Systemic ß adrenergic stimulation/ sympathetic nerve system stimulation influences intraocular RAS through cAMP in the RPE.

Several lines of evidence support the existence of a renin-angiotensin system (RAS) in the retina that is separated from the blood stream by the retinal pigment epithelium (RPE). Under physiological conditions, increased activity of intraretinal RAS regulates neuronal activity of the retina but patho-physiologically participates in retinal degeneration such as hypertensive or diabetic retinopathy. Interestingly, the RPE appears to be a modulator of intraretinal RAS in response to changes in systemic RAS. As increased systemic RAS activity is associated with increased sympathetic tonus, we investigated whether systemic ß-adrenergic stimulation of the RPE also modulates renin expression in the RPE. In vivo, the mouse RPE expresses the ß-adrenergic receptor subtypes 1 and 2. Staining of retina sagittal sections showed tyrosine hydroxylase positive nerve endings in the choroid indicating adrenaline/noradrenaline production sites in close proximity to the RPE. Systemic infusion of isoproterenol increased renin expression in the RPE but not in the retina. This increase was sensitive to concomitant systemic application of the angiotensin-2 receptor-type-1 blocker losartan. In vitro analysis of renin gene expression using polarized porcine RPE showed that the activity of the renin promoter can be increased by cAMP stimulation (IBMX/forskolin) but was not influenced by angiotensin-2. Thus, with the identification of the ß-adrenergic system we added a new regulator of the retinal RAS with relevance for retinal function and pathology. Furthermore, it appears that the RPE is not only a close interaction partner of the photoreceptors but also a regulator or retinal activity in general.

Human bone marrow mesenchymal stem cells secrete endocannabinoids that stimulate in vitro hematopoietic stem cell migration effectively comparable to beta-adrenergic stimulation.

Granulocyte colony-stimulating factor (G-CSF) is a well-known hematopoietic stem cell (HSC)-mobilizing agent used in both allogeneic and autologous transplantation. However, a proportion of patients or healthy donors fail to mobilize a sufficient number of cells. New mobilization agents are therefore needed. Endocannabinoids (eCBs) are endogenous lipid mediators generated in the brain and peripheral tissues and activate the cannabinoid receptors CB1 and CB2. We suggest that eCBs may act as mobilizers of HSCs from the bone marrow (BM) under stress conditions as beta-adrenergic receptors (Adrß). This study demonstrates that BM mesenchymal stem cells (MSCs) secrete anandamide (AEA) and 2-arachidonylglycerol (2-AG) and the peripheral blood (PB) and BM microenvironment contain AEA and 2-AG. 2-AG levels are significantly higher in PB of the G-CSF-treated group compared with BM plasma. BM mononuclear cells (MNCs) and CD34+ HSCs express CB1, CB2, and Adrß subtypes. CD34+ HSCs had higher CB1 and CB2 receptor expression in G-CSF-untreated and G-CSF-treated groups compared with MSCs. MNCs but not MSCs expressed CB1 and CB2 receptors based on qRT-PCR and flow cytometry. AEA- and 2-AG-stimulated HSC migration was blocked by eCB receptor antagonists in an in vitro migration assay. In conclusion, components of the eCB system and their interaction with Adrß subtypes were demonstrated on HSCs and MSCs of G-CSF-treated and G-CSF-untreated healthy donors in vitro, revealing that eCBs might be potential candidates to enhance or facilitate G-CSF-mediated HSC migration under stress conditions in a clinical setting.

Effects of ß-adrenergic receptor drugs on embryonic ventricular cell proliferation and differentiation and their impact on donor cell transplantation.

ß-Adrenergic receptors (ß-ARs) and catecholamines are present in rodents as early as embryonic day (E)10.5. However, it is not known whether ß-AR signaling plays any role in the proliferation and differentiation of ventricular cells in the embryonic heart. Here, we characterized expression profiles of ß-AR subtypes and established dose-response curves for the nonselective ß-AR agonist isoproterenol (ISO) in the developing mouse ventricular cells. Furthermore, we investigated the effects of ISO on cell cycle activity and differentiation of cultured E11.5 ventricular cells. ISO treatment significantly reduced tritiated thymidine incorporation and cell proliferation rates in both cardiac progenitor cell and cardiomyocyte populations. The ISO-mediated effects on DNA synthesis could be abolished by cotreatment of E11.5 cultures with either metoprolol (a ß1-AR antagonist) or ICI-118,551 (a ß2-AR antagonist). In contrast, ISO-mediated effects on cell proliferation could be abolished only by metoprolol. Furthermore, ISO treatment significantly increased the percentage of differentiated cardiomyocytes compared with that in control cultures. Additional experiments revealed that ß-AR stimulation leads to downregulation of Erk and Akt phosphorylation followed by significant decreases in cyclin D1 and cyclin-dependent kinase 4 levels in E11.5 ventricular cells. Consistent with in vitro results, we found that chronic stimulation of recipient mice with ISO after intracardiac cell transplantation significantly decreased graft size, whereas metoprolol protected grafts from the inhibitory effects of systemic catecholamines. Collectively, these results underscore the effects of ß-AR signaling in cardiac development as well as graft expansion after cell transplantation.NEW & NOTEWORTHY ß-Adrenergic receptor (ß-AR) stimulation can decrease the proliferation of embryonic ventricular cells in vitro and reduce the graft size after intracardiac cell transplantation. In contrast, ß1-AR antagonists can abrogate the antiproliferative effects mediated by ß-AR stimulation and increase graft size. These results highlight potential interactions between adrenergic drugs and cell transplantation.

Activation of α2A-adrenergic signal transduction in chondrocytes promotes degenerative remodelling of temporomandibular joint.

This study tested whether activation of adrenoreceptors in chondrocytes has roles in degenerative remodelling of temporomandibular joint (TMJ) and to determine associated mechanisms. Unilateral anterior crossbite (UAC) was established to induce TMJ degeneration in rats. Saline vehicle, α2- and ß-adrenoreceptor antagonists or agonists were injected locally into the TMJ area of UAC rats. Cartilage degeneration, subchondral bone microarchitecture and the expression of adrenoreceptors, aggrecans, matrix metalloproteinases (MMPs) and RANKL by chondrocytes were evaluated. Chondrocytes were stimulated by norepinephrine to investigate signal transduction of adrenoreceptors. Increased α2A-adrenoreceptor expression was observed in condylar cartilage of UAC rats, together with cartilage degeneration and subchondral bone loss. Norepinephrine depresses aggrecans expression but stimulates MMP-3, MMP-13 and RANKL production by chondrocytes through ERK1/2 and PKA pathway; these effects were abolished by an α2A-adrenoreceptor antagonist. Furthermore, inhibition of α2A-adrenoreceptor attenuated degenerative remodelling in the condylar cartilage and subchondral bone, as revealed by increased cartilage thickness, proteoglycans and aggrecan expression, and decreased MMP-3, MMP-13 and RANKL expressions in cartilage, increased BMD, BV/TV, and decreased Tb.Sp in subchondral bone. Conversely, activation of α2A-adrenoreceptor intensified aforementioned degenerative changes in UAC rats. It is concluded that activation of α2A-adrenergic signal in chondrocytes promotes TMJ degenerative remodelling by chondrocyte-mediated pro-catabolic activities.

Exacerbated cardiac fibrosis induced by ß-adrenergic activation in old mice due to decreased AMPK activity.

Senescent hearts exhibit defective responses to ß-adrenergic receptor (ß-AR) over-activation upon stress, leading to more severe pathological cardiac remodelling. However, the underlying mechanisms remain unclear. Here, we investigated the role of adenosine monophosphate-activated protein kinase (AMPK) in protecting against ageing-associated cardiac remodelling in mice upon ß-AR over-activation. 10-week-old (young) and 18-month-old (old) mice were subcutaneously injected with the ß-AR agonist isoproterenol (ISO; 5 mg/kg). More extensive cardiac fibrosis was found in old mice upon ISO exposure than in young mice. Meanwhile, ISO treatment decreased AMPK activity and increased ß-arrestin 1, but not ß-arrestin 2, expression, and the effects of ISO on AMPK and ß-arrestin 1 were greater in old mice than in young mice. Similarly, young AMPKα2-knockout (KO) mice showed more extensive cardiac fibrosis upon ISO exposure than that was observed in age-matched wild-type (WT) littermates. The extent of cardiac fibrosis in WT old mice was similar to that in young KO mice. Additionally, AMPK activities were decreased and ß-arrestin 1 expression increased in KO mice. In contrast, the AMPK activator metformin decreased ß-arrestin 1 expression and attenuated cardiac fibrosis in both young and old mice upon ISO exposure. In conclusion, more severe cardiac fibrosis is induced by ISO in old mice than in young mice. A decrease in AMPK activity, which further increases ß-arrestin 1 expression, is the central mechanism underlying the ageing-related cardiac fibrosis induced by ISO. The AMPK activator metformin is a promising therapeutic agent for treating ageing-related cardiac remodelling upon ß-AR over-activation.

Propranolol represses infantile hemangioma cell growth through the ß2-adrenergic receptor in a HIF-1α-dependent manner.

Propranolol, as a non-selective blocker of the ß-adrenergic receptor (AR), is utilised as the first-line treatment for infantile hemangiomas. However, the underlying mechanism remains poorly understood. The present study was designed to investigate the molecular basis of propranolol on the regression of infantile hemangiomas using a proliferating infantile hemangioma-derived endothelial cell line. In infantile hemangioma patients, we found that propranolol significantly decreased the expression levels of the hypoxia inducible factor (HIF)-1α in serum and urine, as well as in hemangioma tissues. In vitro analysis revealed that propranolol reduces the expression of HIF-1α in hemangioma cells in a dose- and time-dependent manner, mainly by acting on ß2-AR. Interestingly, it was observed that overexpression of HIF-1α apparently abrogated the inhibitory effects of propranolol on vascular endothelial growth factor (VEGF) expression and cell growth. Our data further demonstrated that propranolol inhibited the signal transducer and activator of transcription 3 (STAT3), a critical oncogenic signaling molecule, and the anti-apoptotic protein Bcl-2. Additionally, overexpression of HIF-1α significantly reversed the inhibitory effects of propranolol on STAT3 signaling. In a mouse xenograft hemangioma model, overexpression of HIF-1α significantly attenuated the therapeutic effects of propranolol and inhibited propranolol-induced hemangioma cell apoptosis. Moreover, the protein levels of VEGF, phosphorylated STAT3, total STAT3 and Bcl-2 were significantly upregulated by HIF-1α overexpression in propranolol-treated nude mice bearing hemangiomas. Collectively, our data provide evidence that propranolol may regress infantile hemangiomas by suppressing VEGF and STAT3 signaling pathways in an HIF-1α-dependent manner.

The B2 - adrenoreceptor gene promoter polymorphisms may modulate B2 - agonist and glucocorticoid-induced IgE synthesis

BACKGROUND: β2-adrenoreceptor (β2-AR) agonists and glucocorticoids (GCS) were shown to induce IgE synthesis in human PBMCs. Serum total IgE levels are associated with single nucleotide polymorphisms (SNPs) of the β2-AR gene. We aimed to assess the association of the effect of fenoterol (β2-AR agonist) on IL-4-driven and budesonide-induced IgE synthesis with genetic variants of β2-AR. METHODS: The study included 25 individuals: 13 with allergic asthma and/or allergic rhinitis and 12 healthy volunteers. PBMCs were cultured with IL-4, fenoterol and/or budesonide, and IgE concentrations in supernatants were assessed. Five SNPs in positions: −47, −20, 46, 79 and 252 of β2-AR were determined by direct DNA sequencing. RESULTS: In −47 T/T and −20 T/T patients, incubation with fenoterol resulted in decreased IgE production, whereas in −47 C/T and −47 C/C as well as in −20 C/T and −20 C/C individuals, it was enhanced. In contrast to fenoterol, budesonide-induced IgE synthesis was significantly increased in −47 T/T and −20 T/T patients as compared to −47 C/T, −47 C/C, −20 C/T and −47 C/C individuals. Polymorphisms in positions 46, 79 and 252 were not associated with fenoterol- or budesonide-modulated IgE synthesis. No differences in the distribution of IgE synthesis was seen between atopic and non-atopic individuals carrying the same alleles. CONCLUSIONS: The differential effect of β2-agonists and GCS on IgE synthesis may be associated with genetic variants of promoter region of the β2-AR gene

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Propranolol enhanced adipogenesis instead of induction of apoptosis of hemangiomas stem cells.

Propranolol has been widely used in treating infantile hemangiomas (IHs). But recurrence of IHs was found in some cases on cessation of propranolol treatment. The other is that Chinese individuals reacted to propranolol differently from American Whites. Whether the difference of sensitivity is due to the ß adrenoceptor (ß-AR) expression pattern of hemangioma initiating cells remains unclear. In the present study, we isolated hemangioma-derived stem cells (hemSCs) from proliferative IHs and analyzed the biological characteristics and ß-AR expression pattern of hemSCs by immunostaining, Western blotting and multilineage differentiation assay as well. We also tested the effects of propranolol on hemSCs by evaluating VEGF expression, proliferation and apoptosis related parameters. Our results indicated that CD133(+) hemSCs located pre-vascular in proiferative IH tissues. Both ß1 and ß2-AR were expressed, while ß2-AR was dominant on hemSCs. Propranolol at 100-150 µM inhibited proliferation of hemSCs, not did 50 µM. Propranolol down-regulated VEGF expression of hemSCs, instead of inducing apoptosis. The adipogenic potential was enhanced by propranolol. Therefore, our current results suggested propranolol could not induce apoptosis of hemSCs, but played a curative role though suppressing VEGF synthesis and enhancement of adipogenesis of hemSCs. Our results might partially provide the insight of mechanism of relapse in some cases on cessation of propranolol treatment.

GRK2 in the heart: a GPCR kinase and beyond.

SIGNIFICANCE: Heart failure (HF) is a common end point for many underlying cardiovascular diseases. Down-regulation and desensitization of ß-adrenergic receptors (ß-AR) caused by G-protein-coupled receptor (GPCR) kinase 2 (GRK2) are prominent features of HF. Recent Advances and Critical Issues: Significant progress has been made to understand the pathological role of GRK2 in the heart both as a GPCR kinase and as a molecule that can exert GPCR-independent effects. Inhibition of cardiac GRK2 has proved to be therapeutic in the failing heart and may offer synergistic and additional benefits to ß-blocker therapy. However, the mechanisms of how GRK2 directly contributes to the pathogenesis of HF need further investigation, and additional verification of the mechanistic details are needed before GRK2 inhibition can be used for the treatment of HF. FUTURE DIRECTIONS: The newly identified characteristics of GRK2, including the S-nitrosylation of GRK2 and the localization of GRK2 on mitochondria, merit further investigation. They may contribute to it being a pro-death kinase and result in HF under stressed conditions through regulation of intracellular signaling, including cardiac reduction-oxidation (redox) balance. A thorough understanding of the functions of GRK2 in the heart is necessary in order to finalize it as a candidate for drug development.

Parallel increase in secretory activity between N-glycosylated and nonglycosylated α-amylase without protein synthesis after short-term ß-adrenergic receptor activation in isolated rat parotid acinar cells.

OBJECTIVE: To investigate comparisons of the secretory activity between N-glycosylated and nonglycosylated α-amylase, and α-amylase synthetic activity, after ß-adrenergic receptor activation in rat parotid acinar cells in vitro. STUDY DESIGN: Rat parotid acinar cells were incubated in the presence or absence of (-)-isoproterenol. For ß-adrenergic blocking experiments, acinar cells were pretreated with (±)-propranolol prior to adding agonist. After the time indicated, the "released amylase" and "total amylase" were obtained. Western blotting was applied to identify and quantify the N-glycosylated and nonglycosylated α-amylase. Amylase activity was also measured. RESULTS: The potent ß-adrenergic agonist (-)-isoproterenol induced a dramatic increase (2-3-fold) of α-amylase secretion for 30 minutes (p < 0.05 vs. control), while the effect was completely abolished when cells were pretreated with (±)-propranolol for 15 minutes. Moreover, the N-glycosylated level of released and total amylase among groups was measured accordingly. Our data showed the N-glycosylated level ratios (released amylase/total amylase) did not differ among groups, which indicated that the N-glycosylated form of α-amylase was not secreted more easily than the nonglycosylated one after stimulation. Interestingly, the total amylase concentration remained unchanged after stimulation within 30 minutes, which might indicate no α-amylase synthesized within the time indicated. CONCLUSION: Our findings suggest a parallel increase in secretory activity between N-glycosylated and nonglycosylated α-amylase after ß-adrenergic receptor activation. There seems to be a dissociation of α-amylase synthesis from secretion within 30 minutes.

Influence of glucocorticoids, neuregulin-1ß, and sex on surfactant phospholipid secretion from type II cells.

Glucocorticoids induce lung fibroblasts to produce fibroblast-pneumocyte factor, a peptide that stimulates type II cells to synthesize pulmonary surfactant. This effect is known to be more apparent in cells derived from female fetuses, a characteristic that has been attributed to sex-linked differences in the fibroblasts. In the current study, it has been shown that dexamethasone enhances both ß-adrenergic receptor (ß-AR) activity (1.3- to 1.6-fold increase) and (-)-isoproterenol-induced secretion of surfactant (1.8- to 1.9-fold increase) in type II cells. However, fibroblast-conditioned media (FCM), prepared in the presence of dexamethasone, generates a much greater response to (-)-isoproterenol (3.1- to 3.8-fold increase). Furthermore, each of these effects is more pronounced if both cell types are female-derived. It is hypothesized that the enhanced response to glucocorticoids is the result of a synergistic effect between the steroid and a component of FCM. Neuregulin-1ß (NRG1ß), which is elevated in FCM generated in the presence of dexamethasone, influences not only the rate of surfactant secretion and the ß-AR activity in type II cells, but also enhances in both sexes the cellular response to (-)-isoproterenol. These results suggest that NRG1ß might be more effective than glucocorticoids in treating prematurely born male infants, which are known to respond poorly to glucocorticoids. Given that glucocorticoids are known to induce higher levels of ß-AR mRNA, the effect of NRG1ß, alone and in combination with dexamethasone, on ß-AR gene expression was measured using qRT-PCR. Whereas NRG1ß had no effect alone, in combination with dexamethasone it produced up to a 4.2-fold elevation in the level of ß-AR mRNA.

Influence of regular physical activity and caloric restriction on ß-adrenergic and natriuretic peptide receptor expression in retroperitoneal adipose tissue of OLETF rats.

The mechanisms underlying exercise-induced increases in adipose tissue blood flow and lipolysis involve both ß-adrenergic receptor (ßAR)- and natriuretic peptide receptor (NPR)-dependent processes. We hypothesized that daily wheel running (RUN) would increase the expression of NPR1, NPR2, ßAR2 and ßAR3 in retroperitoneal (RP) and epididymal (EPI) adipose tissues of obese Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Four-week-old OLETF rats were assigned to sedentary (SED, n = 6), calorie-restricted (CR, n = 8; fed 70% of SED) or RUN groups (n = 8). Rats were killed at 40 weeks of age. By design, body weight and adiposity were similar between RUN and CR animals, but each was lower than SED (P < 0.01). Compared with SED, RP depots of RUN rats exhibited 1.7- to 3.2-fold greater NPR1, NPR2, ßAR2 and ßAR3 mRNA levels (all P < 0.05). There were no differences between CR and SED in the expression of these genes in RP adipose tissues, and there were no differences in gene expression among groups in EPI adipose tissues. At the protein level, ßAR2 and ßAR3 were elevated in RUN and CR groups relative to the SED group in RP adipose tissues. In order to gain insight into the mechanisms underlying the activity-induced increases in NPR and ßAR mRNAs, RP adipose tissue explants from Wistar rats were treated with atrial natriuretic peptide (ANP), adrenaline and/or S-nitroso-N-acetyl-dl-penicillamine (SNAP; a nitric oxide donor) in organ culture experiments. SNAP synergistically enhanced adrenaline- and ANP-stimulated increases in NPR2 and ßAR2 mRNA levels. Our data suggest that physical activity-induced increases in nitric oxide interact with adrenaline and ANP to trigger the induction of NPR and ßAR mRNAs in the RP adipose tissue depot of the OLETF rat.

ß-adrenoceptor pathway enhances mitochondrial function in human neural stem cells via rotary cell culture system.

The structure and function of the human nervous system are altered in space when compared with their state on earth. To investigate directly the influence of simulated microgravity conditions which may be beneficial for cultivation and proliferation of human neural stem cells (hNSCs), the rotary cell culture system (RCCS) developed at the National Aeronautics and Space Administration (NASA) was used. RCCS allows the creation of a unique microgravity environment of low shear force, high-mass transfer and enables three-dimensional (3D) cell culture of dissimilar cell types. The results show that simulated microgravity using an RCCS would induce ß-adrenoceptor, upregulate cAMP formation and activate both PKA and CREB (cAMP response element binding protein) pathways. The expression of intracellular mitochondrial genes, including PGC1α (PPAR coactivator 1α), nuclear respiratory factors 1 and 2 (NRF1 and NRF2) and mitochondrial transcription factor A (Tfam), regulated by CREB, were all significantly increased at 72 h after the onset of microgravity. Accordingly and importantly, the ATP level and amount of mitochondrial mass were also increased. These results suggest that exposure to simulated microgravity using an RCCS would induce cellular proliferation in hNSCs via an increased mitochondrial function. In addition, the RCCS bioreactor would support hNSCs growth, which may have the potential for cell replacement therapy in neurological disorders.

Neonatal androgenization affects the efficiency of ß-adrenoceptor-mediated modulation of thymopoiesis.

We tested the hypothesis that neonatal androgenization affects the efficacy of ß-adrenoceptor (ß-AR)-mediated fine tuning of thymopoiesis in adult female rats by modulating the thymic noradrenaline (NA) level and/or ß-AR expression. In adult rats administered with 1000 µg testosterone enanthate at postnatal day 2 a higher density of catecholamine (CA)-synthesizing thymic cells, including thymocytes, and a rise in their CA content was found. In addition, in these animals increased thymic noradrenergic nerve fiber fluorescence intensity, reflecting their increased CA content, was detected. These changes were followed by an increase in thymic NA concentration. The rise in thymic NA content in thymic nerve fibers and cells was associated with changes in the expression of mRNA for enzymes controlling pivotal steps in NA biosynthesis (tyrosine hydroxylase, dopamine-ß-hydroxylase) and inactivation (monoamine oxidase). In contrast, the thymic level of ß(2)-AR mRNA on a per cell basis and the receptor surface density on thymocytes was reduced in testosterone-treated (TT) rats. As a consequence, 14-day-long treatment with propranolol, a ß-AR blocker, was ineffective in modulating T-cell differentiation/maturation in TT rats. In conclusion, the study indicates the importance of the neonatal sex steroid milieu for shaping the immunomodulatory capacity of the thymic NA/ß-AR signaling system in adult rats.

Caveolin-1 deficiency exacerbates cardiac dysfunction and reduces survival in mice with myocardial infarction.

Caveolin (Cav)-1 has been involved in the pathogenesis of ischemic injuries. For instance, modulations of Cav-1 expression have been reported in animal models of myocardial infarction and cerebral ischemia-reperfusion. Furthermore, ablation of the Cav-1 gene in mice has been shown to increase the extent of ischemic injury in models of cerebral and hindlimb ischemia. Cav-1 has also been suggested to play a role in myocardial ischemic preconditioning. However, the role of Cav-1 in myocardial ischemia (MI)-induced cardiac dysfunction still remains to be determined. We determined the outcome of a permanent left anterior descending coronary artery (LAD) ligation in Cav-1 knockout (KO) mice. Wild-type (WT) and Cav-1 KO mice were subjected to permanent LAD ligation for 24 h. The progression of ischemic injury was monitored by echocardiography, hemodynamic measurements, 2,3,5-triphenyltetrazolium chloride staining, ß-binding analysis, cAMP level measurements, and Western blot analyses. Cav-1 KO mice subjected to LAD ligation display reduced survival compared with WT mice. Despite similar infarct sizes, Cav-1 KO mice subjected to MI showed reduced left ventricular (LV) ejection fraction and fractional shortening as well as increased LV end-diastolic pressures compared with their WT counterparts. Mechanistically, Cav-1 KO mice subjected to MI exhibit reduced ß-adrenergic receptor density at the plasma membrane as well as decreased cAMP levels and PKA phosphorylation. In conclusion, ablation of the Cav-1 gene exacerbates cardiac dysfunction and reduces survival in mice subjected to MI. Mechanistically, Cav-1 KO mice subjected to LAD ligation display abnormalities in ß-adrenergic signaling.

Gene therapy for heart failure.

Despite the progress achieved in conventional treatment modalities, heart failure remains a major cause of mortality and morbidity. The identification of novel signaling pathways has provided a solid scientific rationale which has stimulated preclinical development of gene-based therapies for heart failure. Advances in somatic gene transfer technologies have been crucial to the advent of the first human clinical trials which are currently in progress. As these and other trials of gene transfer-based therapies are initiated, these approaches have generated excitement and hope for novel treatments for cardiovascular disease. In this review, we present a summary of advancements in construction of different vectors and methods of delivery that have been used for specific myocardial gene delivery. In addition, we will show results from studies focusing on the use of gene therapy to target heart failure mechanisms in animal models of cardiac dysfunction. Finally, we discuss the limited but highly promising results from clinical studies that have served as catalysts to translate preclinical achievements towards new treatment modalities for heart failure.

Expression and activation of ß-adrenoceptors in the colorectal mucosa of rat and human.

BACKGROUND: The functions of the distal colon are regulated by local and extrinsic neural pathways. In previous studies, we have found that dopamine (DA) and norepinephrine (NE) could evoke colonic ion transport by activating ß-adrenoceptors. The present study aims to investigate the segmental differences in expression and activation of ß-adrenoceptors in the distal colon in physiological and pathophysiological conditions. METHODS: Real-time PCR, immunofluorescence, and Western blotting were used to detect the expression of ß-adrenoceptors in the rat and human distal colon. Short-circuit current measurements (Isc) were used to assess the role of ß-adrenoceptors in ion transport. KEY RESULTS: DA and NE caused greater suppression of baseline Isc in distal colon adjacent to the rectum than in segments further away from the anus. These responses were inhibited by selective antagonists of ß1- and ß2-adrenoceptors, but not ß3-adrenoceptor. The expression levels of ß1- and ß2-adrenoceptors in colonic mucosa were higher in colorectum than the regions away from the anus of rats and humans. In wrap-restraint stress (2 h), DA-, NE-induced ΔIsc and the expression of ß-adrenoceptors in the colorectum were significantly reduced. However, when endogenous catecholamines were depleted by 6-hydroxydopamine (75 mg kg(-1), i.p., 3 days), DA-, NE-induced ΔIsc as well as the expression of ß-adrenoceptors were significantly enhanced in the rat colorectum but not in more proximal regions of the distal colon. CONCLUSIONS & INFERENCES: ß1- and ß2-adrenoceptors are predominantly expressed in the colorectal mucosa. Perturbation of endogenous catecholamine levels influences the expression and activation of ß-adrenoceptors in the colorectal region.

Pineal melatonin synthesis is altered in Period1 deficient mice.

Melatonin is an important endocrine signal for darkness in mammals. Transcriptional activation of the arylalkylamine-N-acetyltransferase gene encoding for the penultimate enzyme in melatonin synthesis drives the daily rhythm of the hormone in the pineal gland of rodents. Rhythmic arylalkylamine-N-acetyltransferase expression is controlled by the cAMP-signal transduction pathway and involves the activation of ß-adrenergic receptors and the inducible cAMP early repressor. In addition, the rat arylalkylamine-N-acetyltransferase promoter contains an E-box element which can interact with clock proteins. Moreover, the pineal gland of mice shows a circadian rhythm in clock proteins such as the transcriptional repressor Period1, which has been shown to control rhythmic gene expression in a variety of tissues. However, the role of Period1 in the regulation of pineal melatonin synthesis is still unknown. Therefore, circadian rhythms in arylalkylamine-N-acetyltransferase, ß-adrenergic receptor, and inducible cAMP early repressor mRNA levels (real time PCR), arylalkylamine-N-acetyltransferase enzyme activity (radiometric assay) and melatonin concentration radio immuno assay (RIA) were analyzed in the pineal gland of mice with a targeted deletion of the Period1 gene (Per1-/-) and the corresponding wildtype. In Per1-/- the amplitude in arylalkylamine-N-acetyltransferase expression was significantly elevated as compared to wildtype. In contrast, ß-adrenergic receptor and inducible cAMP early repressor mRNA levels were not affected by the Period1-deficiency. This indicates that the molecular clockwork alters the amplitude of arylalkylamine-N-acetyltransferase expression. In vitro, pineal glands of Per1-/- mice showed a day night difference in arylalkylamine-N-acetyltransferase expression with high levels at night. This suggests that a deficient in Period1 elicits similar effects as the activation of the cAMP-signal transduction pathway in wildtype mice.

Modulation of beta-adrenergic receptor signaling in heart failure and longevity: targeting adenylyl cyclase type 5.

Despite remarkable advances in therapy, heart failure remains a leading cause of morbidity and mortality. Although enhanced beta-adrenergic receptor stimulation is part of normal physiologic adaptation to either the increase in physiologic demand or decrease in cardiac function, chronic beta-adrenergic stimulation has been associated with increased mortality and morbidity in both animal models and humans. For example, overexpression of cardiac Gsalpha or beta-adrenergic receptors in transgenic mice results in enhanced cardiac function in young animals, but with prolonged overstimulation of this pathway, cardiomyopathy develops in these mice as they age. Similarly, chronic sympathomimetic amine therapy increases morbidity and mortality in patients with heart failure. Conversely, the use of beta-blockade has proven to be of benefit and is currently part of the standard of care for heart failure. It is conceivable that interrupting distal mechanisms in the beta-adrenergic receptor-G protein-adenylyl cyclase pathway may also provide targets for future therapeutic modalities for heart failure. Interestingly, there are two major isoforms of adenylyl cyclase (AC) in the heart (type 5 and type 6), which may exert opposite effects on the heart, i.e., cardiac overexpression of AC6 appears to be protective, whereas disruption of type 5 AC prolongs longevity and protects against cardiac stress. The goal of this review is to summarize the paradigm shift in the treatment of heart failure over the past 50 years from administering sympathomimetic amine agonists to administering beta-adrenergic receptor antagonists, and to explore the basis for a novel therapy of inhibiting type 5 AC.

Characterization of the expression pattern of adrenergic receptors in rat taste buds.

Taste buds signal the presence of chemical stimuli in the oral cavity to the central nervous system using both early transduction mechanisms, which allow single cells to be depolarized via receptor-mediated signaling pathways, and late transduction mechanisms, which involve extensive cell-to-cell communication among the cells in the bud. The latter mechanisms, which involve a large number of neurotransmitters and neuropeptides, are less well understood. Among neurotransmitters, multiple lines of evidence suggest that norepinephrine plays a yet unknown role in the taste bud. This study investigated the expression pattern of adrenergic receptors in the rat posterior taste bud. Expression of alpha1A, alpha1B, alpha1D, alpha2A, alpha2B, alpha2C, beta1, and the beta2 adrenoceptor subtypes was observed in taste buds using RT-PCR and immunocytochemical techniques. Taste buds also expressed the biosynthetic enzyme for norepinephrine, dopamine beta-hydroxylase (DbetaH), as well as the norepinephrine transporter. Further, expression of the epinephrine synthetic enzyme, phenylethanolamine N-methyltransferase (PNMT), was observed suggesting a possible role for this transmitter in the bud. Phenotyping adrenoceptor expression patterns with double labeling experiments to gustducin, synaptosomal-associated protein 25 (SNAP-25), and neural cell adhesion molecule (NCAM) suggests they are prominently expressed in subsets of cells known to express taste receptor molecules but segregated from cells known to have synapses with the afferent nerve fiber. Alpha and beta adrenoceptors co-express with one another in unique patterns as observed with immunocytochemistry and single cell reverse transcription polymerase chain reaction (RT-PCR). These data suggest that single cells express multiple adrenergic receptors and that adrenergic signaling may be particularly important in bitter, sweet, and umami taste qualities. In summary, adrenergic signaling in the taste bud occurs through complex pathways that include presynaptic and postsynaptic receptors and likely play modulatory roles in processing of gustatory information similar to other peripheral sensory systems such as the retina, cochlea, and olfactory bulb.