Differential involvement of N-type calcium channels in transmitter release from vasoconstrictor and vasodilator neurons

1 The effects of calcium channel blockers on co-transmission from different populations of autonomic vasomotor neurons were studied on isolated segments of uterine artery and vena cava from guinea-pigs. 2 Sympathetic, noradrenergic contractions of the uterine artery (produced by 200 pulses at 1 or 10 Hz; 600 pulses at 20 Hz) were abolished by the N-type calcium channel blocker omega-conotoxin (CTX) GVIA at 1-10 nM. 3 Biphasic sympathetic contractions of the vena cava (600 pulses at 20 Hz) mediated by noradrenaline and neuropeptide Y were abolished by 10 nM CTX GVIA. 4 Neurogenic relaxations of the uterine artery (200 pulses at 10 Hz) mediated by neuronal nitric oxide and neuropeptides were reduced < 50% by CTX GVIA 10-100 nM. 5 Capsaicin (3 muM) did not affect the CTX GVIA-sensitive or CTX GVIA-resistant neurogenic relaxations of the uterine artery. 6 The novel N-type blocker CTX CVID (100-300 nM), P/Q-type blockers agatoxin IVA (10-100 nM) or CTX CVIB (100 nM), the L-type blocker nifedipine (10 muM) or the 'R-type' blocker SNX-482 (100 nM), all failed to reduce CTX GVIA-resistant relaxations. The T-type channel blocker NiCl2 (100-300 muM) reduced but did not abolish the remaining neurogenic dilations. 7 Release of different neurotransmitters from the same autonomic vasomotor axon depends on similar subtypes of calcium channels. N-type channels are responsible for transmitter release from vasoconstrictor neurons innervating a muscular artery and capacitance vein, but only partly mediate release of nitric oxide and neuropeptides from pelvic vasodilator neurons.

Ovarian-Steroid Modulation of Locus Coeruleus Activity in Female Rats: Involvement in Luteinising Hormone Regulation

The noradrenergic nucleus locus coeruleus (LC) has been reported to regulate luteinising hormone (LH) secretion in female rats. Both oestrogen and progestin receptors have been demonstrated in LC neurones, suggesting that these cells are possibly responsive to variations in circulating levels of ovarian steroids. We therefore evaluated changes in the activity of LC neurones during the oestrous cycle and after ovarian-steroid treatment in ovariectomised (OVX) rats, as determined by immunoreactivity to Fos-related antigens (FRA), which comprises all of the known members of the Fos family. Effects of ovarian steroids on the firing rate of LC neurones were also determined in a slice preparation. The number of FRA/tyrosine hydroxylase (TH)-immunoreactive (ir) neurones in the LC increased from 14.00-16.00 h on pro-oestrus, coinciding with the onset of the LH surge and rise in plasma progesterone. FRA immunoreactivity was unaltered during dioestrus. Oestradiol-treated OVX rats (OVX+E) displayed marked reduction in FRA/TH-ir neurones in LC compared to oil-treated OVX rats. Accordingly, oestradiol superfusion significantly reduced the spontaneous firing rate of LC neurones in slices from OVX rats. Compared to OVX+E, oestradiol-treated rats injected with progesterone at 08.00 h (OVX+EP) exhibited higher number of FRA/TH-ir neurones in the LC at 10.00 h and 16.00 h, and great amplification of the LH surge. Bath application of progesterone significantly increased the spontaneous firing rate of OVX+E LC neurones. Our data suggest that ovarian steroids may physiologically modulate the activity of LC neurones in females, with possible implications for LH secretion. Moreover, oestradiol and progesterone appear to exert opposite and complementary effects (i.e. whereas oestradiol inhibits, progesterone, after oestradiol priming, stimulates LC activity).

The noradrenergic projection from the locus coeruleus to the cochlear root neurons in rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Enhanced noradrenergic activity in the amygdala contributes to hyperarousal in an animal model of PTSD

Increased activity of the noradrenergic system in the amygdala has been suggested to contribute to the hyperarousal symptoms associated with post-traumatic stress disorder (PTSD). However, only two studies have examined the content of noradrenaline or its metabolites in the amygdala of rats previously exposed to traumatic stress showing inconsistent results. The aim of this study was to investigate the effects of an inescapable foot shock (IFS) procedure 1) on reactivity to novelty in an open-field (as an index of hyperarousal), and 2) on noradrenaline release in the amygdala during an acute stress. To test the role of noradrenaline in amygdala, we also investigated the effects of microinjections of propranolol, a β-adrenoreceptor antagonist, and clenbuterol, a β-adrenoreceptor agonist, into the amygdala of IFS and control animals. Finally, we evaluated the expression of mRNA levels of β-adrenoreceptors (β1 and β2) in the amygdala, the hippocampus and the prefrontal cortex. Male Wistar rats (3 months) were stereotaxically implanted with bilateral guide cannulae. After recovering from surgery, animals were exposed to IFS (10 shocks, 0.86 mA, and 6 seconds per shock) and seven days later either microdialysis or microinjections were performed in amygdala. Animals exposed to IFS showed a reduced locomotion compared to non-shocked animals during the first 5 minutes in the open-field. In the amygdala, IFS animals showed an enhanced increase of noradrenaline induced by stress compared to control animals. Bilateral microinjections of propranolol (0.5 μg) into the amygdala one hour before testing in the open-field normalized the decreased locomotion observed in IFS animals. On the other hand, bilateral microinjections of clenbuterol (30 ng) into the amygdala of control animals did not change the exploratory activity induced by novelty in the open field. IFS modified the mRNA expression of β1 and β2 adrenoreceptors in the prefrontal cortex and the hippocampus. These results suggest that an increased noradrenergic activity in the amygdala contributes to the expression of hyperarousal in an animal model of PTSD.

Evidencia sobre la eficacia de cannabinoides en el dolor neuropático oncológico

Contexto: La eficacia de los cannabinoides en el dolor neuropático es desconocida. El control del dolor es determinante en los pacientes ya que genera un impacto negativo en la calidad de vida de los pacientes. Objetivo: El presente trabajo pretende demostrar la evidencia sobre la eficacia de los medicamentos cannabinoides en el control del dolor neuropático oncológico, mediante la evaluación de la literatura disponible. Metodología: Se realizó una revisión sistemática de literatura incluyendo estudios experimentales, observacionales y revisiones sistemáticas en un periodo de 15 años. Se incluyeron todos los estudios desde el años 2000 con evidencia IB según la escala de evidencia de Oxford. Resultados: Cuatro estudios cumplieron criterios para su inclusión, sin embargo la evidencia es baja y no permite recomendar o descartar los cannabinoides como terapia coadyuvante en control del dolor neuropático oncológico. La combinación de THC/CDB (Sativex®) parece ser un medicamento seguro pues no se reportaron muertes asociadas a su uso, sin embargo la presentación de eventos adversos a nivel gastrointestinal y neurológico podría aumentar el riesgo de interacciones medicamentosas y tener un impacto negativo en la calidad de vida de los pacientes oncológicos. Conclusiones: No hay suficiente literatura y la evidencia no es suficiente para recomendar o descartar el uso de los cannabinoides en dolor neuropático oncológico. Futuros estudios deben realizarse para analizar el beneficio de estos medicamentos. Aunque ética y socialmente hay resistencia para el uso de los cannabinoides, actualmente hay una gran discusión política en el mundo y en Colombia para su aceptación como terapia en el control del dolor.

G protein ss gamma subunits mediate presynaptic inhibition of transmitter release from rat superior cervical ganglion neurones in culture

The activation of presynaptic G protein-coupled receptors (GPCRs) is widely reported to inhibit transmitter release; however, the lack of accessibility of many presynaptic terminals has limited direct analysis of signalling mediators. We studied GPCR-mediated inhibition of fast cholinergic transmission between superior cervical ganglion neurones (SCGNs) in culture. The adrenoceptor agonist noradrenaline (NA) caused a dose-related reduction in evoked excitatory postsynaptic potentials (EPSPs). NA-induced EPSP decrease was accompanied by effects on the presynaptic action potential (AP), reducing AP duration and amplitude of the after-hyperpolarization (AHP), without affecting the pre- and postsynaptic membrane potential. All effects of NA were blocked by yohimbine and synaptic transmission was reduced by clonidine, consistent with an action at presynaptic alpha 2-adrenoceptors. NA-induced inhibition of transmission was sensitive to pre-incubation of SCGNs with pertussis toxin (PTX), implicating the involvement of G alpha(i)/(o)beta y subunits. Expression of G alpha transducin, an agent which sequesters G protein beta gamma (G beta y) subunits, in the presynaptic neurone caused a time-dependent attenuation of NA-induced inhibition. Injection of purified G beta gamma subunits into the presynaptic neurone inhibited transmission, and also reduced the AHP amplitude. Furthermore, NA-induced inhibition was occluded by pre-injection of G beta gamma subunits. The Ca2+ channel blocker Cd2+ mimicked NA effects on transmitter release. Cd2+, NA and G beta gamma subunits also inhibited somatic Ca2+ current. In contrast to effects on AP-evoked transmitter release, NA had no clear action on AP-independent EPSPs induced by hypertonic solutions. These results demonstrate that G beta gamma subunits functionally mediate inhibition of transmitter release by alpha 2-adrenoceptors and represent important regulators of synaptic transmission at mammalian presynaptic terminals.

The neuronal noradrenaline transporter, anxiety and cardiovascular disease

Panic disorder can serve as a clinical model for testing whether mental stress can cause heart disease. Potential neural mechanisms of cardiac risk are the sympathetic activation during panic attacks, continuing release of adrenaline as a co-transmitter in the cardiac sympathetic nerves, and impairment of noradrenaline neuronal reuptake, augmenting sympathetic neural respnses.

The phenotype of impaired neuronal reuptake of noradrenaline: an epigenetic mechanism? We suspect that this phenotype, in sensitizing people to heart symptom development, is a cause of panic disorder, and by magnifying the sympathetic neural signal in the heart, underlies increased cardiac risk. No loss of function mutations of the coding region of the norepinephrine transporter (NET) are evident, but we do detect hypermethylation of CpG islands in the NET gene promoter region. Chromatin immunoprecipitation methodology demonstrates binding of the inhibitory transcription factor, MeCP2, to promoter region DNA in panic disorder patients.

Cardiovascular illnesses co-morbid with panic disorder. Panic disorder commonly coexists with essential hypertension and the postural tachycardia syndrome. In both of these cardiovascular disorders the impaired neuronal noradrenaline reuptake phenotype is also present and, as with panic disorder, is associated with NET gene promoter region DNA hypermethylation. An epigenetic ‘co-morbidity’ perhaps underlies the clinical concordance.

Brain neurotransmitters. Using internal jugular venous sampling, in the absence of a panic attack we find normal norepinephrine turnover, but based on measurements of the overflow of the serotonin metabolite, 5HIAA, a marked increase (six to sevenfold) in brain serotonin turnover in patients with panic disorder. This appears to represent the underlying neurotransmitter substrate for the disorder. Whether this brain serotonergic activation is a prime mover, or consequential on other primary causes of panic disorder, including cardiac sensitization by faulty neuronal noradrenaline reuptake leading to cardiac symptoms and the enhanced vigilance which accompanies them, is unclear at present.

Mechanisms involved in the renal responses to intravenous and renal artery infusions of noradrenaline in conscious dogs

1. The renal haemodynamic and glomerular filtration rate (G.F.R.) responses to intravenous and intrarenal infusions of noradrenaline were studied in conscious dogs, either with or without prior blockade of angiotensin II formation with teprotide.

2. Infusion noradrenaline by either route resulted in dose-related rises in plasma renin activity.

3. Pretreatment with teprotide reduced the rise in mean arterial pressure and abolished the rise in G.F.R. seen during intravenous infusions of noradrenaline (0.1, 0.2 and 0.4 microgram/kg . min). Noradrenaline also reduced filtration fraction more after teprotide pretreatment.

4. Renal blood flow rose and renal vascular resistance fell in response to I.V. noradrenaline infusions. This renal vasodilatation was unaffected by pretreatment of the dogs with teprotide, indomethacin or DL-propranolol. However after pentolinium pretreatment, I.V. noradrenaline infusion caused a dose-related renal vasoconstriction.

5. Infusion of noradrenaline into the renal artery (0.02, 0.05 and 0.1 microgram/kg . min) resulted in rises in mean arterial pressure and G.F.R. which were abolished by teprotide pretreatment. Filtration fraction rose when noradrenaline was administered alone but fell when it was infused after teprotide treatment.

6. Thus angiotensin II formed as the result of increased renin release acted to maintain G.F.R. and filtration fraction during noradrenaline infusion. In addition, I.V. noradrenaline infusions in conscious dogs caused reflex vasodilatation of the renal vasculature.

Noradrenaline, but not neuropeptide Y, is elevated in cerebrospinal fluid from the third cerebral ventricle following audiovisual stress in gonadectomised rams and ewes

There are sex differences in the activation of the hypothalamo-pituitary-adrenal axis in response to stress, but the source of these differences is unknown. The hypothalamo-pituitary-adrenal axis is regulated by corticotropin-releasing hormone and arginine-vasopressin neurones located in the paraventricular nucleus and these, in turn, are regulated by neural systems that include afferent noradrenergic and neuropeptide Y (NPY)-producing neural pathways. We tested the hypothesis that concentrations of noradrenaline and NPY will be elevated in cerebrospinal fluid (CSF) sampled from the third cerebral ventricle in response to stress, and these responses will differ in males and females. We collected concurrent samples of CSF (1 ml) from the third ventricle and blood (5 ml) from the jugular vein from gonadectomised rams (n = 7) and ewes (n = 5) at 10-min intervals for 3 h. This procedure was conducted on a day when no stress was imposed and on a day when audiovisual stress was imposed for 5 min after 1 h of sampling. Following the audiovisual stress, plasma concentrations of cortisol and CSF concentrations of noradrenaline were elevated (p < 0.05), but CSF concentrations of NPY did not change. Adrenaline was not detected in samples of CSF. The rise in plasma cortisol following the stress was greater (p < 0.05) in ewes than in rams, but there were no sex differences in the rise in noradrenaline. Basal concentrations of NPY in the CSF were higher (p < 0.05) in rams than in ewes. We conclude that the sex differences in the stress-induced activity of the hypothalamo-pituitary-adrenal axis in sheep are not likely to be due to differences in the level of noradrenergic and/or NPY input to the hypothalamus.

Cardiovascular effects of noradrenaline microinjected into the insular cortex of unanesthetized rats

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Inhibitory effect of DUP-753 on the drinking responses of rats to central administration of noradrenaline and angiotensin II and to dehydration

We investigated the effect of losartan (DUP-753) on the dipsogenic responses produced by intracerebroventricular (icv) injection of noradrenaline (40 nmol/mu l) and angiotensin II (ANG II) (2 ng/mu l) in male Holtzman rats weighing 250-300 g. The effect of DUP-753 was also studied in animals submitted to water deprivation for 30 h. After control injections of isotonic saline (0.15 M NaCl, 1 mu l) into the lateral ventricle (LV) the water intake was 0.2 +/- 0.01 ml/h. DUP-753 (50 nmol/mu l) when injected alone into the LV of satiated animals had no significant effect on drinking (0.4 +/- 0.02 ml/h) (N = 8). DUP-753 (50 nmol/mu l) injected into the LV prior to noradrenaline reduced the water intake from 2.4 +/- 0.8 to 0.8 +/- 0.2 ml/h (N = 8). The water intake induced by injection of ANG II and water deprivation was also reduced from 9.2 +/- 1.4 and 12.7 +/- 1.4 ml/h to 0.8 +/- 0.2 and 1.7 +/- 0.3 ml/h (N = 6 and N = 8), respectively. These data indicate a correlation between noradrenergic pathways and angiotensinergic receptors and lead us to conclude that noradrenaline-induced water intake may be due to the release of ANG II by the brain. The finding that water intake was reduced by DUP-753 in water-deprived animals suggests that dehydration releases ANG II, and that AT(1) receptors of the brain play an important role in the regulation of water intake induced by deprivation.

Chemical mechanism of controlled nitric oxide release from trans-[RuCl([15]aneN(4))NO](PF6)(2) as a vasorelaxant agent

The nitrosyl ruthenium complex, trans-[RuCl([15]aneN(4))NO](PF6)(2), ([15]aneN(4) = 1,4,8,12-tetraazacyclopentadecane), exhibits vasorelaxation characteristics attributed to its nitric oxide release properties. The observed in vitro and in vivo vasodilation is dependent on noradrenaline concentration. We report here the chemical mechanism of the reaction between noradrenaline and trans-[RuCl([15]aneN(4))NO](PF6)(2) in aqueous phosphate buffer solution at pH 7.40. NO measurement by NO-sensor electrode, cyclic voltammetry, (PNMR)-P-31 and HPLC analysis were used to investigate the reduction process as the fundamental step for NO release characteristic of trans-[RuCl([15]aneN(4))NO](PF6)(2). A supramolecular species containing HPO4 (2-) as a bridging group between noradrenaline and trans-[RuCl([15]aneN(4))NO](PF6)(2) is suggested as an intermediate prior to the reduction of the nitrosyl ruthenium complex.

PARAVENTRICULAR AND SUPRAOPTIC NUCLEI OF THE HYPOTHALAMUS MEDIATE CARDIOVASCULAR RESPONSES EVOKED BY THE MICROINJECTION OF NORADRENALINE INTO THE MEDIAL AMYGDALOID NUCLEUS OF THE RAT BRAIN

The medial amygdaloid nucleus (MeA) is a part of the limbic system and is involved in cardiovascular modulation. We previously reported that microinjection of noradrenaline (NA) into the MeA of unanesthetized rats caused pressor and bradycardiac responses, which were mediated by acute vasopressin release into the systemic circulation. In the present study, we tested the possible involvement of magnocellular neurons of the paraventricular (PVN) and/or supraoptic (SON) of the hypothalamus that synthesize vasopressin in the cardiovascular pathway activated by the microinjection of NA into the MeA. Pressor and bradycardiac responses to the microinjection of NA (27 nmol/100 nL) into the MeA were blocked by pretreatment of either the PVN or the SON with cobalt chloride (CoCl2, 1 mM/100 nL), thus indicating that both hypothalamic nuclei mediate the cardiovascular responses evoked by microinjection of NA Into the MeA. Our results suggest that the pressor and bradycardiac response caused by the microinjection of NA into the MeA is mediated by magnocellular neurons in both the PVN and SON. (C) 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

The medial amygdaloid nucleus is involved in the cardiovascular pathway activated by noradrenaline into the lateral septal area of rats

We have previously reported that noradrenaline (NA) microinjected into the lateral septal area (LSA) caused pressor and bradicardic responses that were mediated by vasopressin release into the circulation through the paraventricular nucleus of hypothalamus (PVN). Although PVN is the final structure involved in the cardiovascular responses caused by NA in the LSA, there is no evidence of direct connections between these areas, suggesting that some structures could be links in this pathway. In the present study, we verified the effect of reversible synaptic inactivation of the medial amygdaloid nucleus (MeA), bed nucleus of stria terminalis (BNST) or diagonal band of Broca (DBB) with Cobalt Chloride (CoCl2) on the cardiovascular response to NA microinjection into the LSA of unanesthetized rats. Male Wistar rats had guide cannulae implanted into the LSA and the MeA, BNST or DBB for drug administration, and a femoral catheter for blood pressure and heart rate recordings. Local microinjection of CoCl2 (1 mm in 100 nL) into the MeA significantly reduced the pressor and bradycardic responses caused by NA microinjection (21 nmol in 200 nL) into the LSA. In contrast, microinjection of CoCl2 into the BNST or DBB did not change the cardiovascular responses to NA into the LSA. The results indicate that synapses within the MeA, but not in BNST or DBB, are involved in the cardiovascular pathway activated by NA microinjection into the LSA.