Modulation of arachidonic Acid metabolism in the rat kidney by sulforaphane: implications for regulation of blood pressure.

Background. We investigated the effects of sulforaphane (SF), the main active isothiocyanate in cruciferous vegetables, on arachidonic acid (AA) metabolism in the kidney and its effect on arterial blood pressure, using spontaneously hypertensive rats (SHR) as models. Methods. Rats were treated for 8 weeks with either drinking water alone (control) or SF (20 or 40 mg/kg) added to drinking water. Mean arterial pressure (MAP) was measured at 7-day intervals throughout the study. At the end of treatment rats were euthanized, and kidneys were harvested to prepare microsomes and measure enzymes involved in regulation of vasoactive metabolites: CYP4A, the key enzyme in the formation of 20-hydroxyeicosatetraenoic acid, and the soluble epoxide hydrolase, which is responsible for the degradation of the vasodilator metabolites such as epoxyeicosatetraenoic acids. Effect of SF on kidney expression of CYP4A was investigated by immunoblotting. Results. We found that treatment with SF leads to significant reductions in both, the expression and activity of renal CYP4A isozymes, as well as the activity of soluble epoxide hydrolase (sEH). Consistent with these data, we have found that treatment with SF resisted the progressive rise in MAP in the developing SHR in a dose-dependent manner. Conclusion. This is the first demonstration that SF modulates the metabolism of AA by both P450 enzymes and sEH in SHR rats. This may represent a novel mechanism by which SF protects SHR rats against the progressive rise in blood pressure.

Heterogeneous intracellular trafficking dynamics of brain-derived neurotrophic factor complexes in the neuronal soma revealed by single quantum dot tracking.

Accumulating evidence underscores the importance of ligand-receptor dynamics in shaping cellular signaling. In the nervous system, growth factor-activated Trk receptor trafficking serves to convey biochemical signaling that underlies fundamental neural functions. Focus has been placed on axonal trafficking but little is known about growth factor-activated Trk dynamics in the neuronal soma, particularly at the molecular scale, due in large part to technical hurdles in observing individual growth factor-Trk complexes for long periods of time inside live cells. Quantum dots (QDs) are intensely fluorescent nanoparticles that have been used to study the dynamics of ligand-receptor complexes at the plasma membrane but the value of QDs for investigating ligand-receptor intracellular dynamics has not been well exploited. The current study establishes that QD conjugated brain-derived neurotrophic factor (QD-BDNF) binds to TrkB receptors with high specificity, activates TrkB downstream signaling, and allows single QD tracking capability for long recording durations deep within the soma of live neurons. QD-BDNF complexes undergo internalization, recycling, and intracellular trafficking in the neuronal soma. These trafficking events exhibit little time-synchrony and diverse heterogeneity in underlying dynamics that include phases of sustained rapid motor transport without pause as well as immobility of surprisingly long-lasting duration (several minutes). Moreover, the trajectories formed by dynamic individual BDNF complexes show no apparent end destination; BDNF complexes can be found meandering over long distances of several microns throughout the expanse of the neuronal soma in a circuitous fashion. The complex, heterogeneous nature of neuronal soma trafficking dynamics contrasts the reported linear nature of axonal transport data and calls for models that surpass our generally limited notions of nuclear-directed transport in the soma. QD-ligand probes are poised to provide understanding of how the molecular mechanisms underlying intracellular ligand-receptor trafficking shape cell signaling under conditions of both healthy and dysfunctional neurological disease models.

Upregulation of brain-derived neurotrophic factor expression in nodose ganglia and the lower brainstem of hypertensive rats.

Hypertension leads to structural and functional changes at baroreceptor synapses in the medial nucleus tractus solitarius (NTS), but the underlying molecular mechanisms remain unknown. Our previous studies show that brain-derived neurotrophic factor (BDNF) is abundantly expressed by rat nodose ganglion (NG) neurons, including baroreceptor afferents and their central terminals in the medial NTS. We hypothesized that hypertension leads to upregulation of BDNF expression in NG neurons. To test this hypothesis, we used two mechanistically distinct models of hypertension, the spontaneously hypertensive rat (SHR) and the deoxycorticosterone acetate (DOCA)-salt rat. Young adult SHRs, whose blood pressure was significantly elevated compared with age-matched Wistar-Kyoto (WKY) control rats, exhibited dramatic upregulation of BDNF mRNA and protein in the NG. BDNF transcripts from exon 4, known to be regulated by activity, and exon 9 (protein-coding region) showed the largest increases. Electrical stimulation of dispersed NG neurons with patterns that mimic baroreceptor activity during blood pressure elevations led to increases in BDNF mRNA that were also mediated through promoter 4. The increase in BDNF content of the NG in vivo was associated with a significant increase in the percentage of BDNF-immunoreactive NG neurons. Moreover, upregulation of BDNF in cell bodies of NG neurons was accompanied by a significant increase in BDNF in the NTS region, the primary central target of NG afferents. A dramatic increase in BDNF in the NG was also detected in DOCA-salt hypertensive rats. Together, our study identifies BDNF as a candidate molecular mediator of activity-dependent changes at baroafferent synapses during hypertension.

Epoxyeicosatrienoic acids are endogenous regulators of vasoactive neuropeptide release from trigeminal ganglion neurons.

Epoxyeicosatrienoic acids (EETs) are bioactive eicosanoids produced from arachidonic acid by cytochrome P450 epoxygenases. We previously described the expression of cytochrome P450-2J epoxygenase in rat trigeminal ganglion neurons and that EETs signaling is involved in cerebrovascular dilation resulting from perivascular nerve stimulation. In this study, we evaluate the presence of the EETs signaling pathway in trigeminal ganglion neurons and their role in modulating the release of calcitonin gene-related peptide (CGRP) by trigeminal ganglion neurons. Liquid chromatography tandem mass spectrometry identified the presence of each of the four EETs regio-isomers within primary trigeminal ganglion neurons. Stimulation for 1 h with the transient receptor potential vanilloid-1 channel agonist capsaicin (100 nmol/L) or depolarizing K(+) (60 mmol/L) increased CGRP release as measured by ELISA. Stimulation-evoked CGRP release was attenuated by 30 min pre-treatment with the EETs antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, 10 µmol/L). K(+) stimulation elevated CGRP release 2.9 ± 0.3-fold above control levels, whereas in the presence of 14,15-EEZE K(+)-evoked CGRP release was significantly reduced to 1.1 ± 0.2-fold above control release (p < 0.01 anova, n = 6). 14,15-EEZE likewise attenuated capsaicin-evoked CGRP release from trigeminal ganglion neurons (p < 0.05 anova, n = 6). Similarly, pre-treatment with the cytochrome P450 epoxygenase inhibitor attenuated stimulation-evoked CGRP release. These data demonstrate that EETs are endogenous constituents of rat trigeminal ganglion neurons and suggest that they may act as intracellular regulators of neuropeptide release, which may have important clinical implications for treatment of migraine, stroke and vasospasm after subarachnoid hemorrhage.

Nitric oxide regulates BDNF release from nodose ganglion neurons in a pattern-dependent and cGMP-independent manner.

Activity of arterial baroreceptors is modulated by neurohumoral factors, including nitric oxide (NO), released from endothelial cells. Baroreceptor reflex responses can also be modulated by NO signaling in the brainstem nucleus tractus solitarius (NTS), the primary central target of cardiovascular afferents. Our recent studies indicate that brain-derived neurotrophic factor (BDNF) is abundantly expressed by developing and adult baroreceptor afferents in vivo, and released from cultured nodose ganglion (NG) neurons by patterns of baroreceptor activity. Using electrical field stimulation and ELISA in situ, we show that exogenous NO nearly abolishes BDNF release from newborn rat NG neurons in vitro stimulated with single pulses delivered at 6 Hz, but not 2-pulse bursts delivered at the same 6-Hz frequency, that corresponds to a rat heart rate. Application of L-NAME, a specific inhibitor of endogenous NO synthases, does not have any significant effect on activity-dependent BDNF release, but leads to upregulation of BDNF expression in an activity-dependent manner. The latter effect suggests a novel mechanism of homeostatic regulation of activity-dependent BDNF expression with endogenous NO as a key player. The exogenous NO-mediated effect does not involve the cGMP-protein kinase G (PKG) pathway, but is largely inhibited by N-ethylmaleimide and TEMPOL that are known to prevent S-nitrosylation. Together, our current data identify previously unknown mechanisms regulating BDNF availability, and point to NO as a likely regulator of BDNF at baroafferent synapses in the NTS.

Brain-derived neurotrophic factor in arterial baroreceptor pathways: implications for activity-dependent plasticity at baroafferent synapses.

Functional characteristics of the arterial baroreceptor reflex change throughout ontogenesis, including perinatal adjustments of the reflex gain and adult resetting during hypertension. However, the cellular mechanisms that underlie these functional changes are not completely understood. Here, we provide evidence that brain-derived neurotrophic factor (BDNF), a neurotrophin with a well-established role in activity-dependent neuronal plasticity, is abundantly expressed in vivo by a large subset of developing and adult rat baroreceptor afferents. Immunoreactivity to BDNF is present in the cell bodies of baroafferent neurons in the nodose ganglion, their central projections in the solitary tract, and terminal-like structures in the lower brainstem nucleus tractus solitarius. Using ELISA in situ combined with electrical field stimulation, we show that native BDNF is released from cultured newborn nodose ganglion neurons in response to patterns that mimic the in vivo activity of baroreceptor afferents. In particular, high-frequency bursting patterns of baroreceptor firing, which are known to evoke plastic changes at baroreceptor synapses, are significantly more effective at releasing BDNF than tonic patterns of the same average frequency. Together, our study indicates that BDNF expressed by first-order baroreceptor neurons is a likely mediator of both developmental and post-developmental modifications at first-order synapses in arterial baroreceptor pathways.

Endomorphin-2 is released from newborn rat primary sensory neurons in a frequency- and calcium-dependent manner.

Recent evidence indicates that endomorphins, endogenous mu-opioid receptor (MOR) agonists, modulate synaptic transmission in both somatic and visceral sensory pathways. Here we show that endomorphin-2 (END-2) is expressed in newborn rat dorsal root ganglion (DRG) and nodose-petrosal ganglion complex (NPG) neurons, and rarely co-localizes with brain-derived neurotrophic factor (BDNF). In order to examine activity-dependent release of END-2 from neurons, we established a model using dispersed cultures of DRG and NPG cells activated by patterned electrical field stimulation. To detect release of END-2, we developed a novel rapid capture enzyme-linked immunosorbent assay (ELISA), in which END-2 capture antibody was added to neuronal cultures shortly before their electrical stimulation. The conventional assay was effective at reliably detecting END-2 only when the cells were stimulated in the presence of CTAP, a MOR-selective antagonist. This suggests that the strength of the novel assay is related primarily to rapid capture of released END-2 before it binds to endogenous MORs. Using the rapid capture ELISA, we found that stimulation protocols known to induce plastic changes at sensory synapses were highly effective at releasing END-2. Removal of extracellular calcium or blocking voltage-activated calcium channels significantly reduced the release. Together, our data provide the first evidence that END-2 is expressed by newborn DRG neurons of all sizes found in this age group, and can be released from these, as well as from NPG neurons, in an activity-dependent manner. These results point to END-2 as a likely mediator of activity-dependent plasticity in sensory pathways.

Calcitonin gene-related peptide enhances release of native brain-derived neurotrophic factor from trigeminal ganglion neurons.

Activity-dependent plasticity in nociceptive pathways has been implicated in pathomechanisms of chronic pain syndromes. Calcitonin gene-related peptide (CGRP), which is expressed by trigeminal nociceptors, has recently been identified as a key player in the mechanism of migraine headaches. Here we show that CGRP is coexpressed with brain-derived neurotrophic factor (BDNF) in a large subset of adult rat trigeminal ganglion neurons in vivo. Using ELISA in situ, we show that CGRP (1-1000 nM) potently enhances BDNF release from cultured trigeminal neurons. The effect of CGRP is dose-dependent and abolished by pretreatment with CGRP receptor antagonist, CGRP(8-37). Intriguingly, CGRP-mediated BDNF release, unlike BDNF release evoked by physiological patterns of electrical stimulation, is independent of extracellular calcium. Depletion of intracellular calcium stores with thapsigargin blocks the CGRP-mediated BDNF release. Using transmission electron microscopy, our study also shows that BDNF-immunoreactivity is present in dense core vesicles of unmyelinated axons and axon terminals in the subnucleus caudalis of the spinal trigeminal nucleus, the primary central target of trigeminal nociceptors. Together, these results reveal a previously unknown role for CGRP in regulating BDNF availability, and point to BDNF as a candidate mediator of trigeminal nociceptive plasticity.

Cellular mechanisms regulating activity-dependent release of native brain-derived neurotrophic factor from hippocampal neurons.

Brain-derived neurotrophic factor (BDNF) plays a critical role in activity-dependent modifications of neuronal connectivity and synaptic strength, including establishment of hippocampal long-term potentiation (LTP). To shed light on mechanisms underlying BDNF-dependent synaptic plasticity, the present study was undertaken to characterize release of native BDNF from newborn rat hippocampal neurons in response to physiologically relevant patterns of electrical field stimulation in culture, including tonic stimulation at 5 Hz, bursting stimulation at 25 and 100 Hz, and theta-burst stimulation (TBS). Release was measured using the ELISA in situ technique, developed in our laboratory to quantify secretion of native BDNF without the need to first overexpress the protein to nonphysiological levels. Each stimulation protocol resulted in a significant increase in BDNF release that was tetrodotoxin sensitive and occurred in the absence of glutamate receptor activation. However, 100 Hz tetanus and TBS, stimulus patterns that are most effective in inducing hippocampal LTP, were significantly more effective in releasing native BDNF than lower-frequency stimulation. For all stimulation protocols tested, removal of extracellular calcium, or blockade of N-type calcium channels, prevented BDNF release. Similarly, depletion of intracellular calcium stores with thapsigargin and treatment with dantrolene, an inhibitor of calcium release from caffeine-ryanodine-sensitive stores, markedly inhibited activity-dependent BDNF release. Our results indicate that BDNF release can encode temporal features of hippocampal neuronal activity. The dual requirement for calcium influx through N-type calcium channels and calcium mobilization from intracellular stores strongly implicates a role for calcium-induced calcium release in activity-dependent BDNF secretion.

Influence of morphine on the activity of low-threshold visceral mechanoreceptors in cats with acute pericarditis.

The purpose of this investigation was to test whether morphine (morphinum hydrochloricum) applied to the receptive field of the thoracic visceral afferent fibres modifies their activity. Experiments were performed on chloralose-anaesthetised cats, paralysed and artificially ventilated, in a state of pericarditis that was induced by intrapericardial injection of lambda-carrageenan and kaolin. Resulting acute inflammation was proven histopathologically and documented electrocardiographically. Single afferent fibres with receptive fields in thoracic viscera were dissected from thoracic sympathetic chain (19 fibres), as well as the vagus nerve (9 fibres). All tested fibres transmitted sensory information from the low-threshold mechanoreceptors. As a final result, it was found that morphine (0.001-1.0 mg/ml) when applied locally activates, depending on the dose, afferent fibres as follows: 12 sympathetic afferents (out of 12 tested), and 7 vagal afferents (out of 9 tested). In examining the specificity of morphine action, the preliminary local application of naloxone (1.0 mg/ml) just before morphine, blocked all excitatory responses. The excitatory response was present whether the receptive field was located in the inflammatory area, or outside it, in group III or IV fibres.