Stimulatory Effect Of Norepinephrine Research Articles

Norepinephrine-Induced Stimulation of Kir4.1/Kir5.1 Is Required for the Activation of NaCl Transporter in Distal Convoluted Tubule.

The stimulation of β-adrenergic receptor increases thiazide-sensitive NaCl cotransporter (NCC), an effect contributing to salt-sensitive hypertension by sympathetic stimulation. We now test whether the stimulation of β-adrenergic receptor-induced activation of NCC is achieved through activating basolateral Kir4.1 in the distal convoluted tubule (DCT). Application of norepinephrine increased the basolateral 40 pS K+ channel (Kir4.1/Kir5.1 heterotetramer) in the DCT. The stimulatory effect of norepinephrine on the K+ channel was mimicked by cAMP analogue but abolished by inhibiting PKA (protein kinase A). Also, the effect of norepinephrine on the K+ channel in the DCT was recapitulated by isoproterenol but not by α-adrenergic agonist and blocked by propranolol, suggesting that norepinephrine effect on the K+ channel was mediated by β-adrenergic receptor. The whole-cell recording shows that norepinephrine and isoproterenol increased DCT K+ currents and shifted the K+ current ( IK) reversal potential to negative range (hyperpolarization). Continuous norepinephrine perfusion (7 days) increased DCT K+ currents, hyperpolarized IK reversal potential, and increased the expression of total NCC/phosphorylated NCC, but it had no significant effect on the expression of NKCC2 (type 2 Na-Cl-K cotransporter) and ENaC-α (epithelial Na channel-α subunit). Renal clearance study demonstrated that norepinephrine perfusion augmented thiazide-induced urinary Na+ excretion only in wild-type but not in kidney-specific Kir4.1 knockout mice, suggesting that Kir4.1 is required for mediating the effect of norepinephrine on NCC. However, norepinephrine perfusion did not affect urinary K+ excretion. We conclude that the stimulation of β-adrenergic receptor activates the basolateral Kir4.1 in the DCT and that the activation of Kir4.1 is required for norepinephrine-induced stimulation of NCC.

Beta-adrenergic regulation of adenylyl cyclase signaling system in the myocardium and brain of rats with obesity and type 2 diabetes mellitus as affected by long-term intranasal insulin administration

The stimulatory effects of norepinephrine, isoproterenol and selective s3-adrenoceptor agonists BRL 37344 and CL 316.243 on the adenylyl cyclase signaling system (ACSS) in the brain and myocardium of young and mature rats with experimental obesity and type 2 diabetes mellitus (DM2) (disease induction at 2 and 4 months of age, respectively) and the influence of the long-term intranasal insulin (I-I) administration were studied. It was shown that the AC-stimulatory effects of s-agonist isoproterenol in animals with obesity and DM2 practically did not change. The respective effects of norepinephrine on the AC activity slackened in the brain of young and mature rats and myocardium of mature rats, while the I-I treatment led to their partial recovery. In the brain and myocardium of mature rats with obesity and DM2 an increase in the AC-stimulatory effects of s3- AR agonists was observed, while in young rats the similar influence of the same pathologies was missing. The I-I treatment resulted in an attenuation of the AC-stimulatory effects of s3-agonists to their control level. Since the disruption of the adrenergic agonist-sensitive ACSS is one of the causes of metabolic syndrome and DM2, the recovery of ACSS dysfunctions by the long-term I-I administration offers one of the ways of correcting these diseases and their complications in the nervous and cardiovascular systems.

Norepinephrine stimulates the epithelial Na+ channel in cortical collecting duct cells via α2-adrenoceptors.

There is good evidence for a causal link between excessive sympathetic drive to the kidney and hypertension. We hypothesized that sympathetic regulation of tubular Na(+) absorption may occur in the aldosterone-sensitive distal nephron, where the fine tuning of renal Na(+) excretion takes place. Here, the appropriate regulation of transepithelial Na(+) transport, mediated by the amiloride-sensitive epithelial Na(+) channel (ENaC), is critical for blood pressure control. To explore a possible effect of the sympathetic transmitter norepinephrine on ENaC-mediated Na(+) transport, we performed short-circuit current (Isc) measurements on confluent mCCDcl1 murine cortical collecting duct cells. Norepinephrine caused a complex Isc response with a sustained increase of amiloride-sensitive Isc by ∼44%. This effect was concentration dependent and mediated via basolateral α2-adrenoceptors. In cells pretreated with aldosterone, the stimulatory effect of norepinephrine was reduced. Finally, we demonstrated that noradrenergic nerve fibers are present in close proximity to ENaC-expressing cells in murine kidney slices. We conclude that the sustained stimulatory effect of locally elevated norepinephrine on ENaC-mediated Na(+) absorption may contribute to the hypertensive effect of increased renal sympathetic activity.

Abstract 406: Mechanisms of stress-buffering effects of dopamine on tumor vasculature

Abstract Chronic stress is known to promote tumor growth by activating the sympathetic nervous system (SNS) with resultant increases in norepinephrine (NE) and epinephrine (E). In contrast to NE and E, dopamine (DA) levels are low under chronic stress conditions. We have recently demonstrated that dopamine replacement in mice exposed to daily restraint stress can reverse the stimulatory effects of NE and E on ovarian cancer growth. Ovarian tumor tissues from stressed mice treated with dopamine showed significant increases in pericyte coverage of tumor microvessels. The focus of the current study was to examine the mechanisms by which dopamine treatment affected pericyte coverage of tumor endothelial cells under stress conditions. Female nude mice were subjected to chronic stress using the restraint-stress procedure. Tumor formation was induced by injecting the SKOV3 ip1 or HeyA8 ovarian cancer cells into the peritoneal cavity. Stressed and non-stressed mice were divided into four treatment groups: Control (PBS), DA, DA plus butaclamol (DR1 antagonist) and DA plus eticlopride (DR2 antagonist). Following therapy, harvested tumors were examined for microvessel density (MVD), vascular maturation (pericyte coverage) and proliferating cell nuclear antigen (PCNA). Expression of DA receptors (DR1-DR5) was analyzed by RT-PCR and Western blotting. Mice exposed to daily restraint stress showed 2-fold increased SKOV3ip1-tumor growth compared to non-stressed mice; treatment with DA alone resulted in 78% (p<0.01) decrease in tumor growth and 68% (p<0.01) decrease in MVD compared to control stressed mice. Similarly, DA/butaclamol combined treatment led to 71% (p<0.01) reduction in tumor growth and a 65% (p<0.01) decrease in MVD. Eticlopride in combination with DA reversed the inhibitory effects of DA on tumor growth. Furthermore, in stressed mice, daily dopamine treatment resulted in significant increase in pericyte coverage (51.4%; p<0.001) compared to controls. This effect was abrogated by butaclamol (44%; p<0.01), but not by eticlopride treatment. Similar results were obtained in stressed mice bearing HeyA8-tumors. In T10.5-pericyte like cells, treatment with DA, DA plus NE, DA agonist: SKF38393 or PDGFBB increased significantly cell migration. No significant changes were noted with NE alone or DA plus butaclamol treatments. In MOEC, no significant changes in cell migration were observed under these experimental conditions. Collectively, our data indicate that DA, acting through DR1, could stimulate recruitment of pericytes to tumor endothelial cells, and promote vascular maturation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 406. doi:10.1158/1538-7445.AM2011-406

D1-like receptors inhibit insulin-induced vascular smooth muscle cell proliferation via down-regulation of insulin receptor expression

Vascular smooth muscle cell (VSMC) proliferation is central to the development of vascular diseases, including hypertension, which is regulated by numerous hormones and humoral factors. Our previous study showed that the stimulatory effect of norepinephrine on VSMC proliferation is inhibited by D1-like receptors and the D3 dopamine receptor, a member of the D2-like receptor family. Insulin is a proliferative hormone but it is not known if there is any interaction between insulin and D1-like receptors. We hypothesized that Dl-like receptors may have an inhibitory effect on the insulin-induced VSMC proliferation; aberrant insulin and Dl-like receptor functions could be involved in the pathogenesis of essential hypertension. VSMC proliferation was determined by [H]-thymidine incorporation; insulin receptor mRNA and protein expressions were determined by RT-PCR, immunoblotting, and immunohistochemistry. Insulin increased VSMC proliferation in immortalized aortic A10 cells, determined by [H]-thymidine incorporation. Although the D1-like receptor, by itself, had no effect on VSMC proliferation, stimulation with fenoldopam, a D1-like receptor agonist, inhibited the stimulatory effect of insulin. The inhibitory effect of fenoldopam on insulin-mediated VSMC proliferation was receptor specific, because its effect could be blocked by SCH23390, a D1-like receptor antagonist. Fenoldopam also inhibited insulin receptor mRNA and protein expression, which was time dependent and concentration dependent. A PKC or MAP kinase inhibitor blocked the inhibitory effect of fenoldopam on insulin receptor expression, indicating that PKC and MAP kinase were involved in the signaling pathway. The inhibitory effect of D1-like receptors on insulin-mediated VSMC proliferation may play an important role in the regulation of blood pressure.

Noradrenaline triggers muscle tone by amplifying glutamate‐driven excitation of somatic motoneurones in anaesthetized rats

Postural muscle tone is potently suppressed during sleep and cataplexy. Since brainstem noradrenergic cell discharge activity is tightly coupled with state-dependent changes in muscle activity, it is assumed that noradrenergic drive on to somatic motoneurones modulates basal muscle tone. However, it has never been determined whether noradrenergic neurotransmission acts to directly regulate motoneurone activity or whether it functions to modulate prevailing synaptic activity. This is an important distinction because noradrenaline regulates cell excitability by both directly depolarizing neurones and by indirectly potentiating glutamate-mediated excitation. We used reverse-microdialysis, electrophysiology, neuro-pharmacological and histological techniques in anaesthetized rats to determine whether strengthening noradrenergic drive (via exogenous noradrenaline application) on to trigeminal motoneurones affects masseter muscle tone by increasing spontaneous motoneurone activity or whether it acts to amplify prevailing glutamate-driven excitation. Although noradrenaline is hypothesized to modulate motor activity, we found that direct stimulation of trigeminal motoneurones by alpha(1)-adrenoceptor activation had no direct effect on basal masseter tone. However, when glutamate-driven excitation was increased at the trigeminal motor pool by either endogenous glutamate release (induced by the monosynaptic masseteric reflex) or exogenous AMPA application, noradrenaline triggered a potent increase in basal masseter tone. The stimulatory effects of noradrenaline were unmasked and rapidly switched on only in the presence of glutamatergic transmission. Blockade of AMPA receptors abolished this excitatory effect, indicating that noradrenergic drive requires ongoing glutamatergic activity. Our data indicate that exogenous noradrenergic drive does not directly affect spontaneous motoneurone discharge activity in anaesthetized rats; rather, it triggers postural muscle tone by amplifying prevailing glutamate-driven excitation.

Stimulatory effects of bioamines norepinephrine and dopamine on locomotion of Pyrrhocoris apterus (L.): Is the adipokinetic hormone involved?

In the present paper we studied the effects of five biogenic amines – norepinephrine, dopamine, octopamine, serotonin and histamine – on the locomotory activity and mobilization of lipids in the adult females of the firebug, Pyrrhocoris apterus (L.). We tested the hypothesis (1) whether the stimulation of walking activity in the bugs injected with the bioamines is associated also with their hyperlipaemic effects, like in the case of adipokinetic hormones (AKHs), and (2) whether these effects are direct or mediated through a release of the AKHs into the hemolymph. The results demonstrated that all five tested biogenic amines mobilized the fat body lipids, but only norepinephrine and dopamine were capable to enhance the walking activity simultaneously with an elevation of the lipid level in the hemolymph. Those two amines had no effect on the level of AKHs in CNS, but modulated the AKHs level in hemolymph: norepinephrine increased it, while dopamine decreased it. The results indicate an apparent feedback between AKH characteristics and dopamine and norepinephrine actions occurring in this insect species. While the stimulatory effects of norepinephrine on lipid mobilization and walking activity could involve the release of bug’s own AKHs, dopamine probably employs an independent stimulatory pathway.

Kyotorphin suppresses proliferation and Ca2+ signaling in brown preadipocytes

A positive correlation was revealed between stimulation of protein and DNA synthesis in preadipocytes by norepinephrine or neokyotorphin and intracellular Ca2+ concentration in these cells. Kyotorphin abolished the stimulatory effect of norepinephrine on proliferation of cultured cells and cold-induced [3H]-thymidine incorporation into DNA of mouse brown adipose tissue in vivo. These changes correlated with peptide-induced suppression of slow calcium signaling in preadipocytes.

Role of protein turnover in the activation of p38 mitogen-activated protein kinase in rat pinealocytes

Differences in the time profiles of activation between p38MAPK and p42/44MAPK by norepinephrine (NE) in rat pinealocytes suggest involvement of mechanisms other than the phosphorylation cascades in their activation. In the present study we investigated whether protein turnover played a role in regulating p38MAPK activation in the rat pineal gland. NE stimulation caused an increase in MAPK kinase3/6 (MKK 3/6) and p38MAPK phosphorylation that occurred in the absence of changes in the mRNA or protein levels of p38MAPK or MKK3/6. The stimulatory effect of NE on phosphorylated MKK3/6 and p38MAPK, but not phosphorylated p42/44MAPK, was blocked by treatment with actinomycin or cycloheximide, indicating a requirement of transcription and translation in activation of the p38MAPK but not the p42/44MAPK pathway. Moreover, inhibition of proteasomes by clasto-lactacystin β-lactone or Z-Leu-Leu-Leu-CHO (MG132) selectively increased basal and NE-stimulated phosphorylated MKK3/6 and p38MAPK levels without affecting the mRNA or protein levels of MKK3 or p38MAPK. In contrast, the effect of proteasomal inhibition on NE-stimulated p42/44MAPK phosphorylation was inhibitory. Treatment with MG132 also reduced the decline in the phosphorylated levels of NE-stimulated MKK3/6 and p38MAPK that normally follows β-adrenergic blockade. Together, our results indicate that p38MAPK but not p42/44MAPK activation in the rat pineal gland is tightly coupled to protein synthesis and degradation. The synthesis of an activator upstream of MKK3/6 is required for the NE-activation of p38MAPK.

Molecular Mechanisms of Modified Sensitivity of the Adenylate Cyclase Signaling System to Biogenic Amines during Streptozotocin-Induced Diabetes

We demonstrated changes in the sensitivity of the adenylate cyclase signaling system to biogenic amines (adrenoceptor agonists and serotonin) underwent a change in skeletal muscles of rats with 30-day streptozotocin-induced diabetes. Isoproterenol had a less significant stimulatory effect on adenylate cyclase in diabetic rats. Hormonal signals via Gi proteins were suppressed in animals with diabetes, which determined a greater stimulatory effect of norepinephrine and serotonin on adenylate cyclase. Hormones less significantly increased guanosine triphosphate-binding activity of G proteins in diabetic rats, which reflects the impairment of their functional coupling with receptors.

Uncoupling Protein 1 Is Necessary for Norepinephrine-Induced Glucose Utilization in Brown Adipose Tissue

Sympathetic stimulation activates glucose utilization in parallel with fatty acid oxidation and thermogenesis in brown adipose tissue (BAT) through the beta-adrenergic receptors. To clarify the roles of the principal thermogenic molecule mitochondrial uncoupling protein 1 (UCP1) in the sympathetically stimulated glucose utilization, we investigated the uptake of 2-deoxyglucose (2-DG) into BAT and some other tissues of UCP1-knockout (KO) mice in vivo. In wild-type (WT) mice, administration of norepinephrine (NE) accelerated the disappearance of plasma 2-DG and increased 2-DG uptake into BAT and heart without any rise of plasma insulin level. In UCP1-KO mice, the stimulatory effect of NE on 2-DG uptake into BAT, but not into heart, disappeared completely. Insulin administration increased 2-DG uptake into BAT and also heart similarly in WT and UCP1-KO mice. NE also increased the activity of AMP-activated protein kinase (AMP kinase) in BAT of WT but not UCP1-KO mice. Our results, together with reports that the activation of AMP kinase increases glucose transport in myocytes, suggest that the sympathetically stimulated glucose utilization in BAT is due to the serial activation of UCP1 and AMP kinase.

Involvement of Endogeneous Glutamate in the Stimulatory Effect of Norepinephrine and Serotonin on the Hypothalamo-Pituitary-Adrenocortical Axis

The effects of ionotropic glutamate receptor antagonists on the pituitary adrenal responses following injections of norepinephrine (NE) and serotonin (5-HT) receptor agonists into the hypothalamic paraventricular nucleus (PVN) or electrical stimulation of central NE and 5-HT pathways were studied in anesthetized male rats. PVN injections of an α₁-adrenergic receptor agonist or a serotonergic 5-HT_1A receptor agonist markedly increased both adrenocorticotropin (ACTH) and corticosterone (CS) serum levels. These responses were significantly inhibited by separate pre-injection of the selective non-NMDA and NMDA glutamate receptor subtype antagonists into the PVN in a dose-dependent manner. Electrical stimulation of either the ventral noradrenergic bundle or the dorsal raphe nucleus markedly increased serum ACTH and CS. These responses were also significantly attenuated by pre-injection of the above glutamate ionotropic receptor antagonists in a dose-dependent manner. These results suggest that glutamatergic interneurons in the PVN, acting via non-NMDA and NMDA receptors, may act as an excitatory mechanism in the NE and 5-HT control of hypothalamic ACTH secretagogues.

Mechanism of action of noradrenaline on secretion of progesterone and oxytocin by the bovine corpus luteum in vitro.

The present studies were conducted: (1) to determine which beta-adrenoceptor subtypes are involved in progesterone and oxytocin (OT) secretion, (2) to examine whether noradrenaline (NA) acts directly on the cytochrome P-450scc and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), and (3) to study the effect of prostaglandin F2 alpha (PGF2 alpha) on NA-stimulated steroidogenesis in luteal cells. The effect of NA on progesterone secretion from luteal slices of heifers on days 8-12 of the oestrous cycle was blocked by both atenolol (beta 1-antagonist) and ICI 118.551 hydrochloride (beta 2-antagonist). OT secretion was blocked only after treatment with ICI 118.551 hydrochloride (P < 0.05). Dobutamine (10(-4)-10(-6) M), a selective beta 1 agonist and salbutamol (10(-4)-10(-6) M), a selective beta 2 agonist, both increased progesterone production (P < 0.01) with an efficiency comparable to that produced by NA (P < 0.01). The increase of OT content in luteal slices was observed only after treatment with salbutamol at the dose of 10(-5) M (P < 0.01). Dobutamine had no effect on OT production at any dose. A stimulatory effect of NA on cytochrome P-450scc activity (P < 0.05) was demonstrated using 25-hydroxycholesterol as substrate. 3 beta-HSD activity also increased following NA (P < 0.01) or pregnenolone (P < 0.05) and in tissue treated with pregnenolone together with NA (P < 0.01). PGF decreased progesterone synthesis (P < 0.05) and 3 beta-HSD activity (P < 0.01) in tissue treated with NA. We conclude that NA stimulates progesterone secretion by luteal beta 1- and beta 2-adrenoceptors, while OT secretion is probably mediated only via the beta 2-receptor. NA also increases cytochrome P-450scc and 3 beta-HSD activity. PGF inhibits the luteotropic effect of NA on the luteal tissue.

Norepinephrine stimulates interleukin-6 mRNA expression in primary cultured rat hepatocytes.

Non-invasive immobilization stress causes an increase in the plasma interleukin (IL)-6 level accompanied by increased IL-6 mRNA expression and IL-6 immunoactivity in the liver [Biochem. Biophys. Res. Commun. (1997) 238, 707-711]. In the present study, using rat primary cultured hepatocytes and non-parenchymal liver cells, the effect of norepinephrine (NE) on IL-6 mRNA expression was determined. IL-6 mRNA expression in hepatocytes, but not in non-parenchymal liver cells, increased when the cells were treated with NE. The stimulatory effect of NE was inhibited by the combined use of alpha- and beta-adrenergic antagonists. IL-6 mRNA expression in hepatocytes also increased on incubation with the culture medium of non-parenchymal liver cells treated with NE. The effect of the medium was blocked by an IL-1 receptor antagonist. Moreover, exogenous IL-1beta stimulated IL-6 mRNA expression in hepatocytes. IL-1beta was present in the medium of non-parenchymal liver cells and increased with NE-treatment. These results suggest that NE released from sympathetic nerve terminals during stress can directly increase IL-6 mRNA expression in hepatocytes and indirectly through IL-1beta production from non-parenchymal liver cells.

Catecholamines and angiotensinogen gene expression in kidney proximal tubular cells.

To investigate the molecular mechanism(s) of action of catecholamines on the expression of the angiotensinogen (ANG) gene in kidney proximal tubular cells, we used opossum kidney (OK) cells with a fusion gene containing the 5'-flanking regulatory sequence of the rat ANG gene fused with a human growth hormone (hGH) gene as a reporter, pOGH (rANG N-1498/+18), permanently integrated into their genomes. The level of expression of the ANG-GH fusion gene was quantified by the amount of immunoreactive-hGH (IR-hGH) secreted into the medium. The addition of norepinephrine (NE), isoproterenol (a beta1/beta2-adrenergic receptor (AR) agonist) and iodoclonidine (an alpha2-AR agonist) stimulated the expression of the ANG-GH fusion gene in a dose-dependent manner, whereas the addition of epinephrine and phenylephrine (alpha1-AR agonist) had no effect. The stimulatory effect of NE was blocked by the presence of propranolol (beta-AR blocker), atenolol (beta1-AR blocker), yohimbine (alpha2-AR blocker), Rp-cAMP (an inhibitor of cAMP-dependent protein kinase AI & AII) and staurosporine (an inhibitor of protein kinase C), but was not blocked by ICI 118, 551 (beta2-AR blocker) and prazosin (alpha1-AR blocker). The addition of a combination of isoproterenol and iodoclonidine or a combination of 8-Bromo-cAMP (8-Br-cAMP) and phorbol 12-myristate (PMA) synergistically stimulated the expression of the ANG-GH fusion gene as compared to the addition of isoproterenol, iodoclonidine, 8-Br-cAMP or PMA alone. Furthermore, the addition of NE, 8-Br-cAMP or PMA stimulated the expression of pOGH (rANG N-806/-779/-53/+18), a fusion gene containing the putative cAMP responsive element (CRE, ANG N-806/-779) upstream of the ANG promoter (ANG N-53/+18) in OK cells, but had no effect on the expression of fusion genes containing the mutant of the CRE. Gel mobility shift assays revealed that the ANG-CRE binds with the DNA-binding domain (bZIP254-327) of the cAMP-responsive binding protein (CREB). The binding of the labeled ANG-CRE to CREB (bZIP254-327) was displaced by unlabeled ANG-CRE and the CRE of the somatostatin gene but not by the mutants of the ANG-CRE. Finally, NE stimulated the phosphorylation of CREB in OK cells. These studies demonstrate that the molecular mechanism(s) of NE action on the expression of the ANG gene in OK cells may be mediated via both the PKA and PKC signalling pathways and via the phosphorylation of CREB. The phosphorylated CREB then interacts with the CRE in the 5'-flanking region of the ANG gene and subsequently stimulates the gene expression.

Noradrenaline stimulates the production of prostaglandin f2alpha in cultured bovine endometrial cells.

The stimulatory effect of noradrenaline (NA) as well as oxytocin (OT) on bovine endometrial prostaglandin (PG) F2alpha production, and the intracellular mechanisms of their actions, were investigated in cultured bovine endometrial cells (a mixture of epithelial, stromal, and glandular cells). The cells were cultured in Dulbecco's Modified Eagle's medium and Ham's F-12 medium (1:1 [v:v]) with 10% calf serum. When the cells reached confluence, the culture medium was replaced with fresh medium with 0.1% BSA and various doses of NA (10(-8)-10(-4) M). NA stimulated PGF2alpha production in a dose-dependent manner (p < 0.05). To evaluate the intracellular mechanisms of NA and OT actions, the cells were treated with forskolin (an activator of adenylate cyclase), phorbol 12-myristate 13-acetate (PMA, an activator of protein kinase [PK] C), Rp-cAMP (a competitive cAMP antagonist and an inhibitor of PKA), U-73122 (an inhibitor of phospholipase [PL] C), or anthranilic acid (ACA, an inhibitor of PLA2). Forskolin and PMA stimulated PGF2alpha production in a dose-dependent manner (p < 0.05). Rp-cAMP completely inhibited (p < 0.001) the NA-induced, but not the OT-induced, PGF2alpha production. Although U-73122 inhibited only OT-induced PGF2alpha production (p < 0.001), ACA completely stopped the actions of NA and OT. The overall results indicate that NA as well as OT is involved in the regulation of the endometrial PGF2alpha production in cattle and that the stimulatory effects of NA and OT on PGF2alpha production are mediated via the PKA and PKC pathways, respectively.

Molecular mechanism(s) of action of norepinephrine on the expression of the angiotensinogen gene in opossum kidney cells

Norepinephrine (NE) is the major endogenous neurotransmitter of the renal sympathetic nerves interacting with both the alpha- and beta-adrenoceptors in the renal proximal tubules. We have previously reported that isoproterenol and iodoclonidine stimulate the expression of the angiotensinogen (ANG) gene in opossum kidney (OK) proximal tubular cells via the beta1-adrenoceptor and alpha2-adrenoceptor, respectively. We hypothesized that NE may interact with the beta- and/or alpha2-adrenoceptors to stimulate the expression of the ANG gene in OK cells. The fusion genes containing the various lengths of the 5'-flanking regulatory sequence of the rat ANG gene fused with a human growth hormone (hGH) gene as a reporter were stably transfected into the OK cells. The stimulatory effect of NE on the expression of the fusion genes was evaluated by the amount of immunoreactive hGH (IR-hGH) secreted into the culture medium. The addition of NE stimulated the expression of the fusion gene, pOGH (ANG N-1498/+18) in a dose-dependent manner. The stimulatory effect of NE was inhibited in the presence of propranolol, atenolol, Rp-cAMP, yohimbine, staurosporine, H-7 and U73122 but not in the presence of ICI 118,551 and prazosin. The addition of a combination of isoproterenol and iodoclonidine synergistically stimulated the expression of pOGH (ANG N-1498/+18) as compared to the addition of isoproterenol and iodoclonidine alone. Furthermore, the addition of NE, forskolin, 8-Br-cAMP or phorbol 12-myristate (PMA) stimulated the expression of pOGH (ANG N-806/-779/-53/+18), a fusion gene containing the putative cAMP responsive element (CRE, ANG N-806/-779) upstream of the ANG promoter (ANG N-53/+ 18) in OK 95 cells, but had no effect on the expression of fusion genes containing the mutant of the CRE. These studies demonstrate that the stimulatory effect of NE on the expression of the ANG gene in OK cells may be mediated via both the beta1- and alpha2-adrenoceptors and via the CRE (ANG N-806/-779) in the 5'flanking region of rat ANG gene.

Glucocorticoids provoke a shift from alpha2- to alpha1-adrenoreceptor activities in cultured hypothalamic slices leading to opposite noradrenaline effect on corticotropin-releasing hormone release.

We have shown previously that noradrenaline (NA) stimulated or inhibited the release of corticotropin-releasing hormone (CRH) according to the availability of adrenal steroids. The aim of the present work was to examine whether the changes in the NA modulation of CRH release from hypothalamic neurons result from a steroid-induced plasticity of the adrenergic transduction pathways. From anterior hypothalamic slices cultured in standard medium (i.e., containing adrenal steroids at a final dilution of 61 +/- 9 ng/ml), (a) the stimulatory effect of NA on CRH release was reversed in a dose-dependent manner by increasing concentrations of the alpha1-adrenoreceptor antagonist prazosin, (b) activation of protein kinase C by acute treatment with phorbol 12-myristate 13-acetate (0.5 microM, 1 h) mimicked NA stimulation of CRH secretion, and (c) the activation of L-type Ca2+ channels by Bay K 8644 also produce an increased CRH secretion. In contrast, the inhibitory effect of NA on CRH secretion from slices cultured in steroid-free medium was markedly reversed by the alpha2-adrenoreceptor antagonist yohimbine, by pretreatment with pertussin toxin, or by the addition of 4-aminopyridine, a K+-channel blocker. Acute treatment with phorbol 12-myristate 13-acetate did not change the inhibitory NA effect. Moreover, all these effects were reversed by daily corticosterone supplementation, for as long as they were tested. These results are consistent with a steroid-dependent change in the nature of adrenergic receptors and its associated transduction pathways involved in the regulation of CRH secretion in the hypothalamus.

Noradrenaline increases glucose transport into brown adipocytes in culture by a mechanism different from that of insulin

Glucose uptake into brown adipose tissue has been shown to be enhanced directly by noradrenaline (norepinephrine) released from sympathetic nerves. In this study we characterized the glucose transport system in cultured brown adipocytes, which responds to noradrenaline as well as insulin, and analysed the mechanism underlying the noradrenaline-induced increase in glucose transport. Insulin increased 2-deoxyglucose (dGlc) uptake progressively at concentrations from 10(-11) to 10(-6) M, with maximal stimulation at 10(-7) M. Noradrenaline concentrations ranging from 10(-8) to 10(-6) M also enhanced dGlc uptake, even in the absence of insulin. The effects of noradrenaline and insulin on dGlc uptake were additive. The stimulatory effect of noradrenaline was mimicked by the beta3-adrenergic agonist, BRL37344, at concentrations two orders lower than noradrenaline. Dibutyryl cyclic AMP also mimicked the stimulatory effect of noradrenaline, and the antagonist of cyclic AMP, cyclic AMP-S Rp-isomer, blocked the enhancement of glucose uptake due to noradrenaline. Furthermore Western blot analysis with an anti-phosphotyrosine antibody revealed that, in contrast with insulin, noradrenaline apparently does not stimulate intracellular phosphorylation of tyrosine, suggesting that the noradrenaline-induced increase in dGlc uptake depends on elevation of the intracellular cyclic AMP level and not on the signal chain common to insulin. When cells were incubated with insulin, the content of the muscle/adipocyte type of glucose transporter (GLUT4) in the plasma membrane increased, with a corresponding decrease in the amount in the microsomal membrane. In contrast, noradrenaline did not affect the subcellular distribution of GLUT4 or that of the HepG2/erythrocyte type of glucose transporter. Although insulin increased Vmax. and decreased the Km value for glucose uptake, the effect of noradrenaline was restricted to a pronounced decrease in Km. These results suggest that the mechanism by which noradrenaline stimulates glucose transport into brown adipocytes is not due to translocation of GLUT but is probably due to an increase in the intrinsic activity of GLUT, which is mediated by a cyclic AMP-dependent pathway.

Expression of beta3-adrenoceptor and stimulation of glucose transport by beta3-agonists in brown adipocyte primary culture.

Precursor cells of brown adipocytes were isolated from the interscapular brown fat of newborn rats and cultured on collagen-coated dishes. When confluent cells were treated with dexamethasone, mRNAs for muscle/adipocyte type of glucose transporter, hormone-sensitive lipase, and CCAAT/enhancer binding protein alpha were increased remarkably, confirming a predominant effect of dexamethasone on the terminal differentiation of the cultured cells. Effects of dexamethasone on the expression of three subtypes of beta-adrenoceptor were also examined. beta1- and beta2-adrenoceptor mRNAs remained constant regardless of dexamethasone-treatment, while beta3-adrenoceptor mRNA was present only in dexamethasone-treated differentiated cells. To assess the metabolic response mediated by beta3-adrenoceptor, glucose transport into the cells was estimated. Norepinephrine enhanced glucose transport in dexamethasone-treated differentiated cells, but not in undifferentiated cells. beta3-Adrenergic agonists mimicked completely the stimulatory effect of norepinephrine at concentrations lower by two orders of magnitude. These results suggest that the beta3-adrenoceptor is expressed during the course of differentiation in brown adipocytes and plays a significant role in the response of glucose transport to adrenergic stimulation.