression in adult rat ventricular myocytes: regulation by pertussis toxin-sensitive Gi protein and cAMP.

Downregulation of the sarcoplasmic reticulum calcium ATPase (SERCA2) is associated with diastolic dysfunction in the failing heart. Elevated plasma endothelin-1 (ET) levels are correlated with congestive heart failure suggesting that ET may play a pathophysiological role. We have investigated the ability of ET to regulate SERCA2 gene expression in isolated adult rat ventricular myocytes. We find that ET enhances net protein synthesis by approximately 40% but significantly downregulates SERCA2 mRNA expression, time dependently, by approximately 30-50%, and the expression of SERCA2 protein by approximately 50%. In myoyctes, ET binds to ET(A) receptor that couples to G(q) and G(i) proteins. Inhibition of G(q)-PLC-induced phosphoinositide (PI) hydrolysis with U73122 (1 muM) or inhibition of G(i) protein with pertussis toxin (PTX) abolishes the ability of ET to downregulate SERCA2 mRNA gene expression. Further investigation suggests that ET coupling to PTX-sensitive G(i) with consequent lowering of cAMP is required for downregulation of SERCA2 mRNA levels. Increasing intracellular cAMP quantity using cAMP-specific PDE inhibitor Ro20-1724 or cAMP analog dibutyryl-cAMP reverses ET-induced downregulation of SERCA2 mRNA levels. The data indicate that, in adult myocytes, ET downregulates SERCA2 mRNA and protein levels, and the effect requires cross-talk between G(q) and PTX-sensitive G(i) pathways.

Lysophosphatidic acid induces hypertrophy of neonatal cardiac myocytes via activation of Gi and Rho.

The effect of the lysophospholipid, lysophosphatidic acid (LPA), on signaling and hypertrophy of neonatal rat ventricular cardiomyocytes was examined. Myocytes express mRNA for all three G-protein-coupled LPA receptor subtypes (LPA(1)/Edg-2, LPA(2)/Edg-4, and LPA(3)/Edg-7) as indicated by RT-PCR analysis. LPA inhibits isoproterenol-stimulated cyclic AMP accumulation with an IC(50) approximately 40 nM and promotes phosphorylation of ERK-1/2. LPA also elicits a small, slow onset, and activation of phosphoinositide hydrolysis with EC(50) approximately 400 nM, and stimulates a marked increase in the extent of Rho activation. Longer-term treatment with LPA induces a hypertrophic response in myocytes as indicated by increases in cell size, actin organization, ANF staining of the perinuclear region and activation of ANF promoter-luciferase gene expression. Pretreatment of myocytes with pertussis toxin (PTX) not only blocks the capacity of LPA to inhibit cyclic AMP formation and stimulate ERK phosphorylation, but also inhibits hypertrophic changes in cell morphology and ANF-luciferase gene expression. Neither phospholipase C nor Rho activation is PTX sensitive. The hypertrophic effects of LPA on myocytes are also inhibited by treatment with C3 exoenzyme or by transfection of plasmids expressing either C3 exoenzyme or dominant-negative Rho to block Rho function. Inhibition of ERK activation with PD98059 blocks LPA-induced hypertrophy while inhibitors of phospholipase C (U73122), PKC (GF109203X), or p38MAPK (SB203580) do not. These data suggest that LPA induces cardiomyocyte hypertrophy via a pathway different from the conventional G(q) pathway utilized by phenylephrine, endothelin, and PGF2 alpha and involving activation of a PTX-sensitive G(i)/ERK pathway in conjunction with activation of Rho-mediated signals.

Balanol analogues probe specificity determinants and the conformational malleability of the cyclic 3',5'-adenosine monophosphate-dependent protein kinase catalytic subunit.

The protein kinase family is a prime target for therapeutic agents, since unregulated protein kinase activities are linked to myriad diseases. Balanol, a fungal metabolite consisting of four rings, potently inhibits Ser/Thr protein kinases and can be modified to yield potent inhibitors that are selective-characteristics of a desirable pharmaceutical compound. Here, we characterize three balanol analogues that inhibit cyclic 3',5'-adenosine monophosphate-dependent protein kinase (PKA) more specifically and potently than calcium- and phospholipid-dependent protein kinase (PKC). Correlation of thermostability and inhibition potency suggests that better inhibitors confer enhanced protection against thermal denaturation. Crystal structures of the PKA catalytic (C) subunit complexed to each analogue show the Gly-rich loop stabilized in an "intermediate" conformation, disengaged from important phosphoryl transfer residues. An analogue that perturbs the PKA C-terminal tail has slightly weaker inhibition potency. The malleability of the PKA C subunit is illustrated by active site residues that adopt alternate rotamers depending on the ligand bound. On the basis of sequence homology to PKA, a preliminary model of the PKC active site is described. The balanol analogues serve to test the model and to highlight differences in the active site local environment of PKA and PKC. The PKA C subunit appears to tolerate balanol analogues with D-ring modifications; PKC does not. We attribute this difference in preference to the variable B helix and C-terminal tail. By understanding the details of ligand binding, more specific and potent inhibitors may be designed that differentiate among closely related AGC protein kinase family members.

In vivo adenoviral transfer of sorcin reverses cardiac contractile abnormalities of diabetic cardiomyopathy.

In many types of heart failure cardiac myocyte Ca(2+) handling is abnormal because of downregulation of key Ca(2+) - handling proteins like sarco(endo)plasmic reticulum Ca(2+) - ATPase (SERCA)2a and ryanodine receptor (RyR)2. The alteration in SERCA2a and RyR2 expression results in altered cytosolic Ca(2+) transients, leading to abnormal contraction. Sorcin is an EF-hand protein that confers the property of caffeine-activated intracellular Ca(2+) release in nonmuscle cells by interacting with RyR2. To determine whether sorcin could improve the contractile function of the heart, we overexpressed sorcin in the heart of either normal or diabetic mice and in adult rat cardiomyocytes with an adenoviral gene transfer approach. Sorcin overexpression was associated with an increase in cardiac contractility of the normal heart and dramatically rescued the abnormal contractile function of the diabetic heart. These effects could be attributed to an improvement of the Ca(2+) transients found in the cardiomyocyte after sorcin overexpression. Viral vector-mediated delivery of sorcin to cardiac myocytes is beneficial, resulting in improved contractile function in diabetic cardiomyopathy.

PDE4: arrested at the border.

Compartmentation plays a critical role in determining the specificity and efficacy of signaling by adenosine 3',5'-monophosphate (cAMP) and other second messengers. It has become apparent that all of the protein components of the cAMP signaling pathway are subject to regulation and may be localized to particular subcellular compartments. This Perspective discusses recent research concerning the importance of cAMP phosphodiesterase localization in cAMP signaling, functional implications of the organization of this pathway's protein components into macromolecular complexes, and common themes in the compartmentation of cAMP- and Ca2+-mediated signaling pathways.

Modeling beta-adrenergic control of cardiac myocyte contractility in silico.

The beta-adrenergic signaling pathway regulates cardiac myocyte contractility through a combination of feedforward and feedback mechanisms. We used systems analysis to investigate how the components and topology of this signaling network permit neurohormonal control of excitation-contraction coupling in the rat ventricular myocyte. A kinetic model integrating beta-adrenergic signaling with excitation-contraction coupling was formulated, and each subsystem was validated with independent biochemical and physiological measurements. Model analysis was used to investigate quantitatively the effects of specific molecular perturbations. 3-Fold overexpression of adenylyl cyclase in the model allowed an 85% higher rate of cyclic AMP synthesis than an equivalent overexpression of beta 1-adrenergic receptor, and manipulating the affinity of Gs alpha for adenylyl cyclase was a more potent regulator of cyclic AMP production. The model predicted that less than 40% of adenylyl cyclase molecules may be stimulated under maximal receptor activation, and an experimental protocol is suggested for validating this prediction. The model also predicted that the endogenous heat-stable protein kinase inhibitor may enhance basal cyclic AMP buffering by 68% and increasing the apparent Hill coefficient of protein kinase A activation from 1.0 to 2.0. Finally, phosphorylation of the L-type calcium channel and phospholamban were found sufficient to predict the dominant changes in myocyte contractility, including a 2.6x increase in systolic calcium (inotropy) and a 28% decrease in calcium half-relaxation time (lusitropy). By performing systems analysis, the consequences of molecular perturbations in the beta-adrenergic signaling network may be understood within the context of integrative cellular physiology.

Angiotensin II enhances adenylyl cyclase signaling via Ca2+/calmodulin. Gq-Gs cross-talk regulates collagen production in cardiac fibroblasts.

Cardiac fibroblasts regulate formation of extracellular matrix in the heart, playing key roles in cardiac remodeling and hypertrophy. In this study, we sought to characterize cross-talk between Gq and Gs signaling pathways and its impact on modulating collagen synthesis by cardiac fibroblasts. Angiotensin II (ANG II) activates cell proliferation and collagen synthesis but also potentiates cyclic AMP (cAMP) production stimulated by beta-adrenergic receptors (beta-AR). The potentiation of beta-AR-stimulated cAMP production by ANG II is reduced by phospholipase C inhibition and enhanced by overexpression of Gq. Ionomycin and thapsigargin increased intracellular Ca2+ levels and potentiated isoproterenol- and forskolin-stimulated cAMP production, whereas chelation of Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid/AM inhibited such potentiation. Inhibitors of tyrosine kinases, protein kinase C, or Gbetagamma did not alter this cross-talk. Immunoblot analyses showed prominent expression of adenylyl cyclase 3 (AC3), a Ca2+-activated isoform, along with AC2, AC4, AC5, AC6, and AC7. Of those isoforms, only AC3 and AC5/6 proteins were detected in caveolin-rich fractions. Overexpression of AC6 increased betaAR-stimulated cAMP accumulation but did not alter the size of the ANG II potentiation, suggesting that the cross-talk is AC isoform-specific. Isoproterenol-mediated inhibition of serum-stimulated collagen synthesis increased from 31 to 48% in the presence of ANG II, indicating that betaAR-regulated collagen synthesis increased in the presence of ANG II. These data indicate that ANG II potentiates cAMP formation via Ca2+-dependent activation of AC activity, which in turn attenuates collagen synthesis and demonstrates one functional consequence of cross-talk between Gq and Gs signaling pathways in cardiac fibroblasts.

Calcium signaling in excystation of the early diverging eukaryote, Giardia lamblia.

Excystation of Giardia lamblia, which initiates infection, is a poorly understood but dramatic differentiation induced by physiological signals from the host. Our data implicate a central role for calcium homeostasis in excystation. Agents that alter cytosolic Ca(2+) levels (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-tetra(acetyloxymethyl) ester, a Ca(2+) channel blocker, Ca(2+) ionophores, and thapsigargin) strongly inhibit excystation. Treatment of Giardia with thapsigargin raised intracellular Ca(2+) levels, and peak Ca(2+) responses increased with each stage of excystation, consistent with the kinetics of inhibition. Fluorescent thapsigargin localized to a likely Ca(2+) storage compartment in cysts. The ability to sequester ions in membrane-bounded compartments is a hallmark of the eukaryotic cell. These studies support the existence of a giardial thapsigargin-sensitive Ca(2+) storage compartment resembling the sarcoplasmic/endoplasmic reticulum calcium ATPase pump-leak system and suggest that it is important in regulation of differentiation and appeared early in the evolution of eukaryotic cells. Calmodulin antagonists also blocked excystation. The divergent giardial calmodulin localized to the eight flagellar basal bodies/centrosomes, like protein kinase A. Inhibitor kinetics suggest that protein kinase A signaling triggers excystation, whereas calcium signaling is mainly required later, for parasite activation and emergence. Thus, the basal bodies may be a cellular control center to coordinate the resumption of motility and cytokinesis in excystation.

Cyclic nucleotide research -- still expanding after half a century.

Since the discovery in 1957 that cyclic AMP acts as a second messenger for the hormone adrenaline, interest in this molecule and its companion, cyclic GMP, has grown. Over a period of nearly 50 years, research into second messengers has provided a framework for understanding transmembrane signal transduction, receptor-effector coupling, protein-kinase cascades and downregulation of drug responsiveness. The breadth and impact of this work is reflected by five different Nobel prizes.