Circulatory Catecholamines Research Articles

Adrenal G protein-coupled Receptor Kinase (GRK)-2 Downregulation And Regulator of G protein Signaling (RGS)-4 Upregulation Mediate The Sympatholytic Effect Of Dapagliflozin

Dapagliflozin is a sodium/­glucose co-­transporter (SGLT)-­2 inhibitor with beneficial cardiovascular effects independent of its anti­diabetic actions. Among these is sympatholysis, i.e., norepinephrine (NE) lowering, beneficial in chronic human heart failure (HF). Adrenal G protein­-coupled receptor kinase (GRK)-­2 upregulation is a major driver of circulating catecholamine (CA) elevation and sympathetic nervous system (SNS) hyperactivity in HF due to dysregulation of adrenal sympatho-­inhibitory α2­-adrenergic receptors (ARs). On the other hand, we recently reported that Regulator of G protein Signaling (RGS)-­4 inhibits free fatty acid receptor (FFAR)-­3-stimulated NE release from sympathetic neurons, while it is also known to inhibit adrenal CA secretion induced by acetylcholine. Thus, we hypothesized herein that dapagliflozin may lower SNS hyperactivity by enhancing adrenal α2AR and RGS4 functions. We used the rat pheochromocytoma PC12 cell line expressing human α2A­AR, as well as freshly isolated adrenal glands from rats treated with dapagliflozin in vivo. Dapagliflozin treatment for 7 consecutive days (20 mg/kg/d in drinking water) led to a significant reduction in blood circulating NE levels (217±67 pg/ml, n=6), compared to vehicle-­treated rats (363±77 pg/ml, n=6, p<.05), suggesting reduced SNS activity. This was accompanied by reduced GRK2 and tyrosine hydroxylase (TH) mRNA and protein levels in dapagliflozin-­treated vs. vehicle­-treated adrenal glands. In contrast, RGS4 mRNA and protein levels were increased by dapagliflozin ­treatment. Adrenal α2AR density was higher in dapagliflozin- ­vs. vehicle-­treated rats (51.3±7.3 vs. 26.1±8.1 fmol/mg of protein, respectively; n=12 glands from 6 animals per group, p<.05). These results (i.e., GRK2 & TH downregulation with RGS4 upregulation) were completely recapitulated in PC12 cells in culture, treated with 5 μM dapagliflozin (or vehicle) for 24 hours. Importantly, α2AR signaling towards inhibition of CA secretion was markedly enhanced, while the cholinergic agonist carbachol-induced CA secretion was reduced post-­dapagliflozin treatment of PC12 cells. GRK2 overexpression and RGS4 siRNA-mediated knockdown partially reversed dapagliflozin‘s effect on CA secretion, confirming the involvement of GRK2 and RGS4 in dapagliflozin‘s sympatholytic effects in PC12 cells. In conclusion, dapagliflozin exerts adrenal sympatholysis via: a) downregulation of TH, which reduces CA biosynthesis, and GRK2, which reduces α2AR desensitization (i.e., enhances α2AR sympatho-inhibitory signaling); and b) upregulation of RGS4, which directly opposes cholinergic-stimulated CA secretion. 1) NIH/NHLBI #HL155718-01. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Ameliorative role of melatonin against adrenaline induced oxidative stress mediated cardiac and hepatic tissue injuries through preserving pyridoxine metabolism in male Wistar rats: A mechanistic insight

Adrenaline (AD) is a naturally occurring catecholamine, synthesised in the adrenal medulla to prepare the organisms for a "fight or flight" response. Under stressful circumstances, the circulatory catecholamine undergoes auto-oxidation, resulting in the formation of free radicals. Chronic stress results in the depletion of micronutrient stores in the body. Pyridoxine, often known as vitamin B6, is an essential water-soluble vitamin that acts as a coenzyme in many metabolic processes. Therefore, our current investigation has prioritized the regulation of pyridoxine metabolism during the period of chronic stress and the precise role of melatonin, as a natural antioxidant, in preventing the alterations generated by adrenaline in cardiac and hepatic tissues. Adrenaline augmented the oxidative stress indices, leading to an imbalance in the antioxidative state resulting in changes in the levels of certain organ-specific serum markers, modifications in the levels of PL (pyridoxal), PLP (pyridoxal-5-phosphate), and the enzymes responsible for their metabolism and breakdown. The foregoing results were corroborated by the histochemical and histological examinations. Melatonin efficiently counteracted all these harmful changes. Besides, the current study demonstrates that both PLP and melatonin show efficacy in scavenging free radicals, including superoxide anion free radicals and hydroxyl radicals, in the chemical system. However, the in vitro studies demonstrated that when administered together, melatonin and PLP more effectively mitigate free radical generation than the individual molecule. These findings were further confirmed by the ITC binding study. These results suggest that a combination of melatonin and PLP could be a better therapeutic approach for the amelioration of stress induced oxidative damages in cardiac and hepatic tissues with an improved pyridoxine metabolism.

Dapagliflozin downregulates G protein-coupled receptor Kinase-2 and upregulates regulator of g protein signaling-4 in adrenals to exert sympatholysis in heart failure

Abstract Dapagliflozin is a sodium-glucose co-transporter (SGLT)-2 inhibitor with beneficial cardiovascular effects independent of its anti-diabetic actions. Among these is sympatholysis, i.e., norepinephrine (NE) lowering which is beneficial in chronic human heart failure (HF). Adrenal G protein-coupled receptor kinase (GRK)-2 upregulation is a major driver of circulating catecholamine (CA) elevation and sympathetic nervous system (SNS) hyperactivity in HF due to dysregulation of adrenal sympatho-inhibitory α2-adrenergic receptors (ARs) [1]. On the other hand, we recently reported that signaling by neuronal free fatty acid receptor (FFAR)-3, which stimulates NE release from sympathetic neurons, is blocked by Regulator of G protein Signaling (RGS)-4 [2,3]. We hypothesized herein that dapagliflozin may lower SNS hyperactivity in the adrenal gland by improving α2AR, and antagonizing FFAR3 signaling in adrenal chromaffin cells. We used the rat pheochromocytoma PC12 cell line expressing human α2A-AR, as well as freshly isolated adrenal glands from rats treated with dapagliflozin in vivo. Dapagliflozin treatment for 7 consecutive days (20 mg/kg/d in drinking water) led to a significant reduction in blood circulating NE levels (217+67 pg/ml, n=6), compared to control, vehicle-treated rats (363+77 pg/ml, n=6, p<.05), suggesting reduced SNS activity. This was accompanied by reduced GRK2 and tyrosine hydroxylase (TH) mRNA and protein levels in dapagliflozin-treated rat adrenals vs. vehicle-treated animal-derived glands. In contrast, RGS4 mRNA levels were increased in dapagliflozin-treated adrenals. Adrenal α2AR density was higher in dapagliflozin- vs. vehicle-treated rats (51.3+7.3 vs. 26.1+8.1 fmol/mg of protein, respectively; n=12 glands from 6 animals per group, p<.05). These results (i.e., GRK2 & TH downregulation with RGS4 upregulation) were completely recapitulated in PC12 cells in culture, treated with 5 microM dapagliflozin (or vehicle) for 24 hours. Importantly, α2AR-dependent G protein-mediated signaling towards inhibition of CA secretion was markedly enhanced, whereas FFAR3-dependent phospholipase C/calcium signaling was reduced, at 24 hrs post-dapagliflozin treatment of PC12 cells. GRK2 overexpression partially reversed dapagliflozin`s effect on cholinergic-induced CA secretion. In conclusion, dapagliflozin exerts a sympatholytic action in the adrenal medulla via a) downregulation of TH, which reduces CA biosynthesis, and GRK2, which reduces α2AR desensitization; and b) upregulation of RGS4 to block FFAR3-stimulated CA secretion (Figure).Figure

Abstract 11977: Dapagliflozin Downregulates Adrenal G Protein-Coupled Receptor-Kinase-2 to Exert Sympatholysis in Heart Failure

Introduction: Dapagliflozin is a sodium-glucose co-transporter (SGLT)-2 inhibitor exerting several cardiorenal protective effects independently of blood glucose regulation. Adrenal G protein-coupled receptor kinase (GRK)-2 upregulation is a major driver of circulating catecholamine (CA) elevation and sympathetic nervous system (SNS) hyperactivity, especially in heart failure (HF). GRK2 severely dysregulates adrenal sympatho-inhibitory α 2 -adrenergic receptors (ARs), leading to unchecked, chronically elevated CA secretion. Hypothesis: SGLT2 inhibition with dapagliflozin lowers SNS hyperactivity in part by opposing adrenal GRK2 actions. Methods: We used the rat pheochromocytoma PC12 cell line expressing human α 2A AR, as well as freshly isolated adrenal glands from rats treated with dapagliflozin in vivo. Results: Dapagliflozin treatment for 7 consecutive days (20 mg/kg/d in drinking water) led to a significant reduction in blood circulating NE levels (217+67 pg/ml, n=6), compared to control, vehicle-treated rats (363+77 pg/ml, n=6, p<.05), suggesting reduced SNS activity. This was accompanied by reduced GRK2 and tyrosine hydroxylase (TH) mRNA and protein levels in dapagliflozin-treated rat adrenals vs. vehicle-treated animal-derived glands, indicating reduced adrenal CA synthesis and secretion. Finally, adrenal α 2 AR density was higher in dapagliflozin- vs. vehicle-treated rats(51.3+7.3 vs. 26.1+8.1 fmol/mg of protein, respectively; n=12 glands from 6 animals per group, p<.05). GRK2 and TH downregulation were also observed in PC12 cells treated with 5 μM dapagliflozin for 24 hours. Importantly, α 2 AR-dependent inhibition of CA secretion was markedly enhanced 24 hrs post-dapagliflozin application. This was due to reduced GRK2-dependent receptor desensitization induced by dapagliflozin, since this effect was abolished in PC12 cells overexpressing GRK2. Conclusions: Dapagliflozin exerts a sympatholytic action in the adrenal medulla via downregulation of both TH, thus reducing CA biosynthesis, and GRK2, thus reducing α 2 AR desensitization to lower CA secretion. This adrenal-mediated sympatholysis may be part of the increasingly apparent cardiovascular benefits of SGLT2 inhibitors in human HF irrespective of diabetic status.

739 Atypical presentation of Takotsubo syndrome early after pacemaker implantation

Abstract Aims Takotsubo syndrome (TTS) is an acute stress-induced cardiomyopathy showing left ventricular (LV) dysfunction without obstructive coronary arteries disease. A sudden massive surge of circulatory catecholamines from an intense physical or emotional stress may play a central role in the pathogenesis of TTS. We report the case of an 87 years-old woman who developed TTS with uncommon presentation after permanent pacemaker (PM) implantation. Methods and results The patient was referred to our hospital for PM implantation because of advanced atrio-ventricular block (3:1). She suffered by rheumatoid arthritis (RA), arterial hypertension, and chronic kidney disease. Echocardiogram, performed before PM implantation, showed normal LV kinesis and normal ejection fraction (EF 60%). She was initially administered with infusion of Isoprenaline 2 mcg/min. The subsequent day, she underwent permanent dual-chamber pacemaker implantation without any complications. After 3 days, the patient complained severe asthenia and fever, together with increase of white blood cells and C reactive protein. Blood cultures were negative. We started antibiotic therapy and, suspecting a reactivation of RA, steroid therapy with infusion of methylprednisolone 40 mg/die. Electrocardiogram showed normal sinus rhythm and paced ventricular rhythm. PM interrogation showed normal function. Surprisingly, echocardiogram showed LV dysfunction with apical and medium segments akinesia, and severe EF reduction (35%). Coronary angiography documented absence of coronary obstructive lesions, assessing diagnosis of TTS. The patient was discharged 1 week after admission in good clinical condition. One week later, an echocardiogram showed apical akinesia, partial recovery of medium segments motility, and slight increase of EF (40%). The excess of catecholamines could lead to decreased cardiac muscular function and to spasm of coronary arteries: these events can lead to acute heart failure and decrease of LVEF. Furthermore, about 90% of patients with TTS are women, especially in postmenopausal period. Peculiarities of this case were the atypical symptoms of TTS and the combination of different predisposing stressors factors: female sex in postmenopausal period, anamnesis of chronic inflammatory disease, use of stress-inducing drugs (methylprednisolone and isoprenaline, the last associated with TTS after PM-implantation), atrio-ventricular block itself, and PM implantation procedure. Our findings remark that even a low-risk procedure could be a possible cause of TTS in patient with such risk factors. In our opinion, in this subset of patients, conscious sedation could be useful to reduce the stress load, together with an early procedure and consequently the minimal use of exogenous stress drugs like Isoprenaline, even if the patient is in a good clinical condition. Conclusions This case highlights TTS as a potential complication after PM implantation, especially in post-menopausal women with high pre-existing stress load.

Dapagliflozin Exerts Adrenal Sympatholysis via G protein-Coupled Receptor Kinase-2 & Tyrosine Hydroxylase Downregulation

Abstract The sodium-glucose co-transporter (SGLT)-2 inhibitor dapagliflozin was recently reported to reduce renal tyrosine hydroxylase (TH) and norepinephrine levels, lowering blood pressure and preventing endothelial dysfunction, in a murine model of neurogenic hypertension. This suggested that dapagliflozin may combat sympathetic nervous system (SNS) hyperactivity, which is known to accompany and aggravate chronic heart failure (CHF). Adrenal G protein-coupled receptor kinase (GRK)-2 upregulation is a major driver of circulating catecholamine (CA) elevation and SNS hyperactivity, especially in CHF. GRK2 severely dysregulates adrenal sympatho-inhibitory α 2-adrenergic receptors (ARs), leading to unchecked, chronically elevated CA secretion. Therefore, we hypothesized herein that SGLT2 inhibition with dapagliflozin may lower SNS hyperactivity in the adrenal gland by antagonizing GRK2 actions on α 2ARs in adrenal chromaffin cells. We used the rat pheochromocytoma PC12 cell line expressing human α 2AAR, as well as freshly isolated adrenal glands from adult male Sprague-Dawley rats treated with dapagliflozin in vivo. We measured circulating norepinephrine (NE) in vivo via RIA, GRK2 & TH expression levels via real-time PCR and immunoblotting, adrenal α 2AR density (Bmax) via saturation radioligand binding with [methyl-3H]-rauwolscine, and G protein activation via the GTPγS assay. Dapagliflozin treatment for 7 consecutive days (20 mg/kg/d in drinking water) led to a significant reduction in blood circulating NE levels (217+67 pg/ml, n=6), compared to control, vehicle-treated rats (363+77 pg/ml, n=6, p<.05), suggesting reduced SNS activity. This was accompanied by reduced GRK2 and TH mRNA and protein levels in dapagliflozin-treated rat adrenals vs. vehicle-treated animal-derived glands, indicating reduced adrenal CA synthesis and secretion. Finally, adrenal α 2AR density was higher in dapagliflozin- vs. vehicle-treated rats (51.3+7.3 vs. 26.1+8.1 fmol/mg of protein, respectively; n=12 glands from 6 animals per group, p<.05). These results (i.e. GRK2 and TH downregulation) were completely recapitulated in PC12 α 2AAR-expressing cells in culture, treated with 5 μM dapagliflozin (or vehicle) for 24 hours. Importantly, α 2AR-dependent G protein-mediated signaling towards inhibition of CA secretion was markedly enhanced at 24 hrs post-dapagliflozin application in PC12 cells. This was the result of reduced GRK2-dependent receptor desensitization, since dapagliflozin lacked this effect in cells co-transfected with a GRK2-encoding adenovirus to acutely overexpress GRK2. In conclusion, dapagliflozin exerts a sympatholytic action in the adrenal medulla via downregulation of both TH, which reduces CA biosynthesis, and GRK2, which reduces α 2AR desensitization in favor of enhanced inhibition of CA secretion.

Comparison of Gabapentin and Pregabalin Premedication for Attenuation of Hemodynamic Changes in Elective Laparoscopic Appendicectomy

Introduction: General anaesthesia is the gold standard anaesthetic technique for laproscopic appendicitis. However, this procedure is not risk free. Carbon dioxide is used to create pneumoperitoneum in laproscopic surgeries causes various hemodynamic changes such as abrupt elevation of arterial pressure, systemic vascular resistance and decreased cardiac output. These changes are well tolerated in healthy patients. There is also an increase in circulatory catecholamines during laryngoscopy and intubation. Many pharmacological techniques were evaluated either in the premedication or during the induction to attenuate the hemodynamic response to pneumoperitoneum such as – deepening the anaesthesia, pretreatment with vasodilators, adrenoceptor blockers, calcium channel blockers and opiods. This study is designed to evaluate the hemodynamic changes associated by laryngoscopy, tracheal intubation and pneumoperitoneum in laparoscopic appendicectomy by premedicating the patients with Gabapentin (900mg) and Pregabalin (150mg). Materials and Methods: The study was carried out as a hospital based double blinded randomized prospective comparative study after obtaining institutional ethics committee approval in the Department of Anaesthesiology, SMVMCH Puducherry. The sample size was calculated as 72 with 36 in each group. Patients scheduled for elective laparoscopic appendicectomy were selected for the study based on predetermined inclusion and exclusion criteria. The study drug, Gabapentin 900mg or Pregabalin 150mg, was sealed in a black covered envelope and was given to the patient with sips of water 1 hour before the induction of anaesthesia by an anaesthetist not involved in study. Anaesthetic and surgical techniques were standardized for all patients. HR, SBP, DBP, MAP, SpO2 were recorded at the following points of time:(i) Prior to induction, (ii) 2-3 minutes after intubation, (iii) Before creating pneumoperitoneum, (iv) After creating pneumoperitoneum, (v) 2 minutes after extubation, (vi) Intra op,every 5 mins till the end of surgery, (vii) Post op, every 10 mins for the 1st 30 minutes and then every 30 minutes till 3 hours, along with Ramsay sedation score.

P3124Predictive value of the residual SYNTAX score following primary PCI in multivessel patients with MI-related cardiogenic shock - a CULPRIT SHOCK sub-analysis

Abstract Background Complete revascularization (CR) – assessed by the residual SYNTAX score (rSS) – following PCI is associated with a better prognosis – in stable coronary disease, acute coronary syndrome and myocardial infarction (MI). Whether, the completeness of revascularization impacts the prognosis of patients in cardiogenic shock (CS) remains unclear. Aim Assess the prognosis value of rSS following primary PCI in multivessel patients undergoing MI-related CS. Methods The CULPRIT SHOCK trial – the largest randomized trial (n=706) to date in CS – compared an immediate multivessel PCI (MVPCI) strategy to a culprit lesion only PCI (with possible staged revascularization) strategy in multivessel patients with MI-related CS. The rSS were retrospectively assessed following last PCI (either index or staged) by a central core laboratory and patients were allocated in 4 different groups according to rSS: CR (rSS=0), 0< rSS ≤5, 5< rSS ≤14, rSS >14. The prognostic impact of rSS on the 30-day composite endpoint (mortality and/or severe renal failure) and 30-day and 1-year mortality were assessed using multivariate logistic regression. Results Among the 604 patients with last rSS available, aged 68.2±11.4, the median rSS was 9.0 [4.0–17.0]. CR was achieved in 75 (25%) patients in the MVPCI strategy and in 31 (10.2%) in the culprit lesion only PCI strategy. One hundred and six (17.5%), 102 (16.9%), 198 (32.8%) and 198 (32.8%) patients had a rSS=0, 0< rss ≤5, 5< rSS ≤14 and rSS >14, respectively. Patients with a higher rSS were older, less active smoker, had more triple vessel disease, chronic total occlusion, post-PCI culprit coronary TIMI flow <3 and require more mechanical circulatory support and catecholamine. Univariate analysis shows a stepwise increase in adverse events according to rSS: patients with 5< rss ≤14 and rSS >14 had higher rates of 30-day primary endpoint (OR [95% CI]: 2.02 [1.24; 329] and 2.75 [1.69; 4.49]), 30-day mortality (OR [95% CI]: 2.13 [1.29; 3.51] and 3.14 [1.90; 5.18]) and 1-year mortality (OR [95% CI]: 2.39 [1.46; 3.90] and 3.47 [2.11; 5.71]) compared to patients with CR. After multiple adjustment, rSS – tested as continuous variable – was independently associated with 30-day primary endpoint, 30-day and one-year mortality (Figure) Conclusion Among multivessel patients with MI-related cardiogenic shock, 1) complete revascularization is achieved only in one fourth of the patients using a MVPCI strategy and, 2) the residual SYNTAX score is independently associated with early and late mortality. Acknowledgement/Funding Funded by the European Union 7th Framework Program and others

Role of catecholamines in the pathogenesis of diabetic cardiomyopathy 1.

Although the sympathetic nervous system plays an important role in the regulation of cardiac function, the overactivation of the sympathetic nervous system under stressful conditions including diabetes has been shown to result in the excessive production of circulating catecholamines as well as an increase in the myocardial concentration of catecholamines. In this brief review, we provide some evidence to suggest that the oxidation products of catecholamines such as aminochrome and oxyradicals, lead to metabolic derangements, Ca2+-handling abnormalities, increase in the availability of intracellular free Ca2+, as well as activation of proteases and changes in myocardial gene expression. These alterations due to elevated levels of circulatory catecholamines are associated with oxidative stress, subcellular remodeling, and the development of cardiac dysfunction in chronic diabetes.

Roles of Na+, Ca2+, and K+ channels in the generation of repetitive firing and rhythmic bursting in adrenal chromaffin cells.

Adrenal chromaffin cells (CCs) are the main source of circulating catecholamines (CAs) that regulate the body response to stress. Release of CAs is controlled neurogenically by the activity of preganglionic sympathetic neurons through trains of action potentials (APs). APs in CCs are generated by robust depolarization following the activation of nicotinic and muscarinic receptors that are highly expressed in CCs. Bovine, rat, mouse, and human CCs also express a composite array of Na+, K+, and Ca2+ channels that regulate the resting potential, shape the APs, and set the frequency of AP trains. AP trains of increasing frequency induce enhanced release of CAs. If the primary role of CCs is simply to relay preganglionic nerve commands to CA secretion, why should they express such a diverse set of ion channels? An answer to this comes from recent observations that, like in neurons, CCs undergo complex firing patterns of APs suggesting the existence of an intrinsic CC excitability (non-neurogenically controlled). Recent work has shown that CCs undergo occasional or persistent burst firing elicited by altered physiological conditions or deletion of pore-regulating auxiliary subunits. In this review, we aim to give a rationale to the role of the many ion channel types regulating CC excitability. We will first describe their functional properties and then analyze how they contribute to pacemaking, AP shape, and burst waveforms. We will also furnish clear indications on missing ion conductances that may be involved in pacemaking and highlight the contribution of the crucial channels involved in burst firing.

Abstract 124: Chronic Beta-Adrenergic Stimulation Prevents Protective Insulin-Induced Increases in Cardiomyocyte Respiratory Capacity

Cardiac injury or stress increases circulating catecholamines (CA), stimulating cardiac β adrenergic receptors (βAR) and adaptive positive inotropy. Under such conditions of increased energy demand, cardiomyocyte metabolism and bioenergetics are altered to maintain cellular function and survival. During chronic CA stimulation, however, receptor desensitization occurs, contributing to loss of contractile reserve and heart failure (HF) development. This progression to HF is again marked by metabolic and bioenergetic changes that remain incompletely understood. Notably, chronic CA exposure induces insulin resistance (IR) in the heart, which decreases substrate uptake and prevents protective signaling. Our laboratory has shown that G protein-coupled receptor kinase 2 induces IR in the heart following myocardial infarction. However, neither the molecular mechanisms of CA-induced IR, nor its effects on cardiomyocyte bioenergetics are well characterized. We hypothesize that βAR stimulation induces IR by decreasing insulin-stimulated mitochondrial respiratory capacity, thus, compromising cardiomyocyte survival during stress. Bioenergetics were evaluated by measuring cellular respiration under coupled (basal) and uncoupled (maximal) conditions. Our results show that acute insulin treatment selectively increases (25%) maximal and reserve respiratory capacity in the presence of glucose, which positively correlates with cellular survival. This effect is completely inhibited with chronic βAR stimulation using isoproterenol or clenbuterol, as is glucose transporter (GLUT4) translocation to the membrane. A similar effect is reproduced by high glucose and/or palmitate-induced IR. Notably, chronic βAR stimulation does not affect basal or maximal respiration, while acute stimulation increases basal respiration (25%). In short, the data demonstrate a unique insulin-induced increase in mitochondrial respiratory capacity in cardiomyocytes that is antagonized by chronic CA stimulation. We propose that this effect contributes to the detrimental effects of βAR stimulation during HF. Thus, understanding this phenomenon and its mechanisms is critical for inhibiting IR and improving cardiac metabolism and function during HF.

GRK2 Up-Regulation Creates a Positive Feedback Loop for Catecholamine Production in Chromaffin Cells.

Elevated sympathetic nervous system (SNS) activity aggravates several diseases, including heart failure. The molecular cause(s) underlying this SNS hyperactivity are not known. We have previously uncovered a neurohormonal mechanism, operating in adrenomedullary chromaffin cells, by which circulating catecholamine (CA) levels increase in heart failure: severe dysfunction of the adrenal α2-adrenergic receptors (ARs) due to the up-regulation of G protein-coupled receptor-kinase (GRK)-2, the kinase that desensitizes them. Herein we looked at the potential signaling mechanisms that bring about this GRK2 elevation in chromaffin cells. We found that chronic CA treatment of either PC12 or rat primary chromaffin cells can in itself result in GRK2 transcriptional up-regulation through α2ARs-Gi/o proteins-Src-ERK1/2. The resultant GRK2 increase severely enhances the α2AR desensitization/down-regulation elevating not only CA release but also CA biosynthesis, as evidenced by tyrosine hydroxylase up-regulation. Finally, GRK2 knockdown leads to enhanced apoptosis of PC12 cells, indicating an essential role for GRK2 in chromaffin cell homeostasis/survival. In conclusion, chromaffin cell GRK2 mediates a positive feedback loop that feeds into CA secretion, thereby enabling the adrenomedullary component of the SNS to turn itself on.

GPCRs of adrenal chromaffin cells & catecholamines: The plot thickens

The circulating catecholamines (CAs) epinephrine (Epi) and norepinephrine (NE) derive from two major sources in the whole organism: the sympathetic nerve endings, which release NE on effector organs, and the chromaffin cells of the adrenal medulla, which are cells that synthesize, store and release Epi (mainly) and NE. All of the Epi in the body and a significant amount of circulating NE derive from the adrenal medulla. The secretion of CAs from adrenal chromaffin cells is regulated in a complex way by a variety of membrane receptors, the vast majority of which are G protein-coupled receptors (GPCRs), including adrenergic receptors (ARs), which act as “presynaptic autoreceptors” in this regard. There is a plethora of CA-secretagogue signals acting on these receptors but some of them, most notably the α2ARs, inhibit CA secretion. Over the past few years, however, a few new proteins present in chromaffin cells have been uncovered to participate in CA secretion regulation. Most prominent among these are GRK2 and β-arrestin1, which are known to interact with GPCRs regulating receptor signaling and function. The present review will discuss the molecular and signaling mechanisms by which adrenal chromaffin cell-residing GPCRs and their regulatory proteins modulate CA synthesis and secretion. Particular emphasis will be given to the newly discovered roles of GRK2 and β-arrestins in these processes and particular points of focus for future research will be highlighted, as well.

Serum renalase inversely related to heart rate variability in dialysis patients

Heart rate variability (HRV) is a noninvasive technique for autonomic nerve activity assessment and an indirect index of adrenergic cardiovascular drive. Renalase is the only known enzyme degrading circulatory catecholamines, participating in regulations of the cardiovascular and sympathetic nervous systems. This study aims at the relation between serum renalase and HRV indexes in dialysis patients. There were 14 hemodialysis (HD) cases, 16 continuous ambulatory peritoneal dialysis (PD) cases, and 16 healthy volunteers enrolled in this study. All the subjects underwent 24 h Holter testing by using a dynamic electocardiogram monitor system to determine HRV parameters, and serum renalase levels were measured. The standard deviation of normal to normal R-R intervals (SDNN, a measure of beat-to-beat variability), root mean square successive difference (RMSSD), and the proportion derived by dividing NN50 (the number of interval differences of successive normal to normal intervals greater than 50ms) by the total number of NN intervals (pNN50) were determined to assess HRV. Both HD and PD patients presented increased maximum heart rate (Max-HR), minimum heart rate (Min-HR) and average heart rate (Ave-HR), and presented decreased SDNN, RMSSD and pNN50 relative to healthy controls. Serum levels of renalase were highly negatively correlated with Ave-HR (correlation coefficient r = −0.68, probability P < 0.01), while positively correlated with SDNN (r = 0.64, P < 0.01) in both the HD and PD groups. Serum level of renalase was positively correlated with 24 h urine volume (r = 0.73, P < 0.01) in PD patients. Renalase might be reciprocally related to HRV and inversely related to Ave-HR in dialysis patients, which will help to probe into renalase’s physiology and may pave the way to explore renalase replacement therapy in patients with end stage renal disease (ESRD).

Adrenal adrenoceptors in heart failure.

Heart failure (HF) is a chronic clinical syndrome characterized by the reduction in left ventricular (LV) function and it represents one of the most important causes of morbidity and mortality worldwide. Despite considerable advances in pharmacological treatment, HF represents a severe clinical and social burden. Sympathetic outflow, characterized by increased circulating catecholamines (CA) biosynthesis and secretion, is peculiar in HF and sympatholytic treatments (as β-blockers) are presently being used for the treatment of this disease. Adrenal gland secretes Epinephrine (80%) and Norepinephrine (20%) in response to acetylcholine stimulation of nicotinic cholinergic receptors on the chromaffin cell membranes. This process is regulated by adrenergic receptors (ARs): α2ARs inhibit CA release through coupling to inhibitory Gi-proteins, and β ARs (mainly β2ARs) stimulate CA release through coupling to stimulatory Gs-proteins. All ARs are G-protein-coupled receptors (GPCRs) and GPCR kinases (GRKs) regulate their signaling and function. Adrenal GRK2-mediated α2AR desensitization and downregulation are increased in HF and seem to be a fundamental regulator of CA secretion from the adrenal gland. Consequently, restoration of adrenal α2AR signaling through the inhibition of GRK2 is a fascinating sympatholytic therapeutic strategy for chronic HF. This strategy could have several significant advantages over existing HF pharmacotherapies minimizing side-effects on extra-cardiac tissues and reducing the chronic activation of the renin–angiotensin–aldosterone and endothelin systems. The role of adrenal ARs in regulation of sympathetic hyperactivity opens interesting perspectives in understanding HF pathophysiology and in the identification of new therapeutic targets.

Acute Stress Differently Modulates Beta 1, Beta 2 and Beta 3 Adrenoceptors in T Cells, but Not in B Cells, from the Rat Spleen

Objectives: Stress-induced rise in circulating catecholamines (CAs), followed by modulation of β-adrenergic receptors (adrenoceptors, ARs), is one of the pathways involved in the stress-mediated effects of immune functions. The spleen is an organ with a high number of lymphocytes and provides a unique microenvironment in which they reside. Thus, lymphocytes may respond differently to CAs in the spleen than in the circulation. No reports exist concerning the involvement of β-ARs in stress-mediated effects on T and B cells isolated from the spleen. Therefore, our aim was to investigate the effect of single stress exposure on gene expression and cellular localization of β-adrenoceptor subtypes in splenic T and B cells. We tried to correlate changes in adrenoceptors with the expression of apoptotic proteins. Methods: Immobilization (IMMO) was used as a stress model. T and B cells were isolated from rat spleen using magnetically labeled antibodies. The gene expression of individual adrenoceptors and apoptotic proteins was evaluated by real-time PCR. Immunofluorescence was used to evaluate localization and adrenoceptor expression. Results: We have found T cells to be more vulnerable to stress compared to B cells, because of increased β₁-, β₂- and β₃-ARs after a single IMMO. Moreover, β₂-ARs translocated from the nucleus to the plasma membrane in T cells after IMMO. The rise in β-ARs most probably led to the rise of Bax mRNA and Bax to Bcl-2 mRNA ratio. This might suggest the induction of an apoptotic process in T cells. Conclusion: Higher susceptibility of T cells to stress via modulation of β-ARs and apoptotic proteins might shift the immune responsiveness in the spleen.

Deficiency of type 1 cannabinoid receptors worsens acute heart failure induced by pressure overload in mice

We investigated the influence of type one cannabinoid receptor (CB1) deficiency on acute heart failure (AHF) and the underlying mechanism. Acute heart failure syndrome is an important clinical problem because of its high morbidity and mortality rates. Activation of CB1 induces vascular dilation and reinforces the properties of morphine, long-standing therapies for AHF syndrome, but the effect of endogenous CB1 activation on AHF is largely unknown. Acute heart failure mouse model characterized by hypertension and pulmonary oedema was created by using transverse aortic constriction (TAC). Mortality, echocardiography, haemodynamic, morphology, and circulatory catecholamine levels in response to TAC were evaluated in CB1 knockout (KO) and wild-type mice. Type one cannabinoid receptor KO mice had a much higher mortality rate at 1 week after TAC attributable to AHF (65 vs. 11%, P< 0.001). One hour after TAC, CB1 KO mice had significant larger lung weight to body weight ratio (LW/BW, 14.53 + 1.09 mg/g in KO vs. 10.42 + 0.36 mg/g in WT, P < 0.01) and higher plasma epinephrine levels (9720 + 1226 pg/mL vs. 6378 + 832 pg/mL, P < 0.05). Pharmacological activation of CB1 reduced LW/BW in wild-type mice. Administration of epinephrine to wild-type TAC mice significantly increased left ventricular end-diastolic pressure and LW/BW, while CB1 agonists reduced the LW/BW and the plasma levels of catecholamine and increased myocardial activity of AMP-activated protein kinase. Endogenous activation of CB1 in mice has cardiac protection in AHF, which is attributable to the inhibition of excessive sympathetic activation.

Modulation of Hypoxia‐Induced Pulmonary Vascular Leakage in Rats by Seabuckthorn (Hippophae rhamnoides L.)

Cerebral and pulmonary syndromes may develop in unacclimatized individuals shortly after ascent to high altitude resulting in high altitude illness, which may occur due to extravasation of fluid from intra to extravascular space in the brain, lungs and peripheral tissues. The objective of the present study was to evaluate the potential of seabuckthorn (SBT) (Hippophae rhamnoides L.) leaf extract (LE) in curtailing hypoxia-induced transvascular permeability in the lungs by measuring lung water content, leakage of fluorescein dye into the lungs and further confirmation by quantitation of albumin and protein in the bronchoalveolar lavage fluid (BALF). Exposure of rats to hypoxia caused a significant increase in the transvascular leakage in the lungs. The SBT LE treated animals showed a significant decrease in hypoxia-induced vascular permeability evidenced by decreased water content and fluorescein leakage in the lungs and decreased albumin and protein content in the BALF. The SBT extract was also able to significantly attenuate hypoxia-induced increase in the levels of proinflammatory cytokines and decrease hypoxia-induced oxidative stress by stabilizing the levels of reduced glutathione and antioxidant enzymes. Pretreatment of the extract also resulted in a significant decrease in the circulatory catecholamines and significant increase in the vasorelaxation of the pulmonary arterial rings as compared with the controls. Further, the extract significantly attenuated hypoxia-induced increase in the VEGF levels in the plasma, BALF (ELISA) and lungs (immunohistochemistry). These observations suggest that SBT LE is able to provide significant protection against hypoxia-induced pulmonary vascular leakage.

Reduction of Sympathetic Activity via Adrenal-targeted GRK2 Gene Deletion Attenuates Heart Failure Progression and Improves Cardiac Function after Myocardial Infarction

Chronic heart failure (HF) is characterized by sympathetic overactivity and enhanced circulating catecholamines (CAs), which significantly increase HF morbidity and mortality. We recently reported that adrenal G protein-coupled receptor kinase 2 (GRK2) is up-regulated in chronic HF, leading to enhanced CA release via desensitization/down-regulation of the chromaffin cell alpha(2)-adrenergic receptors that normally inhibit CA secretion. We also showed that adrenal GRK2 inhibition decreases circulating CAs and improves cardiac inotropic reserve and function. Herein, we hypothesized that adrenal-targeted GRK2 gene deletion before the onset of HF might be beneficial by reducing sympathetic activation. To specifically delete GRK2 in the chromaffin cells of the adrenal gland, we crossed PNMTCre mice, expressing Cre recombinase under the chromaffin cell-specific phenylethanolamine N-methyltransferase (PNMT) gene promoter, with floxedGRK2 mice. After confirming a significant ( approximately 50%) reduction of adrenal GRK2 mRNA and protein levels, the PNMT-driven GRK2 knock-out (KO) offspring underwent myocardial infarction (MI) to induce HF. At 4 weeks post-MI, plasma levels of both norepinephrine and epinephrine were reduced in PNMT-driven GRK2 KO, compared with control mice, suggesting markedly reduced post-MI sympathetic activation. This translated in PNMT-driven GRK2 KO mice into improved cardiac function and dimensions as well as amelioration of abnormal cardiac beta-adrenergic receptor signaling at 4 weeks post-MI. Thus, adrenal-targeted GRK2 gene KO decreases circulating CAs, leading to improved cardiac function and beta-adrenergic reserve in post-MI HF. GRK2 inhibition in the adrenal gland might represent a novel sympatholytic strategy that can aid in blocking HF progression.

Adrenal GRK2 lowering is an underlying mechanism for the beneficial sympathetic effects of exercise training in heart failure

Exercise training has been reported to exert beneficial effects on cardiac function and to reduce morbidity and mortality of chronic heart failure (HF). Augmented sympathetic nervous system (SNS) activity, leading to elevated circulating catecholamine (CA) levels, is a hallmark of chronic HF that significantly aggravates this disease. Exercise training has been shown to also reduce SNS overactivity in HF, but the underlying molecular mechanism(s) remain unidentified. We recently reported that adrenal G protein-coupled receptor kinase-2 (GRK2), an enzyme that regulates the sympathoinhibitory alpha(2)-adrenoceptors (alpha(2)-ARs) present in the CA-producing adrenal medulla, is upregulated in HF, contributing to the chronically elevated CA levels and SNS activity of the disease. In the present study, we tested whether exercise training can affect the adrenal GRK2-alpha(2)-AR-CA production system in the context of HF. For this purpose, a 10-wk-long exercise training regimen of adult male Sprague-Dawley rats starting at 4 wk postmyocardial infarction (post-MI) was employed, and examination at the end of this treatment period revealed significant amelioration of beta-AR-stimulated contractility in response to exercise training, accompanied by cardiac GRK2 reduction and restoration of circulating plasma CA levels. Importantly, adrenal GRK2 expression (72 + or - 5% reduction vs. post-MI untrained) and alpha(2)-AR number were also restored after exercise training in post-MI animals. These results suggest that exercise training restores the adrenal GRK2-alpha(2)-AR-CA production axis, and this might be part of the mechanism whereby this therapeutic modality normalizes sympathetic overdrive and impedes worsening of the failing heart.