Laurence L. Brunton

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.

Small Heat Shock Proteins and Protection Against Ischemic Injury in Cardiac Myocytes

BACKGROUND Overexpression of the inducible hsp70 protects against ischemic cardiac damage. However, it is unclear whether the small heat shock proteins hsp27 and alphaB-crystallin protect against ischemic injury. METHODS AND RESULTS Our aim was to examine whether the overexpression of hsp27 and alphaB-crystallin in neonatal and adult rat cardiomyocytes would protect against ischemic injury. Recombinant adenovirus expressing hsp27 or alphaB-crystallin under the control of the cytomegalovirus promoter was used to infect cardiac myocytes at high efficiency as assessed by immunostaining. Overexpression was confirmed by Western blot analysis. Cardiomyocytes were subjected to simulated ischemic stress, and survival was estimated through assessment of lactate dehydrogenase and creatine phosphokinase release. The hsp27 overexpression decreased lactate dehydrogenase release by 45+/-7.5% in adult cardiomyocytes but had no effect in the neonatal cells. In contrast, alphaB-crystallin overexpression was associated with a decrease in cytosolic enzyme release in both adult (29+/-6.6%) and neonatal (32+/-5.4%) cardiomyocytes. Decreased endogenous hsp25 with an antisense adenovirus produced a 29+/-9.9% increase in damage with simulated ischemia. Overexpression of the inducible hsp70 in adult cardiomyocytes was associated with a 34+/-4.6% decrease in lactate dehydrogenase release and is in line with our previous results in neonatal cardiomyocytes. CONCLUSIONS The increased expression of hsp27 and alphaB-crystallin through an adenovirus vector system protects against ischemic injury in adult cardiomyocytes. Likewise, the overexpression of alphaB-crystallin protects against ischemic damage in neonatal cardiomyocytes. Decreasing the high levels of endogenous hsp25 present in neonatal cardiomyocytes renders them more susceptible to damage caused by simulated ischemia.

Modeling β-Adrenergic Control of Cardiac Myocyte Contractility in Silico

The β-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 β-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 β1-adrenergic receptor, and manipulating the affinity of Gsα 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 2.6× 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 β-adrenergic signaling network may be understood within the context of integrative cellular physiology.

Cellular distribution of phosphodiesterase isoforms in rat cardiac tissue.

We have resolved multiple forms of cyclic nucleotide phosphodiesterase (PDE) in whole rat ventricle and in isolated rat ventricular myocytes by use of anion-exchange high-performance liquid chromatography. One major form, the soluble calmodulin-stimulated PDE, is apparently absent from isolated myocytes. We discern four peaks of PDE activity (designated A-D in the order of their elution) in a soluble fraction obtained from whole rat ventricle. Peak A is stimulated twofold to threefold by the addition of calcium and calmodulin (Ca2+/CalM) and preferentially hydrolyzes cGMP over cAMP (in the presence of Ca2+/CalM, KmcGMP = 1.5 microM, KmcAMP = 17 microM). Peak B has similar affinities for both cAMP and cGMP (half-maximum velocities achieved at 30 microM substrate) and demonstrates positive cooperativity with cAMP but not with cGMP. The hydrolysis of cAMP by peak B is stimulated by cGMP at substrate concentrations up to 20 microM; the maximum effect is seen at 1 microM cAMP (25-fold stimulation by 3 microM cGMP). This pattern of stimulation by cGMP results from two kinetic changes: an increase in the enzymes apparent affinity for cAMP (apparent KmcAMP decreases from 33 to 11 microM) and the abolition of positive cooperativity. Peaks C and D selectively hydrolyze cAMP, are not stimulated by Ca2+/CalM or cGMP, and differ in their affinities for substrate (peak C, apparent KmcAMP = 7.2 microM; peak D, 0.44 microM). In addition, peak D is much more sensitive than peak C to inhibition by cGMP, cilostamide, rolipram, and milrinone. Ro20-1724 is a slightly more potent inhibitor of peak D than of peak C. Peak D appears to consist of two different enzyme activities, one inhibited by cGMP, cilostamide, and cardiotonic drugs and the other potently inhibited by rolipram. In contrast to whole ventricle, the soluble fraction of isolated rat ventricular myocytes lacks peak A. Three major peaks in myocytes are entirely analogous to peaks B, C, and D of whole ventricle in terms of the NaCl concentration at which they elute, substrate affinities, and stimulation or inhibition by various drugs and effectors. We conclude that the soluble Ca2+/CalM-stimulated PDE in whole rat ventricle is present in nonmyocyte cells.

Crystal Structure of the Potent Natural Product Inhibitor Balanol in Complex with the Catalytic Subunit of cAMP-Dependent Protein Kinase †

Endogenous protein kinase inhibitors are essential for a wide range of physiological functions. These endogenous inhibitors may mimic peptide substrates as in the case of the heat-stable protein kinase inhibitor (PKI), or they may mimic nucleotide triphosphates. Natural product inhibitors, endogenous to the unique organisms producing them, can be potent exogenous inhibitors against foreign protein kinases. Balanol is a natural product inhibitor exhibiting low nanomolar Ki values against serine and threonine specific kinases, while being ineffective against protein tyrosine kinases. To elucidate balanols specific inhibitory effects and provide a basis for understanding inhibition-regulated biological processes, a 2.1 A resolution crystal structure of balanol in complex with cAMP-dependent protein kinase (cAPK) was determined. The structure reveals conserved binding regions and displays extensive complementary interactions between balanol and conserved cAPK residues. This report describes the structure of a protein kinase crystallized with a natural ATP mimetic in the absence of metal ions and peptide inhibitor.

Molecular design and biological activity of potent and selective protein kinase inhibitors related to balanol

BACKGROUND The protein kinase C (PKC) family of serine/threonine-specific protein kinases is involved in many cellular processes, and the unregulated activation of PKC has been implicated in carcinogenesis. PKC inhibitors thus have significant potential as chemotherapeutic agents. Recently, the fungal metabolite balanol was shown to be an exceptionally potent inhibitor of PKC. We previously developed a practical and efficient total synthesis of balanol. We set out to use this synthetic molecule, and several synthetic analogs, to probe the mechanism of PKC inhibition and to determine the effect of balanol on the activity of other protein kinases. RESULTS As well as inhibiting PKC, balanol is a potent inhibitor of cyclic AMP-dependent protein kinase (PKA), another protein serine/threonine kinase. Balanol does not, however, inhibit the Src or epidermal growth factor receptor protein tyrosine kinases. The inhibition of both PKC and PKA by balanol can be overcome by high concentrations of ATP, and molecular modeling studies suggest that balanol may function as an ATP structural analog. Although balanol discriminates rather poorly between PKC and PKA, only minor modifications to its molecular structure are required to furnish compounds that are highly specific inhibitors of PKA. CONCLUSIONS A number of balanol analogs have been designed and synthesized that, unlike balanol itself, exhibit dramatic selectivity between PKA and PKC. Thus, despite the substantial homology between the catalytic domains of PKA and PKC, there is enough difference to allow for the development of potent and selective inhibitors acting in this region. These inhibitors should be useful tools for analyzing signal transduction pathways and may also aid in the development of drugs with significant therapeutic potential.

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 pathways protein components into macromolecular complexes, and common themes in the compartmentation of cAMP- and Ca2+-mediated signaling pathways.

[8] cAMP export and its regulation by prostaglandin A1

Publisher Summary This chapter examines cAMP export and its regulation by prostaglandin A 1 . The main requirement for studying export is a method for the rapid separation of the intracellular and extracellular spaces, with collection of both, if possible. Export of cAMP is remarkably sensitive to temperature, occurring only slightly, if at all, below 22° in avian red cells and greatly inhibited by decreased temperatures in S49 lymphoma cells and mammalian reticulocytes. Thus, one may “freeze” the level of exported cAMP by the simple expedient of cooling or by dilution with cold buffer, a handy maneuver in many experimental protocols. Despite the simplicity of these comments and procedures and the relative ease of demonstrating efflux of cellular cAMP, readers should realize that the study of solute export is not subject to rigorous analysis because the exact intracellular activity or concentration of the solute being transported is not known or readily controlled. The export of cAMP seems limited by the cells ability to produce and accumulate the solute and properties such as saturability are not directly demonstrable.

Modulation of lymphocyte activation: II. Alteration of intracellular cyclic nucleotide concentrations by an oxidation product of arachidonic acid☆

Abstract Murine spleen cells incubated with arachidonic acid oxidized with lipoxidase (AA/L) do not respond to mitogenic stimulation. Analysis of the components of AA/L separated by high-pressure liquid chromatography has previously demonstrated that purified 15-hydroperoxyarachidonic acid, the predominant product generated, is the seat of the inhibitory activity observed. Correlative studies were undertaken to determine the relationship between the pervasive inhibitory effect of AA/L and the modulation of intracellular cyclic nucleotide concentration. AA/L elevated intracellular levels of cGMP in murine spleen cells cultured under serum-free conditions and caused a similar (two-fold) increment in intracellular levels of cyclic AMP. These are the first cellular activities observed in this laboratory that are not subject to inhibition by treatment of cells with AA/L. Therefore, studies were conducted to determine if AA/L-mediated inhibition of lymphocyte mitogenesis could be mimicked by elevation of intracellular nucleotide levels by pharmacologic agents. Attempts to recreate this inhibitory phenomenon by use of such agents indicated that inhibition could not be induced by modulation of cyclic AMP alone, cyclic GMP alone, or of both together. Similarly, when the same stimuli of cyclic nucleotide accumulation were employed to determine if modulation of intracellular cyclic nucleotide content could counteract the inhibitory effect of AA/L, these agents did not interfere with AA/L-mediated inhibition. The capability of the cyclic nucleotide agonists to elevate levels of cAMP and cGMP was confirmed by direct assay. Thus, although cyclic nucleotides may play an essential, adjunctive role in inhibition of lymphocyte mitogenesis, their modulation is not of itself sufficient either to evoke or to interfere with the establishment of a state of profound inhibition.

Post-receptor modulation of the effects of cyclic AMP in isolated cardiac myocytes.

SummaryThe actions of cyclic AMP are subject to several levels of post-receptor modulation in cardiac tissue. Isoproterenol and prostaglandin E1 both stimulate cAMP accumulation, but only isoproterenol causes activation of particulate cAMP-dependent protein kinase, leading to activation of phosphorylase kinase and glycogen phosphorylase, and inhibition of glycogen synthase. Through the use of isolated, adult ventricular myocytes, we have determined that the hormone-specific activation of glycogen phosphorylase is due to subcellular compartmentation of cAMP. There is some evidence that cyclic nucleotide phosphodiesterases, whose activity is stimulated by alpha1-adrenergic agonists in isolated myocytes, may have a role in compartmentation. Phosphoinositide hydrolysis is stimulated by alpha, and muscarinic agonists, presumably leading to activation of protein kinase C, which in turn has multiple effects on hormone-sensitive adenylate cyclase.