beta-Adrenergic regulation of cAMP and protein phosphorylation in phospholamban-knockout mouse hearts

1997 ◽  
Vol 272 (2) ◽  
pp. H785-H790 ◽  
Author(s):  
E. Kiss ◽  
I. Edes ◽  
Y. Sato ◽  
W. Luo ◽  
S. B. Liggett ◽  
...  
Keyword(s):  
Protein Phosphorylation ◽  
Troponin I ◽  
Signal Transduction Pathway ◽  
Microsomal Protein ◽  
Mouse Heart ◽  
Wild Type ◽  
Adrenergic Stimulation ◽  
Beta Adrenergic ◽  
C Protein ◽  
Camp Levels

The stimulatory effects of beta-adrenergic agonists reflect increases in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels and phosphorylation of key regulatory proteins in the heart. One of these phosphoproteins is phospholamban (PLB) in sarcoplasmic reticulum, and ablation of PLB is associated with attenuation of the contractile responses to beta-adrenergic stimulation in the mouse heart. To determine whether this attenuation of beta-stimulation is due to altered phosphorylation characteristics of the other key cardiac phosphoproteins and/or to compensatory responses occurring in the absence of PLB, PLB-knockout and wild-type hearts were perfused and their protein phosphorylation patterns examined. The beta-adrenergic receptor density, adenylyl cyclase activity, tissue cAMP levels, and the basal phosphoprotein pattern were similar between PLB-knockout and wild-type hearts. Isoproterenol perfusion resulted in similar increases in the tissue cAMP levels and the degree of phosphorylation of troponin I, C protein, and the 21-kDa microsomal protein in wild-type and PLB-knockout hearts. These findings indicate that the attenuation of isoproterenol-mediated increases in contractility of the PLB-knockout hearts is not due to alterations in the beta-adrenergic signal transduction pathway or the degree of phosphorylation of the key cardiac regulatory phosphoproteins in myofibrils and sarcolemma.

Relation between contractile function and regulatory cardiac proteins in hypertrophied hearts

1996 ◽  
Vol 270 (6) ◽  
pp. H2021-H2028 ◽  
Author(s):  
B. Stein ◽  
S. Bartel ◽  
U. Kirchhefer ◽  
S. Kokott ◽  
E. G. Krause ◽  
...  
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Troponin I ◽  
Contractile Function ◽  
Papillary Muscles ◽  
Camp Accumulation ◽  
Adrenergic Stimulation ◽  
Beta Adrenergic ◽  
C Protein ◽  
Camp Formation ◽  
Phosphate Incorporation

The aim of this study was to examine the mechanism(s) underlying the reduced isoproterenol-induced positive inotropic and lusitropic effects in hypertrophied hearts. Chronic beta-adrenergic stimulation (2.4 mg isoproterenol.kg-1. day-1 for 4 days) induced cardiac hypertrophy by 33 +/- 2% in rats. A parallel downregulation of phospholamban (PLB) and sarcoplasmic reticulum Ca2(+)-ATPase (SERCA2) protein expression by 49 and 40%, respectively, was observed, whereas troponin I (TNI) and C protein remained unchanged. In papillary muscles from chronically beta-adrenergically stimulated rats, the isoproterenol-induced positive inotropic and lusitropic effects, as well as adenosine 3',5'-cyclic monophosphate (cAMP) accumulation, were attenuated compared with those in control animals. Acute exposure to isoproterenol induced phosphate incorporation into PLB, TNI, and C protein of 48 +/- 4.6, 55 +/- 5.0, and 27 +/- 4.9 pmol/mg homogenate protein, respectively, in control animals. In the hypertrophied hearts, phosphate incorporation into PLB was reduced by 76%, whereas phosphate incorporation into TNI or C protein remained unchanged. In conclusion, chronic beta-adrenergic stimulation reduced the isoproterenol-stimulated positive inotropic and lusitropic effects in papillary muscles, which were accompanied by 1) diminished cAMP formation, 2) attenuation of cAMP-mediated PLB phosphorylation, and 3) downregulation of PLB and SERCA2 protein.

Intracellular mechanisms mediating reversal of beta-adrenergic stimulation in intact beating hearts

1993 ◽  
Vol 264 (3) ◽  
pp. H791-H797 ◽  
Author(s):  
L. Talosi ◽  
I. Edes ◽  
E. G. Kranias
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Guinea Pig ◽  
Troponin I ◽  
Adrenergic Stimulation ◽  
Beta Adrenergic ◽  
Dependent Protein ◽  
Camp Levels ◽  
Isoproterenol Infusion ◽  
Phosphate Incorporation ◽  
Stimulation Of

The changes in 32P labeling of phosphoproteins were studied in Langendorff-perfused guinea pig hearts during reversal of the stimulatory effects of isoproterenol. Exposure of the hearts to isoproterenol was associated with significant increases in adenosine 3',5'-cyclic monophosphate (cAMP) levels and in the phosphate incorporation into phospholamban in sarcoplasmic reticulum, the 15-kDa protein in the sarcolemma, and troponin I in the myofibrils. Phospholamban was phosphorylated on serine and threonine residues, both of which are sites for cAMP-dependent and Ca(2+)-calmodulin-dependent protein kinases, respectively. Termination of isoproterenol infusion was associated with reversal of the mechanical effects of isoproterenol stimulation and reversal of the increases in tissue cAMP levels. However, the decreases in cAMP levels correlated only with dephosphorylation of phosphoserine in phospholamban. Dephosphorylation of phosphothreonine in phospholamban, the 15-kDa sarcolemmal protein, and troponin I occurred at a slower rate. These findings suggest that cAMP-dependent phosphorylation of phospholamban (phosphoserine) may play a prominent role during beta-adrenergic stimulation of intact hearts.

scholarly journals Phosphorylation of C-protein, troponin I and phospholamban in isolated rabbit hearts

1988 ◽  
Vol 249 (3) ◽  
pp. 709-714 ◽  
Author(s):  
J L Garvey ◽  
E G Kranias ◽  
R J Solaro
Keyword(s):  
Atpase Activity ◽  
Protein Phosphorylation ◽  
Mechanical Response ◽  
Troponin I ◽  
Dominant Role ◽  
Adrenergic Stimulation ◽  
C Protein ◽  
Drug Free ◽  
Phospholamban Phosphorylation ◽  
Stimulation Of

Phosphorylation of myofibrillar and sacroplasmic-reticulum (SR) proteins was studied in Langendorff-perfused rabbit hearts subjected to various inotropic interventions. Stimulation of hearts with isoprenaline resulted in the phosphorylation of both troponin I (TnI) and C-protein in myofibrils and phospholamban in SR. Phosphorylation of phospholamban could be reversed by a 15 min perfusion with drug-free buffer, after a 1 minute pulse perfusion with isoprenaline, at which time the mechanical effects of isoprenaline stimulation had also been reversed. However, both TnI and C-protein remained phosphorylated at this time. Moreover, the inhibition of Ca2+ activation of the Mg2+-dependent ATPase (Mg-ATPase) activity associated with myofibrillar phosphorylation persisted in myofibrils prepared from hearts frozen after 15 min of washout of isoprenaline. To assess the contribution of C-protein phosphorylation in the decrease of Ca2+ activation of the myofibrillar Mg-ATPase activity, we reconstituted a regulated actomyosin system in which only C-protein was phosphorylated. In this system, C-protein phosphorylation did not contribute to the decrease in Ca2+ activation of Mg-ATPase activity, indicating that TnI phosphorylation is responsible for the diminished sensitivity of the myofibrils to Ca2+. These observations support the hypothesis that phospholamban phosphorylation plays a more dominant role than TnI or C-protein phosphorylation in the mechanical response of the mammalian heart to beta-adrenergic stimulation.

scholarly journals Mutational Activation of a Gαi Causes Uncontrolled Proliferation of Aerial Hyphae and Increased Sensitivity to Heat and Oxidative Stress in Neurospora crassa

Genetics ◽  
1999 ◽  
Vol 151 (1) ◽  
pp. 107-117
Author(s):  
Qi Yang ◽  
Katherine A Borkovich
Keyword(s):  
Signal Transduction ◽  
Neurospora Crassa ◽  
Signal Transduction Pathway ◽  
Sexual Cycle ◽  
Intracellular Camp ◽  
Wild Type ◽  
Independent Functions ◽  
Aerial Hyphae ◽  
Mutational Activation ◽  
Camp Levels

Abstract Heterotrimeric G proteins, consisting of α, β, and γ subunits, transduce environmental signals through coupling to plasma membrane-localized receptors. We previously reported that the filamentous fungus Neurospora crassa possesses a Gα protein, GNA-1, that is a member of the Gαi superfamily. Deletion of gna-1 leads to defects in apical extension, differentiation of asexual spores, sensitivity to hyperosmotic media, and female fertility. In addition, Δgna-1 strains have lower intracellular cAMP levels under conditions that promote morphological abnormalities. To further define the function of GNA-1 in signal transduction in N. crassa, we examined properties of strains with mutationally activated gna-1 alleles (R178C or Q204L) as the only source of GNA-1 protein. These mutations are predicted to inhibit the GTPase activity of GNA-1 and lead to constitutive signaling. In the sexual cycle, gna-1R178C and gna-1Q204L strains are female-fertile, but produce fewer and larger perithecia than wild type. During asexual development, gna-1R178C and gna-1Q204L strains elaborate abundant, long aerial hyphae, produce less conidia, and possess lower levels of carotenoid pigments in comparison to wild-type controls. Furthermore, gna-1R178C and gna-1Q204L strains are more sensitive to heat shock and exposure to hydrogen peroxide than wild-type strains, while Δgna-1 mutants are more resistant. In contrast to Δgna-1 mutants, gna-1R178C and gna-1Q204L strains have higher steady-state levels of cAMP than wild type. The results suggest that GNA-1 possesses several Gβγ-independent functions in N. crassa. We propose that GNA-1 mediates signal transduction pathway(s) that regulate aerial hyphae development and sensitivity to heat and oxidative stresses, possibly through modulation of cAMP levels.

Troponin I phosphorylation in spontaneously hypertensive rat heart: effect of beta-adrenergic stimulation

1997 ◽  
Vol 273 (3) ◽  
pp. H1440-H1451 ◽  
Author(s):  
B. K. McConnell ◽  
C. S. Moravec ◽  
I. Morano ◽  
M. Bond
Keyword(s):  
Troponin I ◽  
Ventricular Myocytes ◽  
Spontaneously Hypertensive Rat ◽  
Left Ventricular ◽  
Adrenergic Stimulation ◽  
Beta Adrenergic ◽  
Spontaneously Hypertensive ◽  
Myosin Light Chain 2 ◽  
Wistar Kyoto ◽  
Phospholamban Phosphorylation

We compared baseline and protein kinase A (PKA)-dependent troponin I (TnI) phosphorylation in 32Pi-labeled left ventricular myocytes from hearts of 26-wk spontaneously hypertensive rats (SHR) and Wistar-Kyoto controls (WKY). TnI phosphorylation was normalized to myosin light chain 2 phosphorylation, which was invariant. There was no difference in baseline TnI phosphorylation in SHR and WKY, but stimulation with isoproterenol, norepinephrine plus prazosin, forskolin, chloroadenosine 3',5'-cyclic monophosphate, or 3-isobutyl-1-methylxanthine caused a greater increase in TnI phosphorylation in the SHR than in the WKY. This was observed both in the presence and absence of the phosphatase inhibitor calyculin A; thus the differences in TnI phosphorylation between SHR and WKY are not due to decreased phosphatase activity in the SHR. After stimulation of the beta-adrenergic pathway, phospholamban phosphorylation was not different in SHR and WKY, indicating that the observed differences may be specific for PKA phosphorylation of TnI. The increased PKA-dependent TnI phosphorylation in the SHR resulted in decreased Ca2+ sensitivity of actomyosin adenosinetriphosphatase activity as compared with the WKY. We conclude that increased PKA-dependent TnI phosphorylation in the SHR may contribute to the impaired response to sympathetic stimulation.

Role of protein phosphorylation in regulating rat submandibular mucin secretion

1983 ◽  
Vol 245 (1) ◽  
pp. G44-G53 ◽  
Author(s):  
D. O. Quissell ◽  
L. M. Deisher ◽  
K. A. Barzen
Keyword(s):  
Plasma Membrane ◽  
Protein Phosphorylation ◽  
Adrenergic Receptor ◽  
Enzyme Analysis ◽  
Dibutyryl Camp ◽  
Adrenergic Stimulation ◽  
Mucin Secretion ◽  
Receptor Stimulation ◽  
Beta Adrenergic ◽  
Camp Stimulation

The possible involvement of protein phosphorylation during beta-adrenergic receptor stimulation in rat submandibular gland was investigated in vitro using a dispersed cell preparation. (-)-Isoproterenol, a beta-adrenergic agonist, or dibutyryl cAMP stimulation was associated with an enhanced phosphorylation of three protein bands having apparent molecular weights of 34,000, 26,000, and 21,000, respectively. (-)-Propranolol, a beta-adrenergic antagonist, inhibited the phosphorylation of the three proteins during beta-adrenergic stimulation but not during dibutyryl cAMP stimulation. The three proteins were not fragments of a higher-molecular-weight protein. Subcellular fractionation using differential centrifugation, fractionation in an aqueous two-phase polymer system, and discontinuous sucrose gradient ultracentrifugation coupled with marker enzyme analysis indicated that all three proteins were enriched in the same subfractions: a heavy plasma membrane fraction and a fraction containing plasma membrane and Golgi membrane material. The extent of protein phosphorylation for all three proteins increased as a function of time and dose after beta-adrenergic stimulation. After 20 min of maximal beta-adrenergic stimulation, the addition of a beta-adrenergic blocker caused a time-dependent decrease in the 32P content of all three proteins. Pure cholinergic or pure alpha-adrenergic receptor stimulation had no effect on the 32P content of the three proteins. These data are consistent with a role for cAMP-mediated protein phosphorylation during mucin secretion from rat submandibular cells.

scholarly journals Measurement of cAMP in the cardiac conduction system of rats.

1995 ◽  
Vol 43 (6) ◽  
pp. 601-605 ◽  
Author(s):  
A Sugiyama ◽  
S McKnite ◽  
P Wiegn ◽  
K G Lurie
Keyword(s):  
Atrioventricular Node ◽  
Conduction System ◽  
Cardiac Conduction ◽  
Cardiac Conduction System ◽  
Camp Production ◽  
Adrenergic Stimulation ◽  
Beta Adrenergic ◽  
His Bundle ◽  
Freeze Dried ◽  
Camp Levels

To characterize differences in regional cAMP production in the cardiac conduction system, 18 rats were anesthetized with pentobarbital (65 mg/kg IP) and randomized into a control (n = 9) and a stimulated group (n = 9). The stimulated group received aminophylline (20 mg/kg SC) and isoproterenol (16 micrograms/kg SC). The concentration of cAMP in freeze-dried, micro dissected pieces (1-3 micrograms) of cardiac tissue was measured using a new microanalytical method. The cAMP contents in right atrium, atrioventricular node, His bundle, and left ventricle (fmol/microgram dry weight, mean +/- SE) were 38.9 +/- 2.5, 39.0 +/- 4.3, 46.4 +/- 6.1, and 41.4 +/- 3.3 in controls and 72.9 +/- 6.7, 86.1 +/- 2.9, 115.0 +/- 11.5, and 79.5 +/- 7.3 in the stimulated group, respectively. Basal cAMP levels were similar throughout the heart, whereas isoproterenol increased cAMP levels in all regions (p < 0.01). Furthermore, cAMP levels in His bundle, after isoproterenol, were higher than in any other region (p < 0.05). These results demonstrate that: (a) cAMP can be measured in discrete portions of the cardiac conduction system; (b) there are significant regional differences of beta-adrenergic control in the cardiac conduction system; and (c) cAMP production after beta-adrenergic stimulation was lower than expected in the AV nodal region, based on previously described beta-adrenoceptor density measurements.

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