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Background: Although the bronchodilatory actions of volatile anesthetics, such as halothane, isoflurane, and sevoflurane, have been well documented in previous studies, the properties of desflurane remain controversial. The aim of this study was to investigate the effects of desflurane at different concentrations and durations in an ovalbumin-sensitized guinea pig model of airway hyper-responsiveness., Methods: Ovalbumin-sensitized animals (n = 176) were randomly assigned to 5 groups according to the minimum alveolar concentration (MAC) of desflurane they received: 0.0, 0.5, 1.0, 1.5, and 2.0 MAC. Total lung resistance in vivo, airway smooth muscle tension in vitro, and intracellular cyclic adenosine monophosphate (AMP) levels were measured to evaluate the effects of desflurane., Results: In 5 sensitized groups, total lung resistance increased from baseline to peak at approximately 8 minutes and then decreased slowly until about 17 minutes with extended administration of desflurane. Desflurane dose-dependently increased total lung resistance with or without incremental doses of acetylcholine and reduced muscle tension with increasing concentrations of carbacholine. Cyclic AMP levels were increased by desflurane: at the 60-minute time point, cyclic AMP concentrations (means ± SD) with 0.5 MAC (1.96 ± 0.40) and 1.0 MAC (2.11 ± 0.50) desflurane were higher than those at the 8-minute time point (1.11 ± 0.23 and 1.32 ± 0.32)., Conclusions: Desflurane exerted time- and dose-dependent effects and could be used at 0.5 and 1.0 MAC concentrations without significant bronchoconstriction in ovalbumin-sensitized guinea pigs. Cyclic AMP-mediated airway smooth muscle relaxation might be one mechanism by which desflurane induces bronchodilation.

cAMP is a universal second messenger that plays central roles in cardiovascular regulation influencing gene expression, cell morphology, and function. A crucial step toward a better understanding of cAMP signaling came 18 years ago with the discovery of the exchange protein directly activated by cAMP (EPAC). The 2 EPAC isoforms, EPAC1 and EPAC2, are guanine-nucleotide exchange factors for the Ras-like GTPases, Rap1 and Rap2, which they activate independently of the classical effector of cAMP, protein kinase A. With the development of EPAC pharmacological modulators, many reports in the literature have demonstrated the critical role of EPAC in the regulation of various cAMP-dependent cardiovascular functions, such as calcium handling and vascular tone. EPAC proteins are coupled to a multitude of effectors into distinct subcellular compartments because of their multidomain architecture. These novel cAMP sensors are not only at the crossroads of different physiological processes but also may represent attractive therapeutic targets for the treatment of several cardiovascular disorders, including cardiac arrhythmia and heart failure., (© 2016 American Heart Association, Inc.)

Rationale: Neointimal hyperplasia characterized by abnormal accumulation of vascular smooth muscle cells (SMCs) is a hallmark of occlusive disorders such as atherosclerosis, postangioplasty restenosis, vein graft stenosis, and allograft vasculopathy. Cyclic nucleotides are vital in SMC proliferation and migration, which are regulated by cyclic nucleotide phosphodiesterases (PDEs)., Objective: Our goal is to understand the regulation and function of PDEs in SMC pathogenesis of vascular diseases., Methods and Results: We performed screening for genes differentially expressed in normal contractile versus proliferating synthetic SMCs. We observed that PDE1C expression was low in contractile SMCs but drastically elevated in synthetic SMCs in vitro and in various mouse vascular injury models in vivo. In addition, PDE1C was highly induced in neointimal SMCs of human coronary arteries. More importantly, injury-induced neointimal formation was significantly attenuated by PDE1C deficiency or PDE1 inhibition in vivo. PDE1 inhibition suppressed vascular remodeling of human saphenous vein explants ex vivo. In cultured SMCs, PDE1C deficiency or PDE1 inhibition attenuated SMC proliferation and migration. Mechanistic studies revealed that PDE1C plays a critical role in regulating the stability of growth factor receptors, such as PDGF receptor β (PDGFRβ) known to be important in pathological vascular remodeling. PDE1C interacts with low-density lipoprotein receptor-related protein-1 and PDGFRβ, thus regulating PDGFRβ endocytosis and lysosome-dependent degradation in an low-density lipoprotein receptor-related protein-1-dependent manner. A transmembrane adenylyl cyclase cAMP-dependent protein kinase cascade modulated by PDE1C is critical in regulating PDGFRβ degradation., Conclusions: These findings demonstrated that PDE1C is an important regulator of SMC proliferation, migration, and neointimal hyperplasia, in part through modulating endosome/lysosome-dependent PDGFRβ protein degradation via low-density lipoprotein receptor-related protein-1., (© 2015 American Heart Association, Inc.)

Background: Pulmonary hypertension (PH) is a life-threatening disorder characterized by increased pulmonary artery pressure, remodeling of the pulmonary vasculature, and right ventricular failure. Loss of endothelium-derived nitric oxide (NO) and prostacyclin contributes to PH pathogenesis, and current therapies are targeted to restore these pathways. Phosphodiesterases (PDEs) are a family of enzymes that break down cGMP and cAMP, which underpin the bioactivity of NO and prostacyclin. PDE5 inhibitors (eg, sildenafil) are licensed for PH, but a role for PDE2 in lung physiology and disease has yet to be established. Herein, we investigated whether PDE2 inhibition modulates pulmonary cyclic nucleotide signaling and ameliorates experimental PH., Methods and Results: The selective PDE2 inhibitor BAY 60-7550 augmented atrial natriuretic peptide- and treprostinil-evoked pulmonary vascular relaxation in isolated arteries from chronically hypoxic rats. BAY 60-7550 prevented the onset of both hypoxia- and bleomycin-induced PH and produced a significantly greater reduction in disease severity when given in combination with a neutral endopeptidase inhibitor (enhances endogenous natriuretic peptides), trepostinil, inorganic nitrate (NO donor), or a PDE5 inhibitor. Proliferation of pulmonary artery smooth muscle cells from patients with pulmonary arterial hypertension was reduced by BAY 60-7550, an effect further enhanced in the presence of atrial natriuretic peptide, NO, and treprostinil., Conclusions: PDE2 inhibition elicits pulmonary dilation, prevents pulmonary vascular remodeling, and reduces the right ventricular hypertrophy characteristic of PH. This favorable pharmacodynamic profile is dependent on natriuretic peptide bioactivity and is additive with prostacyclin analogues, PDE5 inhibitor, and NO. PDE2 inhibition represents a viable, orally active therapy for PH., (© 2014 American Heart Association, Inc.)

Rationale: The sympathetic nervous system plays a fundamental role in the regulation of myocardial function. During chronic pressure overload, overactivation of the sympathetic nervous system induces the release of catecholamines, which activate β-adrenergic receptors in cardiomyocytes and lead to increased heart rate and cardiac contractility. However, chronic stimulation of β-adrenergic receptors leads to impaired cardiac function, and β-blockers are widely used as therapeutic agents for the treatment of cardiac disease. MicroRNA-133 (miR-133) is highly expressed in the myocardium and is involved in controlling cardiac function through regulation of messenger RNA translation/stability., Objective: To determine whether miR-133 affects β-adrenergic receptor signaling during progression to heart failure., Methods and Results: Based on bioinformatic analysis, β1-adrenergic receptor (β1AR) and other components of the β1AR signal transduction cascade, including adenylate cyclase VI and the catalytic subunit of the cAMP-dependent protein kinase A, were predicted as direct targets of miR-133 and subsequently validated by experimental studies. Consistently, cAMP accumulation and activation of downstream targets were repressed by miR-133 overexpression in both neonatal and adult cardiomyocytes following selective β1AR stimulation. Furthermore, gain-of-function and loss-of-function studies of miR-133 revealed its role in counteracting the deleterious apoptotic effects caused by chronic β1AR stimulation. This was confirmed in vivo using a novel cardiac-specific TetON-miR-133 inducible transgenic mouse model. When subjected to transaortic constriction, TetON-miR-133 inducible transgenic mice maintained cardiac performance and showed attenuated apoptosis and reduced fibrosis compared with control mice., Conclusions: miR-133 controls multiple components of the β1AR transduction cascade and is cardioprotective during heart failure., (© 2014 American Heart Association, Inc.)

Background: Prostacyclin (PGI2) is known to be an important mediator of peripheral pain sensation (nociception) whereas little is known about its role in central sensitization., Methods: The levels of the stable PGI2-metabolite 6-keto-prostaglandin F1α (6-keto-PGF1α) and of prostaglandin E2 (PGE2) were measured in the dorsal horn with the use of mass spectrometry after peripheral inflammation. Expression of the prostanoid receptors was determined by immunohistology. Effects of prostacyclin receptor (IP) activation on spinal neurons were investigated with biochemical assays (cyclic adenosine monophosphate-, glutamate release-measurement, Western blot analysis) in embryonic cultures and adult spinal cord. The specific IP antagonist Cay10441 was applied intrathecally after zymosan-induced mechanical hyperalgesia in vivo., Results: Peripheral inflammation caused a significant increase of the stable PGI2 metabolite 6-keto-PGF1α in the dorsal horn of wild-type mice (n = 5). IP was located on spinal neurons and did not colocalize with the prostaglandin E2 receptors EP2 or EP4. The selective IP-agonist cicaprost increased cyclic adenosine monophosphate synthesis in spinal cultures from wild-type but not from IP-deficient mice (n = 5-10). The combination of fluorescence-resonance-energy transfer-based cyclic adenosine monophosphate imaging and calcium imaging showed a cicaprost-induced cyclic adenosine monophosphate synthesis in spinal cord neurons (n = 5-6). Fittingly, IP activation increased glutamate release from acute spinal cord sections of adult mice (n = 13-58). Cicaprost, but not agonists for EP2 and EP4, induced protein kinase A-dependent phosphorylation of the GluR1 subunit and its translocation to the membrane. Accordingly, intrathecal administration of the IP receptor antagonist Cay10441 had an antinociceptive effect (n = 8-11)., Conclusion: Spinal prostacyclin synthesis during early inflammation causes the recruitment of GluR1 receptors to membrane fractions, thereby augmenting the onset of central sensitization.

In the heart, adrenergic stimulation activates the β-adrenergic receptors coupled to the heterotrimeric stimulatory Gs protein, followed by subsequent activation of adenylyl cyclase, elevation of cyclic AMP levels, and protein kinase A (PKA) activation. One of the main targets for PKA modulation is the cardiac L-type Ca²⁺ channel (CaV1.2) located in the plasma membrane and along the T-tubules, which mediates Ca²⁺ entry into cardiomyocytes. β-Adrenergic receptor activation increases the Ca²⁺ current via CaV1.2 channels and is responsible for the positive ionotropic effect of adrenergic stimulation. Despite decades of research, the molecular mechanism underlying this modulation has not been fully resolved. On the contrary, initial reports of identification of key components in this modulation were later refuted using advanced model systems, especially transgenic animals. Some of the cardinal debated issues include details of specific subunits and residues in CaV1.2 phosphorylated by PKA, the nature, extent, and role of post-translational processing of CaV1.2, and the role of auxiliary proteins (such as A kinase anchoring proteins) involved in PKA regulation. In addition, the previously proposed crucial role of PKA in modulation of unstimulated Ca²⁺ current in the absence of β-adrenergic receptor stimulation and in voltage-dependent facilitation of CaV1.2 remains uncertain. Full reconstitution of the β-adrenergic receptor signaling pathway in heterologous expression systems remains an unmet challenge. This review summarizes the past and new findings, the mechanisms proposed and later proven, rejected or disputed, and emphasizes the essential issues that remain unresolved.

Rationale: C2238 atrial natriuretic peptide (ANP) minor allele (substitution of thymidine with cytosine in position 2238) associates with increased risk of cardiovascular events., Objective: We investigated the mechanisms underlying the vascular effects of C2238-αANP., Methods and Results: In vitro, human umbilical vein endothelial cell were exposed to either wild-type (T2238)- or mutant (C2238)-αANP. Cell survival and apoptosis were tested by Trypan blue, annexin V, and cleaved caspase-3 assays. C2238-αANP significantly reduced human umbilical vein endothelial cell survival and increased apoptosis. In addition, C2238-αANP reduced endothelial tube formation, as assessed by matrigel. C2238-αANP did not differentially modulate natriuretic peptide receptor (NPR)-A/B activity with respect to T2238-αANP, as evaluated by intracellular cGMP levels. In contrast, C2238-αANP, but not T2238-αANP, markedly reduced intracellular cAMP levels in an NPR-C-dependent manner. Accordingly, C2238-αANP showed higher affinity binding to NPR-C, than T2238-αANP. Either NPR-C inhibition by antisense oligonucleotide or NPR-C gene silencing by small interfering RNA rescued survival and tube formation of human umbilical vein endothelial cell exposed to C2238-αANP. Similar data were obtained in human aortic endothelial cell with NPR-C knockdown. NPR-C activation by C2238-αANP inhibited the protein kinase A/Akt1 pathway and increased reactive oxygen species. Adenovirus-mediated Akt1 reactivation rescued the detrimental effects of C2238-αANP. Overall, these data indicate that C2238-αANP affects endothelial cell integrity through NPR-C-dependent inhibition of the cAMP/protein kinase A/Akt1 pathway and increased reactive oxygen species production. Accordingly, C2238-αANP caused impairment of acetylcholine-dependent vasorelaxation ex vivo, which was rescued by NPR-C pharmacological inhibition. Finally, subjects carrying C2238 minor allele showed early endothelial dysfunction, which highlights the clinical relevance of our results., Conclusions: C2238-αANP reduces endothelial cell survival and impairs endothelial function through NPR-C signaling. NPR-C targeting represents a potential strategy to reduce cardiovascular risk in C2238 minor-allele carriers.

Background: The involvement of the L-arginine/nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway in antinociception has been implicated as a molecular mechanism of antinociception produced by several antinociceptive agents, including μ-, κ-, or δ-opioid receptor agonists, nonsteroidal analgesics, cholinergic agonist, and α2C adrenoceptor agonist. In this study, we investigated whether ketamine, a dissociative anesthetic N-methyl-D-aspartate receptor antagonist, was also capable of activating the L-arginine/NO/cGMP pathway and eliciting peripheral antinociception., Methods: The rat paw pressure test was used, with hyperalgesia induced by intraplantar injection of prostaglandin E2. All drugs were locally administered into the right hindpaw of male Wistar rats., Results: Ketamine (10, 20, 40, 80 μg/paw) elicited a local antinociceptive effect that was antagonized by the nonselective NOS inhibitor L-NOARG (12, 18, and 24 μg/paw) and by the selective neuronal NOS inhibitor L-NPA (12, 18, and 24 μg/paw). In another experiment, we used the inhibitors L-NIO and L-NIL (24 μg/paw) to selectively inhibit endothelial and inducible NOS, respectively. These 2 drugs were ineffective at blocking the effects of the peripheral ketamine injection. In addition, the level of nitrite in the homogenized paw indicated that exogenous ketamine is able to induce NO release. The soluble guanylyl cyclase inhibitor ODQ (25, 50, and 100 μg/paw) blocked the action of ketamine, and the cGMP-phosphodiesterase inhibitor zaprinast (50 μg/paw) enhanced the antinociceptive effects of low-dose ketamine (10 μg/paw)., Conclusions: Our results suggest that ketamine stimulates the L-arginine/NO/cyclic GMP pathway via neuronal NO synthase to induce peripheral antinociceptive effects.

Objective: Cilostazol, a potent type 3 phosphodiesterase inhibitor, has recently been found to reduce neointimal formation by inhibiting vascular smooth muscle cell (VSMC) proliferation. The aim of this study is to investigate whether cilostazol exerts an action on phenotypic modulation of VSMCs, another important process in the pathogenesis of neointimal formation., Methods and Results: Cilostazol may convert VSMCs from a serum-induced dedifferentiation state to a differentiated state, as indicated by a spindle-shaped morphology and an increase in the expression of smooth muscle cell differentiation marker contractile proteins. The upregulation of contractile proteins by cilostazol involves the cAMP/protein kinase A (PKA) signaling pathway, because the cAMP analog mimicked and specific cAMP/PKA inhibitors opposed the effect of cilostazol. Furthermore, cilostazol-activated cAMP response element (CRE)-binding protein (CREB), including phosphorylation at Ser133 and its nuclear translocation. Deletion and mutational analysis of the contractile protein promoters along with chromatin immunoprecipitation using anti-CREB antibody showed that CRE is essential for cilostazol-induced contractile protein expression. Transfection of dominant-negative CREB (mutated Ser133) plasmid in VSMCs blocked cilostazol-stimulated contractile protein expression. In vivo, cilostazol upregulated contractile proteins and induced the activation of CREB in the neointima of balloon-injured arteries., Conclusions: Cilostazol promotes VSMC differentiation through the cAMP/PKA/CREB signaling cascade.

Rationale: cAMP and cGMP are intracellular second messengers involved in heart pathophysiology. cGMP can potentially affect cAMP signals via cGMP-regulated phosphodiesterases (PDEs)., Objective: To study the effect of cGMP signals on the local cAMP response to catecholamines in specific subcellular compartments., Methods and Results: We used real-time FRET imaging of living rat ventriculocytes expressing targeted cAMP and cGMP biosensors to detect cyclic nucleotides levels in specific locales. We found that the compartmentalized, but not the global, cAMP response to isoproterenol is profoundly affected by cGMP signals. The effect of cGMP is to increase cAMP levels in the compartment where the protein kinase (PK)A-RI isoforms reside but to decrease cAMP in the compartment where the PKA-RII isoforms reside. These opposing effects are determined by the cGMP-regulated PDEs, namely PDE2 and PDE3, with the local activity of these PDEs being critically important. The cGMP-mediated modulation of cAMP also affects the phosphorylation of PKA targets and myocyte contractility., Conclusions: cGMP signals exert opposing effects on local cAMP levels via different PDEs the activity of which is exerted in spatially distinct subcellular domains. Inhibition of PDE2 selectively abolishes the negative effects of cGMP on cAMP and may have therapeutic potential.

Exposure to perinatal hypoxia results in alteration of the adult pulmonary circulation, which is linked among others to alterations in K(+) channels in pulmonary artery (PA) smooth muscle cells. In particular, large conductance Ca(2+)-activated K(+) (BK(Ca)) channels protein expression and activity were increased in adult PA from mice born in hypoxia compared with controls. We evaluated long-term effects of perinatal hypoxia on the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway-mediated activation of BK(Ca) channels, using isoproterenol, forskolin, and dibutyryl-cAMP. Whole-cell outward current was higher in pulmonary artery smooth muscle cells from mice born in hypoxia compared with controls. Spontaneous transient outward currents, representative of BK(Ca) activity, were present in a greater proportion in pulmonary artery smooth muscle cells of mice born in hypoxia than in controls. Agonists induced a greater relaxation in PA of mice born in hypoxia compared with controls, and BK(Ca) channels contributed more to the cAMP/PKA-mediated relaxation in case of perinatal hypoxia. In summary, perinatal hypoxia enhanced cAMP-mediated BK(Ca) channels activation in adult murine PA, suggesting that this pathway could be a potential target for modulating adult pulmonary vascular tone after perinatal hypoxia.

Chemokines are small secreted proteins with chemoattractant properties that play a key role in inflammation, metastasis, and embryonic development. We previously demonstrated a nonchemotactic role for one such chemokine pair, stromal cell-derived factor-1α and its G-protein coupled receptor, CXCR4. Stromal cell-derived factor-1/CXCR4 are expressed on cardiac myocytes and have direct consequences on cardiac myocyte physiology by inhibiting contractility in response to the nonselective β-adrenergic receptor (βAR) agonist, isoproterenol. As a result of the importance of β-adrenergic signaling in heart failure pathophysiology, we investigated the underlying mechanism involved in CXCR4 modulation of βAR signaling. Our studies demonstrate activation of CXCR4 by stromal cell-derived factor-1 leads to a decrease in βAR-induced PKA activity as assessed by cAMP accumulation and PKA-dependent phosphorylation of phospholamban, an inhibitor of SERCA2a. We determined CXCR4 regulation of βAR downstream targets is β2AR-dependent. We demonstrated a physical interaction between CXCR4 and β2AR as determined by coimmunoprecipitation, confocal microscopy, and BRET techniques. The CXCR4-β2AR interaction leads to G-protein signal modulation and suggests the interaction is a novel mechanism for regulating cardiac myocyte contractility. Chemokines are physiologically and developmentally relevant to myocardial biology and represent a novel receptor class of cardiac modulators. The CXCR4-β2AR complex could represent a hitherto unknown target for therapeutic intervention.

Rationale: Sympathetic stimulation of the heart increases the force of contraction and rate of ventricular relaxation by triggering protein kinase (PK)A-dependent phosphorylation of proteins that regulate intracellular calcium. We hypothesized that scaffolding of cAMP signaling complexes by AKAP5 is required for efficient sympathetic stimulation of calcium transients., Objective: We examined the function of AKAP5 in the β-adrenergic signaling cascade., Methods and Results: We used calcium imaging and electrophysiology to examine the sympathetic response of cardiomyocytes isolated from wild type and AKAP5 mutant animals. The β-adrenergic regulation of calcium transients and the phosphorylation of substrates involved in calcium handling were disrupted in AKAP5 knockout cardiomyocytes. The scaffolding protein, AKAP5 (also called AKAP150/79), targets adenylyl cyclase, PKA, and calcineurin to a caveolin 3-associated complex in ventricular myocytes that also binds a unique subpopulation of Ca(v)1.2 L-type calcium channels. Only the caveolin 3-associated Ca(v)1.2 channels are phosphorylated by PKA in response to sympathetic stimulation in wild-type heart. However, in the AKAP5 knockout heart, the organization of this signaling complex is disrupted, adenylyl cyclase 5/6 no longer associates with caveolin 3 in the T-tubules, and noncaveolin 3-associated calcium channels become phosphorylated after β-adrenergic stimulation, although this does not lead to an enhanced calcium transient. The signaling domain created by AKAP5 is also essential for the PKA-dependent phosphorylation of ryanodine receptors and phospholamban., Conclusions: These findings identify an AKAP5-organized signaling module that is associated with caveolin 3 and is essential for sympathetic stimulation of the calcium transient in adult heart cells.

Objectives: That glucagonlike peptide-1 (GLP-1) induces differentiation of primate embryonic stem (ES) cells into insulin-producing cells has been reported by several groups and also confirmed with our observations., Methods: To further elucidate the process in detail and the signaling pathways involved in this differentiation, we induced human ES cells HUES1 differentiated into insulin secretion cells by GLP-1 treatment., Results: A time-dependent pattern of down expression of the stem cell markers (human telomerase reverse transcriptase and octamer-4), and the appearance of multiple beta-cell-specific proteins (insulin, glucokinase, glucose transporter, type 2, and islet duodenal homeobox 1) and hedgehog signal molecules (Indian hedgehog, sonic hedgehog, and hedgehog receptor, patched) have been identified. Cotreatment with hedgehog signal inhibitor cytopamine was able to block this differentiation, providing evidence of the involvement of the hedgehog signaling pathway in GLP-1-induced differentiation. We also observed increased transcripts of the transcription factors of activator protein 1, serum response element-1, DNA-binding transcription factors, and cAMP response element in GLP-1-induced ES cell differentiation. Inhibition profile by its specific inhibitors indicated that the cyclic adenosine monophosphate and phosphatidylinositol-3-kinase pathways, but not the mitogen-activated protein kinase pathway, were required for the induced differentiation of ES cells., Conclusions: These data support that GLP-1 directs human ES cell differentiation into insulin-producing cells via hedgehog, cyclic adenosine monophosphate, and phosphatidylinositol-3-kinase pathways.

Purpose of Review: Olfaction is a field of growing interest in schizophrenia research. This article reviews recent studies on olfactory functions in schizophrenia., Recent Findings: The current literature provides additional insights into olfactory deficits, abnormalities, and olfactory hedonic dysfunction in schizophrenia. Recent findings reinforce particular associations with negative symptoms and deficit syndrome schizophrenia. Studies indicate that abnormalities in patients with schizophrenia extend to more peripheral olfactory structures and functions, including olfactory receptor neuron dysfunction. Olfactory identification ability was found to relate to prodromal disorganization symptoms in young high-risk patients. Further support for the notion of a genetic contribution to olfactory dysfunction in schizophrenia derives from studies reporting physiological olfactory dysfunction (olfactory event-related potentials) in unaffected relatives, and an odor-specific hyposmia, present in both patients with schizophrenia and family members., Summary: Further research is needed to improve our understanding of olfactory dysfunction in schizophrenia. Recent encouraging findings underscore that the olfactory system is a field of research that holds promise for advancing our understanding of the pathophysiology of schizophrenia and possibly as a useful endophenotypic marker of neurodevelopmental vulnerability.

Abstract: Pacemaking is a basic physiological process, and the cellular mechanisms involved in this function have always attracted the keen attention of investigators. The "funny" (I(f)) current, originally described in sinoatrial node myocytes as an inward current activated on hyperpolarization to the diastolic range of voltages, has properties suitable for generating repetitive activity and for modulating spontaneous rate. The degree of activation of the funny current determines, at the end of an action potential, the steepness of phase 4 depolarization; hence, the frequency of action potential firing. Because I(f) is controlled by intracellular cAMP and is thus activated and inhibited by beta-adrenergic and muscarinic M2 receptor stimulation, respectively, it represents a basic physiological mechanism mediating autonomic regulation of heart rate. Given the complexity of the cellular processes involved in rhythmic activity, an exact quantification of the extent to which I(f) and other mechanisms contribute to pacemaking is still a debated issue; nonetheless, a wealth of information collected since the current was first described more than 30 years ago clearly agrees to identify I(f) as a major player in both generation of spontaneous activity and rate control. I(f)- dependent pacemaking has recently advanced from a basic, physiologically relevant concept, as originally described, to a practical concept that has several potentially useful clinical applications and can be valuable in therapeutically relevant conditions. Typically, given their exclusive role in pacemaking, f-channels are ideal targets of drugs aiming to pharmacological control of cardiac rate. Molecules able to bind specifically to and block f-channels can thus be used as pharmacological tools for heart rate reduction with little or no adverse cardiovascular side effects. Indeed a selective f-channel inhibitor, ivabradine, is today commercially available as a tool in the treatment of stable chronic angina. Also, several loss-of-function mutations of HCN4 (hyperpolarization-activated, cyclic-nucleotide gated 4), the major constitutive subunit of f-channels in pacemaker cells, are known today to cause rhythm disturbances, such as for example inherited sinus bradycardia. Finally, gene- or cell-based methods for in situ delivery of f-channels to silent or defective cardiac muscle represent novel approaches for the development of biological pacemakers eventually able to replace electronic devices.

Androgens produce acute vasodilation of systemic, pulmonary, and coronary arteries in several mammal preparations and increase cardiomyocyte contractility. A decrease of the spontaneous beating of sinoatrial cells has also been described. The aim of this study was to characterize the direct effect of 5alpha-dihydrotestosterone on the spontaneous chronotropism and inotropism in the same preparation as an approach to establish the effect on cardiac output and their mechanism of action. The effects were studied on isolated right atria of Wistar rats placed in an organ bath in Tyrode solution at 37 degrees C and bubbled with carbogen. In male rats, the acute administration of 5alpha-dihydrotestosterone, a nonaromatizable derivate of testosterone, elicited a positive inotropism, which was associated with a negative chronotropism. As reported in the left atria, polyamines and beta-adrenoceptors played a role in 5alpha-dihydrotestosterone-elicited positive inotropism because the effect was antagonized by alpha-difluoromethylornithine, an inhibitor of polyamine synthesis, and atenolol, a beta1-adrenoceptor blocker, but not on the negative effect on chronotropism. The androgen increased the sinoatrial node recovery time, suggesting an effect on the mechanisms of spontaneous diastolic depolarization involved in atria pacemaking. These effects of 5alpha-dihydrotestosterone are not hormonally regulated because they are similarly produced in estrogenized females and gonadectomized male and female rats. These results suggest that the androgen could acutely improve cardiac performance.

Objective: To consider the mechanisms that may link asthma and major depressive disorder (MDD). Asthma and MDD co-occur at higher rates than expected, but whether this reflects shared underlying pathophysiological mechanisms is not known., Methods: A review of the epidemiological data linking asthma and MDD was conducted and the possible biological mechanisms that could account for the high rate of this comorbidity were reviewed., Results: MDD occurs in almost half of patients with asthma assessed in tertiary care centers. Dysregulation of the hypothalamic pituitary adrenal axis may predispose people to both MDD and asthma, and similar alterations in the immune, autonomic nervous, and other key systems are apparent and may contribute to this increased risk of co-occurrence., Conclusions: High rates of MDD in asthma may result from the stress of chronic illness, the medications used to treat it, or a combination of the two. The high level of co-occurrence may also reflect dysregulation of certain stress-sensitive biological processes that contribute to the pathophysiology of both conditions.

Vascular smooth muscle (VSM) growth is integral in the pathophysiology of blood vessel diseases, and identifying approaches that have capacity to regulate VSM growth is critically essential. Cyclic nucleotide signaling has been generally considered protective in cardiac and vascular tissues and has been the target of numerous basic science and clinical studies. In this project, the influence of BAY 41-2272 (BAY), a recently described soluble guanylate cyclase stimulator and inducer of cyclic guanosine monophosphate (cGMP) synthesis, on VSM cell growth was analyzed. In rat A7R5 VSM cells, BAY significantly reduced proliferation in a dose- and time-dependent fashion. BAY activated cGMP and cyclic adenosine monophosphate (cAMP) signaling evidenced through elevated cGMP and cAMP content, increased expression of cyclic nucleotide-dependent protein kinases, and differential vasodilator-stimulated phosphoprotein phosphorylation. BAY significantly elevated cyclin E expression, decreased expression of the regulatory cyclin-dependent kinases -2 and -6, increased expression of cell cycle inhibitory p21 WAF1/Cip1 and p27 Kip1, and reduced expression of phosphorylated focal adhesion kinase. These comprehensive findings provide first evidence for the antigrowth cell cycle-regulatory properties of the neoteric agent, BAY 41-2272, in VSM and lend support for its continued study in the clinical and basic cardiovascular sciences.

Objective: The neuropeptide calcitonin gene-related peptide (CGRP) is a migraine trigger that plays a crucial role in migraine pathophysiology, and CGRP antagonism is efficient in the treatment of migraine attacks. Familial hemiplegic migraine (FHM) is a dominantly inherited subtype of migraine with aura associated with several gene mutations. FHM shares many phenotypical similarities with common types of migraine, indicating common neurobiological pathways. We tested the hypothesis that the FHM genotype confers a CGRP hypersensitive phenotype., Methods: We included 9 FHM patients with known mutations in the CACNA1A and ATP1A2 genes and 10 healthy controls. All subjects received i.v. infusion of CGRP (1.5 microg/min). We recorded headache intensity on a verbal rating scale and vascular changes in the middle cerebral artery and the superficial temporal artery., Results: CGRP infusion did not induce an aura in any of the participants. The incidences of reported migraine and migraine-like headache were not different in the two groups, with 22% (2 of 9) reporting migraine in the patient group and 10% (1 of 10) reporting migraine-like headache in the control group (95% CI -0.31 to 0.55; p = 0.58). Headache severity and intensity were not different between the groups., Conclusions: Familial hemiplegic migraine (FHM) patients do not show hypersensitivity of the calcitonin gene-related peptide (CGRP)-cyclic adenosine 3',5'-monophosphate pathway, as characteristically seen in migraine patients without aura. This indicates that the pathophysiologic pathways underlying migraine headache in FHM may be different from the common types of migraine and questions whether CGRP antagonists would be effective in the treatment of FHM patients.

Ablation of the enzyme phosphoinositide 3-kinase (PI3K)gamma (PI3Kgamma(-/-)) in mice increases cardiac contractility by elevating intracellular cAMP and enhancing sarcoplasmic reticulum Ca(2+) handling. Because cAMP is a critical determinant of heart rate, we investigated whether heart rate is altered in mice lacking PI3Kgamma. Heart rate was similar in anesthetized PI3Kgamma(-/-) and wild-type (PI3Kgamma(+/+)) mice. However, IP injection of atropine (1 mg/kg), propranolol (1 mg/kg), or both drugs in combination unmasked elevated heart rates in PI3Kgamma(-/-) mice, suggesting altered sinoatrial node (SAN) function. Indeed, spontaneous action potential frequency was approximately 35% greater in SAN myocytes isolated from PI3Kgamma(-/-) mice compared with PI3Kgamma(+/+) mice. These differences in action potential frequency were abolished by intracellular dialysis with the cAMP/protein kinase A antagonist Rp-cAMP but were unaffected by treatment with ryanodine to inhibit sarcoplasmic reticulum Ca(2+) release. Voltage-clamp experiments demonstrated that elevated action potential frequencies in PI3Kgamma(-/-) SAN myocytes were more strongly associated with cAMP-dependent increases in L-type Ca(2+) current (I(Ca,L)) than elevated hyperpolarization-activated current (I(f)). In contrast, I(Ca,L) was not increased in working atrial myocytes, suggesting distinct subcellular regulation of L-type Ca(2+) channels by PI3Kgamma in the SAN compared with the working myocardium. In summary, PI3Kgamma regulates heart rate by the cAMP-dependent modulation of SAN function. The effects of PI3Kgamma ablation in the SAN are unique from those in the working myocardium.

cAMP regulates integrin-dependent adhesions of vascular endothelial cells (VECs) to extracellular matrix proteins, their vascular endothelial cadherin-dependent intercellular adhesions, and their proliferation and migration in response to growth and chemotactic factors. Previously, we reported that cAMP-elevating agents differentially inhibited migration of human VECs isolated from large vascular structures (macro-VECs, human aortic endothelial cells [HAECs]) or small vascular structures (micro-VECs, human microvascular endothelial cells [HMVECs]) and that cAMP hydrolysis by phosphodiesterase (PDE)3 and PDE4 enzymes was important in coordinating this difference. Here we report that 2 cAMP-effector enzymes, namely protein kinase (PK)A and exchange protein activated by cAMP (EPAC), each regulate extracellular matrix protein-based adhesions of both macro- and micro-VECs. Of interest and potential therapeutic importance, we report that although specific pharmacological activation of EPAC markedly stimulated adhesion of micro-VECs to extracellular matrix proteins when PKA was inhibited, this treatment only modestly promoted adhesion of macro-VECs. Consistent with an important role for cAMP PDEs in this difference, PDE3 or PDE4 inhibitors promoted EPAC-dependent adhesions in micro-VECs when PKA was inhibited but not in macro-VECs. At a molecular level, we identify multiple, nonoverlapping, PKA- or EPAC-based signaling protein complexes in both macro- and micro-VECs and demonstrate that each of these complexes contains either PDE3B or PDE4D but not both of these PDEs. Taken together, our data support the concept that adhesion of macro- and micro-VECs is differentially regulated by cAMP and that these differences are coordinated through selective actions of cAMP at multiple nonoverlapping signaling complexes that contain PKA or EPAC and distinct PDE variants.

Background: Alpha2-adrenoceptor agonist has been reported to produce inhibition of arginine vasopressin release, diuresis, and sympatholytic effects. However, its mechanisms of central action remain incompletely understood. Hypothalamic paraventricular nucleus (PVN) neurons, which are in direct contact with noradrenergic synapses and are controlled by the hyperpolarization-activated currents, are called Ih (H current). The effect of dexmedetomidine, a highly selective and potent agonist, at alpha2 adrenoceptors on Ih is unknown. The purpose of this study was to examine the effects of dexmedetomidine on the PVN neuron, which is involved in the arginine vasopressin release and autonomic regulation., Methods: The authors investigated the effects of dexmedetomidine on the membrane properties in PVN magnocellular neurons and an Ih in PVN parvocellular neurons with a whole cell patch clamp technique using a rat brain slice preparation., Results: Dexmedetomidine dose-dependently hyperpolarized PVN magnocellular neurons. In the voltage clamp mode, dexmedetomidine induced an outward current, with a reversal potential of -94 mV, and this was shown to depend on the external concentration of K. Pretreatment with Ba or peptide toxin tertiapin blocked hyperpolarization induced by dexmedetomidine. The effect of dexmedetomidine was blocked by an alpha2-adrenoceptor antagonist, yohimbine. Ih was suppressed dose dependently by dexmedetomidine in PVN parvocellular neurons. Pretreatment with Cs occluded the Ih suppression by dexmedetomidine. Yohimbine blocked the Ih suppression by dexmedetomidine. The Ih sensitive to dexmedetomidine was weakly modulated by intracellular cyclic adenosine monophosphate., Conclusions: Dexmedetomidine inhibited PVN magnocellular neurons by activation of the G protein-coupled inwardly rectifying K current and inhibited PVN parvocellular neurons by suppression of Ih.

Objectives: Using FM1-43 epifluorescence imaging and electron microscopy, we recently reported that glucagon-like peptide (GLP-1)-mediated cyclic adenosine monophosphate (cAMP) potentiation of insulin secretion markedly promotes the number of plasma membrane (PM) exocytic sites and insulin secretory granule (SG)-to-granule fusions underlying compound and sequential exocytosis., Methods: Here, we used FM1-43 imaging to dissect the distinct contributions of putative GLP-1/cAMP activated substrates--exchange protein directly activated by cAMP (EPAC) and protein kinase A (PKA)--in mediating these exocytic events., Results: Like GLP-1, cAMP activation by forskolin increased the number of PM exocytic sites (2.3-fold), which were mainly of the robust-sustained (55.8%) and stepwise-multiphasic (37.7%) patterns corresponding to compound and sequential SG-SG exocytosis, respectively, with few monophasic hotspots (6.5%) corresponding to single-granule exocytosis. Direct activation of EPAC by 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cAMP also increased the number of exocytic sites, but which were mainly multiphasic (60%) and monophasic (40%) hotspots. Protein kinase A inhibition by H89 blocked forskolin-evoked robust-sustained hotspots, while retaining multiphasic (47%) and monophasic (53%) hotspots. Consistently, PKA activation (N6-benzoyladenosine-3',5'-cAMP) evoked only multiphasic (60%) and monophasic (40%) hotspots. These results suggested that PKA activation is required but alone is insufficient to promote compound SG-SG fusions. 8-(4-Chloro-phenylthio)-2'-O-methyladenosine-3',5'-cAMP plus N6-benzoyladenosine-3',5'-cAMP stimulation completely reconstituted the effects of forskolin, including increasing the number of exocytic sites, with a similar pattern of robust-sustained (42.6%) and stepwise (39.6%) hotspots and few monophasic (17.8%) hotspots., Conclusions: The EPAC and PKA modulate both distinct and common exocytic steps to potentiate insulin exocytosis where (a) EPAC activation mobilizes SGs to fuse at the PM, thereby increasing number of PM exocytic sites; and (b) PKA and EPAC activation synergistically modulate SG-SG fusions underlying compound and sequential exocytoses.

In the present study we hypothesized that endothelial progenitor cells (EPCs) enhance production of vasoprotective substances in cerebral arteries. Isolated mononuclear cells from rabbit peripheral blood were cultured in endothelial growth medium (EGM-2) for 7 days to yield EPCs. Rabbit basilar arteries were exposed to autologous EPCs ( approximately 5x10(5) cells) in vitro or in vivo. Twenty-four hours after intracisternal delivery of autologous EPCs, basilar arteries were isolated and expression of vasoregulatory proteins, production of prostacyclin (PGI(2)), and cAMP were determined. Arteries transplanted with EPCs demonstrated increased protein expression of cyclooxygenase-2 and PGI(2) in adventitia, media, and endothelium. Furthermore, production of PGI(2) and arterial content of cAMP, second messenger for PGI(2), were significantly augmented after transplantation of EPCs. In contrast, production of thromboxane A(2) was significantly reduced, whereas production of prostaglandin E(2) remained unchanged. The increased production of PGI(2) and arterial content of cAMP were inhibited only by a selective cyclooxygenase-2 inhibitor, NS-398. In vitro or in vivo treatment of basilar artery with conditioned media from EPCs also caused increase in cyclooxygenase-2 and PGI(2) synthase protein expression associated with elevation of cAMP. Our results suggest that in cerebral arteries, paracrine effect of EPCs promotes vasoprotection by increasing PGI(2) production and intracellular concentration of cAMP. This effect appears to be mediated by activation of arachidonic acid metabolism via stimulation of cyclooxygenase-2/PGI(2) synthase pathway.

Heterotrimeric G proteins are pivotal regulators of myocardial contractility. In addition to the receptor-induced GDP/GTP exchange, G protein alpha subunits can be activated by a phosphate transfer via a plasma membrane-associated complex of nucleoside diphosphate kinase B (NDPK B) and G protein betagamma-dimers (Gbetagamma). To investigate the physiological role of this phosphate transfer in cardiomyocytes, we generated a Gbeta1gamma2-dimer carrying a single amino acid exchange at the intermediately phosphorylated His-266 in the beta1 subunit (Gbeta1H266Lgamma2). Recombinantly expressed Gbeta1H266Lgamma2 were integrated into heterotrimeric G proteins in rat cardiomyocytes but were deficient in intermediate Gbeta phosphorylation. Compared with wild-type Gbeta1gamma2 (Gbeta1WTgamma2), overexpression of Gbeta1H266Lgamma2 suppressed basal cAMP formation up to 55%. A similar decrease in basal cAMP production occurred when the formation of NDPK B/Gbetagamma complexes was attenuated by siRNA-mediated NDPK B knockdown. In adult rat cardiomyocytes expressing Gbeta1H266Lgamma2, the basal contractility was suppressed by approximately 50% which correlated to similarly reduced basal cAMP levels and reduced Ser16-phosphorylation of phospholamban. In the presence of the beta-adrenoceptor agonist isoproterenol, the total cAMP formation and contractility were significantly lower in Gbeta1H266Lgamma2 than in Gbeta1WTgamma2 expressing cardiomyocytes. However, the relative isoproterenol-induced increased was not affected by Gbeta1H266Lgamma2. We conclude that the receptor-independent activation of G proteins via NDPK B/Gbetagamma complexes requires the intermediate phosphorylation of G protein beta subunits at His-266. Our results highlight the histidine kinase activity of NDPK B for Gbeta and demonstrate its contribution to the receptor-independent regulation of cAMP synthesis and contractility in intact cardiomyocytes.

Contraction and relaxation of vascular smooth muscle and cardiac myocytes are key physiological events in the cardiovascular system. These events are regulated by second messengers, cAMP and cGMP, in response to extracellular stimulants. The strength of signal transduction is controlled by intracellular cyclic nucleotide concentrations, which are determined by a balance in production and degradation of cAMP and cGMP. Degradation of cyclic nucleotides is catalyzed by 3',5'-cyclic nucleotide phosphodiesterases (PDEs), and therefore regulation of PDEs hydrolytic activity is important for modulation of cellular functions. Mammalian PDEs are composed of 21 genes and are categorized into 11 families based on sequence homology, enzymatic properties, and sensitivity to inhibitors. PDE families contain many splice variants that mostly are unique in tissue-expression patterns, gene regulation, enzymatic regulation by phosphorylation and regulatory proteins, subcellular localization, and interaction with association proteins. Each unique variant is closely related to the regulation of a specific cellular signaling. Thus, multiple PDEs function as a particular modulator of each cardiovascular function and regulate physiological homeostasis.

Background: Diminished cardiac S100A1 protein levels are characteristic of ischemic and dilated human cardiomyopathy. Because S100A1 has recently been identified as a Ca2+-dependent inotropic factor in the heart, this study sought to explore the pathophysiological relevance of S100A1 levels in development and progression of postischemic heart failure (HF)., Methods and Results: S100A1-transgenic (STG) and S100A1-knockout (SKO) mice were subjected to myocardial infarction (MI) by surgical left anterior descending coronary artery ligation, and survival, cardiac function, and remodeling were compared with nontransgenic littermate control (NLC) and wild-type (WT) animals up to 4 weeks. Although MI size was similar in all groups, infarcted S100A1-deficient hearts (SKO-MI) responded with acute contractile decompensation and accelerated transition to HF, rapid onset of cardiac remodeling with augmented apoptosis, and excessive mortality. NLC/WT-MI mice, displaying a progressive decrease in cardiac S100A1 expression, showed a later onset of cardiac remodeling and progression to HF. Infarcted S100A1-overexpressing hearts (STG-MI), however, showed preserved global contractile performance, abrogated apoptosis, and prevention from cardiac hypertrophy and HF with superior survival compared with NLC/WT-MI and SKO-MI. Both Gq-protein-dependent signaling and protein kinase C activation resulted in decreased cardiac S100A1 mRNA and protein levels, whereas Gs-protein-related signaling exerted opposite effects on cardiac S100A1 abundance. Mechanistically, sarcoplasmic reticulum Ca2+ cycling and beta-adrenergic signaling were severely impaired in SKO-MI myocardium but preserved in STG-MI., Conclusions: Our novel proof-of-concept study provides evidence that downregulation of S100A1 protein critically contributes to contractile dysfunction of the diseased heart, which is potentially responsible for driving the progressive downhill clinical course of patients with HF.

Background: Cardiac-directed expression of adenylyl cyclase type VI (AC(VI)) in mice results in structurally normal hearts with normal basal heart rate and function but increased responses to catecholamine stimulation. We tested the hypothesis that increased left ventricular (LV) AC(VI) content would increase mortality after acute myocardial infarction (MI)., Methods and Results: Transgenic mice with cardiac-directed AC(VI) expression and their transgene-negative littermates (control) underwent coronary ligation, and survival, infarct size, and LV size and function were assessed 1 to 7 days after MI. Mice with increased AC(VI) expression had increased survival (control 41%, AC(VI) 74%; P = 0.004). Infarct size and myocardial apoptotic rates were similar in AC(VI) and control mice; however, AC(VI) mice had less LV dilation (P < 0.001) and increased ejection fractions (P < 0.03). Three days after MI, studies in isolated perfused hearts showed that basal LV +dP/dt was similar, but graded dobutamine infusion was associated with a more robust LV contractile response in AC(VI) mice (P < 0.05). Increased LV function was associated with increases in cAMP generation (P = 0.0002), phospholamban phosphorylation (P < 0.04), sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) affinity for calcium (P < 0.015), and reduced AV block (P = 0.04)., Conclusions: In acute MI, increased cardiac AC(VI) content attenuates adverse LV remodeling, preserves LV contractile function, and reduces mortality.

Objective: Hyperhomocysteinemia is an independent risk factor for cardiovascular disorders. Our previous studies demonstrated that hyperhomocysteinemia not only elicited inflammatory responses in the vascular endothelium but also induced fatty liver and hypercholesterolemia via transcriptional regulation. One of the transcription factors activated in the liver during hyperhomocysteinemia was cAMP-response element binding protein (CREB). CREB regulates the expression of many genes including those involved in lipid and glucose metabolism. In this study, we investigated the molecular mechanism by which Hcy activated CREB in rat liver and in hepatocytes (HepG2)., Method and Results: Hyperhomocysteinemia was induced in rats by feeding high-methionine diet for 4 weeks. There was a significant increase in hepatic cAMP levels, protein kinase A (PKA) activity and an activation of CREB. Incubation of HepG2 cells with Hcy (50 to 100 micromol/L) significantly enhanced CREB phosphorylation and subsequently increased CREB/DNA binding activity. PKA was activated in Hcy-treated cells as a result of increased cellular cAMP level. Inhibition of adenylyl cyclase not only reduced the intracellular cAMP levels elevated by Hcy treatment but also inhibited PKA activation and prevented Hcy-induced CREB phosphorylation., Conclusions: These results suggest that the cAMP/PKA signaling pathway plays an important role in mediating Hcy-induced CREB activation in hepatocyte.

The activation of the cyclic nucleotide protein kinase A (PKA) and PKG by their respective second messengers is responsible for the modulation of many cellular functions in the heart including cardiac hypertrophy, strength of contraction, and ion flux. However, several studies have revealed that a general increase in cyclic nucleotide concentration in the cell is not sufficient for the specific regulation of target proteins. These studies found that PKA and PKG must be colocalized with their targets to ensure spatial-temporal control of substrate phosphorylation. This compartmentation of cyclic nucleotide signaling is accomplished by tethering the protein kinases with their respective substrates through the association with scaffolding proteins. For cAMP signaling, A-kinase anchoring proteins (AKAPs) provide a molecular mechanism for cAMP compartmentation, allowing for the precise control of PKA-mediated phosphorylation events. (cAMP, PKA, AKAP, PKG).

Compartmentation of cAMP is thought to generate the specificity of Gs-coupled receptor action in cardiac myocytes, with phosphodiesterases (PDEs) playing a major role in this process by preventing cAMP diffusion. We tested this hypothesis in adult rat ventricular myocytes by characterizing PDEs involved in the regulation of cAMP signals and L-type Ca2+ current (I(Ca,L)) on stimulation with beta1-adrenergic receptors (beta1-ARs), beta2-ARs, glucagon receptors (Glu-Rs) and prostaglandin E1 receptors (PGE1-Rs). All receptors but PGE1-R increased total cAMP, and inhibition of PDEs with 3-isobutyl-1-methylxanthine strongly potentiated these responses. When monitored in single cells by high-affinity cyclic nucleotide-gated (CNG) channels, stimulation of beta1-AR and Glu-R increased cAMP, whereas beta2-AR and PGE1-R had no detectable effect. Selective inhibition of PDE3 by cilostamide and PDE4 by Ro 20-1724 potentiated beta1-AR cAMP signals, whereas Glu-R cAMP was augmented only by PD4 inhibition. PGE1-R and beta2-AR generated substantial cAMP increases only when PDE3 and PDE4 were blocked. For all receptors except PGE1-R, the measurements of I(Ca,L) closely matched the ones obtained with CNG channels. Indeed, PDE3 and PDE4 controlled beta1-AR and beta2-AR regulation of I(Ca,L), whereas only PDE4 controlled Glu-R regulation of I(Ca,L) thus demonstrating that receptor-PDE coupling has functional implications downstream of cAMP. PGE1 had no effect on I(Ca,L) even after blockade of PDE3 or PDE4, suggesting that other mechanisms prevent cAMP produced by PGE1 to diffuse to L-type Ca2+ channels. These results identify specific functional coupling of individual PDE families to Gs-coupled receptors as a major mechanism enabling cardiac cells to generate heterogeneous cAMP signals in response to different hormones.

Subtle elevations in cAMP localized to the plasma membrane intensely strengthen endothelial barrier function. Paradoxically, pathogenic bacteria insert adenylyl cyclases (ACs) into eukaryotic cells generating a time-dependent cytosolic cAMP-increase that disrupts rather than strengthens the endothelial barrier. These findings bring into question whether membrane versus cytosolic AC activity dominates in control of cell adhesion. To address this problem, a mammalian forskolin-sensitive soluble AC (sACI/II) was expressed in pulmonary microvascular endothelial cells. Forskolin stimulated this sACI/II construct generating a small cytosolic cAMP-pool that was not regulated by phosphodiesterases or Galphas. Whereas forskolin simultaneously activated the sACI/II construct and endogenous transmembrane ACs, the modest sACI/II activity overwhelmed the barrier protective effects of plasma membrane activity to induce endothelial gap formation. Retargeting sACI/II to the plasma membrane retained AC activity but protected the endothelial cell barrier. These findings demonstrate for the first time that the intracellular location of cAMP synthesis critically determines its physiological outcome.

Prostaglandin E2 exerts an antiinflammatory action by ligation of the heptahelical receptor EP4 in human macrophages. Because the mechanism by which EP4 receptor stimulation suppresses inflammatory activation in macrophages remains undefined, we sought interactors with the carboxyl-terminal cytoplasmic domain of the EP4 receptor. Yeast 2-hybrid screening of the human bone marrow cDNA library with the EP4 receptor as a bait identified a cDNA clone encoding a 669-amino acid protein, designated here as EP4 receptor-associated protein (EPRAP), which contains 8 ankyrin motifs that might recruit other signaling molecules. EPRAP bound to the full-length EP4 receptor in HEK293 cells cotransfected with V5-tagged EPRAP and FLAG-tagged EP4 receptor cDNA, as anti-FLAG antibody coimmunoprecipitated EPRAP with the EP4 receptor from the lysates of cotransfected cells. Human macrophages derived from peripheral blood monocytes expressed an approximately 70-kDa protein detected by Western blotting with a polyclonal anti-EPRAP antibody. Fluorescence immunohistochemistry colocalized EPRAP with the EP4 receptor in human atheromata. Interference with EPRAP function by small interference RNA limited prostaglandin E2-mediated suppression of chemokine expression in macrophages activated with lipopolysaccharide and tumor necrosis factor alpha. In conclusion, the antiinflammatory action of prostaglandin E2 in macrophages involves EPRAP that associates directly with the cytoplasmic tail of EP4 receptor.

Local, rhythmic, subsarcolemmal Ca2+ releases (LCRs) from the sarcoplasmic reticulum (SR) during diastolic depolarization in sinoatrial nodal cells (SANC) occur even in the basal state and activate an inward Na(+)-Ca2+ exchanger current that affects spontaneous beating. Why SANC can generate spontaneous LCRs under basal conditions, whereas ventricular cells cannot, has not previously been explained. Here we show that a high basal cAMP level of isolated rabbit SANC and its attendant increase in protein kinase A (PKA)-dependent phosphorylation are obligatory for the occurrence of spontaneous, basal LCRs and for spontaneous beating. Gradations in basal PKA activity, indexed by gradations in phospholamban phosphorylation effected by a specific PKA inhibitory peptide were highly correlated with concomitant gradations in LCR spatiotemporal synchronization and phase, as well as beating rate. Higher levels of basal PKA inhibition abolish LCRs and spontaneous beating ceases. Stimulation of beta-adrenergic receptors extends the range of PKA-dependent control of LCRs and beating rate beyond that in the basal state. The link between SR Ca2+ cycling and beating rate is also present in vivo, as the regulation of beating rate by local beta-adrenergic receptor stimulation of the sinoatrial node in intact dogs is markedly blunted when SR Ca2+ cycling is disrupted by ryanodine. Thus, PKA-dependent phosphorylation of proteins that regulate cell Ca2+ balance and spontaneous SR Ca2+ cycling, ie, phospholamban and L-type Ca2+ channels (and likely others not measured in this study), controls the phase and size of LCRs and the resultant Na(+)-Ca2+ exchanger current and is crucial for both basal and reserve cardiac pacemaker function.

Background: A consequence of ischemia/reperfusion (IR) is endothelial barrier dysfunction and intravascular volume loss. The purposes of our study are to explore the impact of: 1) cyclic guanosine monophosphate (cGMP) synthesis inhibition, 2) cyclic adenosine monophosphate (cAMP) synthesis inhibition, 3) treatment with endothelin-1, and 4) endothelin-1 (ET-1)-mediated cAMP changes on IR-induced fluid leak. We hypothesize that IR-mediated microvascular fluid leak results from increased cGMP activity and ET-1 decreases IR-induced fluid leak via cAMP., Methods: A micro-cannulation technique was used to determine fluid leak or hydraulic permeability (Lp) in rat mesenteric venules. Lp was measured during IR and after treatment with 1) cGMP synthesis inhibitor (LY83583,10 micromol/L) 2) cAMP synthesis inhibitor (2',5'dideoxyadenosine,10 micromol/L), 3) ET-1 (80 pM), and 4) cAMP synthesis inhibitor plus ET-1 (n=6 in each group; Lp represented as mean+/-standard error of the mean; units 10-cm/sec/cmH2O)., Results: IR resulted in an increase in Lp (Lp=7.07+/-0.20) sevenfold above baseline (1.05+/-0.31) (p

Unlabelled: Systematic investigations of the actions of phosphodiesterase (PDE)-3 inhibitors on different human vascular tissues have not been performed. We investigated the effects of specific PDE-3 inhibitors (olprinone, milrinone, and amrinone) on contracted human gastroepiploic arteries (n = 70), internal mammary arteries (n = 72), and radial arteries (n = 70) harvested from a total of 134 patients, all of whom were undergoing coronary artery bypass surgery. Each of these PDE-3 inhibitors dose-dependently diminished the contractile responses to 10(-6) mol/L norepinephrine and to either 10(-9) or 10(-8) mol/L of the thromboxane A2 analog U46619. In inducing vasorelaxations, these inhibitors were significantly more potent in norepinephrine-contracted rings than in those contracted with U46619. Further, at concentrations similar to the maximum therapeutic plasma concentrations (10(-7) mol/L olprinone; 10(-6) mol/L milrinone; 10(-5) mol/L amrinone) olprinone and milrinone were more potent at inducing relaxations than amrinone in gastroepiploic arteries and radial arteries, whereas in internal mammary arteries milrinone was more potent than the others. These results suggest different activities for the three PDE-3 inhibitors among human arteries located in different regions and may be informative about the effectiveness of these inhibitors in preventing spasms in the various arterial grafts used in revascularization., Implications: Because three phosphodiesterase-3 inhibitors (milrinone, olprinone, and amrinone) differed in their vasodilator potencies (against the contractile response to either norepinephrine or a thromboxane A2 analog) among human arteries removed from different parts of the body, their vascular relaxation profiles should be considered before they are used clinically.

Gap junctions (GJs) constituted by neighboring cardiac myocytes are essential for gating ions and small molecules to coordinate cardiac contractions. cAMP is suggested to be a potent stimulus for enhancement of GJ function. However, it remains elusive how cAMP potentiates the GJ of cardiomyocytes. Here we demonstrated that the gating function of GJ is enhanced by the protein kinase A (PKA)-dependent signal, and that the accumulation of connexin43 (Cx43), the most abundant Cx in myocytes, is enhanced by an exchange protein directly activated by cAMP (Epac) (Rap1 activator)-dependent signal. The gating function of GJs was analyzed by microinjected dye transfer method. The accumulation of Cx43 was analyzed by quantitative immunostaining. Using the PKA-specific activator N6-benzoyladenosine-3',5'-cyclic monophosphate (6Bnz) and Epac-specific activator 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8CPT), we could delineate the two important downstream signals of cAMP for enhanced GJ neoformation. Whereas 6Bnz potentiated gating function of GJs with slight accumulation of Cx43 at cell-cell contacts, 8CPT remarkably enhanced the accumulation of Cx43 with a slight effect on gating. We further noticed that adherens junctions (AJs) were maturated by 8CPT, as marked by increased neural-cadherin immunostaining. Because AJ formation precedes the GJ formation, AJ formation accelerated by Epac-Rap1 signal may result in enhanced GJ formation. The involvement of Epac-Rap1 signal in GJ neoformation was further confirmed by evidence that inactivation of Rap1 by overexpression of Rap1GAP1b perturbed the accumulation of Cx43 at cell-cell contacts. Collectively, PKA and Epac cooperatively enhance functional GJ neoformation in cardiomyocytes.

Intermolecular interactions between members of both similar and divergent G protein-coupled receptor subfamilies have been shown in various experimental systems. Here, we demonstrate heterodimerization of predominant beta-adrenergic receptor (betaAR) subtypes expressed in the heart, beta1AR, and beta2AR, and its physiological relevance. In intact adult-mouse cardiac myocytes lacking native beta1AR and beta2AR, coexpression of both betaAR subtypes led to receptor heterodimerization, as evidenced by their coimmunoprecipitation, colocalization at optical resolution, and markedly increased binding affinity for subtype-selective ligands. As a result, the dose-response curve of myocyte contraction to betaAR agonist stimulation with isoproterenol (ISO) was shifted leftward by approximately 1.5 orders of magnitude, and the response of cellular cAMP formation to ISO was enhanced concomitantly, indicating that intermolecular interactions of betaAR subtypes resulted in sensitization of these receptors in response to agonist stimulation. In contrast, the presence of beta1AR greatly suppressed ligand-independent spontaneous activity of coexisting beta2ARs. Thus, heterodimerization of beta1AR and beta2AR in intact cardiac myocytes creates a novel population of betaARs with distinct functional and pharmacological properties, resulting in enhanced signaling efficiency in response to agonist stimulation while silencing ligand-independent receptor activation, thereby optimizing beta-adrenergic modulation of cardiac contractility.

Background: Endothelial cell receptors involved in post-injury/sepsis fluid extravasation are coupled to G-proteins that stimulate production of cGMP and cAMP. We hypothesize that cGMP and cAMP are endothelial second messengers that control microvascular permeability. The purposes of this series of experiments are to determine microvascular permeability under the following conditions: 1) reduced cGMP levels, 2) elevated cGMP levels, 3) reduced cAMP levels, and 4) elevated cAMP levels., Methods: Rat mesenteric venules were cannulated and hydraulic permeability (Lp) was measured at 3 to 5 minute intervals during 1) cGMP synthesis inhibition, 2) inhibition of cGMP degradation, 3) cAMP synthesis inhibition, and 4) inhibition of cAMP degradation (n = 6 in each study group). Lp units are x10 cm(-7)/sec/cmH2O and represented as mean +/- SEM., Results: Compared with baseline Lp (1.10 +/- 0.06), reduced cGMP levels by inhibiting its synthesis decreased Lp by over 50% (0.50 +/- 0.02, p < 0.001), while elevated cGMP levels by preventing its degradation increased Lp by more than 2-fold (0.91 +/- 0.10 to 2.26 +/- 0.15, p < 0.001). The reduction of cAMP levels by synthesis inhibition elevated Lp over 400% from 0.92 +/- 0.04 to 4.11 +/- 0.54 (p < 0.001), and elevation of cAMP level by blocking its degradation reduced Lp almost 50% from 1.11 +/- 0.04 to 0.59 +/- 0.06 (p < 0.001)., Conclusions: The second messengers, cGMP and cAMP, contribute to the control mechanisms that govern fluid leak across the endothelial barrier: cGMP increases microvascular permeability, while cAMP decreases microvascular permeability. Endothelial cell cyclic nucleotide second messengers are pharmacologically accessible and may be targeted during post-injury/sepsis-associated microvascular fluid leak.

We recently showed that phosphoinositide-3-kinase-gamma-deficient (PI3Kgamma-/-) mice have increased cardiac contractility without changes in heart size compared with control mice (ie, PI3Kgamma+/+ or PI3Kgamma+/-). In this study, we show that PI3Kgamma-/- cardiomyocytes have elevated Ca2+ transient amplitudes with abbreviated decay kinetics compared with control under field-stimulation and voltage-clamp conditions. When Ca2+ transients were eliminated with high Ca2+ buffering, L-type Ca2+ currents (I(Ca,L)), K+ currents, and action potential duration (APD) were not different between the groups, whereas, in the presence of Ca2+ transients, Ca2+-dependent phase of I(Ca,L) inactivation was abbreviated and APD at 90% repolarization was prolonged in PI3Kgamma-/- mice. Excitation-contraction coupling (ECC) gain, sarcoplasmic reticulum (SR) Ca2+ load, and SR Ca(2+) release fluxes measured as Ca2+ spikes, were also increased in PI3Kgamma-/- cardiomyocytes without detectable changes in Ca2+ spikes kinetics. The cAMP inhibitor Rp-cAMP eliminated enhanced ECC and SR Ca2+ load in PI3Kgamma-/- without effects in control myocytes. On the other hand, the beta-adrenergic receptor agonist isoproterenol increased I(Ca,L) and Ca2+ transient equally by approximately 2-fold in both PI3Kgamma-/- and PI3Kgamma+/- cardiomyocytes. Our results establish that PI3Kgamma reduces cardiac contractility in a highly compartmentalized manner by inhibiting cAMP-mediated SR Ca2+ loading without directly affecting other major modulators of ECC, such as AP and I(Ca,L).

I(Ks), the slowly activating component of the delayed rectifier current, plays a major role in repolarization of the cardiac action potential (AP). Genetic mutations in the alpha- (KCNQ1) and beta- (KCNE1) subunits of I(Ks) underlie Long QT Syndrome type 1 and 5 (LQT-1 and LQT-5), respectively, and predispose carriers to the development of polymorphic ventricular arrhythmias and sudden cardiac death. beta-adrenergic stimulation increases I(Ks) and results in rate dependent AP shortening, a control system that can be disrupted by some mutations linked to LQT-1 and LQT-5. The mechanisms by which I(Ks) regulates action potential duration (APD) during beta-adrenergic stimulation at different heart rates are not known, nor are the consequences of mutation induced disruption of this regulation. Here we develop a complementary experimental and theoretical approach to address these questions. We reconstituted I(Ks) in CHO cells (ie, KCNQ1 coexpressed with KCNE1 and the adaptator protein Yotiao) and quantitatively examined the effects of beta-adrenergic stimulation on channel kinetics. We then developed theoretical models of I(Ks) in the absence and presence of beta-adrenergic stimulation. We simulated the effects of sympathetic stimulation on channel activation (speeding) and deactivation (slowing) kinetics on the whole cell action potential under different pacing conditions. The model suggests these kinetic effects are critically important in rate-dependent control of action potential duration. We also investigate the effects of two LQT-5 mutations that alter kinetics and impair sympathetic stimulation of I(Ks) and show the likely mechanism by which they lead to tachyarrhythmias and indicate a distinct role of I(KS) kinetics in this electrical dysfunction. The full text of this article is available online at http://circres.ahajournals.org.

Toward the goal of defining new pharmacological targets for the treatment of chronic pain conditions, in previous studies we established a model, termed 'hyperalgesic priming,' in which an acute inflammatory stimulus causes a long-lasting latent susceptibility to hyperalgesia induced by subsequent exposures to the inflammatory mediator, prostaglandin E2 (PGE2). Those investigations suggested the hypothesis that priming induces a novel linkage between the PGE2-activated second messenger cascade and the epsilon isoform of protein kinase C (PKCepsilon). In the present study, comparison of dose-response relations for hyperalgesia produced by PGE2, forskolin, 8-Br-cAMP, or the protein kinase A (PKA) catalytic subunit, in primed versus normal animals, demonstrated that priming-induced enhancement of the PGE2-activated second messenger cascade occurs downstream to adenylate cyclase and upstream to PKA. Therefore, PGE2-induced hyperalgesia in the primed animal is enhanced by the recruitment of a novel cAMP/PKCepsilon signaling pathway in addition to the usual cAMP/PKA pathway. These observations suggest that pharmacological disruption of the novel interaction between cAMP and PKCepsilon might provide a route toward the development of highly specific methods to reverse cellular processes that underlie chronic pain states.

A tenet of beta1-adrenergic receptor (beta1AR) signaling is that stimulation of the receptor activates the adenylate cyclase-cAMP-protein kinase A (PKA) pathway, resulting in positive inotropic and relaxant effects in the heart. However, recent studies have suggested the involvement of Ca2+/calmodulin-dependent protein kinase II (CaMKII) in beta1AR-stimulated cardiac apoptosis. In this study, we determined roles of CaMKII and PKA in sustained versus short-term beta1AR modulation of excitation-contraction (E-C) coupling in cardiac myocytes. Short-term (10-minute) and sustained (24-hour) beta1AR stimulation with norepinephrine similarly enhanced cell contraction and Ca2+ transients, in contrast to anticipated receptor desensitization. More importantly, the sustained responses were largely PKA-independent, and were sensitive to specific CaMKII inhibitors or adenoviral expression of a dominant-negative CaMKII mutant. Biochemical assays revealed that a progressive and persistent CaMKII activation was associated with a rapid desensitization of the cAMP/PKA signaling. Concomitantly, phosphorylation of phospholamban, an SR Ca2+ cycling regulatory protein, was shifted from its PKA site (16Ser) to CaMKII site (17Thr). Thus, beta1AR stimulation activates dual signaling pathways mediated by cAMP/PKA and CaMKII, the former undergoing desensitization and the latter exhibiting sensitization. This finding may bear important etiological and therapeutical ramifications in understanding beta1AR signaling in chronic heart failure.