Distribution of the Noradrenaline Innervation and Adrenoceptors in the Macaque Monkey Thalamus.

Noradrenaline (NA) in the thalamus has important roles in physiological, pharmacological, and pathological neuromodulation. In this work, a complete characterization of NA axons and Alpha adrenoceptors distributions is provided. NA axons, revealed by immunohistochemistry against the synthesizing enzyme and the NA transporter, are present in all thalamic nuclei. The most densely innervated ones are the midline nuclei, intralaminar nuclei (paracentral and parafascicular), and the medial sector of the mediodorsal nucleus (MDm). The ventral motor nuclei and most somatosensory relay nuclei receive a moderate NA innervation. The pulvinar complex receives a heterogeneous innervation. The lateral geniculate nucleus (GL) has the lowest NA innervation. Alpha adrenoceptors were analyzed by in vitro quantitative autoradiography. Alpha-1 receptor densities are higher than Alpha-2 densities. Overall, axonal densities and Alpha adrenoceptor densities coincide; although some mismatches were identified. The nuclei with the highest Alpha-1 values are MDm, the parvocellular part of the ventral posterior medial nucleus, medial pulvinar, and midline nuclei. The nucleus with the lowest Alpha-1 receptor density is GL. Alpha-2 receptor densities are highest in the lateral dorsal, centromedian, medial and inferior pulvinar, and midline nuclei. These results suggest a role for NA in modulating thalamic involvement in consciousness, limbic, cognitive, and executive functions.

Downregulation of connexin43 potentiates noradrenaline-induced expression of brain-derived neurotrophic factor in primary cultured cortical astrocytes.

Decreased expression of brain-derived neurotrophic factor (BDNF) is involved in the pathology of depressive disorders. Astrocytes produce BDNF following antidepressant treatment or stimulation of adrenergic receptors. Connexin43 (Cx43) is mainly expressed in central nervous system astrocytes and its expression is downregulated in patients with major depression. How changes in Cx43 expression affect astrocyte function, including BDNF production, is poorly understood. The current study examined the effect of Cx43 knockdown on BDNF expression in cultured cortical astrocytes after stimulation of adrenergic receptors. The expression of Cx43 in rat primary cultured cortical astrocytes was downregulated with RNA interference. Levels of messenger RNAs (mRNAs) or proteins were measured by real-time PCR and western blotting, respectively. Knockdown of Cx43 potentiated noradrenaline (NA)-induced expression of BDNF mRNA in cultured astrocytes. NA treatment induced proBDNF protein expression in astrocytes transfected with small interfering RNA (siRNA) targeting Cx43, but not with control siRNA. This potentiation was mediated by the Src tyrosine kinase-extracellular signal-regulated kinase (ERK) pathway through stimulation of adrenergic α1 and ß receptors. Furthermore, the Gq/11 protein-Src-ERK pathway and the G-protein coupled receptor kinase 2-Src-ERK pathway were involved in α1 and ß adrenergic receptor-mediated potentiation of BDNF mRNA expression, respectively. The current studies demonstrate a novel mechanism of BDNF expression in cortical astrocytes mediated by Cx43, in which downregulation of Cx43 increases, through adrenergic receptors, the expression of BDNF. The current findings indicate a potentially novel mechanism of action of antidepressants, via regulation of astrocytic Cx43 expression and subsequent BDNF expression.

Bed nucleus of the stria terminalis modulates baroreflex cardiac activity: an interaction between alpha-1 receptors and NMDA/nitric oxide pathway.

The bed nucleus of the stria terminalis (BNST) is a forebrain structure, involved in the modulation of neuroendocrine, cardiovascular and autonomic responses. One of the responses is baroreflex activity, which consists in a neural mechanism responsible for keeping the blood pressure within a narrow range of variation. It has been reported that blockade of BNST α1-adrenoceptors increased the bradycardic component of baroreflex. In addition, such receptors are able to modulate glutamate release in this structure. Interestingly, BNST NMDA receptor antagonism and neuronal nitric oxide synthase (nNOS) inhibition led to the same effect of the α1-adrenoceptors blockade on baroreflex bradycardic response. Therefore, the hypothesis of the present study is that BNST noradrenergic transmission interacts with NMDA/NO pathway through α1 adrenoceptors to modulate the baroreflex activity. Male Wistar rats had stainless steel guide cannulas bilaterally implanted in the BNST. Subsequently, a catheter was inserted into the femoral artery for cardiovascular recordings, and into the femoral vein for assessing baroreflex activation. Injection of the noradrenaline reuptake inhibitor reboxetine in the BNST did not modify the tachycardic, but significantly decreased the bradycardic component of baroreflex. Administration of an α1, but not an α2 antagonist into the BNST prior to reboxetine prevented this effect. Likewise, previous injection of NMDA/NO pathway blockers inhibited the effect of reboxetine on bradycardic response. In conclusion, it was demonstrated for the first time the existence of an interaction between BNST noradrenergic, glutamatergic and nitrergic neurotransmissions in the modulation of bradycardic baroreflex response.

Effect of ingesting a meal and orthostasis on the regulation of splanchnic and systemic hemodynamics and the responsiveness of cardiovascular α1-adrenoceptors.

Postprandial orthostasis activates mechanisms of cardiovascular homeostasis to maintain normal blood pressure (BP) and adequate blood flow to vital organs. The underlying mechanisms of cardiovascular homeostasis in postprandial orthostasis still require elucidation. Fourteen healthy volunteers were recruited to investigate the effect of an orthostatic challenge (60°-head-up-tilt for 20 min) on splanchnic and systemic hemodynamics before and after ingesting an 800-kcal composite meal. The splanchnic circulation was assessed by ultrasonography of the superior mesenteric and hepatic arteries and portal vein. Systemic hemodynamics were assessed noninvasively by continuous monitoring of BP, heart rate (HR), cardiac output (CO), and the pressor response to an intravenous infusion on increasing doses of phenylephrine, an α1-adrenoceptor agonist. Neurohumoral regulation was assessed by spectral analysis of HR and BP, plasma catecholamine and aldosterone levels and plasma renin activity. Postprandial mesenteric hyperemia was associated with an increase in CO, a decrease in SVR and cardiac vagal tone, and reduction in baroreflex sensitivity with no change in sympathetic tone. Arterial α1-adrenoceptor responsiveness was preserved and reduced in hepatic sinusoids. Postprandial orthostasis was associated with a shift of 500 mL of blood from mesenteric to systemic circulation with preserved sympathetic-mediated vasoconstriction. Meal ingestion provokes cardiovascular hyperdynamism, cardiac vagolysis, and resetting of the baroreflex without activation of the sympathetic nervous system. Meal ingestion also alters α1-adrenoceptor responsiveness in the hepatic sinusoids and participates in the redistribution of blood volume from the mesenteric to the systemic circulation to maintain a normal BP during orthostasis.NEW & NOTEWORTHY A unique integrated investigation on the effect of meal on neurohumoral mechanisms and blood flow redistribution of the mesenteric circulation during orthostasis was investigated. Food ingestion results in cardiovascular hyperdynamism, reduction in cardiac vagal tone, and baroreflex sensitivity and causes a decrease in α1-adrenoceptor responsiveness only in the venous intrahepatic sinusoids. About 500-mL blood shifts from the mesenteric to the systemic circulation during orthostasis. Accordingly, the orthostatic homeostatic mechanisms are better understood.

Cardiac α1A-adrenergic receptors: emerging protective roles in cardiovascular diseases.

α1-Adrenergic receptors (ARs) are catecholamine-activated G protein-coupled receptors (GPCRs) that are expressed in mouse and human myocardium and vasculature, and play essential roles in the regulation of cardiovascular physiology. Though α1-ARs are less abundant in the heart than ß1-ARs, activation of cardiac α1-ARs results in important biologic processes such as hypertrophy, positive inotropy, ischemic preconditioning, and protection from cell death. Data from the Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT) indicate that nonselectively blocking α1-ARs is associated with a twofold increase in adverse cardiac events, including heart failure and angina, suggesting that α1-AR activation might also be cardioprotective in humans. Mounting evidence implicates the α1A-AR subtype in these adaptive effects, including prevention and reversal of heart failure in animal models by α1A agonists. In this review, we summarize recent advances in our understanding of cardiac α1A-ARs.

The α1-adrenergic receptors in the amygdala regulate the induction of learned despair through protein kinase C-beta signaling.

Hyperactivity of amygdala is observed in patients with major depressive disorder. Although the role of α1-adrenoceptor in amygdala on fear memory has been well studied, the role of α1-adrenoceptor in amygdala on depression-like behaviors remains unclear. Therefore, we investigated the effect of α1A-adrenoreceptor in amygdala on despair behavior, evaluated by the immobility time during tail suspension test (TST), pharmacological intervention, and immunohistological methods. C57BL6/J mice given a bilateral intra-amygdala injection of artificial cerebrospinal fluid exhibited an increased duration of immobility in the latter half of both trials of TST with a 24-h interval, a phenomenon known as learned despair. Intra-amygdala injection of WB4101 (1.7 nmol/0.1 µl), an α1 adrenoreceptor antagonist, but not propranolol (250 pmol/0.1 µl), a ß-adrenoreceptor antagonist, blocked the induction of learned despair during TST. Immunostaining experiments revealed that ~61-75% of α1A-adrenoreceptor-positive neurons were colocalized with GAD65/67 in amygdala, implying that the α1-adrenoceptors in amygdala may enormously regulate the GABA release. Protein kinase C-beta (PKCß) was predominantly expressed in the α1A-adrenoreceptor-positive neurons in the BLA, whereas protein kinase C-epsilon (PKCε) was highly expressed with the α1A-adrenoreceptor in the Central nucleus of amygdala. Intra-amygdala injection of ruboxistaurin (10 pmol/0.1 µl), a PKCß inhibitor, blocked the induction of learned despair during TST, whereas neither TAT-εV1-2 (500 ng/0.1 µl), a cell-permeant PKCε inhibitory peptide, nor HBDDE (50 pmol/0.1 µl), an inhibitor of PKCα and -γ, affected the duration of immobility during TST. These data suggest that the α1-adrenoreceptor in amygdala regulates the induction of learned despair via PKCß.

In Silico and in Vitro Study of Trace Amines (TA) and Dopamine (DOP) Interaction with Human Alpha 1-Adrenergic Receptor and the Bacterial Adrenergic Receptor QseC.

BACKGROUND/AIMS: Trace amines (TA) are small organic compounds that have neuromodulator activity due to their interaction with some neuron-related receptors, such as trace amine associated receptors (TAARs), α2-adrenergic receptor (α2-AR) and ß-adrenergic receptor (ß-AR). However, there is little information on whether TA and dopamine (DOP) can interact with other adrenergic receptors (ARs) such as the mammalian α1-AR and the bacterial counterpart QseC, which is involved in quorum sensing of some Gram-negative pathogens. The aim of this study was to investigate the interaction of TA and DOP with α1-AR and QseC. METHODS: We performed an in silico study using 3D structure from SWISS MODEL and analyzed the protein interaction via molecular docking using PyMol, PoseView and PyRX 8.0. For the in vitro study, we investigated the QseC kinase activity by measuring the remaining ATP in a reaction containing QseC-enriched membrane incubated together with purified QseB and EPI, TA, DOP, or PTL respectively. We also measured the intracellular Ca++ levels, which represents the α1-AR activation, in LNCAP (pancreatic cell line) cells treated with EPI, TA, DOP and PTL respectively using a fluorescence-based assay. The LNCAP cell proliferation was measured using an MTT-based assay. RESULTS: Our in silico analysis revealed that TAs and DOP have high binding affinity to the human α1-AR and the bacterial adrenergic receptor (QseC), comparable to epinephrine (EPI). Both are membrane-bound kinases. Experimental studies with pancreatic cell line (LNCAP) showed that the TAs and DOP act as α1-AR antagonist by counteracting the effect of EPI. In the presence of EPI, TA and DOP trigger an increase of the intracellular Ca++ levels in the LNCAP cells leading to an inhibition of cell proliferation. Although in silico data suggest an interaction of TA and DOP with QseC, they do not inhibit the kinase activity of QseC, a histidine kinase receptor involved in quorum sensing which is also sensitive to EPI. CONCLUSION: Our study showed that the TAs and DOP act as α1-AR antagonist but no effect was observed for QseC.

Increased vascular α1-adrenergic receptor sensitivity in older adults with posttraumatic stress disorder.

Posttraumatic stress disorder (PTSD) is an independent risk factor for the development of hypertension and cardiovascular disease. Patients with PTSD have heightened blood pressure and sympathetic nervous system reactivity; however, it is unclear if patients with PTSD have exaggerated vasoconstriction in response to sympathetic nerve activation that could also contribute to increased blood pressure reactivity. Therefore, we hypothesized that patients with PTSD have increased sensitivity of vascular α1-adrenergic receptors (α1ARs), the major mediators of vasoconstriction in response to release of norepinephrine at sympathetic nerve terminals. To assess vascular α1AR sensitivity, we measured the degree of venoconstriction in a dorsal hand vein in response to exponentially increasing doses of the selective α1AR agonist, phenylephrine (PE), in 9 patients with PTSD (age = 59 ± 2 yr) and 10 age-matched controls (age = 60 ± 1 yr). Individual dose-response curves were generated to determine the dose of PE that induces 50% of maximal venoconstriction (i.e., PE ED50) reflective of vascular α1AR sensitivity. In support of our hypothesis, PE ED50 values were lower in PTSD compared with controls (245 ± 54 ng/min vs. 1,995 ± 459 ng/min, P = 0.012), indicating increased vascular α1AR sensitivity in PTSD. The PTSD group also had an increase in slope of rise in venoconstriction, indicative of an altered venoconstrictive reactivity to PE compared with controls (19.8% ± 1.2% vs. 15.1% ± 1.2%, P = 0.009). Heightened vascular α1AR sensitivity in PTSD may contribute to augmented vasoconstriction and blood pressure reactivity to sympathoexcitation and to increased cardiovascular disease risk in this patient population.

Activation of noradrenergic terminals in the reticular thalamus delays arousal from propofol anesthesia in mice.

Electroencephalogram monitoring during propofol (PRO) anesthesia typically features low-frequency oscillations, which may be involved with thalamic reticular nucleus (TRN) modulation. TRN receives noradrenergic inputs from the locus coeruleus (LC). We hypothesized that specific noradrenergic connections in the TRN may contribute to the emergence from PRO anesthesia. Intranuclei norepinephrine (NE) injections (n = 10) and designer receptors exclusively activated by designer drugs (DREADDs) (n = 10) were used to investigate the role of noradrenergic inputs from the LC to the TRN during PRO anesthesia. Whole-cell recording in acute brain slice preparations was used to identify the type of adrenoceptor that regulates noradrenergic innervation in the TRN. An intracerebral injection of NE into the TRN delays arousal in mice recovering from PRO anesthesia (means ± sd; 486.6 ± 57.32 s for the NE injection group vs. 422.4 ± 48.19 s for the control group; P = 0.0143) and increases the cortical-δ (0.1-4 Hz, 25.4 ± 2.9 for the NE injection group vs. 21.0 ± 1.7 for the control group; P = 0.0094) oscillation. An intra-TRN injection of NE also decreased the EC50 of PRO-induced unconsciousness (57.05 ± 1.78 mg/kg for the NE injection group vs. 72.44 ± 3.23 mg/kg for the control group; P = 0.0096). Moreover, the activation of LC-noradrenergic nerve terminals in the TRN using DREADDs increased the recovery time [466.1 ± 44.57 s for the clozapine N-oxide (CNO) injection group vs. 426.1 ± 38.75 s for the control group; P = 0.0033], decreased the EC50 of PRO-induced unconsciousness (64.77 ± 3.40 mg/kg for the CNO injection group vs. 74.00 ± 2.08 mg/kg for the control group; P = 0.0081), and increased the cortical-δ oscillation during PRO anesthesia (23.29 ± 2.58 for the CNO injection group vs. 19.56 ± 1.9 for the control group; P = 0.0213). In addition, whole-cell recording revealed that NE augmented the inhibitory postsynaptic currents in the TRN neurons via the α1-adrenoceptor. Our data indicated that enhanced NE signaling at the noradrenergic terminals of the LC-TRN projection delays arousal from general anesthesia, which is likely mediated by the α1-adrenoceptor activation. Our findings open a door for further understanding of the functions of various LC targets in both anesthesia and arousal.-Zhang, Y., Fu, B., Liu, C., Yu, S., Luo, T., Zhang, L., Zhou, W., Yu, T. Activation of noradrenergic terminals in the reticular thalamus delays arousal from propofol anesthesia in mice.

Effects of Carvedilol and Thyroid Hormones Co-administration on Apoptotic and Survival Proteins in the Heart After Acute Myocardial Infarction.

Cellular death and survival signaling plays a key role in the progress of adverse cardiac remodeling after acute myocardial infarction (AMI). Therapeutic strategies, such as co-treatment with beta-blocker carvedilol and thyroid hormones (THs), give rise to new approaches that can sustain the cellular homeostasis after AMI. Therefore, we sought to investigate the effects of carvedilol and TH co-administration on apoptosis and survival proteins and on cardiac remodeling after AMI. Male Wistar rats were distributed in 5 groups as follows: sham-operated group (SHAM), infarcted group (MI), infarcted plus carvedilol group (MI+C), infarcted plus TH group (MI+TH), and infarcted plus carvedilol and TH co-treatment group (MI+C+TH). Echocardiographic analysis was performed, and hearts were collected for western blot evaluation. The MI group presented systolic posterior wall thickness loss, an increase in the wall tension index, and an increase in atrial natriuretic peptide tissue levels than the SHAM group. However, in the MI+C+TH group, these parameters were equally to the SHAM group. Moreover, whereas the MI group showed Bax protein expression elevated in relation to the SHAM group, the MI+C+TH group presented Bax reduction and also Akt activation compared with the MI group. In addition, the MI+TH group revealed beta-1 adrenergic receptor (ß1AR) upregulation compared with the MI and MI+C groups, whereas the MI+C+TH group presented lower levels of ß1AR in relation to the SHAM and MI+TH groups. In conclusion, we suggest that carvedilol and TH co-administration may mediate its cardioprotective effects against adverse cardiac remodeling post-AMI through the Bax reduction, Akt activation, and ß1AR decrease.

Activation of α-adrenoceptors depresses synaptic transmission of myelinated afferents and inhibits pathways mediating primary afferent depolarization (PAD) in the in vitro mouse spinal cord.

Somatosensory afferent transmission strength is controlled by several presynaptic mechanisms that reduce transmitter release at the spinal cord level. We focused this investigation on the role of α-adrenoceptors in modulating sensory transmission in low-threshold myelinated afferents and in pathways mediating primary afferent depolarization (PAD) of neonatal mouse spinal cord. We hypothesized that the activation of α-adrenoceptors depresses low threshold-evoked synaptic transmission and inhibits pathways mediating PAD. Extracellular field potentials (EFPs) recorded in the deep dorsal horn assessed adrenergic modulation of population monosynaptic transmission, while dorsal root potentials (DRPs) recorded at root entry zone assessed adrenergic modulation of PAD. We found that noradrenaline (NA) and the α1-adrenoceptor agonists phenylephrine and cirazoline depressed synaptic transmission (by 15, 14 and 22%, respectively). DRPs were also depressed by NA, phenylephrine and cirazoline (by 62, 30, and 64%, respectively), and by the α2-adrenoceptor agonist clonidine, although to a lower extent (20%). We conclude that NA depresses monosynaptic transmission of myelinated afferents onto deep dorsal horn neurons via α1-adrenoceptors and inhibits interneuronal pathways mediating PAD through the activation of α1- and α2-adrenoceptors. The functional significance of these modulatory actions in shaping cutaneous and muscle sensory information during motor behaviors requires further study.

Comparison of the Effects of Tadalafil and α1-Adrenoceptor Antagonists on Spontaneous Seminal Emission and Electrical Field Stimulation-Induced Seminal Vesicle Contraction in Rats.

BACKGROUND: Although numerous reports have shown that α1-adrenoceptor (α1-AR) antagonists, which are used to treat benign prostatic hyperplasia (BPH), can cause ejaculatory disorders, few studies have investigated whether the phosphodiesterase 5 (PDE5) inhibitor tadalafil has such adverse effects. In this study, we compared the effects of tadalafil and α1-AR antagonists on seminal emission and their mechanisms of action. AIM: To evaluate in normal rats the possible effects of tadalafil on spontaneous seminal emission (SSE) and seminal contraction evoked by hypogastric nerve stimulation. METHODS: Male Sprague-Dawley rats were used. To assess SSE, plastic corsets were fitted around the thorax and upper abdomen of male Sprague-Dawley rats to prevent genital autogrooming. Rats were treated orally with tadalafil or an α1-AR antagonist (silodosin, naftopidil, or tamsulosin) for 3 days and housed in wire-bottomed cages. Ejaculatory plugs dropped on the bottoms of the cages were counted and weighed. To assess the intraluminal pressure of seminal vesicles, the hypogastric nerve of urethane-anesthetized rats was isolated and electrically stimulated. After stabilization of seminal vesicle contraction, the rats were intravenously administered test drugs. The expression of PDE5, endothelial nitric oxide synthetase (eNOS), and neuronal NOS (nNOS) in the seminal vesicle and vas deferens were measured by reverse-transcription polymerase chain reaction. MAIN OUTCOME MEASURE: The number and weight of the ejaculatory plugs produced by corset-fitted rats and the intraluminal pressure of the seminal vesicle were evaluated. RESULTS: Tadalafil did not affect the number or weight of the ejaculatory plugs of corset-fitted rats, whereas all α1-AR antagonists decreased both in a dose-dependent manner. The α1-AR antagonists, but not tadalafil, inhibited the seminal vesicle contraction evoked by electrical stimulation of the hypogastric nerve. The seminal vesicle and vas deferens expressed higher levels of PDE5 and eNOS mRNA and lower levels of nNOS mRNA relative to the urethra. CLINICAL IMPLICATIONS: Tadalafil can be a treatment option in cases where there is concern about negative effects on seminal emission. STRENGTHS AND LIMITATIONS: We demonstrated different effects of tadalafil and 3 α1-AR antagonists on rat SSE and their mechanisms of action by measuring seminal vesicle contractility in vivo. A limitation is that we used normal rats, not BPH model rats, and so our results might not apply to human BPH patients. CONCLUSION: Tadalafil did not inhibit spontaneous seminal emission or electrical field stimulation-induced seminal vesicle contraction in normal rats. The NO-cyclic guanosine monophosphate pathway is unlikely to be involved in the inhibition of seminal vesicle contraction in normal rats. Yoshinaga R, Fukui T, Yoshifuji M, et al. Comparison of the Effects of Tadalafil and α1-Adrenoceptor Antagonists on Spontaneous Seminal Emission and Electrical Field Stimulation-Induced Seminal Vesicle Contraction in Rats. J Sex Med 2019;16:680-690.

TRPV4 (Transient Receptor Potential Vanilloid 4) Channel-Dependent Negative Feedback Mechanism Regulates Gq Protein-Coupled Receptor-Induced Vasoconstriction.

OBJECTIVE: Several physiological stimuli activate smooth muscle cell (SMC) GqPCRs (Gq protein-coupled receptors) to cause vasoconstriction. As a protective mechanism against excessive vasoconstriction, SMC GqPCR stimulation invokes endothelial cell vasodilatory signaling. Whether Ca2+ influx in endothelial cells contributes to the regulation of GqPCR-induced vasoconstriction remains unknown. Ca2+ influx through TRPV4 (transient receptor potential vanilloid 4) channels is a key regulator of endothelium-dependent vasodilation. We hypothesized that SMC GqPCR stimulation engages endothelial TRPV4 channels to limit vasoconstriction. APPROACH AND RESULTS: Using high-speed confocal microscopy to record unitary Ca2+ influx events through TRPV4 channels (TRPV4 sparklets), we report that activation of SMC α1ARs (alpha1-adrenergic receptors) with phenylephrine or thromboxane A2 receptors with U46619 stimulated TRPV4 sparklets in the native endothelium from mesenteric arteries. Activation of endothelial TRPV4 channels did not require an increase in Ca2+ as indicated by the lack of effect of L-type Ca2+ channel activator or chelator of intracellular Ca2+ EGTA-AM. However, gap junction communication between SMCs and endothelial cells was required for phenylephrine activation or U46619 activation of endothelial TRPV4 channels. Lowering inositol 1,4,5-trisphosphate levels with phospholipase C inhibitor or lithium chloride suppressed phenylephrine activation of endothelial TRPV4 sparklets. Moreover, uncaging inositol 1,4,5-trisphosphate profoundly increased TRPV4 sparklet activity. In pressurized arteries, phenylephrine-induced vasoconstriction was followed by a slow, TRPV4-dependent vasodilation, reflecting activation of negative regulatory mechanism. Consistent with these data, phenylephrine induced a significantly higher increase in blood pressure in TRPV4-/- mice. CONCLUSIONS: These results demonstrate that SMC GqPCR stimulation triggers inositol 1,4,5-trisphosphate-dependent activation of endothelial TRPV4 channels to limit vasoconstriction.

Impact of Racial Discrimination and Hostility on Adrenergic Receptor Responsiveness in African American Adults.

OBJECTIVE: Racial discrimination is increasingly recognized as a contributor to increased cardiovascular disease (CVD) risk among African Americans. Previous research has shown significant overlap between racial discrimination and hostility, an established predictor of CVD risk including alterations in adrenergic receptor functioning. The present study examined the associations of racial discrimination and hostility with adrenergic receptor responsiveness. METHODS: In a sample (N = 57) of young to middle-aged African American adults (51% female) with normal and mildly elevated blood pressure, a standardized isoproterenol sensitivity test (CD25) was used to evaluate ß-AR responsiveness, whereas the dose of phenylephrine required to increase mean arterial pressure by 25 mm Hg (PD25) was used to assess α1-AR responsiveness. Racial discrimination was measured using the Perceived Racism Scale and hostility was assessed using the Cook-Medley Hostility Scale. RESULTS: In hierarchical regression models, greater racial discrimination, but not hostility, emerged as a significant predictor of decreased ß-adrenergic receptor responsiveness (ß = .38, p = .004). However, moderation analysis revealed that the association between racial discrimination and blunted ß-adrenergic receptor responsiveness was strongest among those with higher hostility (ß = .49, 95% confidence interval = .17-.82, p = .004). In addition, hostility, but not racial discrimination, significantly predicted α1-AR responsiveness. CONCLUSIONS: These findings suggest racial discrimination was associated with blunted ß-adrenergic receptor responsiveness, providing further evidence of the potential contribution of racial discrimination to increased CVD risk among African Americans. The adverse effects of discrimination on cardiovascular health may be enhanced in individuals with higher levels of hostility.

Human Myocardium Has a Robust α1A-Subtype Adrenergic Receptor Inotropic Response.

Recent studies report that a single subtype of α1-adrenergic receptor (α1-AR), the α1A-subtype, mediates robust cardioprotective effects in multiple experimental models of heart failure, suggesting that the α1A-subtype is a potential therapeutic target for an agonist to treat heart failure. Moreover, we recently found that the α1A-subtype is present in human heart. The goal of this study was to assess the inotropic response mediated by the α1A-subtype in human myocardium, and to determine whether the response is downregulated in myocardium from failing human heart. We measured in vitro contractile responses of cardiac muscle preparations (trabeculae) isolated from the right ventricle from nonfailing and failing human hearts. Addition of the α1A-subtype agonist A61603 (100 nM) resulted in a large positive inotropic response (force increased ≈ 2-fold). This response represented ≈70% of the response mediated by the ß-adrenergic receptor agonist isoproterenol (1 µM). Moreover, in myocardium from failing hearts, α1A-subtype responses remained robust, and only slightly reduced relative to nonfailing hearts. We conclude that α1A-subtype-mediated inotropy could represent a significant source of inotropic support in the human heart. Furthermore, the α1A-subtype remains functional in myocardium from failing human hearts and thus, might be a therapeutic target to support cardioprotective effects in patients with heart failure.

Adreno-muscarinic synergy in the male human urinary outflow tract.

AIM: To examine putative interaction between adrenergic and muscarinic contractile activation in the human urinary outflow tract. METHODS: Tissue from the trigone and prostatic urethra was obtained from 12 cystectomy and 16 prostatectomy specimen. Contractions were elicited by exposure to exogenous agonists before and after inhibition of Rho kinase and protein kinase c (PKC). Immunofluorescence and Western-blot studies were performed using antibodies to muscarinic M3-receptors (M3-R) and alpha1A-adrenoreceptors (alpha1A-AR). The study is registered with ClinicalTrials.gov, number NCT01227447. RESULTS: There was strong co-localization of M3-R and alpha1A-AR on trigonal and urethral myocytes. Western blot analysis revealed a significantly higher expression of alpha1A-AR in the superficial than in the deep trigone. Phenylephrine (PE, 1 µm) augmented contractions induced by carbachol (CA, 3 µm) to more than threefold control in the male superficial trigone, and to about sevenfold control in the proximal urethra. No such potentiation could be detected in female bladder outlet. Both PKC inhibitor GF 109203X and Rho kinase inhibitor Y-27632 reduced responses to 1 µM PE as well as to 3 µM CA significantly. However, the synergistic effect of the combined intervention remained proportionally unaffected. CONCLUSIONS: Muscarinic and adrenergic receptor activation exerts a strong synergistic effect in the male human bladder trigone and proximal urethra.

How important is the α1-adrenoceptor in primate and rodent proximal urethra? Sex differences in the contribution of α1-adrenoceptor to urethral contractility.

Urethral smooth muscle (USM) contributes to urinary continence by contracting during the urine storage phase, which is mainly mediated by activation of postjunctional α1-adrenoceptors. Males and females show differences in the functioning of the lower urinary tract and the most common urinary tract symptoms (LUTS). LUTS in men typically occur in association with bladder outlet obstruction, whereas in women urinary urge-incontinence symptoms are more common. Therefore, this study aimed to evaluate sex differences in α1-adrenoceptor subtype expression and their importance in proximal urethra contraction in the mouse (C57BL6/J) and marmoset (Callithrix jacchus). Contractile responses to phenylephrine, norepinephrine, potassium chloride (KCl), and electrical-field stimulation (EFS) were evaluated. Phenylephrine, norepinephrine, KCl, and EFS produced markedly greater contractions in male mice and marmoset USM compared with females. The sex differences remained unchanged by Nω-nitro-l-arginine (l-NAME; nitric oxide synthase inhibitor), atropine (muscarinic receptor antagonist), and PPADS (P2X1-purinoceptor antagonist). Additionally, selective α1A (but not α1B- and α1D-)-adrenoceptor antagonists significantly reduced phenylephrine-induced USM contractions. qRT-PCR for α1A-, B-, and D-adrenoceptor subtypes revealed a marked presence of the α1A-adrenoceptor subtype in male USM, but not females. Male mouse urethra also exhibited a higher tyrosine hydroxylase mRNA expression. Histomorphometric analysis showed a greater USM area in male than female mice. In conclusion, male mouse and marmoset proximal USM shows strong α1A- adrenoceptor-induced contractions and abundant α1A-adrenoceptor expression, whereas α1A-adrenoceptor-mediated mechanisms are much less important in females. The differential expression of α1-adrenoceptors in the proximal urethra may contribute to the higher incidence of urinary incontinence in women and obstructed voiding in men.

Maternal separation diminishes α-adrenergic receptor density and function in renal vasculature from male Wistar-Kyoto rats.

Adult rats exposed to maternal separation (MatSep) are normotensive but display lower glomerular filtration rate and increased renal neuroadrenergic drive. The aim of this study was to determine the renal α-adrenergic receptor density and the renal vascular responsiveness to adrenergic stimulation in male rats exposed to MatSep. In addition, baroreflex sensitivity was assessed to determine a component of neural control of the vasculature. Using tissue collected from 4-mo-old MatSep and control rats, α1-adrenergic receptors (α1-ARs) were measured in renal cortex and isolated renal vasculature using receptor binding assay, and the α-AR subtype gene expression was determined by RT-PCR. Renal cortical α1-AR density was similar between MatSep and control tissues (Bmax = 44 ± 1 vs. 42 ± 2 fmol/mg protein, respectively); however, MatSep reduced α1-AR density in renal vasculature (Bmax = 47 ± 4 vs. 62 ± 4 fmol/mg protein, P < 0.05, respectively). In a separate group of rats, the pressor, bradycardic, and renal vascular constrictor responses to acute norepinephrine injection (NE, 0.03-0.25 µg/µl) were determined under anesthesia. Attenuated NE-induced renal vasoconstriction was observed in rats exposed to MatSep compared with control (P < 0.05). A third group of rats was infused at steady state with the α1 agonist phenylephrine (10 µg/min iv) and vasodilator sodium nitroprusside (5 µg/min iv). The difference between the change in heart rate/mean arterial pressure slopes was indicative of reduced baroreflex sensitivity in MatSep vs. control rats (-0.45 ± 0.04 vs. -0.95 ± 0.07 beats·min-1·mmHg-1, P < 0.05). These data support the notion that reduced α-adrenergic receptor expression and function in the renal vasculature could develop secondary to MatSep-induced overactivation of the renal neuroadrenergic tone.

α1A-Subtype adrenergic agonist therapy for the failing right ventricle.

Failure of the right ventricle (RV) is a serious disease with a poor prognosis and limited treatment options. Signaling by α1-adrenergic receptors (α1-ARs), in particular the α1A-subtype, mediate cardioprotective effects in multiple heart failure models. Recent studies have shown that chronic treatment with the α1A-subtype agonist A61603 improves function and survival in a model of left ventricular failure. The goal of the present study was to determine if chronic A61603 treatment is beneficial in a RV failure model. We used tracheal instillation of the fibrogenic antibiotic bleomycin in mice to induce pulmonary fibrosis, pulmonary hypertension, and RV failure within 2 wk. Some mice were chronically treated with a low dose of A61603 (10 ng·kg-1·day-1). In the bleomycin model of RV failure, chronic A61603 treatment was associated with improved RV fractional shortening and greater in vitro force development by RV muscle preparations. Cell injury markers were reduced with A61603 treatment (serum cardiac troponin I, RV fibrosis, and expression of matrix metalloproteinase-2). RV oxidative stress was reduced (using the probes dihydroethidium and 4-hydroxynonenal). Consistent with lowered RV oxidative stress, A61603 was associated with an increased level of the cellular antioxidant superoxide dismutase 1 and a lower level of the prooxidant NAD(P)H oxidase isoform NOX4. In summary, in the bleomycin model of RV failure, chronic A61603 treatment reduced RV oxidative stress, RV myocyte necrosis, and RV fibrosis and increased both RV function and in vitro force development. These findings suggest that in the context of pulmonary fibrosis, the α1A-subtype is a potential therapeutic target to treat the failing RV.NEW & NOTEWORTHY Right ventricular (RV) failure is a serious disease with a poor prognosis and no effective treatments. In the mouse bleomycin model of RV failure, we tested the efficacy of a treatment using the α1A-adrenergic receptor subtype agonist A61603. Chronic A61603 treatment improved RV contraction and reduced multiple indexes of RV injury, suggesting that the α1A-subtype is a therapeutic target to treat RV failure.

The Protein Acyl Transferase ZDHHC21 Modulates α1 Adrenergic Receptor Function and Regulates Hemodynamics.

OBJECTIVE: Palmitoylation, the reversible addition of the lipid palmitate to a cysteine, can alter protein localization, stability, and function. The ZDHHC family of protein acyl transferases catalyzes palmitoylation of numerous proteins. The role of ZDHHC enzymes in intact tissue and in vivo is largely unknown. Herein, we characterize vascular functions in a mouse that expresses a nonfunctional ZDHHC21 (F233Δ). APPROACH AND RESULTS: Physiological studies of isolated aortae and mesenteric arteries from F233Δ mice revealed an unexpected defect in responsiveness to phenylephrine, an α1 adrenergic receptor agonist. In vivo, F233Δ mice displayed a blunted response to infusion of phenylephrine, and they were found to have elevated catecholamine levels and elevated vascular α1 adrenergic receptor gene expression. Telemetry studies showed that the F233Δ mice were tachycardic and hypotensive at baseline, consistent with diminished vascular tone. In biochemical studies, ZDHHC21 was shown to palmitoylate the α1D adrenoceptor and to interact with it in a molecular complex, thus suggesting a possible molecular mechanism by which the receptor can be regulated by ZDHHC21. CONCLUSIONS: Together, the data support a model in which ZDHHC21 F233Δ diminishes the function of vascular α1 adrenergic receptors, leading to reduced vascular tone, which manifests in vivo as hypotension and tachycardia. This is to our knowledge the first demonstration of a ZDHHC isoform affecting vascular function in vivo and identifies a novel molecular mode of regulation of vascular tone and blood pressure.