Inhibitory Effect Of Noradrenaline Research Articles

The inhibitory effect of norepinephrine on the migration of ES-2 ovarian carcinoma cells involves a Rap1-dependent pathway

Our previous studies have shown that norepinephrine induces the migratory activity of human PC-3 prostate, SW 480 colon and MDA-MB-468 breast carcinoma cells. In contrast to these results, we show here that human ES-2 ovarian carcinoma cells have a reduced migratory activity after norepinephrine treatment. This inhibitory effect is possibly mediated by a cAMP-dependent activation of the small GTPase Rap1 via Epac. Furthermore, a key signalling event of the promigratory effect of norepinephrine in the above mentioned carcinoma cells is the activation of phospholipase C enzymes. In ES-2 cells, this part of the signalling cascade is constitutively active.

Vasopressinergic neurons in rats in ontogenesis: Responses to salt loading and their modulation by noradrenergic afferents

Salt loading in adult mammals leads to increased vasopressin secretion by vasopressinergic neurons in the supraoptic nucleus, which is mediated by the actions of a number of hormones and neurotransmitters, including noradrenaline. The present study addressed identification of the stage of ontogenesis at which vasopressinergic neurons start to respond to salt loading and when the noradrenalinergic regulation of this process begins. Studies were performed on rats at embryonic day 21 (E21), postnatal day 3 (P3), and postnatal day 13 (P13) using immunocytochemical and in situ hybridization. Animals were subjected to salt loading, in some cases on the background of the alpha1-adrenoceptor inhibitor prazosin. Salt loading in rats of all age groups induced increases in the synthesis of vasopressin mRNA, probably accompanied by increased synthesis of vasopressin peptide. At E21 and P3, intraneuronal vasopressin levels were increased; there was no change at P13. In salt loading on the background of prazosin administration, vasopressin mRNA and vasopressin contents at E21 showed no change, while at P3 they were increased, which is evidence of the inhibitory effect of noradrenaline on vasopressin expression in the early postnatal period. Thus, vasopressinergic neurons start to respond to salt loading at the end of the prenatal period with increases in vasopressin expression; noradrenergic afferents have inhibitory influences on vasopressin expression in the early postnatal period.

Sympathetic (Adrenergic) Innervation Modulates But Does Not Generate Basal Tone in the Internal Anal Sphincter Smooth Muscle

Sympathetic (Adrenergic) Innervation Modulates But Does Not Generate Basal Tone in the Internal Anal Sphincter Smooth Muscle

Adrenergic receptor modulation of hippocampal CA3 network activity

Norepinephrine (NE) has demonstrated proconvulsant and antiepileptic properties; however, the specific pharmacology of these actions has not been clearly established. To address this, we studied the effect of NE on hippocampal CA3 epileptiform activity. Frequency changes of burst discharges in response to NE were biphasic; low concentrations increased the number of bursts, while higher concentrations reduced their frequency, suggesting the involvement of multiple adrenergic receptor (AR) types. This hypothesis was confirmed when, in the presence of βAR blockade, increasing concentrations of NE caused a monophasic decrease in epileptiform activity. Antagonists selective for α 1 or α 2ARs were then used to determine which αAR type was involved. While discriminating concentrations of the α 1AR antagonists prazosin and terazosin had no effect, selective amounts of the α 2AR antagonists RS79948 and RX821002 significantly reduced the potency of NE in decreasing epileptiform activity. Furthermore, this antiepileptic action of NE persisted when all GABA-mediated inhibition was blocked. This data suggests that, under conditions of impaired GABAergic inhibition, the excitatory and inhibitory effects of NE on hippocampal CA3 epileptiform activity are mediated primarily via β and α 2ARs, respectively. Moreover, our results imply that the antiepileptic effect of α 2AR activation in CA3 is not dependent on the GABAergic system.

Alpha2 antagonist yohimbine suppresses maintained firing of rat prefrontal neurons in vivo.

As a general alpha2 adrenergic antagonist, yohimbine has been widely used for research needs and in the therapy of malfunction of the noradrenergic system. In this study we analysed the effects of iontophoretically applied yohimbine on 79 prefrontal cortical (PFC) neurons of the rat in vivo. Our results indicate the inhibitory effect of norepinephrine on PFC neurons, which was simulated by yohimbine. The effect of yohimbine was not blocked by known alpha1 or postsynaptic alpha2 receptor agents. These results indicate that yohimbine exerts its antagonising and hyperpolarising effect on presynaptic rather than postsynaptic alpha2 receptors. Since these receptors have a permissive role in maintaining neural activity, their malfunction may contribute to the behavioural deficits observed in prefrontal disorders.

Opposite effects of alpha 1- and beta-adrenoceptor stimulation on both glutamate- and gamma-aminobutyric acid-mediated spontaneous transmission in cultured rat hippocampal neurons.

The effects of adrenergic receptor stimulation on spontaneous synaptic transmission were investigated in cultured rat hippocampal neurons by recording spontaneous excitatory and inhibitory postsynaptic currents (sEPSC and sIPSC). Noradrenaline (NA) inhibited sEPSC in a concentration-dependent manner, with maximal effect at 10 microM. The alpha(1)- and alpha(2)-adrenoceptor-selective agonists cirazoline and clonidine induced an inhibition of sEPSC appearance, whereas the beta-adrenoceptor agonist isoproterenol elicited an increase. The inhibitory effect of NA was reversed by alpha(1)-adrenoceptor blockade. The participation of gamma-aminobutyric acid (GABA)(B)-receptor stimulation in the inhibitory effect of NA was further examined. GABA(B)-receptor stimulation with baclofen induced a strong inhibition of bursting activity, which was fully reversed by the GABA(B) antagonist CGP 55845. By itself, CGP 55845 exerted a stimulatory effect on sEPSC frequency. In the presence of CGP 55845, the inhibitory effects of cirazoline and clonidine were maintained. NA (1, 10, and 100 microM) and alpha-adrenoceptor agonists decreased miniature EPSC and IPSC occurrence, whereas beta-adrenergic stimulation increased it. In 50% of the cells examined, NA (1, 10 microM) had a stimulatory effect on sIPSC, whereas, in the remaining 50% of cells, NA (1, 10 microM) had an inhibitory effect. In all the cells, 100 microM NA induced an inhibition of sIPSC. The inhibitory effect of NA was due to alpha(1)-receptor stimulation, whereas the excitatory effect was due to beta-receptor stimulation. In cultured hippocampal neurons, spontaneous excitatory and inhibitory synaptic transmissions are both similarly altered by adrenoceptor stimulation. However, in a subset of cells, low concentrations of NA mediate an increase of sIPSC via beta-adrenoceptor activation.

Mechanisms of leptin secretion from white adipocytes.

The mechanisms regulating leptin secretion were investigated in isolated rat white adipocytes. Insulin (1-100 nM) linearly stimulated leptin secretion from incubated adipocytes for at least 2 h. The adrenergic agonists norepinephrine, isoproterenol (two nonselective beta-agonists), or CL-316243 (potent beta3) all inhibited insulin (10 nM)-stimulated leptin release. The inhibitory effects of norepinephrine and isoproterenol could be reversed not only by the nonselective antagonist propranolol but also by the selective antagonists ICI-89406 (beta1) or ICI-118551 (beta2), the beta2-antagonist being less effective than the beta1. Insulin-stimulated leptin secretion could also be inhibited by a series of agents increasing intracellular cAMP levels, such as lipolytic hormones (ACTH and thyrotropin-stimulating hormone), various nonhydrolyzable cAMP analogs, pertussis toxin, forskolin, methylxanthines (caffeine, theophylline, IBMX), and specific inhibitors of phosphodiesterase III (imazodan, milrinone, and amrinone). Significantly, antilipolytic agents other than insulin (adenosine, nicotinic acid, acipimox, and orthovanadate) did not mimic the acute stimulatory effects of insulin on leptin secretion under these conditions. We conclude that norepinephrine specifically inhibits insulin-stimulated leptin secretion not only via the low-affinity beta3-adrenoceptors but also via the high-affinity beta1/beta2-adrenoceptors. Moreover, it is suggested that 1) activation of phosphodiesterase III by insulin represents an important metabolic step in stimulation of leptin secretion, and 2) lipolytic hormones competitively counterregulate the stimulatory effects of insulin by activating the adenylate cyclase system.

Modulation of volume-sensitive chloride current by noradrenaline in rabbit portal vein myocytes.

The effect of noradrenaline on the volume-sensitive chloride current (I(Cl(swell))) was studied with conventional whole-cell recording techniques in freshly dispersed isolated smooth muscle cells of the rabbit portal vein. In the absence of receptor antagonists, noradrenaline produced an increase in the amplitude of I(Cl(swell)) in some cells and a decrease in others. In the presence of the beta-adrenoceptor antagonist propranolol, noradrenaline increased I(Cl(swell)) and in the presence of the alpha(1)-adrenoceptor antagonist prazosin, noradrenaline reduced I(Cl(swell).) The phospholipase C (PLC) inhibitor U73122 reduced the amplitude of I(Cl(swell)) whereas the inactive analogue U73343 had no effect. The phorbol esters phorbol-12-myristate-13-acetate (PMA) and phorbol-12,13-dibutyrate (PDBu) increased the amplitude of I(Cl(swell)) by approximately 60 and 100 %, respectively, in a voltage-independent fashion. Inhibitors of protein kinase C (PKC) chelerythrine and calphostin-C decreased the amplitude of I(Cl(swell)) in a concentration-dependent but voltage-independent manner. Bath application of 8-Br-cAMP decreased I(Cl(swell)) by about 60 % whereas the inhibitor of protein kinase A (PKA) KT5720 increased the amplitude of I(Cl(swell)) by approximately 80-90 %. In the presence of propranolol, chelerythrine prevented the increase of I(Cl(swell)) by noradrenaline; in the presence of prazosin, KT5720 blocked the inhibitory action of noradrenaline. The results show that in rabbit portal vein myocytes noradrenaline enhances I(Cl(swell)) by acting on alpha(1)-adrenoceptors and reduces I(Cl(swell)) by stimulating beta-adrenoceptors. The data suggest that the potentiating and inhibitory effects of noradrenaline are mediated, respectively, by PKC and PKA.

Control of proteolysis by norepinephrine and insulin in brown adipocytes: role of ATP, phosphatidylinositol 3-kinase, and p70 S6K.

The objective of this study was to evaluate some of the mechanisms by which norepinephrine (NE) and insulin may influence protein degradation in mouse brown adipocytes differentiated in cultures. The effects of NE and insulin, alone or in combination, on three factors known to influence proteolysis (maintenance of cell ATP and 1-phosphatidylinositol 3-kinase (PI 3-kinase) and p70 ribosomal S6-kinase (p70 S6K) activities) were examined. It was proposed that NE affects proteolysis indirectly by decreasing cell ATP from activation of uncoupling protein-1 (UCP1)-dependent mitochondrial respiration. This was tested by comparing the effects of NE and fatty acids (which directly activate UCP1) on proteolysis in brown adipocytes, as well as in pre-adipocytes and 3T3-L1 adipocytes, which do not express UCP1. An inhibitory effect of insulin on proteolysis is observed in both pre-adipocytes and differentiated cells, whereas NE and exogenously added fatty acids inhibit proteolysis only in brown adipocytes. There is a linear relationship between reductions in cell ATP and proteolysis in response to increasing concentrations of NE or fatty acids. PI 3-kinase activity is required for proteolysis, because two selective inhibitors (wortmannin and LY294002) reduce proteolysis in both pre-adipocytes and differentiated cells. This effect is not additive to that of NE, which suggests they affect the same proteolytic pathway. In contrast to NE, insulin increases PI 3-kinase activity and phosphorylation of p70 S6K. Rapamycin, which prevented insulin-dependent increase in phosphorylation of p70 S6K, increases proteolysis in brown adipocytes and antagonizes the inhibitory effect of insulin on proteolysis, but not the inhibitory effect of NE. Thus, insulin inhibits proteolysis via rapamycin-sensitive activation of p70 S6K, whereas the effect of NE appears largely to be a function of decreasing cell ATP content.

Noradrenaline excites and inhibits GABAergic transmission in parvocellular neurons of rat hypothalamic paraventricular nucleus.

Noradrenaline (NA) is a major neurotransmitter that regulates many neuroendocrine and sympathetic autonomic functions of the hypothalamic paraventricular nucleus (PVN). Previously NA has been shown to increase the frequency of excitatory synaptic activity of parvocellular neurons within the PVN, but little is known about its effects on inhibitory synaptic activity. In this work, we studied the effects of NA (1-100 microM) on the spontaneous inhibitory synaptic currents (sIPSC) of type II PVN neurons in brain slices of the rat using the whole cell patch-clamp technique. Spontaneous IPSCs were observed from most type II neurons (n = 121) identified by their anatomical location within the PVN and their electrophysiological properties. Bath application of NA (100 microM) increased sIPSC frequency by 256% in 59% of the neurons. This effect was blocked by prazosin (2-20 microM), the alpha(1)-adrenoceptor antagonist and mimicked by phenylephrine (10-100 microM), the alpha(1)-adrenoceptor agonist. However, in 33% of the neurons, NA decreased sIPSC frequency by 54%, and this effect was blocked by yohimbine (2-20 microM), the alpha(2)-adrenoceptor antagonist and mimicked by clonidine (50 microM), the alpha(2)-adrenoceptor agonist. The Na(+) channel blocker, tetrodotoxin (0.1 microM) blocked the alpha(1)-adrenoceptor-mediated effect, but not the alpha(2)-adreonoceptor-mediated one. Both of the stimulatory and inhibitory effects of NA on sIPSC frequency were observed in individual neurons when tested with NA alone, or both phenylephrine and clonidine. Furthermore, in most neurons that showed the stimulatory effects, the inhibitory effects of NA were unmasked after blocking the stimulatory effects by prazosin or tetrodotoxin. These data indicate that tonic GABAergic inputs to the majority of type II PVN neurons are under a dual noradrenergic modulation, the increase in sIPSC frequency via somatic or dendritic alpha(1)-adrenoceptors and the decrease in sIPSC frequency via axonal terminal alpha(2)-adrenoceptors on the presynaptic GABAergic neurons.

Modulation of rat medial vestibular nucleus neurone activity by vasopressin and noradrenaline in vitro

In the present study, we examined the effects of bath application of vasopressin and noradrenaline on the spontaneous tonic discharge of medial vestibular nucleus (MVN) neurones and investigated if there is an interaction between the two drugs in an in vitro slice preparation of the rat brainstem containing the MVN. The results showed that vasopressin did not affect the spontaneous discharge rate of MVN neurones when applied either as a 60 s pulse or when the drug continuously perfused the slice for a period of 10 min. In contrast, noradrenaline affected the spontaneous discharge rate of the majority of cells tested (53/60, 88%). Noradrenaline excited the majority (46/53, 87%) of MVN neurones through both α1 and β noradrenergic receptor-linked mechanisms. The remaining cells (7/53, 13%) were inhibited by noradrenaline through an α2 noradrenergic receptor-linked mechanism. Neither the excitatory nor inhibitory effects of noradrenaline were modified by vasopressin when the two drugs were applied together.

Norepinephrine regulation of growth hormone release from goldfish pituitary cells. II. Intracellular sites of action.

Previous results suggest that norepinephrine decreases growth hormone (GH) release in goldfish by means of alpha-2 adrenoceptor activation. The intracellular mechanisms by which norepinephrine inhibits GH release were examined in the present study using dispersed goldfish pituitary cells. In 2-h static incubation experiments, norepinephrine and the alpha-2 agonist clonidine decreased basal GH release and the GH responses to stimulation by the dopamine D1 agonist SKF38393 and two native gonadotropin-releasing hormones (GnRH). Norepinephrine also reduced GH responses to the adenylate cyclase activator forskolin, two protein kinase C (PKC) activators (phorbol ester and synthetic diacylglycerol), and two Ca2+ ionophores (ionomycin and A23187). Similarly, norepinephrine applied as a 1-h pulse in cell column perifusion experiments reduced basal GH release and abolished the GH response to a 5-min pulse of arachidonic acid. In goldfish, D1-stimulated GH release is mediated by AC-, arachidonic acid-and Ca2+-dependent pathways, whereas GnRH action is coupled to PKC-and Ca2+-dependent mechanisms. These results suggest that norepinephrine activation of alpha-2 receptors inhibits ligand-induced GH secretion by actions subsequent to activation of these second messenger cascades. To further characterize norepinephrine mechanisms of action on unstimulated hormone release, the ability of norepinephrine and an alpha-2 agonist to affect activation of two second messenger cascades under basal conditions was also investigated. Static incubation with clonidine reduced cAMP production in a time-and dose-dependent manner, suggesting that norepinephrine inhibitory action can also be expressed at the level of cAMP production. Resting intracellular free calcium levels in single, identified goldfish somatotropes was unaffected by norepinephrine. However, the inhibitory effects of norepinephrine on basal GH secretion was not observed in the presence of a voltage-sensitive Ca2+ channel agonist. Whether these channels are targets for norepinephrine action on unstimulated GH release requires further investigation.

Differentiation-dependent inhibition of proteolysis by norepinephrine in brown adipocytes.

The objective was to evaluate whether norepinephrine (NE) and other hormonal factors have direct effects on protein degradation in brown fat cells. NE inhibited proteolysis by 35-45% in mouse brown adipocytes differentiated in culture. Insulin also inhibited protein degradation but significantly less than NE, whereas glucagon and leptin had no effect. The inhibitory effect of NE was partially antagonized by propranolol but not by prazosin, and dose-response curves with BRL-37344 (a beta(3)-agonist), isoproterenol (a beta(1)/beta(2)-agonist) and dobutamide (a beta(1)-agonist) were consistent with the involvement of a beta(3)-adrenergic receptor. Furthermore, forskolin mimicked the effects of NE, whereas additions of A-23187 or phorbol esters had no effect, alone or in combination with NE or forskolin. Thus inhibition of proteolysis by NE likely involves a beta(3)-adrenergic receptor-mediated increase in cAMP. In contrast, NE, BRL-37344, and dobutamide had no effect on proteolysis in preadipocytes. Inhibition of proteolysis by NE was due at least in part to inhibition of autophagy. Thus inhibition of proteolysis by NE and insulin in mature brown adipocytes is likely an important process contributing to brown fat growth and atrophy under many physiological or pathological conditions.

Demonstration of action-potential-producing cells in the rat pineal gland in vitro and their regulation by norepinephrine and nitric oxide.

There is evidence that sympathetically innervated mammalian pineal glands contain cells that exhibit action potentials. It is unknown whether ex vivo pineal glands deprived of their nervous input are still capable of firing. In the present study, multiple-unit recordings from rat pineals revealed spontaneously active cell clusters with a mean firing frequency of 1.5 +/- 0.3 Hz which could be abolished by tedrodotoxin. Regularly firing clusters showed no inherent periodicity in the minute range, whereas rhythmical clusters with periodically repeated bursts had period lengths of 12.6 min (day) and 9.5 min (night). Superfusion of norepinephrine reduced the firing frequency of both cluster types, or had no effect, and the rhythmical clusters became regular. The effects of norepinephrine appear to be mediated via beta-adrenoceptors because isoproterenol, a beta-agonist, had the same effect as norepinephrine and as the alpha-agonist phenylephrine was without effect. Evidence was obtained that the inhibitory effect of norepinephrine may be mediated by nitric oxide, which appears to affect the electrical discharge of the cells directly or indirectly through cGMP. It is discussed whether the spontaneously active pineal cells represent an intrapineal oscillator or are driven by an intrinsic oscillator which has yet to be defined.

Inhibition of spontaneous inhibitory postsynaptic currents (IPSC) by noradrenaline in rat supraoptic neurons through presynaptic α 2-adrenoceptors

It has been shown that noradrenergic activation has great influence on the activities of hypothalamic supraoptic neurons. No direct evidence has been reported on the presynaptic effects of adrenoceptors in the actions of noradrenaline on supraoptic neurons, although postsynaptic mechanisms have been studied extensively. In the present study, we explored presynaptic effects of noradrenaline on the supraoptic neurons by measuring spontaneous inhibitory postsynaptic currents (IPSC) with the whole-cell patch-clamp technique. Noradrenaline reduced the frequency of IPSCs in a dose-dependent (10 −9 to 10 −3 M) and reversible manner. Noradrenaline did not affect the amplitude of IPSCs at concentrations of 10 −9 to 10 −5 M, but reduced the amplitude of IPSCs at high concentrations (10 −4 and 10 −3 M). The inhibitory effects of noradrenaline were mimicked by the α 2-agonist clonidine (10 −4 M), but not by the α 1-agonist methoxamine (10 −4 M) nor by the β-agonist isoproterenol (10 −4 M). Moreover, the inhibitory effects of noradrenaline on IPSCs were blocked by the non-selective α antagonist phentolamine (10 −4 M) or the selective α 2-antagonist yohimbine (10 −4 M), but not by the α 1-antagonist prazosin (10 −4 M). These results suggest that noradrenaline inhibits release of GABA from the presynaptic GABAergic terminals of the supraoptic neurons by activating presynaptic α 2-adrenoceptors and such presynaptic mechanisms may play a role in the excitatory control of SON neurons by noradrenergic neurons.

Norepinephrine inhibits neurons of the intermediate subnucleus of the lateral septum via α 2-adrenoceptors

The physiological and pharmacological actions of norepinephrine (NE) on neurons of the intermediate subnucleus of the lateral septum (LSI) were examined using intracellular recordings in rat brain-slices. Bath-applied NE inhibited 72.5%, excited 5.5% and had no effect on 22% of LSI neurons tested; this study focused on the inhibitory effects of NE. In current clamp recordings, 100 μM NE produced a hyperpolarization of 10.82±0.72 mV ( n=84) with a decrease in input resistance. In voltage-clamp, NE produced a direct, post-synaptic outward current of 206.8±22 pA ( n=37) with a 64.3±4.9% increase in input conductance (IC 50—17.7±4 μM). The NE-induced inhibition was mimicked by the α 2-agonist, UK14,304, but not by the α 1- or β-adrenoceptor agonists. The α 2-agonist, clonidine, had a weak effect in LSI neurons. Interestingly, the magnitude of the UK14,304-induced response varied between cells (ranging from 29.5 to 320% of the maximal NE inhibition), possibly suggesting the involvement of α 2A-(high affinity for UK14,304) and non-α 2A (low affinity for UK14,304) adrenoceptor subtypes. While the α 2-antagonists, yohimbine, rauwolscine and idazoxan blocked NE-induced inhibition in all neurons tested, the prototypical α 1-antagonist, prazosin produced a variable degree of block (9–58%), further indicating the possible involvement of α 2A (prazosin-insensitive) and non-α 2A (prazosin-sensitive) receptors. However a lack of more selective pharmacological tools precludes definitive classification of the α 2-receptor-mediated responses into different subtypes. The α 2-receptor-mediated current in LSI neurons displayed Ba 2+-sensitive inward rectification, reversed polarity near E K and was sensitive to external K +. In conclusion, NE inhibits LSI neurons via α 2-adrenoceptor subtypes.

Effect of norepinephrine on intracellular pH in kidney proximal tubule: role of Na+-(HCO-3)n cotransport.

We examined the effect of norepinephrine (NE) on intracellular pH (pHi) and activity of Na+ (aNai) in the isolated perfused kidney proximal tubule of Ambystoma, using single-barreled voltage and ion-selective microelectrodes. In control HCO-3 Ringer, addition of 10(-6) M NE to the bath reversibly depolarized the basolateral membrane potential (V1), the luminal membrane potential (V2), and the transepithelial potential difference (V3) and increased pHi by 0. 14 +/- 0.02. These effects were mimicked by isoproterenol but were abolished after pretreatment with SITS or in the absence of CO2/HCO-3. Removal of bath Na+ depolarized V1 and V2, hyperpolarized V3, and decreased pHi. These effects are largely mediated by the electrogenic Na+-(HCO-3)n cotransporter. In the presence of NE, the effects of Na+ removal on membrane potential differences and the rate of change of pHi were significantly smaller. Reducing bath HCO-3 concentration from 10 to 2 mM at constant CO2 (pH 6.8) depolarized V1 and V2, decreased pHi, and lowered aNai. These changes are also due to Na+-(HCO-3)n. In the presence of NE, reducing bath [HCO-3] caused a smaller depolarizations of V1 and V2, and the rate of pHi decrease was significantly reduced. Our results indicate: 1) NE causes an increase in pHi; 2) the NE-induced alkalinization is mediated by a SITS-sensitive and HCO-3-dependent transporter on the basolateral membrane; and 3) in the presence of NE, the reduced effects caused by basolateral HCO-3 changes or Na+ removal are indicative of an inhibitory effect of NE on Na+-(HCO-3)n cotransport.

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.

Voltage-Dependent Calcium Currents in Bulbospinal Neurons of Neonatal Rat Rostral Ventrolateral Medulla: Modulation by α2-Adrenergic Receptors

The properties and modulation by norepinephrine (NE) of voltage-dependent calcium currents were studied in bulbospinal neurons (n = 116) of the rostral ventrolateral medulla (RVLM) using whole cell patch-clamp techniques in neonatal rat brain stem slices. RVLM bulbospinal neurons were identified visually by their location in slices and by the presence of flourescein isothiocyanate-tagged microbeads, which were injected into the spinal cord before the experiment; RVLM neurons were filled with Lucifer yellow during recordings, and the slice was processed for detection of tyrosine hydroxylase immunoreactivity (TH-IR). Thirty-four of 42 recovered cells (81%) were positive for TH-IR, indicating that most recorded cells were C1 neurons. Bulbospinal RVLM neurons expressed a prominent high-voltage-activated (HVA) calcium current, which began to activate at -30 to -40 mV (from a holding potential of -60 or -70 mV), and peaked at approximately 0 mV (0.8 +/- 0.1 nA;mean +/- SE). HVA current comprised predominantly omega-conotoxin GVIA-sensitive, N-type and omega-agatoxin IVA-sensitive, P/Q-type components, with smaller dihydropyridine-sensitive, L-type, and residual current components. Most RVLM bulbospinal neurons (n = 44/52, including 12/14 histologically identified C1 cells) also expressed low-voltage-activated (LVA) calcium current. LVA current began to activate at approximately -60 mV (from a holding potential of -100 mV) and was nearly completely inactivated at -50 mV with a half-inactivation potential of -70 +/- 2 mV. The amplitude of LVA current at -50 mV was 78 +/- 24 pA with Ba2+ and 156 +/- 38 pA with Ca2+ as a charge carrier. NE inhibited HVA current in most bulbospinal RVLM neurons (n = 70/77) with an EC50 of 1.2 muM; NE had no effect on LVA current. Calcium current inhibition by NE was mediated by alpha2-adrenergic receptors (alpha2-ARs) as the effect was mimicked by the selective alpha2-AR agonist, UK-14,304, and blocked by idazoxan, an alpha2-AR antagonist, but unaffected by prazosin and propranolol (alpha1- and beta-AR antagonists, respectively). Most of the NE-sensitive calcium current was N- and P/Q-type. NE-induced inhibition of calcium current evoked by action potential waveforms (APWs) was significantly larger than that evoked by depolarizing steps (34 +/- 2.5 vs. 23 +/- 2.7%; P < 0.05). Although inhibition of calcium current was voltage dependent and partially relieved by strong depolarizations, when calcium currents were evoked with a 10-Hz train of APWs as a voltage command, the inhibitory effect of NE was maintained throughout the train. In conclusion, bulbospinal RVLM neurons, including C1 cells, express multiple types of calcium currents. Inhibition of HVA calcium current by NE may modulate input-output relationships and release of transmitters from C1 cells.

NE inhibits cerebrovascular mast cell exocytosis induced by cholinergic and peptidergic agonists.

Autonomic and sensory nerves frequently contact mast cells contained in rabbit leptomeningeal arteries. We have previously shown that parasympathetic and peptidergic neurotransmitters can stimulate mast cell granule exocytosis and serotonin (5-HT) release. In the present study, we examined ex vivo the possible action of the main sympathetic neurotransmitter, norepinephrine (NE), on this exocytotic process. NE, which had no effect on mast cell 5-HT content per se, totally inhibited carbachol-induced 5-HT release and partially reduced neuropeptide-induced 5-HT release. Pretreatment with the alpha 1-adrenergic blocker did not affect the inhibitory effect of NE. Pretreatment with specific beta 1- or beta 2-adrenergic blockers antagonized this action, but the beta 2-blocker exerts a more specific dose-dependent antagonism. Together with our previous data, these results indicate that the equilibrium between autonomic and sensory nerves may determine the release of 5-HT from mast cells (parasympathetic and sensory nerves can trigger exocytosis while the sympathetics can inhibit it). Such a mechanism could be implicated in pathophysiological events in which autonomic dysfunction is likely to be involved, such as vascular headache or other phenomena involving inflammation.