Small conductance calcium-activated potassium channel contributes to stress induced endothelial dysfunctions.

Patients with Takotsubo syndrome displayed endothelial dysfunction, but underlying mechanisms have not been fully clarified. This study aimed to explore molecular signalling responsible for catecholamine excess induced endothelial dysfunction. Human cardiac microvascular endothelial cells were challenged by epinephrine to mimic catecholamine excess. Patch clamp, FACS, ELISA, PCR, and immunostaining were employed for the study. Epinephrine (Epi) enhanced small conductance calcium-activated potassium channel current (ISK1-3) through activating α1 adrenoceptor. Phenylephrine enhanced edothelin-1 (ET-1) and reactive oxygen species (ROS) production, and the effects involved contribution of ISK1-3. H2O2 enhanced ISK1-3 and ET-1 production. Enhancing ISK1-3 caused a hyperpolarization, which increases ROS and ET-1 production. BAPTA partially reduced phenylephrine-induced enhancement of ET-1 and ROS, suggesting that α1 receptor activation can enhance ROS/ET-1 generation in both calcium-dependent and calcium-independent ways. The study demonstrates that high concentration catecholamine can activate SK1-3 channels through α1 receptor-ROS signalling and increase ET-1 production, facilitating vasoconstriction.

Effect of midodrine on HVPG in advanced chronic liver disease and acute-on-chronic liver failure-A pilot study.

BACKGROUND AND AIMS: Nonselective beta-blockers (NSBB) are the mainstay for treatment of portal hypertension (PH), but require caution in decompensated cirrhosis (DC) or acute-on-chronic liver failure (ACLF) with hypotension, hyponatremia, acute kidney injury (AKI) or type 2 hepatorenal syndrome (HRS). Midodrine is oral, rapidly acting, α1-adrenergic agonist. We evaluated acute effects of midodrine on hepatic venous pressure gradient (HVPG) in DC and ACLF with contraindications to NSBB. METHODS: Patients of DC (n = 30) with grade III ascites and serum sodium (Na) <130/systolic blood pressure (SBP) <90/type II HRS (group I) and ACLF patients (n = 30) with Na <130/SBP <90/AKI (group II) were included. HVPG was done at baseline and repeated 3 h after 10 mg midodrine. Primary outcome was HVPG response (reduction by >20% or to <12 mmHg). RESULTS: In group I, midodrine significantly reduced HVPG (19.2 ± 4.6 to 17.8 ± 4.2, p = .02) and heart rate (HR) (86.3 ± 11.6 to 77.9 ± 13.1, p < .01) and increased mean arterial pressure (MAP) (74.1 ± 6.9 to 81.9 ± 6.6 mmHg, p < .01). In group II also, midodrine reduced HVPG (19.1 ± 4.1 to 17.0 ± 4.2) and HR (92.4 ± 13.7 to 84.6 ± 14.1) and increased MAP (85.4 ± 7.3 to 91.2 ± 7.6 mmHg), p < .01 for all. HVPG response was achieved in 3/30 (10%) in group I and 8/30 (26.7%) in group II. On logistic regression analysis, prerenal AKI (OR 11.04, 95% CI 1.83-66.18, p < .01) and increase in MAP (OR 1.22, 95% CI 1.03-1.43, p = .02) were independent predictors of response. Increase in MAP by 8.5 mmHg with midodrine had best cut-off with AUROC of .76 for response. CONCLUSION: In decompensated cirrhosis and ACLF patients with contraindications to NSBB, midodrine is useful in decreasing HVPG. Dose of midodrine should be titrated to increase MAP atleast by 8.5 mmHg.

Phenylephrine Enhances the Mitogenic Effect of S-Allyl-L-cysteine on Primary Cultured Hepatocytes through Protein Kinase C-Induced B-Raf Phosphorylation.

The co-mitogenic effects of the α1-adrenoceptor agonist phenylephrine on S-allyl-L-cysteine (SAC)-induced hepatocyte proliferation were examined in primary cultures of adult rat hepatocytes. The combination of phenylephrine (10-10-10-6 M) and SAC (10-6 M) exhibited a significant dose-dependent increase in the number of hepatocyte nuclei and viable cells compared to SAC alone. This combination also increased the progression of hepatocyte nuclei into the S-phase. The potentiating effect of phenylephrine on SAC-induced cell proliferation was counteracted by prazosin (an α1-adrenergic receptor antagonist) and GF109203X (selective protein kinase C (PKC) inhibitor). In addition, PMA (direct PKC activator) potentiated the proliferative effects of SAC similarly to phenylephrine. In essence, these findings suggest that PKC activity plays a crucial role in enhancing SAC-induced cell proliferation. Moreover, the effects of phenylephrine on SAC-induced Ras activity, Raf phosphorylation, and extracellular signal-regulated kinase 2 (ERK2) phosphorylation were investigated. Phenylephrine (or PMA) in combination with SAC did not augment Ras activity, but further increased ERK2 phosphorylation and its upstream B-Raf phosphorylation. These results indicate that PKC activation, triggered by stimulating adrenergic α1 receptors, further amplifies SAC-induced cell proliferation through enhanced ERK2 phosphorylation via increased B-Raf-specific phosphorylation in primary cultured hepatocytes.

Development of vascular regulation in the zebrafish embryo.

The thin endothelial wall of a newly formed vessel is under enormous stress at the onset of blood flow, rapidly acquiring support from mural cells (pericytes and vascular smooth muscle cells; vSMCs) during development. Mural cells then develop vasoactivity (contraction and relaxation) but we have little information as to when this first develops or the extent to which pericytes and vSMCs contribute. For the first time, we determine the dynamic developmental acquisition of vasoactivity in vivo in the cerebral vasculature of zebrafish. We show that pericyte-covered vessels constrict in response to α1-adrenergic receptor agonists and dilate in response to nitric oxide donors at 4 days postfertilization (dpf) but have heterogeneous responses later, at 6 dpf. In contrast, vSMC-covered vessels constrict at 6 dpf, and dilate at both stages. Using genetic ablation, we demonstrate that vascular constriction and dilation is an active response. Our data suggest that both pericyte- and vSMC-covered vessels regulate their diameter in early development, and that their relative contributions change over developmental time.

Combined inorganic nitrate/nitrite supplementation blunts α-mediated vasoconstriction during exercise in patients with type 2 diabetes.

AIMS: Patients with type 2 diabetes mellitus (T2DM) have reduced vasodilatory responses during exercise partially attributable to low nitric oxide (NO) levels. Low NO contributes to greater α-adrenergic mediated vasoconstriction in contracting skeletal muscle. We hypothesized boosting NO bioavailability via 8wks of active beetroot juice (BRA, 4.03 mmol nitrate, 0.29 mmol nitrite, n = 19) improves hyperemia, via reduced α-mediated vasoconstriction, during handgrip exercise relative to nitrate/nitrite-depleted beetroot juice (BRP, n = 18) in patients with T2DM. METHODS: Forearm blood flow (FBF) and vascular conductance (FVC) were calculated at rest and during handgrip exercise (20%max, 20contractions·min-1). Phenylephrine (α1-agonist) and dexmedetomidine (α2-agonist) were infused intra-arterially during independent trials to determine the influence of α-mediated vasoconstriction on exercise hyperemia. Vasoconstriction was quantified as the percent-reduction in FVC during α-agonist infusion, relative to pre-infusion, as well as the absolute change in %FVC during exercise relative to the respective rest trial (magnitude of sympatholysis). RESULTS: ΔFBF (156 ± 69 to 175 ± 73 ml min-1) and ΔFVC (130 ± 54 to 156 ± 63 ml min-1·100 mmHg-1, both P < 0.05) during exercise were augmented following BRA, but not BRP (P = 0.96 and 0.51). Phenylephrine-induced vasoconstriction during exercise was blunted following BRA (-17.1 ± 5.9 to -12.6 ± 3.1%, P < 0.01), but not BRP (P = 0.58) supplementation; the magnitude of sympatholysis was unchanged by either (beverage-by-time P = 0.15). BRA supplementation reduced dexmedetomidine-induced vasoconstriction during exercise (-23.3 ± 6.7 to -19.7 ± 5.2%) and improved the corresponding magnitude of sympatholysis (25.3 ± 11.4 to 34.4 ± 15.5%, both P < 0.05). CONCLUSIONS: BRA supplementation improves the hyperemic and vasodilatory responses to exercise in patients with T2DM which appears to be attributable to reduced α-adrenergic mediated vasoconstriction in contracting skeletal muscle.

Altered stress hormone levels affect in vivo vascular function in the hAPP23+/- overexpressing mouse model of Alzheimer's disease.

Alzheimer's disease (AD) has long been considered a brain-specific dementia syndrome. However, in recent decades, the occurrence of cardiovascular (CV) disease in the progression of AD has been confirmed by increasing epidemiological evidence. In this study, we conducted an in-depth cardiovascular characterization of a humanized amyloid precursor protein (APP) overexpressing mouse model (hAPP23+/-), which overexpresses the Swedish mutation (KM670/671NL). At the age of 6 mo, hAPP23+/- mice had a lower survival, lower body weight, and increased corticosterone and VMA levels compared with C57BL/6 littermates. Systolic blood pressure was increased in hAPP23+/- animals compared with C57BL/6 littermates, but diastolic blood pressure was not statistically different. Pulse pressure remained unchanged but abdominal and carotid pulse-wave velocity (aPWV and cPWV) were increased in hAPP23+/- compared with C57BL/6 mice. Echocardiography showed no differences in systolic or diastolic cardiac function. Ex vivo evaluation of vascular function showed decreased adreno receptor dependent vasoconstriction of hAPP23+/- aortic segments, although the isobaric biomechanics of the aortic wall were similar to C57BL/6 aortic segments. In conclusion, hAPP23+/- mice exhibited high serum corticosterone levels, elevated systolic blood pressure, and increased arterial stiffness in vivo. However, ex vivo aortic stiffness of hAPP23+/- aortic segments was not changed and vascular reactivity to α1-adrenoceptor stimulation was attenuated. These findings highlight the need for more frequent assessment of circulating stress hormone levels and PWV measurements in daily clinical practice for people at risk of AD.NEW & NOTEWORTHY We showed that male amyloid precursor protein (APP) transgenic mice have higher circulating stress hormone levels. As a result, higher systolic blood pressure and pulse-wave velocity were measured in vivo in addition to a smaller α-adrenergic receptor-dependent contraction upon ex vivo stimulation with phenylephrine. Our findings highlight the need for more frequent assessment of circulating stress hormone levels and PWV measurements in daily clinical practice for people at risk of Alzheimer's disease.

Long-term electronic cigarette exposure induces cardiovascular dysfunction similar to tobacco cigarettes: role of nicotine and exposure duration.

Electronic cigarette (e-cig) vaping (ECV) has been proposed as a safer alternative to tobacco cigarette smoking (TCS); however, this remains controversial due to a lack of long-term comparative studies. Therefore, we developed a chronic mouse exposure model that mimics human vaping and allows comparison with TCS. Longitudinal studies were performed to evaluate alterations in cardiovascular function with TCS and ECV exposure durations of up to 60 wk. For ECV, e-cig liquid with box-mod were used and for TCS, 3R4F-cigarettes. C57/BL6 male mice were exposed 2 h/day, 5 days/wk to TCS, ECV, or air control. The role of vape nicotine levels was evaluated using e-cig-liquids with 0, 6, or 24 mg/mL nicotine. Following 16-wk exposure, increased constriction to phenylephrine and impaired endothelium-dependent and endothelium-independent vasodilation were observed in aortic segents, paralleling the onset of systemic hypertension, with elevations in systemic vascular resistance. Following 32 wk, TCS and ECV induced cardiac hypertrophy. All of these abnormalities further increased out to 60 wk of exposure, with elevated heart weight and aortic thickness along with increased superoxide production in vessels and cardiac tissues of both ECV and TCS mice. While ECV-induced abnormalities were seen in the absence of nicotine, these occurred earlier and were more severe with higher nicotine exposure. Thus, long-term vaping of e-cig can induce cardiovascular disease similar to TCS, and the severity of this toxicity increases with exposure duration and vape nicotine content.NEW & NOTEWORTHY A chronic mouse exposure model that mimics human e-cigarette vaping and allows comparison with tobacco cigarette smoking was developed and utilized to perform longitudinal studies of alterations in cardiovascular function. E-cigarette exposure led to the onset of cardiovascular disease similar to that with tobacco cigarette smoking. Impaired endothelium-dependent and endothelium-independent vasodilation with increased adrenergic vasoconstriction were observed, paralleling the onset of systemic hypertension and subsequent cardiac hypertrophy. This cardiovascular toxicity was dependent on exposure duration and nicotine dose.

Phenylephrine increases tear cathepsin S secretion in healthy murine lacrimal gland acinar cells through an alternative secretory pathway.

Little is known about the relationship between stimulation of lacrimal gland (LG) tear protein secretion by parasympathetic versus sympathetic nerves, particularly whether the spectrum of tear proteins evoked through each innervation pathway varies. We have previously shown that activity and abundance of cathepsin S (CTSS), a cysteine protease, is greatly increased in tears of Sjögren's syndrome (SS) patients and in tears from the male NOD mouse of autoimmune dacryoadenitis that recapitulates SS-associated dry eye disease. Beyond the increased synthesis of CTSS detected in the diseased NOD mouse LG, increased tear CTSS secretion in NOD mouse tears was recently linked to increased exocytosis from a novel endolysosomal secretory pathway. Here, we have compared secretion and trafficking of CTSS in healthy mouse LG acinar cells stimulated with either the parasympathetic acetylcholine receptor agonist, carbachol (CCh), or the sympathetic α1-adrenergic agonist, phenylephrine (PE). In situ secretion studies show that PE significantly increases CTSS activity and protein in tears relative to CCh stimulation by 1.2-fold (***, p = 0.0009) and ∼5-fold (*, p-0.0319), respectively. A similar significant increase in CTSS activity with PE relative to CCh is observed when cultured LGAC are stimulated in vitro. CCh stimulation significantly elevates intracellular [Ca2+], an effect associated with increases in the size of Rab3D-enriched vesicles consistent with compound fusion, and subsequently decreases in their intensity of labeling consistent with their exocytosis. PE stimulation induces a lower [Ca2+] response and has minimal effects on Rab3D-enriched SV diameter or the intensity of Rab3D-enriched SV labeling. LG deficient in Rab3D exhibit a higher sensitivity to PE stimulation, and secrete more CTSS activity. Significant increases in the colocalization of endolysosomal vesicle markers (Lamp1, Lamp2, Rab7) with the subapical actin suggestive of fusion of endolysosomal vesicles at the apical membrane occur both with CCh and PE stimulation, but PE demonstrates increased colocalization. In conclusion, the α1-adrenergic agonist, PE, increases CTSS secretion into tears through a pathway independent of the exocytosis of Rab3D-enriched mature SV, possibly representing an alternative endolysosomal secretory pathway.

Coupling to Gq Signaling Is Required for Cardioprotection by an Alpha-1A-Adrenergic Receptor Agonist.

RATIONALE: Gq signaling in cardiac myocytes is classically considered toxic. Targeting Gq directly to test this is problematic, because cardiac myocytes have many Gq-coupled receptors. OBJECTIVE: Test whether Gq coupling is required for the cardioprotective effects of an alpha-1A-AR (adrenergic receptor) agonist. METHODS AND RESULTS: In recombinant cells, a mouse alpha-1A-AR with a 6-residue substitution in the third intracellular loop does not couple to Gq signaling. Here we studied a knockin mouse with this alpha-1A-AR mutation. Heart alpha-1A receptor levels and antagonist affinity in the knockin were identical to wild-type. In wild-type cardiac myocytes, the selective alpha-1A agonist A61603-stimulated phosphoinositide-phospholipase C and myocyte contraction. In myocytes with the alpha-1A knockin, both A61603 effects were absent, indicating that Gq coupling was absent. Surprisingly, A61603 activation of cardioprotective ERK (extracellular signal-regulated kinase) was markedly impaired in the KI mutant myocytes, and A61603 did not protect mutant myocytes from doxorubicin toxicity in vitro. Similarly, mice with the α1A KI mutation had increased mortality after transverse aortic constriction, and A61603 did not rescue cardiac function in mice with the Gq coupling-defective alpha-1A receptor. CONCLUSIONS: Gq coupling is required for cardioprotection by an alpha-1A-AR agonist. Gq signaling can be adaptive.

Phenylephrine impairs host defence mechanisms to infection: a combined laboratory study in mice and translational human study.

BACKGROUND: Immunosuppression after surgery is associated with postoperative complications, mediated in part by catecholamines that exert anti-inflammatory effects via the ß-adrenergic receptor. Phenylephrine, generally regarded as a selective α-adrenergic agonist, is frequently used to treat perioperative hypotension. However, phenylephrine may impair host defence through ß-adrenergic affinity. METHODS: Human leukocytes were stimulated with lipopolysaccharide (LPS) in the presence or absence of phenylephrine and α- and ß-adrenergic antagonists. C57BL/6J male mice received continuous infusion of phenylephrine (30-50 µg kg-1 min-1 i.v.) or saline via micro-osmotic pumps, before LPS administration (5 mg kg-1 i.v.) or caecal ligation and puncture (CLP). Twenty healthy males were randomised to a 5 h infusion of phenylephrine (0.5 µg kg-1 min-1) or saline before receiving LPS (2 ng kg-1 i.v.). RESULTS: In vitro, phenylephrine enhanced LPS-induced production of the anti-inflammatory cytokine interleukin (IL)-10 (maximum augmentation of 93%) while attenuating the release of pro-inflammatory mediators. These effects were reversed by pre-incubation with ß-antagonists, but not α-antagonists. Plasma IL-10 levels were higher in LPS-challenged mice infused with phenylephrine, whereas pro-inflammatory mediators were reduced. Phenylephrine infusion increased bacterial counts after CLP in peritoneal fluid (+42%, P=0.0069), spleen (+59%, P=0.04), and liver (+35%, P=0.09). In healthy volunteers, phenylephrine enhanced the LPS-induced IL-10 response (+76%, P=0.0008) while attenuating plasma concentrations of pro-inflammatory mediators including IL-8 (-15%, P=0.03). CONCLUSIONS: Phenylephrine exerts potent anti-inflammatory effects, possibly involving the ß-adrenoreceptor. Phenylephrine promotes bacterial outgrowth after surgical peritonitis. Phenylephrine may therefore compromise host defence in surgical patients and increase susceptibility towards infection. CLINICAL TRIAL REGISTRATION: NCT02675868 (Clinicaltrials.gov).

A positive feedback loop between alpha1-adrenoceptors and inflammatory cytokines in keratinocytes.

A positive feedback loop between inflammatory cytokines and alpha1-adrenoceptors (α1-AR) (a target of the sympathetic nervous system neurotransmitter norepinephrine) influences inflammatory responses in immune cells. This cross-talk between the sympathetic nervous system and immune system may play a role in promoting chronic inflammation. Emerging evidence shows that α1-AR interact with inflammatory cytokines in keratinocytes, and this epidermal adrenergic signalling may contribute to skin inflammatory responses following injury, disease or stress. In this study, utilizing an in vitro approach, we hypothesized that α1-AR interact in a positive feedback loop with inflammatory mediators in keratinocytes. The pro-inflammatory cytokine tumor necrosis factor α (TNFα) was used to induce an inflammatory state in cultured keratinocytes. TNFα increased interleukin (IL)-1ß, IL-6, IL-8 and nerve growth factor (NGF) production and gene expression levels of α1-AR subtype B (α1B-AR). Additional stimulation of α1-AR further increased IL-6 levels, while maintaining high levels of IL-8 and decreasing levels of IL-1ß and NGF. Our results suggest that reciprocal influences between α1-ARs and inflammatory cytokines may play a role in normal inflammatory responses. However, if unchecked, this cycle could contribute to pathology (e.g. chronic inflammatory diseases, chronic pain conditions, and stress-induced cancer progression).

Noradrenergic α1-Adrenoceptor Actions in the Primate Dorsolateral Prefrontal Cortex.

Noradrenergic (NE) α1-adrenoceptors (α1-ARs) contribute to arousal mechanisms and play an important role in therapeutic medications such as those for the treatment of posttraumatic stress disorder (PTSD). However, little is known about how α1-AR stimulation influences neuronal firing in the dorsolateral prefrontal cortex (dlPFC), a newly evolved region that is dysfunctional in PTSD and other mental illnesses. The current study examined the effects of α1-AR manipulation on neuronal firing in dlPFC of rhesus monkeys performing a visuospatial working memory task, focusing on the "delay cells" that maintain spatially tuned information across the delay period. Iontophoresis of the α1-AR antagonist HEAT (2-{[ß-(4-hydroxyphenyl)ethyl]aminomethyl}-1-tetralone) had mixed effects, reducing firing in a majority of neurons but having nonsignificant excitatory effects or no effect in remaining delay cells. These data suggest that endogenous NE has excitatory effects in some delay cells under basal conditions. In contrast, the α1-AR agonists phenylephrine and cirazoline suppressed delay cell firing and this was blocked by coadministration of HEAT. These results indicate an inverted-U dose response for α1-AR actions, with mixed excitatory actions under basal conditions and suppressed firing with high levels of α1-AR stimulation such as with stress exposure. Immunoelectron microscopy revealed α1-AR expression presynaptically in axons and axon terminals and postsynaptically in spines, dendrites, and astrocytes. It is possible that α1-AR excitatory effects arise from presynaptic excitation of glutamate release, whereas postsynaptic actions suppress firing through calcium-protein kinase C opening of potassium channels on spines. The latter may predominate under stressful conditions, leading to loss of dlPFC regulation during uncontrollable stress.SIGNIFICANCE STATEMENT Noradrenergic stimulation of α1-adrenoceptors (α1-ARs) is implicated in posttraumatic stress disorder (PTSD) and other mental disorders that involve dysfunction of the prefrontal cortex, a brain region that provides top-down control. However, the location and contribution of α1-ARs to prefrontal cortical physiology in primates has received little attention. This study found that α1-ARs are located near prefrontal synapses and that α1-AR stimulation has mixed effects under basal conditions. However, high levels of α1-AR stimulation, as occur with stress, suppress neuronal firing. These findings help to explain why we lose top-down control under conditions of uncontrollable stress when there are high levels of noradrenergic release in brain and why blocking α1-AR, such as with prazosin, may be helpful in the treatment of PTSD.

PKCßII specifically regulates KCNQ1/KCNE1 channel membrane localization.

The slow voltage-gated potassium channel (IKs) is composed of the KCNQ1 and KCNE1 subunits and is one of the major repolarizing currents in the heart. Activation of protein kinase C (PKC) has been linked to cardiac arrhythmias. Although PKC has been shown to be a regulator of a number of cardiac channels, including IKs, little is known about regulation of the channel by specific isoforms of PKC. Here we studied the role of different PKC isoforms on IKs channel membrane localization and function. Our studies focused on PKC isoforms that translocate to the plasma membrane in response to Gq-coupled receptor (GqPCR) stimulation: PKCα, PKCßI, PKCßII and PKCε. Prolonged stimulation of GqPCRs has been shown to decrease IKs membrane expression, but the specific role of each PKC isoform is unclear. Here we show that stimulation of calcium-dependent isoforms of PKC (cPKC) but not PKCε mimic receptor activation. In addition, we show that general PKCß (LY-333531) and PKCßII inhibitors but not PKCα or PKCßI inhibitors blocked the effect of cPKC on the KCNQ1/KCNE1 channel. PKCß inhibitors also blocked GqPCR-mediated decrease in channel membrane expression in cardiomyocytes. Direct activation of PKCßII using constitutively active PKCßII construct mimicked agonist-induced decrease in membrane expression and channel function, while dominant negative PKCßII showed no effect. This suggests that the KCNQ1/KCNE1 channel was not regulated by basal levels of PKCßII activity. Our results indicate that PKCßII is a specific regulator of IKs membrane localization. PKCßII expression and activation are strongly increased in many disease states, including heart disease and diabetes. Thus, our results suggest that PKCßII inhibition may protect against acquired QT prolongation associated with heart disease.

Bidirectional modulation of TNF-α transcription via α- and ß-adrenoceptors in cultured astrocytes from rat spinal cord.

Noradrenaline (NA) suppresses TNF-α production via ß-adrenoceptors (ARs) in brain astrocytes. However, the downstream pathways from ß-ARs, and the involvement of α-ARs, remains unknown. In this study, we investigated the AR-mediated regulation of TNF-α mRNA levels in cultured astrocytes from rat spinal cord. NA, the α1-agonist phenylephrine, and the ß-agonist isoproterenol decreased the TNF-α mRNA level, while the α2-agonist dexmedetomidine increased it. The isoproterenol-induced TNF-α mRNA decrease was accompanied by a decrease in ERK phosphorylation. An adenylyl cyclase activator and an ERK inhibitor mimicked these effects. These results indicate that the transcriptional regulation of TNF-α by ß-ARs is mediated via cAMP pathways followed by the ERK pathway inhibition. The dexmedetomidine-induced TNF-α mRNA increase was accompanied by phosphorylation of JNK and ERK, which was blocked by a JNK inhibitor. Furthermore, the LPS-induced increase in the TNF-α mRNA level was accompanied by NF-κB nuclear translocation, and both these effects were blocked by phenylephrine. An NF-κB inhibitor suppressed the LPS-induced increase in the TNF-α mRNA level. These results suggest that α1-ARs suppress the LPS-induced increase in the TNF-α mRNA level via inhibition of NF-κB nuclear translocation. Taken together, our study reveals that both α- and ß-ARs are involved in the transcriptional regulation of TNF-α in astrocytes.

The relative contribution of α- and ß-adrenergic sweating during heat exposure and the influence of sex and training status.

While human eccrine sweat glands respond to adrenergic agonists, there remains a paucity of information on the factors modulating this response. Thus, we assessed the relative contribution of α- and ß-adrenergic sweating during a heat exposure and as a function of individual factors of sex and training status. α- and ß-adrenergic sweating was assessed in forty-eight healthy young men (n = 35) and women (n = 13) including endurance-trained (n = 12) and untrained men (n = 12) under non-heat exposure (temperate, 25°C; n = 17) and heat exposure (hot, 35°C; n = 48) conditions using transdermal iontophoresis of phenylephrine (α-adrenergic agonist) and salbutamol (ß-adrenergic agonist) on the ventral forearm, respectively. Adrenergic sweating was also measured after iontophoretic administration of atropine (muscarinic receptor antagonist) or saline (control) to evaluate how changes in muscarinic receptor activity modulate the adrenergic response to a heat exposure (n = 12). α- and ß-adrenergic sweating was augmented in hot compared with temperate conditions (both P ≤ .014), albeit the relative increase was greater in ß (~5.4-fold)- as compared to α (~1.5-fold)-adrenergic-mediated sweating response. However, both α- and ß-adrenergic sweating was abolished by atropinization (P = .001). Endurance-trained men showed an augmentation in α- (P = .043) but not ß (P = .960)-adrenergic sweating as compared to untrained men. Finally, a greater α- and ß-adrenergic sweating response (both P ≤ .001) was measured in habitually active men than in women. We show that heat exposure augments α-and ß-adrenergic sweating differently via mechanisms associated with altered muscarinic receptor activity. Sex and training status modulate this response.

Dysfunctional bladder neurophysiology in urofacial syndrome Hpse2 mutant mice.

AIMS: Urofacial syndrome (UFS) is an autosomal recessive disease characterized by detrusor contraction against an incompletely dilated outflow tract. This dyssynergia causes dribbling incontinence and incomplete voiding. Around half of individuals with UFS have biallelic mutations of HPSE2 that encodes heparanase 2, a protein found in pelvic ganglia and bladder nerves. Homozygous Hpse2 mutant mice have abnormal patterns of nerves in the bladder body and outflow tract, and also have dysfunctional urinary voiding. We hypothesized that bladder neurophysiology is abnormal Hpse2 mutant mice. METHODS: Myography was used to study bladder bodies and outflow tracts isolated from juvenile mice. Myogenic function was analyzed after chemical stimulation or blockade of key receptors. Neurogenic function was assessed by electrical field stimulation (EFS). Muscarinic receptor expression was semi-quantified by Western blot analysis. RESULTS: Nitrergic nerve-mediated relaxation of precontracted mutant outflow tracts was significantly decreased vs littermate controls. The contractile ability of mutant outflow tracts was normal as assessed by KCl and the α1-adrenoceptor agonist phenylephrine. EFS of mutant bladder bodies induced significantly weaker contractions than controls. Conversely, the muscarinic agonist carbachol induced significantly stronger contractions of bladder body than controls. CONCLUSIONS: The Hpse2 model of UFS features aberrant bladder neuromuscular physiology. Further work is required to determine whether similar aberrations occur in patients with UFS.

α1-AR overactivation induces cardiac inflammation through NLRP3 inflammasome activation.

Acute sympathetic stress causes excessive secretion of catecholamines and induces cardiac injuries, which are mainly mediated by ß-adrenergic receptors (ß-ARs). However, α1-adrenergic receptors (α1-ARs) are also expressed in the heart and are activated upon acute sympathetic stress. In the present study, we investigated whether α1-AR activation induced cardiac inflammation and the underlying mechanisms. Male C57BL/6 mice were injected with a single dose of α1-AR agonist phenylephrine (PE, 5 or 10 mg/kg, s.c.) with or without pretreatment with α-AR antagonist prazosin (5 mg/kg, s.c.). PE injection caused cardiac dysfunction and cardiac inflammation, evidenced by the increased expression of inflammatory cytokine IL-6 and chemokines MCP-1 and MCP-5, as well as macrophage infiltration in myocardium. These effects were blocked by prazosin pretreatment. Furthermore, PE injection significantly increased the expression of NOD-like receptor protein 3 (NLRP3) and the cleavage of caspase-1 (p20) and interleukin-18 in the heart; similar results were observed in both Langendorff-perfused hearts and cultured cardiomyocytes following the treatment with PE (10 µM). Moreover, PE-induced NLRP3 inflammasome activation and cardiac inflammation was blocked in Nlrp3-/- mice compared with wild-type mice. In conclusion, α1-AR overactivation induces cardiac inflammation by activating NLRP3 inflammasomes.

Effects of anaesthesia-induced hypotension and phenylephrine on plasma volume expansion by hydroxyethyl starch: A randomised controlled study.

BACKGROUND: Changes in blood haemoglobin concentration indicate plasma volume expansion following hydroxyethyl starch (HES) infusion, but may be affected by vascular tone and HES-induced shedding of the endothelial surface layer (ESL). We hypothesised that anaesthesia-induced hypotension enhances changes in plasma volume as assessed by blood haemoglobin concentration (ΔPVHb , %) following HES infusion. METHODS: Fifty-two patients undergoing abdominal surgery were randomised to receive a continuous infusion of saline (S group) or phenylephrine to restore vascular tone (P group) (n = 26 each). Both groups received an infusion of 8 mL/kg 6% HES solution after induction of general anaesthesia. We compared ΔPVHb at the end of fluid infusion (15 minutes) and 15 minutes later (30 minutes) between the two groups. We assessed changes in ESL structure by measuring plasma concentrations of hyaluronate and syndecan-1. P < .05 was considered statistically significant. RESULTS: Mean arterial blood pressure was lower in the S group approximately by 30-40% compared to the P group (P < .001). ΔPVHb was larger in the S group compared to the P group at 15 minutes (24.9 [5.2] % vs 19.0 [5.2] %; P < .001) and 30 minutes (26.5 [5.9] % vs 16.9 [6.6] %; P < .001). There were no clinically significant differences in plasma concentrations of hyaluronate and syndecan-1 with time and between the groups. CONCLUSIONS: Increased volume expansion of circulating plasma following HES infusion in anaesthesia-induced hypotension compared to when blood pressure is restored by phenylephrine may result from an attenuation of transcapillary fluid filtration, rather than ESL shedding. UMIN Clinical Trial Registration Number: UMIN000017394 (http://www.umin.ac.jp/ctr/index.htm).

Effects of Mydriatics on Rod/Cone- and Melanopsin-driven Pupil Responses.

SIGNIFICANCE: Pupillometry protocols evaluating rod/cone- and melanopsin-driven responses often use mydriatics to ensure maximal stimulus exposure; however, retinal effects of mydriatics are not fully understood. We demonstrate that dilation with either atropine or phenylephrine results in similar enhancements of rod/cone- and melanopsin-driven pupil responses. PURPOSE: The purposes of this study were to compare the effects of atropine, a muscarinic antagonist, and phenylephrine, an adrenergic agonist, on consensual pupil responses and to assess the repeatability of pupil metrics without mydriasis. METHODS: Right eye pupil responses of 20 adults aged 21 to 42 years were recorded before and 45 minutes after instillation of 0.5% atropine or 2.5% phenylephrine in the left eye. Stimuli were presented to the left eye and included six alternating 1-second 651-nm "red" and 456-nm "blue" flashes. Metrics included baseline pupil diameter, maximal constriction, 6- and 30-second post-illumination pupil responses, and early (0 to 10 seconds) and late (10 to 30 seconds) areas under the curve. RESULTS: Dilation of the stimulated eye with either mydriatic significantly increased the 6-second post-illumination pupil response and early and late areas under the curve for blue stimuli, and early area under the curve for red stimuli (P < .05 for all). Melanopsin-driven post-illumination pupil responses, achieved with either phenylephrine or atropine, did not significantly differ from each other (P > .05 for all). Without mydriasis, intersession intraclass correlation coefficients for pupil metrics were 0.63 and 0.50 (6- and 30-second post-illumination pupil responses, respectively) and 0.78 and 0.44 (early and late areas under the curve, respectively) for blue stimuli, with no significant difference between sessions (P > .05 for all). CONCLUSIONS: Dilation with phenylephrine or atropine resulted in similar enhancements of the rod/cone- and melanopsin-driven pupil responses, despite differing mechanisms. Early pupil metrics without mydriasis demonstrated moderate to good intersession repeatability.

Effect of Adrenergic Agonists on High-Fat Diet-Induced Hepatic Steatosis in Mice.

The autonomic nervous system, consisting of sympathetic and parasympathetic branches, plays an important role in regulating metabolic homeostasis. The sympathetic nervous system (SNS) regulates hepatic lipid metabolism by regulating adrenergic receptor activation, resulting in the stimulation of hepatic very-low-density lipoprotein-triglyceride (TG) production in vivo. However, only a few studies on the relationship between SNS and hepatic steatosis have been reported. Here, we investigate the effect of adrenergic receptor agonists on hepatic steatosis in mice fed a high-fat diet (HFD). The α-adrenergic receptor agonist phenylephrine (10 mg/kg/d) or the ß-adrenergic receptor agonist isoproterenol (30 mg/kg/d) was coadministered with HFD to male mice. After five weeks, hepatic steatosis, TG levels, and hepatic fat metabolism-related biomarkers were examined. HFD treatment induced hepatic steatosis, and cotreatment with phenylephrine, but not isoproterenol, attenuated this effect. Phenylephrine administration upregulated the mRNA levels of hepatic peroxisome proliferator-activated receptor alpha and its target genes (such as carnitine palmitoyltransferase 1) and increased hepatic ß-hydroxybutyrate levels. Additionally, phenylephrine treatment increased the expression of the autophagosomal marker LC3-II but decreased that of p62, which is selectively degraded during autophagy. These results indicate that phenylephrine inhibits hepatic steatosis through stimulation of ß-oxidation and autophagy in the liver.