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Laurence L Brunton

Publications and source records attributed to Laurence L Brunton.

12 recordsLinked to original sources
PubMed2004

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.

Amino Acid Sequence↗
PubMed2004

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.

Adenylyl Cyclases↗
PubMed2003

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.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2003

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.

Adenylyl Cyclases↗
PubMed2003

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.

Adenoviridae↗
PubMed2003

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.

Adenylyl Cyclases↗
PubMed2002

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.

Adenosine Triphosphatases↗
PubMed2002

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.

Animals↗
PubMed2002

Attenuation of cAMP accumulation in adult rat cardiac fibroblasts by IL-1beta and NO: role of cGMP-stimulated PDE2.

Treatment of cultured adult rat cardiac fibroblasts with interleukin-1beta (IL-1beta) induces the inducible nitric oxide synthase (iNOS) expression, increases nitric oxide (NO) and cGMP production, and attenuates cAMP accumulation in response to isoproterenol by ~50%. Reduced cAMP accumulation is due to NO production: the effect is mimicked by NO donors and prevented by N(G)-monomethyl-L-arginine, an NOS inhibitor. Effects of NO are not restricted to the beta-adrenergic response; the response to forskolin is similarly diminished. NO donors only slightly (12%) decrease forskolin-stimulated adenylyl cyclase (AC) activity in cardiac fibroblast plasma membranes, suggesting that the main effect of NO is not a direct one on AC. An inhibitor of soluble guanylyl cyclase inhibits the effects of IL-1beta and NO donors; inhibition of cGMP-dependent protein kinase is without effect. 3-Isobutyl-1-methylxanthine, a nonspecific phosphodiesterase (PDE) inhibitor, and erythro-9-(2-hydroxy-3-nonyl)adenine, a specific inhibitor of the cGMP-stimulated PDE (PDE2), completely restore cAMP accumulation in sodium nitroprusside-treated fibroblasts and largely reverse the attenuated response in IL-1beta-treated fibroblasts. Although NO reportedly acts by reducing AC activity in some cells, in cardiac fibroblasts NO production decreases cAMP accumulation largely by the cGMP-mediated activation of PDE2.

Adenylyl Cyclases↗