The neuropeptide VIP regulates the expression of the tyrosine hydroxylase gene in cultured avian sympathetic neurons

The neuropeptide vasoactive intestinal polypeptide (VIP) increases the activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of catecholamines, in cultured chicken sympathetic neurons. We report here that VIP acts by increasing TH mRNA levels in these cells. Induction of TH mRNA is transient and reaches maximal values 6-8 h after the addition of the peptide to the cultures. TH mRNA levels return to control values after 1-2 days. The quail cDNA probe detects a single mRNA species of approximately 9 kb in RNA extracted both from embryonic chicken sympathetic neurons and adult quail adrenal medulla.     

Effect of neurocatin on the activity of monoamine oxidase B in rat brain synaptosomes

When the phenotype of neurons in pre- and paravertebral sympathetic ganglia are compared, there are marked differences in NGF dependence, neuropeptide content, connectivity and electrophysiological properties. The trophic interactions that induce these differences are currently poorly understood. One explanation is that prevertebral neurons receive a second neurotrophic signal, other than NGF, from their target of innervation. If this is the case, neurons in the prevertebral ganglia should express another neurotrophin receptor, in addition to the NGF receptor (trkA). To test this prediction, the level of expression of three neurotrophin receptors, trkA, trkB and trkC, were examined in one paravertebral sympathetic ganglia, the SCG, and two prevertebral ganglia, the celiac and superior mesenteric ganglia. It was found that mRNA encoding the full-length form of the trkB receptor was barely expressed in the SCG. Significantly higher levels of full-length trkB mRNA expression were found in the prevertebral ganglia. Ligands of the trkB receptor may, therefore, contribute to the differentiation and/or survival of some prevertebral sympathetic neurons.     

Functional Ca2+ and Na+ channels on mouse Schwann cells cultured in serum-free medium: regulation by a diffusible factor from neurons and by cAMP

Regulation of expression of functional voltage-gated ion channels for inward currents was studied in Schwann cells in organotypic cultures of dorsal root ganglia from E19 mouse embryos maintained in serum-free medium. Of the Schwann cells that did not contact axons, 46.5% expressed T-type Ca2+ conductances (ICaT). Two days or more after excision of the ganglia, and consequent disappearance of neurites, ICaT were detectable in only 10.9% of the cells, and the marker 04 disappeared. On Schwann cells deprived of neurons, T- (but not L-) type Ca2+ conductances were re-induced by weakly hydrolysable analogues of cAMP, and by forskolin (an activator of adenylyl cyclase) after long-term treatment (4 days). With CPT cAMP (0.1-2 mM), 8Br cAMP, db cAMP or forskolin (0.01 or 0.1 mM), the proportion of cells with ICaT was not significantly different from the proportion in the cultures with neurons. These agents also induced expression in some cells of tetrodotoxin-resistant Na+ currents, which were rarely induced by neurons, but 04 was not re-induced by cAMP analogue treatments that re-induced ICaT. Inward currents (Ba2+ or Na+) were partly restored (P < 0.05) on Schwann cells cultured for 6-7 days beneath a filter bearing cultured neurons. In contrast, addition of neuron-conditioned medium was ineffective. The results suggest that neurons activate, via diffusible and degradable factors, a subset of Schwann cell cAMP pathways leading to expression of IcaT, and activate additional non-cAMP pathways that lead to expression of 04.     

All prepro-VIP-derived peptides, except PHI/PHV, are expressed in the female rat anterior pituitary and increased by estrogen

The expression of VIP precursor products: prepro-VIP(22-79), peptide histidine isoleucine (PHI), peptide histidine valine (PHV), prepro-VIP(111-122), VIP, prepro-VIP(156-170), and prepro-VIP mRNA in the anterior pituitary of estrogen-treated, ovariectomized rats, of ovariectomized controls, and of sham-operated controls was examined. Using radioimmunoassays based on antisera against each of the prepro-VIP sequences, we found that all sequences were expressed and markedly induced by estrogen, except PHI and PHV, which both were undetectable. By immunohistochemistry, it appeared that the number of cells immunoreactive for each of these sequences was increased in the estrogen-treated animals. However, PHI/PHV-immunoreactive cells could not be detected, despite the use of four different PHI antisera with different specificities. Estrogen treatment increased the prepro-VIP mRNA as judged by Northern blotting. In situ hybridization signals for both VIP mRNA and PHI mRNA were observed in few pituitary cells from control animals whereas strong positive signals were observed in a larger number of cells after estrogen treatment. The findings show that estrogen causes activation of the VIP gene expression in anterior pituitary cells, and that the absence of PHI and PHV probably is due to translational or posttranslational events.     

Bone morphogenetic protein-2 and retinoic acid induce neurotrophin-3 responsiveness in developing rat sympathetic neurons

Expression of the receptor tyrosine kinase, Trk, determines the specificity of neurotrophin responsiveness of different neuronal populations during development. Recently it has become apparent that sympathetic neurons of rat superior cervical ganglia (SCG) acquire sensitivity to neurotrophin-3 (NT3) before they become dependent on the target-derived nerve growth factor (NGF) for their survival by sequential induction of TrkC and TrkA. The mechanism controlling the expression of TrkC as well as the source of NT3 at their initial developmental stage has, however, not been clarified. Here we show that the treatment of the perinatal rat SCG neurons which express high levels of trkA mRNA with bone morphogenetic protein-2 (BMP2) induced the expression of trkC mRNA. Induction of the functional TrkC receptor by BMP2 was confirmed by the enhancement of the survival response of these neurons to NT3. Treatment of SCG neurons with retinoic acid (RA) promoted the effect of BMP2 on the induction of trkC mRNA levels. BMP2 treatment, on the other hand, promoted the effect of RA on the suppressions of trkA mRNA levels and the NGF-dependent survival of the SCG neurons. Furthermore, BMP2/RA treatment induced the endogenous expression of NT3. These results indicate that specific environmental signals can regulate neurotrophin responsiveness of developing sympathetic neurons by differential alteration of the trk and neurotrophin expressions.     

Signals triggering the induction of leukemia inhibitory factor in sympathetic superior cervical ganglia and their nerve trunks after axonal injury

Leukemia inhibitory factor (LIF) plays an important role in regulating neuropeptide expression in sympathetic and sensory neurons after axonal transection. By 2 h after axotomy, LIF mRNA increased in nonneuronal cells in sympathetic ganglia and peripheral nerve. In addition, within 1 h of explanting sympathetic ganglia or segments of sympathetic nerve trunks, a protein factor(s) that was able to induce LIF mRNA both in sympathetic cultures and in intact ganglia in vivo was released. This factor(s) appeared to be present in sympathetic ganglia and their nerve trunks under normal conditions and to be activated and/or released after axonal injury. Since the factor(s) has a molecular weight(s) greater than 66 kDa, and no other proteins of such high molecular weight have been previously identified with LIF-inducing activity, it appears to be a novel inducer of LIF.     

Differential and coordinated regulation of expression of norepinephrine transporter in catecholaminergic cells in culture

The norepinephrine transporter (NET) terminates noradrenergic neurotransmission at synapse by high-affinity sodium-dependent reuptake into presynaptic terminals, and thus serves as a marker of differentiation of noradrenergic neurons. In the present study, we studied the regulatory mechanism of the expression of NET-mRNA in cultured neurons from newborn rat superior cervical ganglia (SCG) and in clonal rat pheochromocytoma cells (PC12) SCG neurons in culture expressed a high level of NET-mRNA, which was further increased 2.5-5 fold from day 1 to day 13. Treatment of SCG neurons with the cholinergic differentiation factor (CDF)/leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF), neurokines known to induce the switch from adrenergic to cholinergic phenotype in SCG neurons, led to the suppression of the level of NET-mRNA in a concentration dependent manner, concomitantly with the suppression of mRNA for tyrosine hydroxylase (TH), an adrenergic marker enzyme in cultured SCG neurons. On the other hand, retinoic acid, a compound which is also known to increase the expression of choline acetyltransferase, a cholinergic marker enzyme, and suppress the expression of TH in the cultured SCG neurons and PCI2 cells, rather increased the level of NET-mRNA in these two cell populations. Alterations of the Na(+)-dependent norepinephrine transport activity which paralleled the changes in the NET-mRNA levels were confirmed by the [3H]norepinephrine uptake assay. These results indicate that cell extrinsic factors regulate the expressions of NET and TH genes by a common as well as by distinct mechanisms.     

The developmental expression of choline acetyltransferase (ChAT) and the neuropeptide VIP in chick sympathetic neurons: evidence for different regulatory events in cholinergic differentiation

Cholinergic properties in chick sympathetic neurons are detectable early during development of paravertebral ganglia and mature after target contact. The cholinergic marker choline acetyltransferase (ChAT) is first detectable at embryonic day 6 and its expression partly overlaps with that of the noradrenergic marker tyrosine hydroxylase (TH). At late embryonic stages, when sympathetic neurons have established target contact, ganglia consist of two major neuronal populations, TH-positive noradrenergic neurons and cholinergic neurons that at this stage express vasoactive intestinal peptide (VIP) in addition to ChAT. The maturation of sympathetic neurons is paralleled by changes in their response to the neurokine ciliary neurotrophic factor (CNTF). These findings suggest that expression of neurotransmitter properties is controlled differentially before and during target innervation.     

Oil blends containing partially hydrogenated or interesterified fats: differential effects on plasma lipids

The mechanism of adenosine-induced inhibition of ganglionic transmission was investigated in the isolated superior cervical ganglion (SCG) of the rat. The inhibitory effect of adenosine on the postganglionic compound action potential (CAP) was antagonized by pretreatment of ganglia with forskolin, isoproterenol (IPNE), arginine vasopressin (AVP), or papaverine, all of which are known to increase tissue cAMP level by different mechanisms. Furthermore, pretreatment of ganglia with the adenylate cyclase inhibitor SQ 22, 536, or the phosphodiesterase activator imidazole reversed the effects of IPNE and forskolin. Pretreatment with 8-bromo-cAMP, resulted in a marked antagonism of the adenosine-induced inhibition. By themselves, none of these drugs had any significant effect on the CAP. Adenosine slightly but significantly decreased the basal level of cAMP in untreated ganglia. Formation of cAMP induced by IPNE was markedly reduced by adenosine. This was largely reversed in the presence of the adenosine A1 receptor antagonist 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX) but not the A2 receptor antagonist 3, 7-dimethyl-1-propargylxanthine (DPMX). We conclude that the inhibition of ganglionic transmission by adenosine involves reduction of cAMP formation through activation of A1 receptors.     

The specification of sympathetic neurotransmitter phenotype depends on gp130 cytokine receptor signaling

Sympathetic ganglia are composed of noradrenergic and cholinergic neurons. The differentiation of cholinergic sympathetic neurons is characterized by the expression of choline acetyltransferase (ChAT) and vasoactive intestinal peptide (VIP), induced in vitro by a subfamily of cytokines, including LIF, CNTF, GPA, OSM and cardiotrophin-1 (CT-1). To interfere with the function of these neuropoietic cytokines in vivo, antisense RNA for gp130, the common signal-transducing receptor subunit for neuropoietic cytokines, was expressed in chick sympathetic neurons, using retroviral vectors. A strong reduction in the number of VIP-expressing cells, but not of cells expressing ChAT or the adrenergic marker tyrosine hydroxylase (TH), was observed. These results reveal a physiological role of neuropoietic cytokines for the control of VIP expression during the development of cholinergic sympathetic neurons.     

Endothelins inhibit cyclic-AMP induced renin gene expression in cultured mouse juxtaglomerular cells

We have recently described that endothelins-1 to -3 equipotently inhibit cAMP stimulated renin secretion from cultured mouse juxtaglomerular cells by a process involving phospholipase C activation. This study examined the influence of endothelin-2 on renin gene expression in renal juxtaglomerular cells. To this end we semiquantitated renin mRNA levels by competitive RT-PCR in primary cultures of mouse renal juxtaglomerular cells after 20 hours of incubation. We found that endothelin-2 (0.1 to 100 nmol/liter) did not change basal renin gene expression. The adenylate cyclase activator forskolin (3 mumol/ liter) increased renin mRNA levels to 400% of the controls and this stimulation was dose-dependently attenuated by ET-2 to 250% of the control value. The effect of ET-2 was mimicked by the ETB-receptor agonist sarafotoxin S6c. The kinase inhibitor staurosporine (100 nmol/ liter) increased renin secretion and renin mRNA levels. Combination of staurosporine with forskolin produced the same effects on renin secretion and renin mRNA levels as did staurosporine alone. In the presence of both forskolin and staurosporine ET-2 had no significant effect on renin secretion and renin gene expression. The phorbol ester PMA (30 nmol/ liter), which was used to stimulate protein kinase C activity, attenuated cAMP stimulated renin secretion and renin mRNA levels. Lowering the extracellular concentration of calcium by the addition of 1 mmol/liter EGTA did not inhibit the effect of ET-2 on cAMP induced renin secretion and renin gene expression. These findings suggest that endothelins inhibit cAMP stimulated renin gene expression by an event that is mediated via ETB receptors. This inhibitory effect may in part involve protein kinase C activation.     

Neural cell classification by wavelets and multiscale curvature

It has been postulated that the aberrant projection of sympathetic axons to individual primary sensory neurons may provide the morphological basis for pain-related behaviors in rat models of chronic pain syndrome. Since nerve growth factor (NGF) can elicit the collateral sprouting of noradrenergic sympathetic terminals, it might be predicted that NGF plays a role in mediating the sprouting of sympathetic axons into sensory ganglia. Using a line of transgenic mice overexpressing NGF among glial cells, it was first found that trigeminal ganglia from adult transgenic mice possessed significantly higher levels of NGF protein in comparison to age-matched wild-type mice; as well, detectable levels of NGF mRNA transgene expression were present in both the ganglia and brain stem. Within the trigeminal ganglia, a small proportion of the sensory neuronal population stained immunohistochemically for NGF; a higher percentage of NGF-positive neurons was evident in transgenic mice. New sympathetic axons extended into the trigeminal ganglia of transgenic mice only and formed perineuronal plexuses surrounding only those neurons immunostained for NGF. In addition, such plexuses were accompanied by glial processes from nonmyelinating Schwann cells. From these data, we propose that accumulation of glial-derived NGF by adult sensory neurons and its putative release into the ganglionic environment induce the directional growth of sympathetic axons to the source of NGF, namely, the cell bodies of primary sensory neurons.     

Preferential accumulation of single-stranded regions in telomeres of human fibroblasts

In vertebrate neuromuscular junctions, post-synaptic specialization includes aggregation of acetylcholine receptors (AChRs) and acetylcholinesterase (AChE). The motor nerve provides soluble factors and electrical activity to achieve this striking localization of AChRs/AChE. Calcitonin gene-related peptide (CGRP), a neuropeptide synthesized by motor neurons, is able to stimulate the expression of AChR in cultured myotubes. Similar to AChR regulation, synthesis of AChE in cultured chick myotubes is also stimulated by CGRP. Application of CGRP onto cultured myotubes stimulated the accumulation of intracellular cyclic AMP (cAMP) as well as the expression of AChE mRNA and protein. However, the enzymatic activity of AChE remained unchanged. In cultured myotubes, various drugs affecting the intracellular level of cAMP, such as N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate, cholera toxin, and forskolin, could mimic the effect of CGRP in stimulating the expression of AChE. When myotubes were transfected with cDNA encoding constitutively active mutant Galpha(s), the intracellular cAMP synthesis was increased. The increase in cAMP level was in parallel with an increase in the expression of AChE, whereas transfection of active mutant Galpha(i) cDNA decreased the cAMP level as well as the AChE expression. In addition, expression of collagen-tailed AChE was up-regulated by the cAMP pathway. These findings indicated that CGRP-induced AChE regulation is mediated by the cAMP pathway and represented the first evidence to suggest that the regulation of mRNA synthesis of AChR and AChE can be mediated by the same neuron-derived factor.     

Response and level of beta-adrenergic, vasoactive intestinal peptide, and PACAP receptors during the circadian cycle

PURPOSE: To determine whether a nocturnal increase of ciliary process beta-adrenergic receptor responsiveness can explain the observation that timolol decreased the aqueous flow rate and intraocular pressure (IOP) during the night but not during the day in rabbits. METHODS: Rabbits were housed in alternating 12-hour periods of light and dark. In vitro stimulation of tissue cyclic adenosine monophosphate (cAMP) levels by isoproterenol (ISO), vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating polypeptide (PACAP), or a soluble derivative of forskolin (sFSK) was measured in ciliary processes harvested at mid-light phase and early and late dark phase. Inhibition of ISO and VIP stimulation of ciliary process cAMP by an alpha 2-adrenergic agonist and maximal binding of [125I]I-pindolol, [125I]I-VIP, and [125I]I-PACAP in ciliary process membranes were measured at the same three times. RESULTS: Although there may have been a nocturnal increase in the sensitivity of ciliary process cAMP levels to stimulation by ISO, this was not observed consistently, VIP, but not PACAP, stimulation increased at night, but there was no change in the response to sFSK. Inhibition by apraclonidine of elevated ciliary process cAMP levels was constant at all three times. Ligand-binding studies showed little change in ciliary process beta-adrenergic, VIP-, or PACAP-receptor levels at the three times. CONCLUSIONS: There is no convincing evidence for a nocturnal increase in beta-adrenergic receptor sensitivity in rabbit ciliary processes that could explain the difference between day and night effects of timolol on aqueous flow and IOP.     

Effects of ethanol on basal and prostaglandin E1-induced increases in beta-endorphin release and intracellular cAMP levels in hypothalamic cells

Our recent studies determining the effect of cAMP-elevating agents forskolin and dibutyryl cAMP on ethanol-induced immunoreactive beta-endorphin (IR-beta-EP) release from hypothalamic cells in primary cultures suggested the possibility that both stimulatory and adaptive secretory responses of beta-EP neurons after ethanol exposure may involve the cAMP system. To determine further the role of cAMP, the effects of prostaglandin E1 (PGE1) on basal and ethanol-regulated IR-beta-EP secretion and cAMP productions were determined in primary cultures of hypothalamic cells. The results presented in this study show that a 50 mM dose of ethanol, which is within the EC50 dose of ethanol required to elevate IR-beta-EP release from hypothalamic cells, increased cellular levels of cAMP and elevated IR-beta-EP release simultaneously from the cultured neurons for a period of 6 hr. The cAMP and IR-beta-EP secretory responses developed desensitization to ethanol challenge after 24 hr of constant ethanol incubation. The cAMP-elevating agent PGE1 increased the cellular content of cAMP and IR-beta-EP release in a dose-dependent manner. The EC50 dose of PGE1 for elevation of IR-beta-EP and cAMP was approximately 0.5 microM. As with ethanol, chronic treatment with PGE1 desensitized the cAMP and IR-beta-EP responses of hypothalamic neurons to PGE1. Acute exposure to ethanol increased the PGE1-stimulated levels of cAMP and IR-beta-EP, whereas chronic exposure to ethanol resulted in diminished cAMP responses to PGE1. These data provide evidence that the cAMP system may be involved in controlling hypothalamic beta-EP secretion, as well in regulating the stimulatory and adaptive responses of beta-EP neurons to ethanol.