Gene-related Peptide Messenger Research Articles

Circulating calcitonin gene-related peptide and its placental origins in normotensive and preeclamptic pregnancies

The present study was designed to determine plasma calcitonin gene-related peptide concentration in both maternal and fetal circulations in normotensive and pre-eclamptic pregnancies and investigate whether placenta is 1 of its origins. Maternal blood, cord blood, and villous tissue were collected from women in normotensive pregnancies and complicated with pre-eclampsia. Calcitonin gene-related peptide concentrations were determined by radioimmunoassay. Cellular localizations of calcitonin gene-related peptide messenger ribonucleic acid and protein expressions in placental villi were determined by in situ hybridization and immunohistochemistry. The following results were reached: (1) maternal plasma calcitonin gene-related peptide concentrations increased with advancing gestation but fell after delivery; (2) both maternal and cord plasma calcitonin gene-related peptide concentrations were positively correlated with the infant birth weights; (3) compared with normotensive pregnancies, calcitonin gene-related peptide levels in both maternal and cord plasma decreased in pregnancies with pre-eclampsia; (4) in normotensive pregnancies, the plasma calcitonin gene-related peptide of the umbilical vein was higher than the umbilical artery, but no significant differences between vein and artery in pre-eclampsia; (5) calcitonin gene-related peptide messenger ribonucleic acid and protein were expressed by syncytiotrophoblast cells and villous vascular endothelial cells in normotensive pregnancies, but only weak or absent staining was observed in pre-eclamptic placentas; and (6) calcitonin gene-related peptide is secreted by villous tissue in explant culture in a time-dependent manner, but less calcitonin gene-related peptide was produced by villous tissues from patients with pre-eclampsia. Calcitonin gene-related peptide may play potential roles in maternal hemodynamic adaptation and fetal growth. Decreased circulating calcitonin gene-related peptide levels may be involved in maternal-fetal pathophysiology of pre-eclampsia. It is novel that placenta villous tissues might be one of the potential sources of calcitonin gene-related peptide during pregnancy.

Calcitonin gene-related peptide gene expression in collagen-induced arthritis is differentially regulated in primary afferents and motoneurons: influence of glucocorticoids

Calcitonin gene-related peptide is involved in peripheral and spinal mechanisms of inflammatory pain. In this paper, we used collagen II-induced arthritis in the rat as a model to investigate the influence of chronic arthritic pain on calcitonin gene-related peptide gene expression in sensory and motor pathways. Additionally, we examined the effect of the glucocorticoid drug budesonide on arthritis-induced changes of calcitonin gene-related peptide expression and constitutive calcitonin gene-related peptide expression. Thirteen days after the immunization with native rat collagen type II rats developed a progressive and chronic polyarthritis which was scored with respect to the degree of swelling and/or redness of the paw and ankle joints. Budesonide significantly attenuated the extent of arthritis. Changes in calcitonin gene-related peptide expression were evaluated by semiquantitative in situ hybridization and immunocytochemistry on day 21 post-immunization. In sensory neurons of dorsal root ganglia of arthritic rats, a significant increase in calcitonin gene-related peptide messenger RNA and protein levels was seen. These increases were completely blocked by budesonide. Also in dorsal root ganglia of non-arthritic rats, budesonide had an effect, with reduced calcitonin gene-related peptide messenger RNA levels below constitutive concentrations. Image analysis of calcitonin gene-related peptide immunoreactivity revealed that changes in calcitonin gene-related peptide expression were due to alterations in calcitonin gene-related peptide expression levels rather than to de novo synthesis or changes in the numbers of calcitonin gene-related peptide expressing neurons. In spinal motoneurons of arthritic rats, marked decreases in calcitonin gene-related peptide messenger RNA and protein levels were measured. These reductions were attenuated by budesonide. The changes in calcitonin gene-related peptide expression in motoneurons correlated with the severity of arthritis in the ipsilateral hind paw. Budesonide had no effects on calcitonin gene-related peptide messenger RNA levels in motoneurons of non-arthritic rats. The opposite regulation of calcitonin gene-related peptide gene expression in primary sensory and spinal somatomotor pathways in collagen-induced arthritis suggests that calcitonin gene-related peptide plays a specific role in both chronic inflammatory pain and arthritis-induced motor dysfunction. The sensitivity of constitutive and inflammation-induced sensory calcitonin gene-related peptide expression to budesonide treatment may indicate that the beneficial effects of steroid treatment in inflammation is partly mediated by down-regulation of calcitonin gene-related peptide in sensory neurons involved in neurogenic inflammation.

Ultrastructural localization of increased neuropeptide immunoreactivity in the axons and cells of the gracile nucleus following chronic constriction injury of the sciatic nerve

Neuropeptide plasticity in the gracile nucleus is thought to play a role in the development of neuropathic pain following nerve injury. Two weeks after chronic constriction injury of adult rat sciatic nerve, galanin, neuropeptide Y and calcitonin gene-related peptide immunoreactivities were increased in fibers and cells in the gracile nucleus ipsilateral to injury. At the electron microscopic level, this increased neuropeptide immunoreactivity was localized in myelinated axons, boutons, dendrites, neurons and glial cells. Galanin-, neuropeptide Y- and calcitonin gene-related peptide-immunoreactive boutons were frequently presynaptic to dendrites of both immunoreactive and non-immunoreactive neurons. However, no neuropeptide Y, galanin and calcitonin gene-related peptide messenger RNA was detected in the injured side gracile nuclei by in situ hybridization. These results show that partial nerve injury to the sciatic nerve induces increases in the content of galanin, neuropeptide Y and calcitonin gene-related peptide immunoreactivities in synaptic terminals within the gracile nucleus, which suggests that there may be increased release of these neuropeptides following sensory or spontaneous stimulation of large-diameter primary afferents following partial nerve injury, perhaps one mechanism involved in neuropathic pain. We also show an apparent transfer of these neuropeptides to the cells of the gracile nucleus, both neurons and glial cells, an intriguing phenomenon of unknown functional significance.

Expression of the genes for α-type and β-type calcitonin gene-related peptide during rat embryogenesis

Throughout rat embryogenesis we analysed the expression patterns of the three mature transcripts generated from the two calcitonin gene-related peptide genes: calcitonin, α-calcitonin gene-related peptide, and β-calcitonin gene-related peptide messenger RNAs. In addition, we examined in parallel the distribution of calcitonin gene-related peptide and calcitonin immunoreactivity. Of the three transcripts, β-calcitonin gene-related peptide messenger RNA was first detected in sensory ganglia on embryonic day 14, and by embryonic day 15 was seen to a lesser degree in motor neurons and autonomic ganglia. Starting at embryonic day 16, however, the highest levels of β-calcitonin gene-related peptide messenger RNA were found in motor neurons rather than sensory ganglia. α-calcitonin gene-related peptide messenger RNA was first detected on embryonic day 16 in both sensory ganglia and motor neurons, but at lower levels than β-calcitonin gene-related peptide, particularly in the motor neurons of the spinal cord. By embryonic day 20, transcripts for α- and β-calcitonin gene-related peptide were expressed in distinct brain regions. High levels of α-calcitonin gene-related peptide messenger RNA were detected in hypoglossal, facial, and parabrachial nuclei, and moderate levels in the trigeminal motor and ambiguus nuclei. By contrast, β-calcitonin gene-related peptide messenger RNA was detected at low levels in hypoglossal, ambiguus, facial, and parabrachial nuclei, and at high levels in the trigeminal nucleus. In the oculomotor-trochlear nucleus, β-calcitonin gene-related peptide messenger RNA was the sole isotype expressed. Low levels of messenger RNA for both calcitonin gene-related peptide transcripts were appreciated in the inferior olive. Outside the nervous system, α-calcitonin gene-related peptide messenger RNA was weakly expressed in the thyroid gland and β-calcitonin gene-related peptide messenger RNA in the thymus. Throughout embryogenesis, calcitonin gene-related peptide immunoreactivity usually followed the expression of either α- or β-calcitonin gene-related peptide messenger RNA. Calcitonin messenger RNA and protein were detected only in the thyroid gland from embryonic day 18 onward. This work shows that of the three mature transcripts produced by the two calcitonin gene-related peptide genes, β-calcitonin gene-related peptide messenger RNA is the predominant transcript produced early in rat embryogenesis. However, by perinatal stages α-calcitonin gene-related peptide shows the highest expression in the brain and spinal cord. In autonomic ganglia, β-calcitonin gene-related peptide is either the sole or the predominant transcript. Unlike the chick embryo in which calcitonin messenger RNA is expressed early in the CNS, in rat it was only expressed outside the nervous system in the thyroid gland during the last days of embryogenesis.

Calcitonin gene-related peptide promotes differentiation, but not survival, of rat mesencephalic dopaminergic neurons in vitro

The aim of this study was to investigate putative effects of calcitonin gene-related peptide on developing dopaminergic neurons in the ventral mesencephalon. To determine a time-point for a physiological role of calcitonin gene-related peptide in the development of this system, we first investigated calcitonin gene-related peptide messenger RNA expression in the ventral mesencephalon of Wistar rats at embryonic days (E) 11–19. Calcitonin gene-related peptide messenger RNA was not detectable at E11, i.e. prior to the appearance of dopaminergic neurons in this area. From E14 to E19, calcitonin gene-related peptide messenger RNA was expressed in increasing amounts. We therefore investigated the effects of calcitonin gene-related peptide on serum-free cell cultures established from the E14 midbrain floor. Addition of calcitonin gene-related peptide (200 ng/ml) every other day significantly increased neuronal differentiation, including longer tyrosine hydroxylase-positive neurites, enhanced immunoreactivity for growth-associated protein-43 and increased dopaminergic uptake per neuron. These effects were maximal after seven to eight days. Calcitonin gene-related peptide acted synergistically with fibroblast growth factor-2 on these parameters. In contrast to fibroblast growth factor-2, however, calcitonin gene-related peptide did not promote survival of tyrosine hydroxylase-immunoreactive neurons. Lack of calcitonin gene-related peptide expression in the mesencephalon at E11 was paralleled by a lack of effect of calcitonin gene-related peptide on early presumptive dopaminergic neurons in terms of eliciting this phenotype. Our data suggest that calcitonin gene-related peptide may act physiologically as a differentiation-promoting factor for phenotypically defined dopaminergic neurons during a time period when dopaminergic neurons assemble in the ventral mesencephalon and grow axons towards their targets.

Changes in the mRNA expression pattern, with special reference to calcitonin gene-related peptide, after axonal injuries in rat motoneurons depends on age and type of injury.

The axotomy reaction in motoneurons after a peripheral nerve transection in the adult animal is characterized by a robust upregulation of alpha-calcitonin gene-related peptide (CGRP) messenger RNA (mRNA) together with mRNAs encoding cytoskeletal and growth-related proteins. Here we have examined whether the nature of the lesion and the age of the animal have any impact on the mRNA regulation in severed cells. Thus, the effect of a sciatic nerve transection in the adult rat was compared with, on the one hand, ventral root avulsions in the adult animal and, on the other hand, sciatic nerve transection in the immature animal. In the two latter cases, a proportion of the lesioned cells die and overall chances of regeneration are small. In the adult animal a sciatic nerve transection induced an upregulation of alpha-CGRP mRNA from the 3rd day after surgery and throughout the first 3 weeks (the time span of the study). Also low-affinity nerve growth factor receptor (p75) and growth-associated protein-43 (GAP-43) mRNAs were upregulated during the entire 3-week period. In contrast, after ventral root avulsion, the expression of alpha-CGRP, c-jun, and p75 mRNAs were normalized within the 1st postoperative week, while GAP-43 mRNA was still upregulated at 3 weeks. Galanin message-associated peptide (GMAP) mRNA became upregulated preferentially in motoneurons subjected to ventral root avulsion, while nitric oxide synthase (NOS) mRNA was expressed exclusively after the latter type of injury. In the immature animal, alpha-CGRP mRNA was downregulated after sciatic nerve transection in rats aged 3 days or 7 days at the time of surgery; while, in contrast, an upregulation was seen in 12- or 21-day-old animals. GAP-43 and c-jun mRNAs were upregulated in lesioned motoneurons of all ages, while GMAP mRNA was upregulated preferentially in lesioned motoneurons of early postnatal animals. p75 mRNA was expressed in unlesioned immature motoneurons until the age of 7-10 days. The downregulation of p75 mRNA in intact cells at this age coincided with a developmental switch in the ability of axotomized cells to express increased levels of p75 mRNA. No expression of NOS mRNA was detectable in lesioned cells of any of the age groups. These results show that the age of the animal and the type of axonal injury are indeed to a high degree influencing the changes seen in the protein expression pattern in axotomized rat motoneurons. The different responses in these paradigms suggest differences in the trophic response from surrounding glia or the trophic responsiveness of lesioned motoneurons. Also, the results may indicate different roles for the studied substances during the regenerative response of lesioned neurons. Of the substances studied here, upregulation of alpha-CGRP and p75 mRNAs best correlated with a possibility of axon regeneration.

Up-regulation of preprotachykinin and calcitonin gene-related peptide messenger RNA in L4 dorsal root ganglion neurons following L5 spinal nerve ligation; a rat neuropathic pain model

The anti-emetic mechanism of action of fentanyl to inhibit nicotine (5 mg/kg, s.c.)-induced emesis was investigated in Suncus murinus. The anti-emetic action of fentanyl (40 μg/kg, s.c.) was antagonised by the opioid receptor antagonists naltrexone (1 mg/kg, s.c.), naloxone (1 mg/kg, s.c.), M8008 (16S-methylcyprenorphine; 1 mg/kg, s.c.) and MR 2266 (5,9-diethyl-2-(3-furylmethyl)2′-hydroxy-7,7-benzomorphan; 1 mg/kg) but not by naloxone methylbromide (1 mg/kg, s.c.), naloxone methyliodide (1 mg/kg, s.c.), naltrindole (1 mg/kg, s.c.), DIPPA (2-(3,4-dichlorophenyl)-N-methyl-N-[1S)-1-(3-isothiocyanatophenyl)-2-(1-pyrrolidinyl)ethyl]acetamide; 3 mg/kg, i.p.) or naloxonazine (35 mg/kg, i.p.). This indicates an involvement of μ2-opioid receptors within the brain to mediate the anti-emetic effect of fentanyl. In other studies, naloxone 10–60 mg/kg, s.c. induced dose-related emesis but naltrexone was only emetic at 60 mg/kg, s.c. and naloxone methylbromide failed to induce emesis at doses up to 60 mg/kg, s.c. The emesis induced by a high dose of naloxone 60 mg/kg, s.c. was antagonized by CP-99,994 ((+)-(2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine; 3–30 mg/kg, i.p.), 8-OH-DPAT, ((±)-8-hydroxy-dipropylaminotetralin; 0.003–0.3 mg/kg, s.c.), buspirone (3 mg/kg, s.c.) and fluphenazine (1–3 mg/kg, i.p.) but not by naltrexone (1–30 mg/kg, s.c.), metoclopramide (0.3–3 mg/kg, i.p.), sulpiride (0.3–3 mg/kg, i.p.), domperidone (0.1–3 mg/kg, i.p.), ondansetron (0.3–3 mg/kg, i.p.), granisetron (0.3–3 mg/kg, i.p.), scopolamine (0.3–3 mg/kg, i.p.) or promethazine (0.3–3 mg/kg, i.p.). The data is discussed in relation to opioid receptor mechanisms moderating emesis and the identification of potential sites of drug action available to inhibit the emetic reflex.

Calcitonin gene-related peptide increase in the rat spinal dorsal horn and dorsal column nucleus following peripheral nerve injury: Up-regulation in a subpopulation of primary afferent sensory neurons

Calcitonin gene-related peptide in sensory primary afferent neurons has an excitatory effect on postsynaptic neurons and potentiates the effect of substance P in the rat spinal dorsal horn. It has been established that calcitonin gene-related peptide expression in dorsal root ganglion neurons is depressed, and the effect of calcitonin gene-related peptide on dorsal horn neurons is attenuated, following peripheral nerve injury. We report here that a subpopulation of injured dorsal root ganglion neurons show increased expression of calcitonin gene-related peptide. Using in situ hybridization and the retrograde tracer, FluoroGold, we detected an increased number of medium- to large-sized rat dorsal root ganglion neurons projecting to the gracile nucleus that expressed α-calcitonin gene-related peptide messenger RNA following spinal nerve transection. Immunohistochemistry revealed a significant increase in calcitonin gene-related peptide immunoreactivity in the gracile nucleus and in laminae III–IV of the spinal dorsal horn.These results indicate that a subpopulation of dorsal root ganglion neurons express α-calcitonin gene-related peptide messenger RNA in response to peripheral nerve injury, and transport this peptide to the gracile nucleus and to laminae III–IV of the spinal dorsal horn. The increase of the excitatory neuropeptide, calcitonin gene-related peptide, in sites of primary afferent termination may affect the excitability of postsynaptic neurons, and have a role in neuronal plasticity following peripheral nerve injury.

Systemic capsaicin in the adult rat differentially affects gene expression for neuropeptides and neurotrophin receptors in primary sensory neurons

While systemic capsaicin in adult rats is known to reduce substance P and somatostatin in primary sensory nerves, it is still unknown if it also affects the production of these peptides at the genetic level. Therefore, we examined the effects of systemically administered capsaicin on the expression of the β-preprotachykinin, γ-preprotachykinin, somatostatin, calcitonin gene-related peptide, vasoactive intestinal polypeptide, galanin, neuropeptide Y and neurotrophin receptor family (trkA, trkB, trkC) genes in dorsal root ganglion neurons by in situ hybridization in adult rats. Nerve growth factor is thought to be involved in the regulation of some of these genes. In the control animals, β-preprotachykinin, γ-preprotachykinin, calcitonin gene-related peptide, somatostatin, trkA, trkB and trkC messenger RNAs were found in about 30%, 30%, 40%, 10%, 40%, 5% and 20% of the lumbar dorsal root ganglion neurons, respectively. The number of neurons expressing β/ γ-preprotachykinin and calcitonin gene-related peptide messenger RNAs decreased to about 50% and 70% of the control values, respectively, six days after subcutaneous administration of capsaicin (950 mg/kg). Simultaneously, the number of trkA messenger RNA-expressing neurons also decreased to about 70% of the control level, while the number of neurons expressing trkB and trkC messenger RNAs was unaffected. On the other hand, vasoactive intestinal polypeptide and galanin messenger RNAs, but not neuropeptide Y messenger RNA, began to be expressed in about 10% of dorsal root ganglion neurons after administration of capsaicin, although their messenger RNAs were not detected in the controls. However, the expression of somatostatin messenger RNA was unaffected by the systemic administration of capsaicin. The somatostatin messenger RNA was not co-expressed with vasoactive intestinal polypeptide and galanin messenger RNAs in the sensory neurons of rats given capsaicin. Electron microscopic analysis revealed a few degenerating unmyelinated afferents in sural nerves of the treated rats. The number of small-sized dorsal root ganglion cells labeled with Fluoro-Gold, a retrograde-tracing dye which was injected into the sural nerve of the treated rats, decreased to half of the control number. Our results suggest that systemic administration of capsaicin in adult rats depresses the expression of β/ γ-preprotachykinin, calcitonin gene-related peptide and trkA messenger RNAs, and induces expression of vasoactive intestinal polypeptide and galanin messenger RNAs in sensory neurons, which may be due to the capsaicin-induced degeneration of a subpopulation of sensory afferents. We also demonstrated that the regulation of somatostatin gene expression in mature sensory neurons is not affected by systemic capsaicin.

Pituitary adenylate cyclase activating peptide expression in the rat dorsal root ganglia: up-regulation after peripheral nerve injury.

Pituitary adenylate cyclase activating peptide (PACAP) is expressed in a population of capsaicin-sensitive primary sensory neurons of small to medium size in the rat. In the present report we have examined the effect of sciatic nerve injury (unilateral transection) on PACAP expression (immunocytochemistry, radioimmunoassay, in situ hybridization and northern blot analysis) in dorsal root ganglia at the lumbar level and on immunoreactive PACAP in the spinal cord and in the sciatic nerve stump. For comparison, calcitonin gene-related peptide was examined. In dorsal root ganglia of the intact side immunoreactive PACAP and PACAP messenger RNA were localised to a population of nerve cell bodies of small to medium size. In dorsal root ganglia on the injured side, PACAP-immunoreactive nerve cell bodies were more numerous and PACAP messenger RNA was considerably more abundant as studied 14 days after sciatic nerve transection. By contrast, calcitonin gene-related peptide-containing nerve cell bodies were numerous and rich in calcitonin gene-related peptide messenger RNA in dorsal root ganglia on the intact side, while after transection both the number of immunoreactive nerve cell bodies and their content of messenger RNA were markedly reduced. There were indications of axotomy-induced expression of PACAP messenger RNA in larger neurons. In the dorsal horn of the spinal cord on the intact side PACAP and calcitonin gene-related peptide-immunoreactive fibres were densely accumulated in the superficial layers. On the transected side the densities of both PACAP and calcitonin gene-related peptide-immunoreactive nerve fibres were reduced in the medial part. The data obtained indicate a marked up-regulation of PACAP in sensory neurons following peripheral nerve injury. Since PACAP depresses a C-fibre evoked flexion reflex, this may have implications for sensory transmission. Further, in view of the known promoting effects of PACAP on neuronal survival and differentiation and non-neuronal cell growth as well as its proinflammatory effects a role of PACAP in the neuronal and periaxonal tissue restoration after injury is not inconceivable.

Pituitary adenylate cyclase activating peptide expression in the rat dorsal root ganglia: up-regulation after peripheral nerve injury

Pituitary adenylate cyclase activating peptide (PACAP) is expressed in a population of capsaicin-sensitive primary sensory neurons of small to medium size in the rat. In the present report we have examined the effect of sciatic nerve injury (unilateral transection) on PACAP expression (immunocytochemistry, radioimmunoassay, in situ hybridization and northern blot analysis) in dorsal root ganglia at the lumbar level and on immunoreactive PACAP in the spinal cord and in the sciatic nerve stump. For comparison, calcitonin gene-related peptide was examined. In dorsal root ganglia of the intact side immunoreactive PACAP and PACAP messenger RNA were localised to a population of nerve cell bodies of small to medium size. In dorsal root ganglia on the injured side, PACAP-immunoreactive nerve cell bodies were more numerous and PACAP messenger RNA was considerably more abundant as studied 14 days after sciatic nerve transection. By contrast, calcitonin gene-related peptide-containing nerve cell bodies were numerous and rich in calcitonin gene-related peptide messenger RNA in dorsal root ganglia on the intact side, while after transection both the number of immunoreactive nerve cell bodies and their content of messenger RNA were markedly reduced. There were indications of axotomy-induced expression of PACAP messengerRNA in larger neurons. In the dorsal horn of the spinal cord on the intact side PACAP and calcitonin gene-related peptide-immunoreactive fibres were densely accumulated in the superficial layers. On the transected side the densities of both PACAP and calcitonin gene-related peptide-immunoreactive nerve fibres were reduced in the medial part. The data obtained indicate a marked up-regulation of PACAP in sensory neurons following peripheral nerve injury. Since PACAP depresses a C-fibre evoked flexion reflex, this may have implications for sensory transmission. Further, in view of the known promoting effects of PACAP on neuronal survival and differentiation and non-neuronal cell growth as well as its proinflammatory effects a role of PACAP in the neuronal and periaxonal tissue restoration after injury is not inconceivable.

Developmental regulation of two distinct neuronal phenotypes in rat dorsal root ganglia.

In a previous study we described two distinct neuronal phenotypes in rat dorsal root ganglia based on immunocytochemical assays for the neuronal intermediate filament proteins, peripherin and low-molecular-weight neurofilaments [Goldstein M. E. et al. (1991) J. Neurosci. Res. 30, 92-104]. In this paper we have extended this classification by using in situ hybridization to localize and evaluate the levels of various cytoskeletal and neuropeptide messenger RNAs within the peripherin-immunoreactive and peripherin-immunoreactive-negative neurons found in embryonic day 15 and 20, postnatal day 2 and adult dorsal root ganglia. We found in postnatal and adult dorsal root ganglia in vivo that the large, peripherin-immunoreactive-negative neurons, which are intensely stained by low-molecular-weight neurofilament antibodies, also contain high levels of low, medium and high-molecular-weight neurofilament messenger RNAs, whereas the smaller peripherin-immunoreactive neurons do not. On the other hand, both cell types contained comparable levels of peripherin and alpha-tubulin messenger RNA. The presence of peripherin messenger RNA but no peripherin immunoreactivity in the large cells suggested either a translational or post-translational regulation of this polypeptide, or rapid clearance of this protein from the perikaryon into the axon. In adult dorsal root ganglia, more than 50% of the peripherin-immunoreactive neurons also contained high levels of substance P and/or calcitonin gene-related peptide messenger RNAs, while less than 20% of the large peripherin-immunoreactive-negative neurons did. The attainment of these phenotypic characteristics during development in vivo was studied by northern blot and in situ hybridization histochemistry. In early embryonic stages (embryonic days 15-16), virtually all neurons were peripherin-immunoreactive and were positive for peripherin, alpha-tubulin and low-molecular-weight neuro-filament messenger RNAs, suggesting a homogeneous population. By embryonic day 20, the two adult phenotypes became clearly evident, and were fully established by postnatal day 2. In cultures of embryonic day 15 dorsal root ganglion neurons grown in the presence of nerve growth factor, peripherin and low-molecular-weight neurofilament messenger RNAs were expressed in all neurons, even after nine days in vitro, similar to embryonic dorsal root ganglia in vivo. Nerve growth factor supplemented by skeletal and heart muscle extracts did up-regulate neurofilament gene expression, but not to the extent characteristic of the peripherin-immunoreactive-negative adult phenotype. These results suggest that development of the mature phenotype of dorsal root ganglion neurons occurs by postnatal day 2 in vivo and is dependent upon target contact and/or target-derived factors.

Coexpression of preprotachykinin-A, alpha-calcitonin gene-related peptide, somatostatin, and neurotrophin receptor family messenger RNAs in rat dorsal root ganglion neurons.

Syntheses of substance P, somatostatin, and calcitonin gene-related peptide in sensory neurons have been suggested to be regulated by neurotrophic factors retrogradely transported from target tissues. In this study, we re-examined this idea by investigating the coexpression of neurotrophin receptor (trk family proto-oncogene) messenger RNAs, and preprotachykinin-A (a precursor peptide of substance P), alpha-calcitonin gene-related peptide and somatostatin messenger RNAs in lumbar dorsal root ganglion neurons by means of in situ hybridization histochemistry in rats. Approximately 35-40%, 5% and 15-20% of sensory neurons displayed signals for trkA, trkB, and trkC messenger RNAs, respectively. Approximately 28% of dorsal root ganglion neurons were positive for preprotachykinin-A messenger RNA, and were divided into two groups; those labeled strongly and those labeled weakly by in situ hybridization. All the strongly-labeled neurons (78% of preprotachykinin-A-positive cells) expressed trkA messenger RNA at the same time, while the weakly-labeled neurons did not. Thirty-seven per cent of dorsal root ganglion neurons expressed alpha-calcitonin gene-related peptide messenger RNA, and most of these neurons (84%) also expressed trkA messenger RNA. No or few preprotachykinin-A messenger RNA- and/or alpha-calcitonin gene-related peptide messenger RNA-expressing neurons were also positive for trkB or trkC messenger RNAs. Nine per cent of dorsal root ganglion neurons expressed somatostatin messenger RNA, and these neurons lacked all three trk messenger RNAs. Furthermore, most of these neurons (about 90%) showed positive, albeit weak, signals for preprotachykinin-A and alpha-calcitonin gene-related peptide messenger RNAs. The results suggest that expression of preprotachykinin-A and alpha-calcitonin gene-related peptide messenger RNAs is mediated by nerve growth factor via trkA receptor but not by brain-derived neurotrophic factor or neurotrophin-3, and that somatostatin gene transcription is not regulated by any member of the neurotrophin family in rat sensory neurons.

Local anaesthesia prevents acute inflammatory changes in neuropeptide messenger RNA expression in rat dorsal root ganglia neurons

Sensory neuropeptides, synthesised in dorsal root ganglia (DRG), are implicated in neurogenic inflammation and nociception in arthritis. Adjuvant monoarthritis increases primary afferent activity and alters expression of neuropeptide genes in DRG We investigated the role of neural discharge in the early changes in neuropeptide gene expression. Adjuvant injection increased preprotachykinin (PPT) and calcitonin gene-related peptide (CGRP) messenger RNA (mRNA) after 8 h, whereas somatostatin mRNA expression remained unchanged, in innervating L5 DRG neurons. The changes in PPT mRNA expression were prevented by concurrent local anaesthesia of the sciatic nerve. Our results suggest that electrical activity mediates, in part, the changes in DRG gene expression in response to acute inflammation.

Reduced sciatic nerve substance P and calcitonin gene-related peptide in rats with short-term diabetes or central hypoxaemia co-exist with normal messenger RNA levels in the lumbar dorsal root ganglia

Endoneurial hypoxia of ischaemic origin is believed to cause the reduction in sciatic nerve substance P levels in experimentally diabetic rats. The first part of this study was designed to determine whether the changes seen extended to another neuropeptide, calcitonin gene-related peptide, and to reveal any correlation between substance P and calcitonin gene-related peptide levels in the sciatic nerve of both diabetic and centrally hypoxaemic rats. Comparison of streptozotocin diabetic rats (four-week duration) with their control group showed clear reductions in both substance P-like immunoreactivity (control= 225 ± 20pg/mg protein,diabetic= 139 ± 19; P < 0.01) and calcitonin gene-related peptide-like immunoreactivity (control= 9.08 ± 0.65ng/mg protein,diabetic= 4.43 ± 0.44; P < 0.001). A similar pattern was seen with the comparison of five-week centrally hypoxaemic rats (housed in 10% O 2) with their controls for both substance P-like immunoreactivity (control= 222 ± 10pg/mg protein,hypoxic= 148 ± 13; P < 0.001) and calcitonin gene-related peptide-like immunoreactivity (control= 6.58 ± 0.42ng/mg protein,hypoxic= 3.01 ± 0.45; P < 0.001). Calcitonin gene-related peptide levels correlated closely with substance P levels in both the diabetes and central hypoxaemia studies ( r 2 = 0.69 and 0.62, respectively). The second part of this study measured the messenger RNA levels of the substance P precursor, preprotachykinin-A, and calcitonin gene-related peptide messenger RNA in the L 4 and L 5 dorsal root ganglia of control, diabetic and centrally hypoxaemic rats. There was no change in preprotachykinin-A or calcitonin gene-related peptide messenger RNA levels between any of the groups, suggesting that the sciatic nerve decreases in substance P and calcitonin gene-related peptide described above are post-transcriptional in origin.

Differential regulation of α-calcitonin gene-related peptide and preprocholecystokinin messenger rna expression in α-motoneurons: Effects of testosterone and inactivity induced factors

α-Calcitonin gene-related peptide expression in α-motoneurons is regulated by spinal cord transection, axotomy and testosterone, but to date there are no studies which examine the regulation of cholecystokinin expression in motoneurons. In the present study, we compared the regulation of preprocholecystokinin and α-calcitonin gene-related peptide messenger RNA levels in motoneurons of the spinal nucleus of the bulbocavernosus. Previously, we demonstrated that manipulations which decrease activity in target muscles of the spinal nucleus of the bulbocavernosus motoneurons increase α-calcitonin gene-related peptide message and peptide levels in spinal nucleus of the bulbocavernosus motoneurons. This muscle-nerve interaction is mediated by a soluble factor which is increased by castration. We now report that decreasing plasma testosterone levels decreased preprocholecystokinin messenger RNA levels. Testosterone replacement at the time of castration restored preprocholecystokinin messenger RNA levels to intact values. Injections of crude extracts prepared from denervated bulbocavernosus/ levator ani into the homologous muscles of gonadally intact rats increased the levels of α-calcitonin gene-related peptide messenger RNA in spinal nucleus of the bulbocavernosus motoneurons. The levels of preprocholecystokinin messenger RNA did not differ in rats injected with denervated bulbocavernosus/ levator ani extract or buffer, both of which were significantly higher than in intact, untreated rats. The results of the present experiments imply that levels of preprocholecystokinin and α-calcitonin gene-related peptide messenger ribonucleic acid are differentially regulated in spinal nucleus of the bulbocavernosus motoneurons.

Molecular mechanisms involved in the tissue-specific production of calcitonin and calcitonin gene-related peptide messenger RNAs

Molecular mechanisms involved in the tissue-specific production of calcitonin and calcitonin gene-related peptide messenger RNAs

Localisation of calcitonin gene-related peptide immunoreactivity and messenger RNA in the rat anterior pituitary and the effect of gonadal steroid manipulations

Localisation of calcitonin gene-related peptide immunoreactivity and messenger RNA in the rat anterior pituitary and the effect of gonadal steroid manipulations

Calcitonin gene-related peptide messenger RNA is expressed in sensory neurones of the dorsal root ganglia and also in spinal motoneurones in man and rat

Calcitonin gene-related peptide (CGRP) mRNA was localised to neurones of the dorsal root ganglia and motoneurones of the ventral horn in man and rat. Presence of α- and β-CGRP mRNA was confirmed by Northern blot analysis of rat tissues which showed α-CGRP was the predominant gene. The distribution of CGRP gene transcripts corresponded with neurones displaying CGRP immunoreactivity in the ganglia of both species and in the rat ventral horn. In man few motoneurones were immunoreactive despite many expressing CGRP mRNA. In situ hybridisation revealed not only sensory but also motor neurones are sites of CGRP manufacture. Thus in conjunction with other evidence the present study reinforces the proposed muscle trophic role for this peptide.

Identification of calcitonin and calcitonin gene-related peptide messenger ribonucleic acid in medullary thyroid carcinomas by hybridization histochemistry.

Synthesis and secretion of calcitonin and calcitonin gene-related peptide (CGRP) were studied in medullary thyroid carcinomas (MTC) by hybridization histochemistry on tissue sections and by Northern gel analysis of mRNA. Five patients with MTC and elevated serum levels of calcitonin and CGRP were studied. Surgically obtained tumor samples (four primary and three lymph node metastases) were extracted after freezing, and the RNA was fractionated on Northern gels. Hybridization was carried out with 32P-labeled synthetic oligodeoxyribonucleotides coding specifically for calcitonin and CGRP. Calcitonin- and CGRP-specific mRNAs approximately 1000 nucleotides in length were demonstrated in all 7 tumor samples. However, neither calcitonin nor CGRP mRNA was detected in a pheochromocytoma from 1 of the patients who had multiple endocrine neoplasia type II. A series of unselected lung carcinomas yielded the same result. Hybridization histochemistry was carried out on sections from the same tumors using the same probes. The mRNAs for calcitonin and CGRP were located in all cells of neoplastic MTC appearance, with CGRP mRNA at significantly lower levels. This demonstrated that both calcitonin and CGRP mRNA were present within the same tumor cells. The lung tumors and pheochromocytoma were negative with both probes. Hybridization histochemistry is likely to be of use in diagnosis of medullary thyroid cancer and in studying the calcitonin-CGRP mRNA processing mechanism in whole cells.