Adrenoceptor sub-type involvement in Ca2+ current stimulation by noradrenaline in human and rabbit atrial myocytes.

Atrial fibrillation (AF) from elevated adrenergic activity may involve increased atrial L-type Ca2+ current (ICaL) by noradrenaline (NA). However, the contribution of the adrenoceptor (AR) sub-types to such ICaL-increase is poorly understood, particularly in human. We therefore investigated effects of various broad-action and sub-type-specific α- and ß-AR antagonists on NA-stimulated atrial ICaL. ICaL was recorded by whole-cell-patch clamp at 37 °C in myocytes isolated enzymatically from atrial tissues from consenting patients undergoing elective cardiac surgery and from rabbits. NA markedly increased human atrial ICaL, maximally by ~ 2.5-fold, with EC75 310 nM. Propranolol (ß1 + ß2-AR antagonist, 0.2 microM) substantially decreased NA (310 nM)-stimulated ICaL, in human and rabbit. Phentolamine (α1 + α2-AR antagonist, 1 microM) also decreased NA-stimulated ICaL. CGP20712A (ß1-AR antagonist, 0.3 microM) and prazosin (α1-AR antagonist, 0.5 microM) each decreased NA-stimulated ICaL in both species. ICI118551 (ß2-AR antagonist, 0.1 microM), in the presence of NA + CGP20712A, had no significant effect on ICaL in human atrial myocytes, but increased it in rabbit. Yohimbine (α2-AR antagonist, 10 microM), with NA + prazosin, had no significant effect on human or rabbit ICaL. Stimulation of atrial ICaL by NA is mediated, based on AR sub-type antagonist responses, mainly by activating ß1- and α1-ARs in both human and rabbit, with a ß2-inhibitory contribution evident in rabbit, and negligible α2 involvement in either species. This improved understanding of AR sub-type contributions to noradrenergic activation of atrial ICaL could help inform future potential optimisation of pharmacological AR-antagonism strategies for inhibiting adrenergic AF.

Opposing functions of α- and ß-adrenoceptors in the formation of processes by cultured astrocytes.

Astrocytes are glial cells with numerous fine processes which are important for the functions of the central nervous system. The activation of ß-adrenoceptors induces process formation of astrocytes via cyclic AMP (cAMP) signaling. However, the role of α-adrenoceptors in the astrocyte morphology has not been elucidated. Here, we examined it by using cultured astrocytes from neonatal rat spinal cords and cortices. Exposure of these cells to noradrenaline and the ß-adrenoceptor agonist isoproterenol increased intracellular cAMP levels and induced the formation of processes. Noradrenaline-induced process formation was enhanced with the α1-adrenoceptor antagonist prazosin and α2-adrenoceptor antagonist atipamezole. Atipamezole also enhanced noradrenaline-induced cAMP elevation. Isoproterenol-induced process formation was not inhibited by the α1-adrenoceptor agonist phenylephrine but was inhibited by the α2-adrenoceptor agonist dexmedetomidine. Dexmedetomidine also inhibited process formation induced by the adenylate cyclase activator forskolin and the membrane-permeable cAMP analog dibutyryl-cAMP. Moreover, dexmedetomidine inhibited cAMP-independent process formation induced by adenosine or the Rho-associated kinase inhibitor Y27632. In the presence of propranolol, noradrenaline inhibited Y27632-induced process formation, which was abolished by prazosin or atipamezole. These results demonstrate that α-adrenoceptors inhibit both cAMP-dependent and -independent astrocytic process formation.

Catecholamines Induce Endoplasmic Reticulum Stress Via Both Alpha and Beta Receptors.

Severely burned patients suffer from a hypermetabolic syndrome that can last for years after the injury has resolved. The underlying cause of these metabolic alterations most likely involves the persistent elevated catecholamine levels that follow the surge induced by thermal injury. At the cellular level, endoplasmic reticulum (ER) stress in metabolic tissues is a hallmark observed in patients following burn injury and is associated with several detrimental effects. Therefore, ER stress could be the underlying cellular mechanism of persistent hypermetabolism in burned patients. Here, we show that catecholamines induce ER stress and that adreno-receptor blockers reduce stress responses in the HepG2 hepatocyte cell line. Our results also indicate that norepinephrine (NE) significantly induces ER stress in HepG2 cells and 3T3L1 mouse adipocytes. Furthermore, we demonstrate that the alpha-1 blocker, prazosin, and beta blocker, propranolol, block ER stress induced by NE. We also show that the effects of catecholamines in inducing ER stress are cell type-specific, as NE treatment failed to evoke ER stress in human fibroblasts. Thus, these findings reveal the mechanisms used by catecholamines to alter metabolism and suggest inhibition of the receptors utilized by these agents should be further explored as a potential target for the treatment of ER stress-mediated disease.

No Protective Effect of Constitutive Activation of AMPK in Endothelial Cells on Vascular Function in Aged Obese Mice but Augmented α1-Adrenergic Contractions in Renal Arteries Reversible by Weight Loss.

BACKGROUND: Aging, obesity, and diabetes favor vascular dysfunction. Endothelial activation of adenosine monophosphate-activated protein kinase (AMPK) has protective effects in diabetes. METHODS: Mice with constitutive endothelial activation of AMPK (CA-AMPK) were given a high fat diet to induce obesity or kept on standard chow as lean controls for up to 2 years. A subset of obese animals was changed to standard chow after 30 weeks of high fat feeding. En-dothelium-dependent and endothelium-independent responses were examined by isometric tension recording. RESULTS AND CONCLUSION: Endothelium-dependent nitric oxide (NO)- and apamin plus charybdotoxin-sensitive relaxations were preserved and similar between aortic or renal arterial preparations of lean and obese CA-AMPK mice and their wild-type littermates. Despite comparable release of vasoconstrictor prostanoids, cyclooxygenase-dependent contractions were enhanced during NO synthase inhibition in carotid arterial rings of obese CA-AMPK mice. Contractions to the α1-adrenoceptor agonist phenylephrine were augmented in renal arteries of obese animals, a genotype-independent phenomenon reversible by weight loss. These data indicate a higher α1-adrenergic reactivity in renal arteries of aged mice with obesity. The current results highlight the potential of weight loss to alleviate vascular dysfunction. However, endothelial activation of the AMPK pathway in obesity may not be sufficient to prevent vascular dysfunction without lifestyle changes.

Participation of hepatic α/β-adrenoceptors and AT1 receptors in glucose release and portal hypertensive response induced by adrenaline or angiotensin II

It has been previously demonstrated that the hemodynamic effect induced by angiotensin II (AII) in the liver was completely abolished by losartan while glucose release was partially affected by losartan. Angiotensin II type 1 (AT1) and adrenergic (∝1- and β-) receptors (AR) belong to the G-proteins superfamily, which signaling promote glycogen breakdown and glucose release. Interactive relationship between AR and AT1-R was shown after blockade of these receptors with specific antagonists. The isolated perfused rat liver was used to study hemodynamic and metabolic responses induced by AII and adrenaline (Adr) in the presence of AT1 (losartan) and ∝1-AR and β-AR antagonists (prazosin and propranolol). All antagonists diminished the hemodynamic response induced by Adr. Losartan abolished hemodynamic response induced by AII, and AR antagonists had no effect when used alone. When combined, the antagonists caused a decrease in the hemodynamic response. The metabolic response induced by Adr was mainly mediated by ∝1-AR. A significant decrease in the hemodynamic response induced by Adr caused by losartan confirmed the participation of AT1-R. The metabolic response induced by AII was impaired by propranolol, indicating the participation of β-AR. When both ARs were blocked, the hemodynamic and metabolic responses were impaired in a cumulative effect. These results suggested that both ARs might be responsible for AII effects. This possible cross-talk between β-AR and AT1-R signaling in the hepatocytes has yet to be investigated and should be considered in the design of specific drugs.

Endothelium-dependent vasodilatory signalling modulates α1 -adrenergic vasoconstriction in contracting skeletal muscle of humans.

KEY POINTS: 'Functional sympatholysis' describes the ability of contracting skeletal muscle to attenuate sympathetic vasoconstriction, and is critical to ensure proper blood flow and oxygen delivery to metabolically active skeletal muscle. The signalling mechanism responsible for sympatholysis in healthy humans is unknown. Evidence from animal models has identified endothelium-derived hyperpolarization (EDH) as a potential mechanism capable of attenuating sympathetic vasoconstriction. In this study, increasing endothelium-dependent signalling during exercise significantly enhanced the ability of contracting skeletal muscle to attenuate sympathetic vasoconstriction in humans. This is the first study in humans to identify endothelium-dependent regulation of sympathetic vasoconstriction in contracting skeletal muscle, and specifically supports a role for EDH-like vasodilatory signalling. Impaired functional sympatholysis is a common feature of cardiovascular ageing, hypertension and heart failure, and thus identifying fundamental mechanisms responsible for sympatholysis is clinically relevant. ABSTRACT: Stimulation of α-adrenoceptors elicits vasoconstriction in resting skeletal muscle that is blunted during exercise in an intensity-dependent manner. In humans, the underlying mechanisms remain unclear. We tested the hypothesis that stimulating endothelium-dependent vasodilatory signalling will enhance the ability of contracting skeletal muscle to blunt α1 -adrenergic vasoconstriction. Changes in forearm vascular conductance (FVC; Doppler ultrasound, brachial intra-arterial pressure via catheter) to local intra-arterial infusion of phenylephrine (PE; α1 -adrenoceptor agonist) were calculated during (1) infusion of the endothelium-dependent vasodilators acetylcholine (ACh) and adenosine triphosphate (ATP), the endothelium-independent vasodilator (sodium nitroprusside, SNP), or potassium chloride (KCl) at rest; (2) mild or moderate intensity handgrip exercise; and (3) combined mild exercise + ACh, ATP, SNP, or KCl infusions in healthy adults. Robust vasoconstriction to PE was observed during vasodilator infusion alone and mild exercise, and this was blunted during moderate intensity exercise (ΔFVC: -34 ± 4 and -34 ± 3 vs. -13 ± 2%, respectively, P < 0.05). Infusion of ACh or ATP during mild exercise significantly attenuated PE vasoconstriction similar to levels observed during moderate exercise (ACh: -3 ± 4; ATP: -18 ± 4%). In contrast, infusion of SNP or KCl during mild exercise did not attenuate PE-mediated vasoconstriction (-32 ± 5 and -46 ± 3%). To further study the role of endothelium-dependent hyperpolarization (EDH), ACh trials were repeated with combined nitric oxide synthase and cyclooxygenase inhibition. Here, PE-mediated vasoconstriction was blunted at rest (blockade: -20 ± 5 vs. CONTROL: -31 ± 3% vs.; P < 0.05) and remained blunted during exercise (blockade: -15 ± 5 vs. CONTROL: -14 ± 5%). We conclude that stimulation of EDH-like vasodilatation can blunt α1 -adrenergic vasoconstriction in contracting skeletal muscle of humans.

Role of Opioid Receptors Signaling in Remote Electrostimulation--Induced Protection against Ischemia/Reperfusion Injury in Rat Hearts.

AIMS: Our previous studies demonstrated that remote electro-stimulation (RES) increased myocardial GSK3 phosphorylation and attenuated ischemia/ reperfusion (I/R) injury in rat hearts. However, the role of various opioid receptors (OR) subtypes in preconditioned RES-induced myocardial protection remains unknown. We investigated the role of OR subtype signaling in RES-induced cardioprotection against I/R injury of the rat heart. METHODS & RESULTS: Male Spraque-Dawley rats were used. RES was performed on median nerves area with/without pretreatment with various receptors antagonists such as opioid receptor (OR) subtype receptors (KOR, DOR, and MOR). The expressions of Akt, GSK3, and PKCε expression were analyzed by Western blotting. When RES was preconditioned before the I/R model, the rat's hemodynamic index, infarction size, mortality and serum CK-MB were evaluated. Our results showed that Akt, GSK3 and PKCε expression levels were significantly increased in the RES group compared to the sham group, which were blocked by pretreatment with specific antagonists targeting KOR and DOR, but not MOR subtype. Using the I/R model, the duration of arrhythmia and infarct size were both significantly attenuated in RES group. The mortality rates of the sham RES group, the RES group, RES group + KOR antagonist, RES group + DOR/MOR antagonists (KOR left), RES group + DOR antagonist, and RES group + KOR/MOR antagonists (DOR left) were 50%, 20%, 67%, 13%, 50% and 55%, respectively. CONCLUSION: The mechanism of RES-induced myocardial protection against I/R injury seems to involve multiple target pathways such as Akt, KOR and/or DOR signaling.

Ingestive and locomotor behaviours induced by pharmacological manipulation of -adrenoceptors into the median raphe nucleus.

The present study evaluated the involvement of α-adrenoceptors of the median raphe nucleus (MRN) in satiated rats, in food and water intake and motor behaviour. Control groups were treated with saline (SAL) or adrenaline (ADR), injected into the MRN seven minutes after injection of the vehicle used to solubilize the antagonists, propylene glycol (PLG) or SAL. Experimental groups were treated with an α-adrenoceptor antagonist, prazosin (α1, 20 or 40 nmol) or yohimbine (α2, 20 or 40 nmol) or phentolamine (non-selective α, 20 or 40 nmol), followed (later) by injection of ADR or SAL. Behaviour was recorded for 30 min. The injection of ADR and the blockade of α1 receptors resulted in hyperphagia whereas blocking α2 or α1 and α2 simultaneously did not change feeding behaviour. Pre-treatment with prazosin, followed by injection of ADR was not able to cause an increase in the amount of food ingested, while the higher dose of the α1 antagonist reduced the latency to start feeding. Pre-treatment with prazosin also caused hyperactivity. However, pre-treatment with phentolamine or yohimbine was able to block ADR-induced feeding. The present study supports the hypothesis that there is a tonic activation of α1-adrenoceptors in the MRN in satiated rats, which activates an inhibitory influence in areas that control food intake. Injection of ADR seems to activate α2 receptors, resulting in a decrease in the availability of endogenous catecholamines, which reduces the release of the signal that inhibits food intake, leading to hyperphagia.

Role of alpha- and beta-adrenoreceptors in rat monocyte/macrophage function at rest and acute exercise.

Previous studies from our laboratory have demonstrated that a single bout of moderate exercise stimulates macrophage function, increasing phagocytic capacity, and production of hydrogen peroxide and nitric oxide (NO˙) through nuclear factor kappa B activation. In this work, we investigated the role of α- and ß-adrenoreceptors on the function of monocyte/macrophages during rest and exercise. Adult male Wistar rats were i.p. administered (100 µL/100 g) with specific adrenergic antagonists before an acute moderate exercise bout: prazosin (α1-specific antagonist 2 mg/kg), propranolol (unspecific ß1/ß2 antagonist 10 mg/kg), double blockade (α1 and ß1/ß2), or phosphate-buffered saline (control). Acute exercise consisted in a single swimming session of moderate intensity (5% body weight overload on the chest) for 60 min. Control groups (rest) received the same antagonists and were killed 60 min after drug administration. Exercise increased phagocytic capacity (1.7-fold, p < 0.05), NO˙ production (5.24 fold, p < 0.001), and inducible nitric oxide synthase (NOS2) expression (by 58.1%), thus suggesting macrophage activation. The ß-adrenoreceptor blockade did not change this behavior. In resting animals, α1 antagonist, as well as the double (α1/ß) blockade, however, further increased phagocytic capacity (by up to 261%, p < 0.001), NO˙ production (by up to 328%, p < 0.001), and the expressions of NOS2 (by 182%, p < 0.001) and HSP70 (by 42.5%, p < 0.01) suggesting a tonic inhibitory effect of α1 stimulation on macrophage activation. In exercised animals, α1-blockade showed similar enhancing effect on phagocytic indices and expressions of NOS and HSP70, particularly in double-blocked groups, although NO˙ production was found to be reduced in exercised animals submitted to both α- and ß-blockade. Redox (glutathione) status and lipoperoxidation were evaluated in all test groups and approximately paralleled macrophage NO˙ production. We suggest the prevalence of a peripheral α1-adrenoreceptor inhibitory tonus that limits macrophage responsiveness but operates differently after physical exercise.

Non-hormonal management of vasomotor symptoms.

Vasomotor symptoms are the most common indication for the prescription of hormone replacement therapy since it is effective in over 80% of cases. In 1995, 37% of American women took hormone replacement therapy, principally for this purpose. However, following the publication of results from the Women's Health Initiative, as many as half of these women in the US and in the UK and New Zealand discontinued hormone therapy. Discontinuation of estrogen is often accompanied by a return of vasomotor symptoms; however, only a small number (18%) of women report restarting hormone therapy. Alternatives are available, but limited knowledge on etiology and mechanisms of hot flushing represents a major obstacle for the development of new, targeted, non-hormonal treatments, and no current alternatives are as effective as estrogen.

Exposure of human seminal vesicle tissue to phosphodiesterase (PDE) inhibitors antagonizes the contraction induced by norepinephrine and increases production of cyclic nucleotides.

OBJECTIVES: To investigate further the role of phosphodiesterase (PDE) isoenzymes in the control of human seminal vesicle (SV) smooth muscle contractility, we examined the functional responses of isolated SV tissue to various PDE inhibitors. It has been suggested that the application of inhibitors of the PDE type 5 may facilitate SV smooth muscle relaxation and, subsequently, retard ejaculatory response. METHODS: Using the organ bath technique, strip preparations of human SV were exposed for 5 minutes to 1 µM of the PDE inhibitors milrinone (PDE3 inhibitor), rolipram, Ro 20-1724 (PDE4 inhibitors), and sildenafil (PDE5 inhibitor). Norepinephrine (NE, alpha agonist) was then added (0,1 µM, 1 µM, and 10 µM) and isometric responses were recorded. A contraction-response curve to NE in the absence of PDE inhibitors was also generated. Drug effects on the production of cyclic adenosine monophosphate (AMP) and cyclic guanosine monophosphate (GMP) were measured by means of radioimmunometric assays. RESULTS: The contraction induced by NE was effectively antagonized by 1 µM of rolipram (83.3% inhibition), Ro 20-1724 (72.3% inhibition), sildenafil (41.6% inhibition), and milrinone (37.5% inhibition). The inhibition of force generation was paralleled by a 1.6-fold to 2.8-fold increase in tissue cyclic AMP (induced by milrinone, rolipram, Ro 20-1724), and a 12-fold rise in cyclic GMP (induced by sildenafil). CONCLUSION: The findings demonstrate that PDE inhibitors can counteract the contraction of human SV mediated by alpha-adrenergic receptors and enhance levels of cyclic nucleotides. This might be of importance with regard to the identification of new options for the pharmacological treatment of premature ejaculation.

Functional study of mucus secretion of the eustachian tube in Guinea pigs.

OBJECTIVES: Our aim was to verify neural regulation of submucous gland mucus secretions in the Eustachian tubes of guinea pigs. STUDY DESIGN: Prospective animal study. METHODS: Eustachian tubes harvested from 12 guinea pigs were used for this study. For real-time resolution of pure glandular secretion, we used a modified method of single-gland optical measurement. Secretory monitoring was undertaken after each preparation with phenylephrine, isoproterenol, forskolin, and substance P. To confirm the viability of each tissue, we examined glandular secretion after treatment with carbachol. Secretory effects of each agonist were evaluated by comparing with basal secretion using a Student's t test (p < 0.01). RESULTS: The Ca-elevating agonists carbachol and substance P showed greater effects on submucous gland secretions of the Eustachian tube than the cyclic adenosine monophosphate (cAMP)-elevating agonists forskolin and isoproterenol. However, phenylephrine, although it belongs to the Ca-elevating agonist group, did not show any significant secretory effect. CONCLUSION: The optical measurement method used in this study had the merit of real-time resolution of submucous glandular secretion. Submucous glandular secretion in the Eustachian tube was regulated by both Ca- and cAMP-elevating agonists, and Ca-elevating agonists seemed to be more potent than cAMP-elevating agonists except phenylephrine. Our results suggest that not only the autonomic nerve system but also the neuropeptides such as substance P are closely related to glandular secretion in the Eustachian tube, and beta-adrenergic receptors seem to be more related to submucous glandular secretion of the Eustachian tube in guinea pig than alpha-adrenergic receptors.

Adrenergic regulation of the rapid component of delayed rectifier K+ current: implications for arrhythmogenesis in LQT2 patients.

BACKGROUND: KCNH2 gene mutations disrupting rapid component of I(K) (I(Kr)) underlie type 2 congenital long QT syndrome (LQT2). Startled auditory stimuli are specific symptomatic triggers in LQT2, thus suggesting fast arrhythmogenic mechanism. OBJECTIVE: We investigated acute alpha(1A)- and cyclic adenosine monophosphate (cAMP)-related beta-adrenergic modulation of I(Kr) in HL-1 cardiomyocytes, wild type (WT)- and 2 LQT2-associated mutant Kv11.1 channels (Y43D- and K595E-Kv11.1) reconstituted in Chinese hamster ovary (CHO) cells. METHODS: I(Kr) and Kv11.1 currents were recorded using the whole-cell patch-clamp technique and confocal microscopy of HL-1 cardiomyocytes transfected with green fluorescent protein (GFP)-tagged pleckstrin homology domain of phospholipase C-delta(1) visualized fluctuations of membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) content. RESULTS: In HL-1 cardiomyocytes expressing human alpha(1A)-adrenoceptor, superfusion with phenylephrine significantly reduced I(Kr) amplitude, shifted current activation to more positive potentials, and accelerated kinetics of deactivation. Confocal images showed a decline of membrane PIP(2) content during phenylephrine exposure. Simultaneous application of adenylyl cyclase activator forskolin and phosphodiesterase inhibitor 3-isobutyl-1-methylxantine (IBMX) shifted I(Kr) activation to more negative potentials and decreased tail current amplitudes after depolarizations between +10 and +50 mV. In CHO cells, alpha(1A)-adrenoceptor activation downregulated WT-Kv11.1 channels and forskolin/IBMX produced a dual effect. Expressed alone, the Y43D-Kv11.1 or K595E-Kv11.1 channel had no measurable function. However, co-expression of WT-Kv11.1 and each mutant protein evoked currents with loss-of-function alterations but identical to WT-Kv11.1 alpha(1A)- and forskolin/IBMX-induced regulation. CONCLUSION: Acute adrenergic regulation of at least 2 Kv11.1 mutant channels is preserved as in WT-Kv11.1 and native I(Kr). Suppression of alpha(1A)-adrenoceptor-related transduction might have therapeutic implications in some cases of LQT2.

Alpha1-adrenoceptors mediate dihydroxyphenylalanine-induced activity in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned macaques.

The mechanisms underlying actions of dihydroxyphenylalanine (L-DOPA) in Parkinson's disease remain to be fully elucidated. Noradrenaline formed from L-DOPA may stimulate alpha(1)-adrenoceptors. We assessed the involvement of alpha(1)-adrenoceptors in actions of L-DOPA in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned macaques. In each animal, the minimal dose of L-DOPA required to alleviate parkinsonian symptoms was defined (12.5-25 mg/kg p.o.). The effects of coadministration of the alpha(1)-adrenoceptor antagonist prazosin ([4-(4-amino-6,7-dimethoxy-quinazolin-2-yl) piperazin-1-yl]-(2-furyl)methanone) on motor activity, parkinsonism, and dyskinesia were assessed. Antiparkinsonian benefit was accompanied by mild dyskinesia. L-DOPA also elicited hyperactivity, i.e., activity greater than that seen in normal animals. Coadministration of prazosin (0.16-0.63 mg/kg p.o.) with L-DOPA did not significantly affect either its antiparkinsonian actions or dyskinesia. However, prazosin significantly and dose-dependently attenuated L-DOPA-induced activity, reducing it to a level equivalent to that of normal animals. More specifically, during periods of pronounced L-DOPA-induced activity, prazosin attenuated the total and duration of activity by 80 and 76%, respectively. These actions of prazosin were expressed in the absence of sedation. Although activation of alpha(1)-adrenoceptors plays no major role in the antiparkinsonian and dyskinetic effects of L-DOPA per se, it does contribute to the induction of hyperactivity. alpha(1)-Adrenoceptors may be involved in pathological responses to L-DOPA treatment, including the dopamine dysregulation syndrome.

Graded sympatholytic effect of exogenous ATP on postjunctional alpha-adrenergic vasoconstriction in the human forearm: implications for vascular control in contracting muscle.

Recent evidence suggests that adenosine triphosphate (ATP) can inhibit vasoconstrictor responses to endogenous noradrenaline release via tyramine in the skeletal muscle circulation, similar to what is observed in contracting muscle. Whether this involves direct modulation of postjunctional alpha-adrenoceptor responsiveness, or is selective for alpha(1)- or alpha(2)-receptors remains unclear. Therefore, in Protocol 1, we tested the hypothesis that exogenous ATP can blunt direct postjunctional alpha-adrenergic vasoconstriction in humans. We measured forearm blood flow (FBF; Doppler ultrasound) and calculated the vascular conductance (FVC) responses to local intra-arterial infusions of phenylephrine (alpha(1)-agonist) and dexmedetomidine (alpha(2)-agonist) during moderate rhythmic handgrip exercise (15% maximum voluntary contraction), during a control non-exercise vasodilator condition (adenosine), and during ATP infusion in eight young adults. Forearm hyperaemia was matched across all conditions. Forearm vasoconstrictor responses to direct alpha(1)-receptor stimulation were blunted during exercise versus adenosine (DeltaFVC = -11 +/- 3% versus -39 +/- 5%; P< 0.05), and were abolished during ATP infusion (-3 +/- 2%). Similarly, vasoconstrictor responses to alpha(2)-receptor stimulation were blunted during exercise versus adenosine (-13 +/- 4% versus -40 +/- 8%; P< 0.05), and were abolished during ATP infusion (-4 +/- 4%). In Prototol 2 (n = 10), we tested the hypothesis that graded increases in ATP would reduce alpha(1)-mediated vasoconstriction in a dose-dependent manner compared with vasodilatation evoked via adenosine. Forearm vasoconstrictor responses during low dose adenosine (-38 +/- 3%) and ATP (-33 +/- 2%) were not significantly different from rest (-40 +/- 3%; P> 0.05). In contrast, vasoconstrictor responses during moderate (-22 +/- 6%) and high dose ATP (-8 +/- 5%) were significantly blunted compared with rest, whereas the responses during adenosine became progressively greater (moderate = -48 +/- 4%, P = 0.10; high = -53 +/- 6%, P< 0.05). We conclude that exogenous ATP is capable of blunting direct postjunctional alpha-adrenergic vasoconstriction, that this involves both alpha(1)- and alpha(2)-receptor subtypes, and that this is graded with ATP concentrations. Collectively, these data are consistent with the conceptual framework regarding how muscle blood flow and vascular tone are regulated in contracting muscles of humans.

Neurotransmission and cancer: implications for prevention and therapy.

Published evidence compiled in this review supports the hypothesis that the development, progression, and responsiveness to prevention and therapy of the most common human cancers is strongly influenced, if not entirely orchestrated, by an imbalance in stimulatory and inhibitory neurotransmission. The neurotransmitters acetylcholine, adrenaline, and noradrenaline of the autonomic nervous system act as powerful upstream regulators that orchestrate numerous cell and tissue functions, by releasing growth factors, angiogenesis factors and metastasis factors, arachidonic acid, proinflammatory cytokines, and local neurotransmitters from cancer cells and their microenvironment. In addition, they modulate proliferation, apoptosis, angiogenesis, and metastasis of cancer directly by intracellular signaling downstream of neurotransmitter receptors. Nicotine and the tobacco-specific nitrosamines have the documented ability to hyperstimulate neurotransmission by both branches of the autonomic nervous system. The expression and function of these neurotransmitter pathways are cell type specific. Lifestyle, diet, diseases, stress, and pharmacological treatments modulate the expression and responsiveness of neurotransmitter pathways. Current preclinical testing systems fail to incorporate the modulating effects of neurotransmission on the responsiveness to anticancer agents and should be amended accordingly. The neurotransmitter gamma-aminobutyric acid has a strong inhibitory function on sympathicus-driven cancers whereas stimulators of cyclic adenosine monophosphate/protein kinase A signaling have strong inhibitory function on parasympathicus-driven cancers. Marker-guided restoration of the physiological balance in stimulatory and inhibitory neurotransmission represents a promising and hitherto neglected strategy for the prevention and therapy of neurotransmitter-responsive cancers.

Physiological regulation of penile arteries and veins.

Recent experimental evidence suggests that arterial insufficiency precedes the structural and functional changes in corpora cavernosa (CC) leading to organic erectile dysfunction (ED). The present review gives an overview of the physiological factors involved in the regulation of penile vasculature. Sympathetic nerves maintain flaccidity and tonically released noradrenaline induces vasoconstriction of both arteries and veins through alpha(1)- and alpha(2)-postsynaptic receptors and downregulates its own release and that of nitric oxide (NO) through alpha(2)-presynaptic receptors. The sympathetic cotransmitter neuropeptide Y (NPY) modulates noradrenergic vasoconstriction in penile small arteries by both enhancing and depressing noradrenaline contractions through Y(1)- and Y(2)-postsynaptic and a NO-independent atypical endothelial receptor, respectively. Activation of alpha(1)-adrenoceptors involves both Ca(2+) influx through L-type and receptor-operated Ca(2+) channels (ROC) and Ca(2+) sensitization mechanisms mediated by protein kinase C (PKC), tyrosine kinases (TKs) and Rho kinase (RhoK). In addition, RhoK can regulate Ca(2+) entry in penile arteries upon receptor stimulation. Vasodilatation of penile arteries and large veins during erection is mediated by neurally released NO. The subsequent increased arterial inflow to the cavernosal sinoids and shear stress on the endothelium lining penile arteries activates endothelial NO production through Akt phosphorylation of endothelial NO synthase (eNOS). NO stimulates guanylate cyclase and increased cyclic guanin 3'-monophosphate (cGMP) levels in turn activate protein kinase G (PKG), which enhances K(+) efflux through Ca(2+)-activated (K(Ca)) and voltage-dependent Ca(2+) (K(v)) channels in penile arteries and veins, respectively. PKG-mediated decrease in Ca(2+) sensitivity and its regulation by RhoK remains to be clarified in penile vasculature. Phosphodiesterase type 5 (PDE5) inhibitors are potent vasodilators of penile resistance arteries and increase the content and effects of basally released endothelial NO. Endothelium-dependent relaxations of penile small arteries also include an endothelium-derived hyperpolarizing factor (EDHF)-type response, which is impaired in diabetes and hypertension-associated ED. Locally produced contractile and relaxant prostanoids regulate penile venous and arterial tone, respectively. The latter activates prostaglandin I (IP) and prostaglandin E (EP) receptors coupled to adenylate cyclase and to the increase of cyclic adenosine monophosphate (cAMP) levels, which in turn stimulates K(+) efflux through ATP-sensitive K(+) (K(ATP)) channels. There is a crosstalk between the cGMP and cAMP signaling pathways in penile small arteries. Relevant issues such as the mechanisms underlying the excitation-secretion coupling of the endothelial cells, as well as those involved in cell proliferation and vascular remodeling of the penile vasculature remain to be elucidated. In addition, only few studies have investigated the changes in structure and function of penile arteries in cardiovascular risk situations leading to ED.

Common approach to managing lower urinary tract symptoms and erectile dysfunction.

The present paper serves as a review of the associations between lower urinary tract symptoms (LUTS) and erectile dysfunction (ED), with a focus on common and combined pathways for treatment. LUTS and ED are common conditions seen in general urologic practice. Research has started to establish epidemiologic and pathophysiologic links between the two conditions and a strong association confirmed across multiple studies. Men seeking care for one condition should always be interviewed for complaints of the other condition. Proposed common pathways include alpha-1 adrenergic receptor imbalance, Rho-kinase overactivity, endothelial cell dysfunction and atherosclerosis-induced ischemia. Medical therapy has replaced surgery as the first-line treatment for LUTS in most patients, with the incorporation of alpha-adrenergic receptor antagonists (alpha-ARAs) and 5-alpha-reductase inhibitors (5-ARIs) into everyday practice. Treatment with alpha-ARAs contributes to some improvement in ED, whereas use of 5-ARIs results in worsened sexual function in some patients. Phosphodiesterase-5 (PDE-5) inhibitors have revolutionized the treatment of ED with a simple oral regimen, and new insights demonstrate a benefit of combined use of PDE-5 inhibitors and alpha-ARAs. The mechanisms of action of these medications support these observed benefits, and they are being studied in the basic science and clinical settings. In addition, novel mechanisms for therapy have been proposed based on clinical and research observations. The minimally invasive and surgical treatments for LUTS are known to have adverse effects on ejaculatory function, while their effects on erectile function are still debated. Much remains to be investigated, but it is clear that the associations between LUTS and ED lay the foundation for future therapies and possible preventative strategies.

Characteristics of adrenaline-driven receptor-mediated signals in human microvessel-derived endothelial cells.

Adrenaline (0.001-1,000 muM) strongly stimulated adenosine-3',5'cyclic monophosphate (cAMP) generation in cultured human microvascular-derived endothelial cells (HMEC-1). Isoprenaline mimicked the action of adrenaline, whereas noradrenaline appeared to be decisively less potent. Experiments carried out with an array of compounds acting selectively on different types/subtypes of adrenergic receptors revealed that the adrenaline cAMP effect in HMEC-1 cells did not possess either an alpha(1) or alpha(2) component. However, the effect may have been mediated through a receptor that did not fit beta(1)-, beta(2)-, or beta(3)-receptor classification. Supporting this assertion, various double and triple beta-subtype selective drug combinations maximally inhibited the adrenaline effect by 50-60%, whereas the non-selective antagonist propranolol totally prevented the hormone-evoked cAMP effect. Based on results utilizing the phosphodiesterase (PDE)-isoform nonselective inhibitor 3-isobutyl-1-methylxanthine (IBMX) and the PDE-4-selective inhibitor rolipram, the adrenaline-driven cAMP signal appeared to be regulated by PDE-4. In addition, the present study demonstrated that phenylephrine, a presumed selective alpha(1)-adrenoceptor agonist, was capable of stimulating cAMP generation in HMEC-1 cells in a prazosin-insensitive and propranolol-sensitive manner. This result indicated that in at least this cell model system, phenylephrine may act nonspecifically. Microvessel-derived endothelial cells such as HMEC-1 exhibit functional differences when compared with macrovessel-derived endothelial cells (e.g. HUVEC sensitivity to adrenaline). Accordingly, these cell cultures represent a useful model system to study the biological effects of endogenous catecholamines, including adrenaline, as well as potential therapeutics targeting adrenergic receptors.