microM Norepinephrine Research Articles

Norepinephrine exacerbates the suppressive action of intermittent hypoxia on synaptic transmission

While acute intermittent hypoxia (IH) has the potential to be harnessed for therapeutic rehabilitation, IH experienced with sleep apnea causes a deterioration in neurophysiology that contributes to cardiorespiratory dysfunction and cognitive decline. A previous investigation in respiratory control demonstrated that the impact of acute IH on neuronal activity is dependent on increased noradrenergic tone—a common feature in sleep apnea. This ongoing study tests the hypothesis that norepinephrine (NE) influences the impact of IH on synaptic transmission in the hippocampus. Electrophysiological recordings of the field excitatory postsynaptic potential (fEPSP) were made in the CA1 neuronal population of adult mouse hippocampal brain slices. Acute IH was achieved by exposing preparations to three hypoxic intervals (95% N2, duration=180 sec) separated by periods of recovery (95% O2, duration=180 sec). During acute IH, the initial hypoxic interval did not suppress synaptic transmission, yet subsequent intervals suppressed the fEPSP (second interval by 16%, third interval by 22%; n=5). 2 microM NE did not affect the fEPSP during baseline conditions or during the initial hypoxic interval (n=5). However, the increased noradrenergic tone augmented fEPSP suppression during the subsequent intervals of hypoxia (second interval by 46%, third interval by 65%; n=5). These findings suggest that the efficacy of IH to suppress glutamatergic synaptic transmission is dependent on the state of oxygenation and may contribute to disrupted neurophysiology observed with sleep apnea.

Endothelium-dependent vascular responses in 1,3-dipropyl-8-sulphophenylxanthine (DPSPX) hypertensive rats.

The study was undertaken to test the endothelium-mediated vascular responses in rats rendered hypertensive by chronic administration of 1,3-dipropyl-8-sulphophenylxanthine (DPSPX). The relaxant effect of carbachol (an endothelium-dependent relaxing drug) and of sodium nitroprusside (an endothelium-independent relaxing drug) as well as the potentiation of the contractile effect of noradrenaline by NG-nitro-L-arginine methyl ester (L-NAME) were compared in aortic rings from normotensive and DPSPX-hypertensive rats. Carbachol and sodium nitroprusside caused concentration-dependent relaxations in aortic rings precontracted by 1 microM noradrenaline. The relaxant effect being of carbachol was significantly reduced in tissues of DPSPX-hypertensive rats: the maximal relaxant effect being 86 +/- 3% and 64 +/- 4% (of the pre-existing tone) in normal and hypertensive rats, respectively, while there were no significant differences in the relaxant effect of sodium nitroprusside. L-NAME (100 microM) significantly reduced the EC50 values of noradrenaline (3.71 +/- 0.28 times, n = 8 and 2.96 +/- 0.27 times, n = 7, in normal and hypertensive rats, respectively) and significantly enhanced the maximal contractile effect of noradrenaline (46 +/- 8%, n = 8 and 35 +/- 6%, n = 7, in normal and hypertensive rats respectively): the factors of reduction of EC50 values and the percentages of enhancement of the maximal contractile effect in the aorta of normal and hypertensive rats were not significantly different. The results obtained provide evidence of functional impairment of the endothelium in DPSPX-hypertensive rats.

The neuroendocrine hormone norepinephrine increases Pseudomonas aeruginosa PA14 virulence through the las quorum-sensing pathway

It has been proposed that the gastrointestinal tract environment containing high levels of neuroendocrine hormones is important for gut-derived Pseudomonas aeruginosa infections. In this study, we report that the hormone norepinephrine increases P. aeruginosa PA14 growth, virulence factor production, invasion of HCT-8 epithelial cells, and swimming motility in a concentration-dependent manner. Transcriptome analysis of P. aeruginosa exposed to 500 microM, but not 50 microM, norepinephrine for 7 h showed that genes involved in the regulation of the virulence determinants pyocyanin, elastase, and the Pseudomonas quinolone signal (PQS, 2-heptyl-3-hydroxy-4-quinolone) were upregulated. The production of rhamnolipids, which are also important in P. aeruginosa infections, was not significantly altered in suspension cultures upon exposure to 500 microM norepinephrine but decreased on semisolid surfaces. Swarming motility, a phenotype that is directly influenced by rhamnolipids, was also decreased upon 500 microM norepinephrine exposure. The increase in the transcriptional activation of lasR but not that of rhlR and the increase in the levels of PQS suggest that the effects of norepinephrine are mediated primarily through the las quorum-sensing pathway. Together, our data strongly suggest that norepinephrine can play an important role in gut-derived infections by increasing the pathogenicity of P. aeruginosa PA14.

Testosterone Relaxes Rabbit Seminal Vesicle by Calcium Channel Inhibition

Recent studies have documented that testosterone relaxes several smooth muscles by modulating K(+) channel activities. Smooth muscles of seminal vesicles play a fundamental role in ejaculation, which might involve testosterone. This study was aimed to assess the role of testosterone in seminal vesicular motility by studying its effects on contractile agents and on the ion channels of single vesicular myocytes in a rabbit model. The contractile responses of circular smooth muscle strips of rabbit seminal vesicles to norepinephrine (10 microM), a high concentration of KCl (70 mM), and testosterone (10 microM) were observed. Single vesicular myocytes of rabbit were isolated using proteolytic enzymes including collagenase and papain. Inside-out, attached, and whole-cell configurations were examined using the patch clamp technique. The applications of 10 microM norepinephrine or 70 mM KCl induced tonic contractions, and 10 microM testosterone (pharmacological concentration) evoked dose-dependent relaxations of these precontracted strips. Various K(+) channel blockers, such as tetraethylammonium (TEA; 10 mM), iberiotoxin (0.1 microM), 4-aminopyridine (4-AP, 10 microM), or glibenclamide (10 microM) rarely affected these relaxations. Single channel data (of inside-out and attached configurations) of BK channel activity were also hardly affected by testosterone (10 microM). On the other hand, however, testosterone reduced L-type Ca(2+) currents significantly, and found to induce acute relaxation of seminal vesicular smooth muscle and this was mediated, at least in part, by Ca(2+) current inhibition in rabbit.

Activation of Large-Conductance Calcium-Activated Potassium Channels by Puerarin: The Underlying Mechanism of Puerarin-Mediated Vasodilation

Puerarin is the main isoflavone found in Pueraria lobata (Willd) Ohwi, which has been used in therapy for various cardiovascular diseases. The present study examined the effects of puerarin on the large-conductance voltage- and Ca2+-activated potassium (BK(Ca)) channel and on rat thoracic aortas. BK(Ca) channels encoded with either alpha (BK-alpha) or alpha/beta subunits (BK-alpha+beta1) were heterologously expressed in Xenopus oocytes or human embryonic kidney 293 cells. The activities of BK(Ca) channels were measured using excised patch-clamp recordings. Puerarin activated BK-alpha+beta1 currents with a half-maximal concentration (EC50) of 0.8 nM and a Hill coefficient of 1.11 at 10 microM Ca2+ and with an EC50 of 12.6 nM and a Hill coefficient of 1.08 at 0 microM Ca2+. Puerarin (1 nM) induced a 16-mV leftward shift in the conductance-voltage curve for BK-alpha+beta1 currents at 10 microM Ca2+ and at 100 nM induced a 26-mV leftward shift at 0 microM Ca2+. Puerarin mainly increased the BK-alpha+beta1 channel open probability without changing the unitary conductance. Activation was also detected in the absence of the beta1 subunit. A deglycosylated analog of puerarin, daidzein, also activated BK(Ca) channels with weaker potency. In addition, puerarin (0.1 to 1000 microM) caused concentration-dependent relaxations of rat thoracic aortic rings contracted with 1 microM noradrenaline bitartrate (EC50 = 1.1 microM). These were significantly inhibited by 50 nM iberiotoxin, a specific blocker of BK(Ca) channels. This is the first study demonstrating that puerarin activates BK(Ca) channels, especially BK-alpha+beta1 channels. The activation of the BK(Ca) channel probably contributes to the puerarin-mediated vasodilation action.

Functional expression of kinin B1 and B2 receptors in mouse abdominal aorta

Previous studies have shown that the vascular reactivity of the mouse aorta differs substantially from that of the rat aorta in response to several agonists such as angiotensin II, endothelin-1 and isoproterenol. However, no information is available about the agonists bradykinin (BK) and DesArg(9)BK (DBK). Our aim was to determine the potential expression of kinin B(1) and B(2) receptors in the abdominal mouse aorta isolated from C57BL/6 mice. Contraction and relaxation responses to BK and DBK were investigated using isometric recordings. The kinins were unable to induce relaxation but concentration-contraction response curves were obtained by applying increasing concentrations of the agonists BK and DBK. These effects were blocked by the antagonists Icatibant and R-715, respectively. The potency (pD(2)) calculated from the curves was 7.0 +/- 0.1 for BK and 7.3 +/- 0.2 for DBK. The efficacy was 51 +/- 2% for BK and 30 +/- 1% for DBK when compared to 1 microM norepinephrine. The concentration-dependent responses of BK and DBK were markedly inhibited by the arachidonic acid inhibitor indomethacin (1 microM), suggesting a mediation by the cyclooxygenase pathway. These contractile responses were not potentiated in the presence of the NOS inhibitor L-NAME (1 mM) or endothelium-denuded aorta, indicating that the NO pathway is not involved. We conclude that the mouse aorta constitutively contains B(1) and B(2) subtypes of kinin receptors and that stimulation with BK and DBK induces contractile effect mediated by endothelium-independent vasoconstrictor prostanoids.

Norepinephrine induces apoptosis in neonatal rat endothelial cells via a ROS-dependent JNK activation pathway

Our previous study demonstrated that norepinephrine (NE) induces endothelial apoptosis mainly through down-regulation of Bcl-2 protein and activation of the beta-adrenergic and caspase-2 pathways. However, whether reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPKs) are involved in this signal transduction remains unknown. Endothelial cells cultured from neonatal rat heart were treated with 100 microM NE. Proteins of MAPKs and Bcl-2 family were assayed by Western blotting. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated nick end-labeling assay. ROS was analyzed with flow cytometry. Caspase activity was measured using specific fluorogenic substrates. Treatment with NE increased intracellular ROS level and extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 phosphorylation. Whereas the phosphorylated form of Akt was decreased. The NE-induced apoptosis was abrogated by SP600125 (a specific inhibitor of JNK). Antioxidants such as vitamin C and N-acetyl cysteine inhibited NE-induced ROS production, JNK phosphorylation, caspase activation and apoptosis. Exogenously added superoxide dismutase or catalase markedly diminished NE-induced ROS production and cell death. In conclusions, our study is the first report documenting that NE induces apoptosis in neonatal rat endothelial cells via a ROS-dependent JNK activation pathway. Antioxidants may be useful in the prevention and management of NE-mediated endothelial apoptosis during heart failure.

Regulation of the crossbridge cycle in vascular smooth muscle by cAMP signalling

Urocortin, a novel vasodilatory peptide related to the corticotropin-releasing factor (CRF) increased cAMP levels to 220.8 +/- 27.6% of control in rat tail arteries. The effect was completely abolished by the adenylyl cyclase inhibitor, SQ22536 (100 microM). Urocortin also decreased phosphorylation of the regulatory light chains of myosin (MLC20) in rat tail arteries stimulated with high K+ from 27.5 +/- 0.9% (control) to 13 +/- 2% (n = 5). This suggests that urocortin relaxes blood vessels via cAMP-mediated dephosphorylation of MLC20. Previously we have shown that urocortin-induced vasodilation can be ascribed to a decrease in Ca2+ -sensitivity of tension and activation of smooth muscle myosin phosphatase (SMPP-1M). In this study, we provide evidence that urocortin-induced Ca2+ -desensitization does not affect agonist-induced Ca2+ -sensitization. Urocortin relaxed alpha-toxin permeabilized mouse tail arteries preconstricted with pCa 6.1, but did not prevent the Ca2+ -sensitization induced by 10 microM 5-HT, 100 microM norepinephrine (NE) or 1 microM GTPgammaS. In keeping, the maximally relaxing concentration of urocortin (100 nM) had no effect on the concentration dependence of the phenylephrine-induced Ca2+ -sensitization. By contrast, treatment with the cAMP analogue, cBIMPS (100 microM), or the Rho kinase inhibitor, H-1152 (3 microM) relaxed the mouse vessels to a greater extend and completely inhibited phenylephrine (PE) induced sensitization. The lack of effect of urocortin on agonist-induced sensitization could be due to a alpha-adrenergic receptor mediated inhibition of cAMP generation. Furthermore PE induced Ca2+ -sensitization was reported to occur independent of changes in MLC20 phosphorylation involving caldesmon. Our results are compatible with a model in which urocortin/cAMP signalling only affects the myosin linked regulation of vascular tone while cBIMPS may inactivate in addition the MLC20 phosphorylation independent pathway.

NITRIC OXIDE MODULATES BIPHASIC CONTRACTILE RESPONSE OF GUINEA PIG VAS DEFERENS TO ELECTRICAL FIELD STIMULATION

Electrical field stimulation (EFS) produced a biphasic contractile response; viz. initial rapid phasic contraction and second slow tonic contraction, in isolated guinea pig vas deferens. Pretreatment with the substrate of nitric oxide (NO) synthase (NOS), 1 mM L-arginine (L-ARG), augmented both the initial rapid and the second slow contractile responses to EFS (5 Hz, 0.5 msec, 30 V, for 30 sec). The increase of stimulation frequency from 5 Hz to 10 Hz or 20 Hz tended to attenuate the augmented responses. On the contrary, pretreatment with an inhibitor of NOS, 0.1 mM NG-nitro-L-arginine (L-NNA) suppressed both the initial rapid and the second slow contractile responses to EFS. The suppressive effect on the initial rapid contraction was also attenuated by the increase of stimulation frequency from 5 Hz or 10 Hz to 20 Hz. Contractile response to exogenously administered 1 mM adenosine triphosphate (ATP) tended to be slightly increased and decreased by the treatment with 1 mM L-ARG and 0.1 mM L-NNA, respectively. Contractile response to exogenously administered 10 microM noradrenaline (NA) was almost unaffected by the treatment with 1 mM L-ARG, while the treatment with 0.1 mM L-NNA slightly depressed the response. Potentiated contractile response to 1 mM ATP in the presence of 10 microM NA was further potentiated by the treatment with 1 mM L-ARG, while the response was almost unaffected by the treatment with 0.1 mM L-NNA. These findings may indicate that NO acts mainly on presynaptic site and increases the release of chemical transmitter, ATP or prevents the inactivation of ATP. Also, NO may act, at least in part, on postsynaptic site and potentiates the contractile response to ATP in the presence of NA.

Induction of β3-Adrenergic Receptor Functional Expression following Chronic Stimulation with Noradrenaline in Neonatal Rat Cardiomyocytes

This study aimed to characterize beta(3)-adrenergic receptors (ARs) in rat neonatal cardiomyocytes using the noradrenaline (NOR) properties to modulate the expression and function of the three beta-ARs. We assessed the effect of NOR (physiological nonselective agonist), isoprenaline (ISO, beta-nonselective agonist), dobutamine (DOB, beta(1)-selective agonist), and procaterol (PROC, beta(2)-selective agonist) on cAMP accumulation using cardiomyocytes untreated or treated with 100 microM NOR for 24 h. The inhibition of forskolin-stimulated cAMP accumulation was determined using NOR, isoprenaline, and the beta(3)-selective agonists 4-[2-[(2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxyacetic acid (BRL 37344) and 5-[-2-([-2-(3-chlorophenyl)-2-hydroxyethyl]amino)propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL 316243). The experiments were performed in the absence or presence of propranolol or 2-hydroxy-5-[2-[[2-hydroxy-3-[4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy]propyl]amino]ethoxy]-benzamide methanesulfonate (CGP 20712A) and/or 1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride (ICI 118551) to inhibit beta(1)- and beta(2)-AR stimulation and 1-(2-ethylphenoxy)-3-[[1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino-(2S)-2-propanol hydrochloride (SR 59230A) (beta(3)-selective antagonist). In addition, the level of the three subtypes was determined by reverse transcription polymerase chain reaction and Western blotting. NOR pretreatment decreased the activation of cAMP induced by NOR, isoprenaline, and DOB, whereas PROC response was abolished. The inhibition of NOR response by CGP 20712A or ICI 118551 demonstrated that beta(1)- and beta(2)-ARs are down-regulated and that beta(2)-AR functional activity was also abolished in cardiomyocytes exposed to chronic stimulation. beta(3)-AR function was observed with NOR and ISO when beta(1)-/beta(2)-ARs were blocked and with both beta(3)-selective agonists in NOR-treated cells only. This response was completely inhibited by SR 59230A and involved G(i) protein. Furthermore, the results from functional studies agree well with those from expression experiments. In conclusion, these data provide strong evidence that beta(3)-ARs are functionally up-regulated and coupled to G(i) protein in rat neonatal cardiomyocytes following chronic exposure to NOR when beta(1)- and beta(2)-ARs are down-regulated.

Influence of low molecular weight heparin preparations on human internal thoracic artery contraction

Low molecular weight heparins (LMWHs) offer practical and potential pharmacological advantages over unfractionated heparin in multiple applications but have not been studied as vasoactive agents. The purpose of this study was to investigate the effects of two commercial preparations of LMWHs, enoxaparin sodium and nadroparin calcium, on vasoconstriction in the human internal thoracic artery (ITA) in vitro. Samples of redundant ITA segments obtained from 36 patients who underwent coronary artery bypass surgery were cut into 3mm wide rings and suspended in 20 ml organ bath. Activity of ITA rings precontracted with 80 mM KCl, 0.1 microM endothelin-1 (ET-1) and 1 microM norepinephrine (NE) after administration of enoxaparin and nadroparin in accumulative concentration ranging from 0.1 to 13.2 UI AXa/ml were recorded under isometric conditions by means of force transducers with digital output. The contraction after 80 mmol KCl, 0.1 microM ET-1 and 1 microM NE administration was treated as a control. Both studied LMWHs in concentration ranging from 0.12 to 13.2 UI AXa/ml did not change basal tonus and KCl precontracted ITA rings. When used in concentrations higher than 13.2 UI AXa/ml nadroparin but not enoxaparin significantly increased the tension in KCl precontracted arterial rings. In NE and ET-1 precontracted rings enoxaparin and nadroparin caused dose dependent relaxation without significant differences between both preparations. Incubation with nitric oxide blocker-Nomega-NITRO-L-ARGININE (L-NNA) in concentration 0.2 mM caused a significant attenuation of relaxant responses to both studied LMWHs in NE and ET-1 precontracted rings. LMWHs can have vasorelaxant effects on the receptor-mediated ITA vasoconstriction. The results suggest that LMWHs-induced relaxation in the human ITA is at least partially caused by nitric oxide release. Although the vasoactive effects are not the primary advantage of these drugs used as antithrombotics, such effects might have some clinical importance in the treatment and prophylaxis of graft spasm.

Tannins and catechin gallate mediate the vasorelaxant effect of Arbutus unedo on the rat isolated aorta

This study examined the vascular effect of Arbutus leaves (aqueous extract) and described the isolation of several fractions responsible for their vasorelaxant activity. The aqueous extract (AE) of leaves was tested on rat aortic rings precontracted with 0.1 microm noradrenaline. At 10(-2) g/L, AE produced an endothelium dependent relaxation of 66% +/- 5%, (n = 8). The leaves of Arbutus were then extracted successively with different solvents and the methanol extract was the most active. When tannins (primarily condensed tannins) were precipitated from the methanol extract, they showed a strong vasorelaxant activity (87% +/- 4%, n = 5), whereas the elimination of tannins in the methanol extract reduced significantly its vasorelaxant activity (42% +/- 8%, n = 8, p < 0.005). The methanol extract was further separated semi-preparatively by reversed-phase HPLC. Four fractions (Fr2, Fr3, Fr4 and Fr6) were the most active and produced 88% +/- 2% (n = 5), 75% +/- 6% (n = 5), 76% +/- 3% (n = 7) and 77% +/- 3% (n = 10) relaxation, respectively. These four fractions mainly correspond to polyphenol compounds. Analysis of Fr6 indicated that this fraction contained catechin gallate. In conclusion, the vasorelaxant activity of Arbutus is likely to be due to polyphenol compounds, primarily condensed tannins and catechin gallate.

Simultaneous alpha2B- and beta2-adrenoceptor activation sensitizes the alpha2B-adrenoceptor for agonist-induced down-regulation.

We recently reported that alpha(2A)-adrenoceptor (AR) desensitization and down-regulation occurs after 24-h treatment with epinephrine (EPI) (0.3 microM) in BE(2)-C cells that express both alpha(2)- and beta(2)-ARs. The same concentration of norepinephrine (NE) has no effect. The effect of EPI is prevented by beta(2)-AR blockade and is associated with an increase in G protein-coupled receptor kinase 3 (GRK3) expression. Because differences in agonist-induced down-regulation of the alpha(2A)-versus alpha(2B)-ARs have been reported, the present study examines the effects of simultaneous activation of alpha(2B)- and beta(2)-ARs on alpha(2B)-AR number and signaling. We studied NG108 cells that naturally express alpha(2B)-ARs, and BN17 cells, NG108 cells transfected to express the human beta(2)-AR. In NG108 cells, alpha(2B)-AR desensitization and down-regulation require treatment with 20 microM EPI or NE; GRK expression was not changed. In BN17 cells expressing beta(2)-ARs, the threshold EPI concentration for alpha(2B)-AR desensitization and down-regulation was reduced to 0.3 microM; 10 microM NE was required for the same effect. Furthermore, 24-h EPI or NE treatments that produced desensitization also resulted in a selective 2-fold up-regulation of GRK3; GRK2 was unchanged. The beta-AR antagonist alprenolol (1 microM) and GRK3 antisense (but not sense) DNA blocked 0.3 microM EPI- and 10 microM NE-induced desensitization and down-regulation of the alpha(2B)-AR as well as GRK3 up-regulation. In conclusion, simultaneous activation of alpha(2B)- and beta(2)-ARs results in a 67-fold decrease in the threshold concentration of EPI required for alpha(2B)-AR down-regulation. This lower threshold for down-regulation is associated with alpha(2B)- and beta(2)-AR dependent up-regulation of GRK3 expression.

Interactions between cGMP- and cAMP-pathways are involved in the regulation of penile smooth muscle tone.

Nitric oxide (NO)/cyclic GMP (cGMP)-mediated mechanisms have a pivotal function in reducing the tone of the penile smooth musculature during normal erectile responses. The cyclic AMP (cAMP) signaling pathway is also involved in the adjustment of smooth muscle contractility, and suggestions for interactions between cGMP- and cAMP-mediated mechanisms have been presented. Using activators of the cGMP- or the cAMP-pathway, as well as inhibitors of protein kinase A (PKA; cAMP-dependent kinase) and protein kinase G (PKG; cGMP-dependent kinase), the present study was undertaken to further delineate the functional relation between these pathways in the penis. In addition, the distribution of PKA and some cAMP-binding phosphodiesterases (cAMP-PDEs) were investigated in human erectile tissue. Functional experiments were performed on isolated human corpus cavernosum (HCC). The effects of an inhibitor of the PKA, Rp-8CPT-cAMPS (10 microM), or the PKG, Rp-8-pCPT-cGMPS (10 microM), on relaxation induced by the cumulative administration of sodium nitroprusside (SNP), forskolin, sildenafil or tadalafil (IC351) were studied in preparations of HCC precontracted with 1 microM norepinephrine (NE). Using immunohistochemical procedures, the presence of immunoreactivity for cAMP-PDEs PDE3, PDE4, and PDE4A, as well as for PKA was investigated in specimens of HCC from which preparations were also used in the functional experiments. Forskolin, SNP, sildenafil, and IC 351 dose-dependently reversed NE-induced tension of isolated HCC preparations. The relaxing effects of SNP were significantly attenuated by Rp-8-pCPT-cGMPS, but not by Rp-8CPT-cAMPS. In contrast, relaxation induced by forskolin, sildenafil and tadalafil were significantly reversed by both Rp-8-pCPT-cGMPS and Rp-8CPT-cAMPS. Abundant immunoreactivity for PDE3 and PKA was observed in the corpus cavernosum smooth muscle cells. Immunoreactivity for PDE4 was also detected in the smooth musculature and in the cytoplasm of endothelial cells lining the cavernous sinusoids, as well as in nerve fibres interspersing the trabecular stroma. The present results support the hypothesis of interactions between cGMP- and cAMP-mediated signals in the HCC, and suggest that the effects of inhibitors of PDE5 on isolated erectile tissue may also partly or indirectly include actions of the cAMP second messenger system. The exact mechanism by which such an interaction occurs is not clear, but it may involve altered activity of the cGMP-inhibited PDE3 brought about by a change in the intracellular levels of cGMP by the inhibition of PDE5. This will in turn lead to increasing levels of cAMP, facilitating the interaction of cAMP with the PKA. The immunoreactivity specific for PDE3, PDE4, PDE4A and PKA registered in HCC section is also in support of an important role for the cAMP/PKA-system for penile smooth muscle function.

Non-contractile cells with thin processes resembling interstitial cells of Cajal found in the wall of guinea-pig mesenteric arteries.

Arterial interstitial cells of Cajal (ICC)-like cells (AIL cells) with a multipolar, irregular, elongated shape and with numerous thin (often less than 1 microm), sometimes branching, processes with lengths up to approximately 60 microm were isolated enzymatically from 1st to 7th order branches of guinea-pig mesenteric artery. Some of the processes of AIL cells were growing (average speed approximately 0.15 microm min-1) and their growth was blocked by 10 microM latrunculin B, an inhibitor of actin polymerisation. Staining with BODIPY phalloidin, a fluorescent dye selective for F-actin, showed the presence of F-actin in the processes of AIL cells. Voltage clamp of single AIL cells revealed an inward current that was four times more dense than in myocytes and was abolished by 10 microM nicardipine, and an outward current carried exclusively by potassium ions that was reduced by 1 mM 4-aminopyridine and/or 100 nM iberiotoxin but unaffected by 10 nM dendrotoxin-K. Imaging of intracellular ionised calcium with fluo-4 using a laser scanning confocal microscope showed local or global calcium transients lasting several seconds in approximately 28 % of AIL cells. When membrane current was recorded simultaneously, the calcium transients were found to correspond to long-lasting transient outward currents, which occurred at potentials positive to -40 mV. Unlike myocytes, AIL cells did not contract in response to 1 mM caffeine or 5 microM noradrenaline, although they responded with a [Ca2+]i increase. The segments of intact arteries did not stain for c-kit, a marker of ICCs. Single AIL cells stained positive for vimentin, desmin and smooth muscle myosin. The presence of ICC-like cells is demonstrated for the first time in the media of resistance arteries.

Role of Ca2+-activated Cl- current during proarrhythmic early afterdepolarizations in sheep and human ventricular myocytes.

The proarrhythmic early afterdepolarizations (EADs) during phase-2 of the cardiac action potential (phase-2 EADs) are associated with secondary Ca2+-release of the sarcoplasmic reticulum. This makes it probable that the Ca2+-activated Cl- current [ICl(Ca)] is present during phase-2 EADs. Activation of ICl(Ca) during phase-2 of the action potential will result in an outwardly directed, repolarizing current and may thus be expected to prevent excessive depolarization of phase-2 EADs. The present study was designed to test this hypothesis. The contribution of ICl(Ca) during phase-2 EADs was studied in enzymatically isolated sheep and human ventricular myocytes using the patch-clamp methodology. EADs were induced by a combination of a low stimulus frequency (0.5 Hz) and exposure to 1 microm noradrenaline. In sheep myocytes, the ICl(Ca) blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, 0.5 mm) abolished phase-1 repolarization of the action potential in all myocytes tested. This indicates that ICl(Ca) is present in all sheep myocytes. However, DIDS had no effect on phase-2 EAD characteristics. In human myocytes, DIDS neither affected phase-1 repolarization nor phase-2 EAD characteristics. In sheep ventricular myocytes, but not in human ventricular myocytes, ICl(Ca) contributes to phase-1 repolarization of the action potential. In both sheep and human myocytes, ICl(Ca) plays a limited role during phase-2 EADs.

The xanthones gentiacaulein and gentiakochianin are responsible for the vasodilator action of the roots of Gentiana kochiana.

Gentiana kochiana Perr. et Song. (Gentianaceae), a plant used in the traditional medicine of Tuscany (Italy) as antihypertensive remedy, exerts a vasodilator action on in vitro aortic rings that is probably linked to the blocking of the ryanodine-sensitive Ca++ channels. In the present study, three known xanthones were isolated from the crude methanolic extract of the roots: gentiacaulein, gentiakochianin, and swertiaperennin. The first two showed a vasorelaxing activity in rat aortic preparations, pre-contracted by 3 microM norepinephrine (pIC50 = 5.00 +/- 0.032 for gentiacaulein, pIC50 = 4.95 +/- 0.068 for gentiakochianin), 20 mM KCl (pIC50 = 4.90 +/- 0.15 for gentiacaulein; 4.59 +/- 0.069 for gentiakochianin), or 5 mM caffeine; on the contrary, in the same conditions, swertiaperennin did not show any vasodilator effect. In conclusion, gentiacaulein and gentiakochianin seem to be the compounds responsible for the vasorelaxing properties of the crude extract of Gentiana kochiana roots.

Extracellular nucleotides induce vasodilatation in human arteries via prostaglandins, nitric oxide and endothelium‐derived hyperpolarising factor

1. The present study was aimed at examining P2 receptor-mediated vasodilatation in human vessels. The isometric tension was recorded in isolated segments of the human left internal mammary artery branches precontracted with 1 microM noradrenaline. 2. Endothelial denudation abolished the dilator responses. 3. The selective P2Y(1) agonist, 2-MeSADP, induced a potent vasodilatation (pEC(50)=6.9+/-0.1). The P2Y(1) antagonist of 10 microM, MRS 2216, shifted the 2-MeSADP concentration-response curve 1.1 log units to the right. The combined P2Y(1) and P2X agonist, 2-MeSATP, stimulated a dilatation with a potency similar to that of 2-MeSADP. Furthermore, MRS 2216 had a similar antagonistic effect on both 2-MeSATP and 2-MeSADP indicating that P2X receptors do not mediate vasodilatation. 4. Both the P2Y(2/4) agonist, UTPgammaS and the P2Y(6) agonist, UDPbetaS, stimulated potent dilatations (pEC(50)=7.8+/-0.4 for UTPgammaS and 8.4+/-0.2 for UDPbetaS). 5. The 2-MeSADP-induced nitric oxide (NO)-mediated dilatation was studied in the presence of 10 micro M indomethacin, 50 nM charybdotoxin and 1 microM apamin. The involvement of the endothelium-derived hyperpolarising factor (EDHF) was investigated in the presence of 0.1 mM L-NOARG and indomethacin. The involvement of prostaglandins was investigated in the presence of L-NOARG, charybdotoxin and apamin. Both NO, EDHF and prostaglandins mediated 2-MeSADP dilatation with similar efficacy (E(max)=25+/-5% for NO, 25+/-6% for EDHF and 27+/-5% for prostaglandins). 6. In conclusion, extracellular nucleotides induce endothelium-derived vasodilatation in human vessels by stimulating P2Y(1), P2Y(2/4) and P2Y(6) receptors, while P2X receptors are not involved. Endothelial P2Y receptors mediate dilatation by release of EDHF, NO and prostaglandins.

Hypoxia inhibits contraction but not calcium channel currents or changes in intracellular calcium in arteriolar muscle cells.

We tested the hypothesis that hypoxia inhibits currents through L-type Ca(2+) channels and inhibits norepinephrine-induced rises in intracellular Ca(2+) in cremasteric arteriolar muscle cells, thus accounting for the inhibitory effect of hypoxia on norepinephrine-induced contraction of these cells. Single smooth muscle cells were enzymatically isolated from second-order and third-order arterioles from hamster cremaster muscles. The effects of hypoxia (partial pressure of oxygen: 10-15 mm Hg) were examined on Ba(2+) (10 mM) currents through L-type Ca(2+) channels by use of the perforated patch clamp technique. Also, the effect of hypoxia on norepinephrine-induced calcium changes was studied using Fura 2 microfluorimetry. Hypoxia inhibited the norepinephrine-induced (10 microM) contraction of single arteriolar muscle cells by 32.9 +/- 5.6% (mean +/- SE, n = 4). However, hypoxia had no significant effect on whole-cell currents through L-type Ca(2+) channels: the peak current densities measured at +20 mV were -3.83 +/- 0.40 pA/pF before hypoxia and -3.97 +/- 0.36 pA/pF during hypoxia (n = 15; p > 0.05). In addition, hypoxia did not inhibit Ca(2+) transients in arteriolar muscle cells elicited by 10 microM norepinephrine. Instead, hypoxia increased basal Ca(2+) (13.8 +/- 3.2%) and augmented peak Ca(2+) levels (29.4 +/- 7.3%) and steady-state Ca(2+) levels (15.2 +/- 5.4%) elicited by 10 microM norepinephrine (n = 21; p < 0.05). These data indicate that hypoxia inhibits norepinephrine-induced contraction of single cremasteric arteriolar muscle cells by a mechanism that involves neither L-type Ca(2+) channels nor norepinephrine-induced Ca(2+) mobilization. Instead, our findings suggest that hypoxia must inhibit norepinephrine-induced contraction by affecting a component of the signaling pathway that lies downstream from the increases in Ca(2+) produced by this neurotransmitter.

Stress and the periodontal diseases: effects of catecholamines on the growth of periodontal bacteria in vitro.

Microorganisms possess the ability to recognize hormones within the host and utilize them to adapt to their surroundings. Noradrenaline and adrenaline, which are released during human stress responses, may act as environmental cues to alter the growth of individual organisms within subgingival biofilms. The aims of this study were to modify, for anaerobic culture, existing methodology used in determining microorganism catecholamine responses and to investigate the growth responses to noradrenaline and adrenaline of 43 microorganisms found within subgingival microbial complexes. We established initial inocula for each strain using anaerobic culture, re-inoculated into a minimal serum-based medium and grown anaerobically at 35 degrees C. We assessed organism growth by optical density (OD(600nm)) readings, with test and control cultures performed in triplicate. Test cultures were supplemented with 50 microm noradrenaline or 100 microm adrenaline. We observed significant growth effects for supplementation with noradrenaline (20 species responding positively) and adrenaline (27 species responding positively), with differences in growth response observed within bacterial species and within and between microbial complexes. The most pronounced positive growth effects of noradrenaline were demonstrated in Actinomyces naeslundii (+ 49.4%), Actinomyces gerenscseriae (+ 57.2%), Eikenella corrodens (+ 143.3%) and Campylobacter gracilis (+ 79.9%). We also observed inhibitory effects of noradrenaline supplementation for Porphyromonas gingivalis (- 11.9%) and Bacteroides forsythus (- 22.2%). Responses to adrenaline tended to mirror the responses seen with noradrenaline. Individual organisms from different microbial complexes vary in their in vitro growth responses to noradrenaline and adrenaline. Such variation may influence the in vivo composition of the subgingival biofilm in response to stress-induced changes in local catecholamine levels and play a significant role in the aetiology and pathogenesis of the periodontal diseases.