Increased adrenergic tone resulting from cardiovascular stress leads to development of heart failure, in part, through chronic stimulation of β1 adrenergic receptors (βARs) on cardiac myocytes. Blocking these receptors is part of the basis for β-blocker therapy for heart failure. Recent data demonstrate that G protein-coupled receptors (GPCRs), including βARs, are activated intracellularly, although the biological significance is unclear. Here we investigated the functional role of Golgi βARs in rat cardiac myocytes and found they activate Golgi localized, prohypertrophic, phosphoinositide hydrolysis, that is not accessed by cell surface βAR stimulation. This pathway is accessed by the physiological neurotransmitter norepinephrine (NE) via an Oct3 organic cation transporter. Blockade of Oct3 or specific blockade of Golgi resident β1ARs prevents NE dependent cardiac myocyte hypertrophy. This clearly defines a pathway activated by internal GPCRs in a biologically relevant cell type and has implications for development of more efficacious β-blocker therapies.

Summary.  Background and objectives:  Src family kinases (SFKs) play a critical role in initiating and propagating signals in platelets. The aims of this study were to quantitate SFK members present in platelets and to analyze their contribution to platelet regulation using glycoprotein VI (GPVI) and intregrin αIIbβ3, and in vivo. Methods and Results:  Mouse platelets express four SFKs, Fgr, Fyn, Lyn and Src, with Lyn expressed at a considerably higher level than the others. Using mutant mouse models, we demonstrate that platelet activation by collagen‐related peptide (CRP) is delayed and then potentiated in the absence of Lyn, but only marginally reduced in the absence of Fyn or Fgr, and unaltered in the absence of Src. Compound deletions of Lyn/Src or Fyn/Lyn, but not of Fyn/Src or Fgr/Lyn, exhibit a greater delay in activation relative to Lyn‐deficient platelets. Fibrinogen‐adherent platelets show reduced spreading in the absence of Src, potentiation in the absence of Lyn, but no change in the absence of Fyn or Fgr. In mice double‐deficient in Lyn/Src or Fgr/Lyn, the inhibitory role of Lyn on spreading on fibrinogen is lost. Lyn is the major SFK‐mediating platelet aggregation on collagen at arterial shear and its absence leads to a reduction in thrombus size in a laser injury model. Conclusion:  These results demonstrate that SFKs share individual and overlapping roles in regulating platelet activation, with Lyn having a dual role in regulating GPVI signaling and an inhibitory role downstream of αIIbβ3, which requires prior signaling through Src.

Increased adrenergic tone resulting from cardiovascular stress leads to development of heart failure, in part, through chronic stimulation of b1 adrenergic receptors (bARs) on cardiac myocytes. Blocking these receptors is part of the basis for b-blocker therapy for heart failure. Recent data demonstrate that G protein-coupled receptors (GPCRs), including bARs, are activated intracellularly, although the biological significance is unclear. Here we investigated the functional role of Golgi bARs in cardiac myocytes and found they activate Golgi localized, prohypertrophic, phosphoinositide hydrolysis, that is not accessed by cell surface bAR stimulation. This pathway is accessed by the physiological neurotransmitter norepinephrine (NE) via an Oct3 organic cation transporter. Blockade of Oct3 or specific blockade of Golgi resident b1ARs prevents NE dependent cardiac myocyte hypertrophy. This clearly defines a physiological pathway activated by internal GPCRs in a biologically relevant cell type and has implications for development of more efficacious b-blocker therapies.

The C-type lectin-like receptor CLEC-2 signals via phosphorylation of a single cytoplasmic YXXL sequence known as a hem-immunoreceptor tyrosine-based activation motif (hemITAM). In this study, we show that phosphorylation of CLEC-2 by the snake toxin rhodocytin is abolished in the absence of the tyrosine kinase Syk but is not altered in the absence of the major platelet Src family kinases, Fyn, Lyn, and Src, or the tyrosine phosphatase CD148, which regulates the basal activity of Src family kinases. Further, phosphorylation of CLEC-2 by rhodocytin is not altered in the presence of the Src family kinase inhibitor PP2, even though PLCγ2 phosphorylation and platelet activation are abolished. A similar dependence of phosphorylation of CLEC-2 on Syk is also seen in response to stimulation by an IgG mAb to CLEC-2, although interestingly CLEC-2 phosphorylation is also reduced in the absence of Lyn. These results provide the first definitive evidence that Syk mediates phosphorylation of the CLEC-2 hemITAM receptor with Src family kinases playing a critical role further downstream through the regulation of Syk and other effector proteins, providing a new paradigm in signaling by YXXL-containing receptors.

In cardiac myocytes activation of an exchange factor activated by cAMP (Epac) leads to activation of phospholipase Cε (PLCε)-dependent hydrolysis of phosphatidylinositol 4-phosphate (PI4P) in the Golgi apparatus a process critical for development of cardiac hypertrophy. Here we show that β-adrenergic receptor (βAR) stimulation does not stimulate this pathway in the presence of the broad spectrum phosphodiesterase (PDE) inhibitor IBMX, but selective PDE3 inhibition revealed βAR-dependent PI4P depletion. On the other hand, selective inhibition of PDE2 or PDE9A blocked endothelin-1 (ET-1) and cAMP-dependent PI4P hydrolysis by PLCε. Direct activation of protein kinase A (PKA), protein kinase G (PKG), or the atrial natriuretic factor (ANF) receptor abolished PI4P hydrolysis in response to multiple upstream stimuli. These results reveal distinct pools of cyclic nucleotides that either inhibit PLCε at the Golgi through PKA/PKG, or activate PLCε at the Golgi through Epac. These data together reveal a new mechanism by which ANF and selective PDE inhibitors can protect against cardiac hypertrophy.