Tissue distribution of messenger RNAs coding for opioid peptide precursors and related RNA

All of the endogenous opioid peptides thus far identified are derived from three types of precursors, i.e. the corticotropin/beta-lipotropin precursor, preproenkephalin A and preproenkephalin B. Poly(A)-containing RNA from various bovine and porcine tissues has been subjected to blot hybridization analysis with the use of cDNA probes specific for the three opioid peptide precursors. Analysis with a corticotropin/beta-lipotropin precursor cDNA probe has revealed, in addition to the pituitary mRNA, a smaller hybridizable RNA species present in bovine extrapituitary tissues, such as the adrenal medulla, thyroid, thymus, duodenum and lung. The hypothalamus contains both these RNA species. DNA complementary to the smaller RNA species from the bovine adrenal medulla has been cloned. Analysis of the cloned cDNA, in conjunction with endonuclease S1 mapping of poly(A)-rich RNA from the adrenal medulla, has indicated that the smaller RNA species represents the 3'-terminal 712-729 nucleotides, excluding the poly(A) tail, of the pituitary corticotropin/beta-lipotropin precursor mRNA, having heterogeneous start sites. Analysis with a preproenkephalin A cDNA probe has shown the presence of hybridizable RNA in the bovine hypothalamus, duodenum and pituitary neurointermediate lobe in addition to the adrenal medulla. The hybridizable RNA species from all these tissues are indistinguishable in size. RNA hybridizable with a preproenkephalin B cDNA probe has been found in the porcine spinal cord and ileum besides the hypothalamus, and these RNA species exhibit an indistinguishable size. The results presented indicate that each opioid peptide precursor is synthesized in different tissues.     

Quantitation of proenkephalin A messenger RNA in bovine brain, pituitary and adrenal medulla: correlation between mRNA and peptide levels.

Concentrations of mRNA coding for the opioid peptide precursor proenkephalin A were measured in bovine brain areas, pituitary and adrenal medulla. In all tissues, a single hybridizable species of 1400 bases in size was found by Northern blot analysis using as a probe a single-stranded (ss) cDNA complementary to bovine proenkephalin A mRNA. In solution hybridization experiments the distribution of the mRNA was quantified. Considerable differences were found for the abundance of proenkephalin A mRNA in the various tissues: from 0.023% in the adrenal medulla to 0.00002% in the adenohypophysis. Relative abundance in the various brain areas varied greater than 20-fold, being highest in the caudate nucleus (0.0025%) and lowest in the thalamus and substantia nigra (0.0001%). Comparison with immunoreactive peptide concentrations in these tissues showed a close correlation between the levels of proenkephalin A mRNA and the immunoreactive peptides.     

Regional Distribution of the GABAA/Benzodiazepine Receptor (α Subunit) mRNA in Rat Brain

A human cDNA clone containing the 5' coding region of the GABAA/benzodiazepine receptor alpha subunit was used to quantify and visualize receptor mRNA in various regions of the rat brain. Using a [32P]CTP-labelled antisense RNA probe (860 bases) prepared from the alpha subunit cDNA, multiple mRNA species were detected in Northern blots using total and poly A rat brain RNA. In all brain regions, mRNAs of 4.4 and 4.8 kb were observed, and an additional mRNA of 3.0 kb was detected in the cerebellum and hippocampus. The level of GABAA/benzodiazepine receptor mRNA was highest in the cerebellum followed by the thalamus = frontal cortex = hippocampus = parietal cortex = hypothalamus much greater than pons = striatum = medulla. In situ hybridization revealed high levels of alpha subunit mRNA in cerebellar gray matter, olfactory bulb, thalamus, hippocampus/dentate gyrus, and the arcuate nucleus of the hypothalamus. These data suggest the presence of multiple GABAA/benzodiazepine receptor alpha subunit mRNAs in rat brain and demonstrate the feasibility of studying the expression of genes encoding the GABAA/benzodiazepine receptor after pharmacological and/or environmental manipulation.     

The rat gene encoding neurotensin and neuromedin N. Structure, tissue-specific expression, and evolution of exon sequences

Recombinant DNA clones encoding the neurotensin/neuromedin N precursor protein have been isolated from both bovine hypothalamus cDNA and rat genomic libraries using a heterologous canine cDNA probe. Nucleotide sequence analysis of these clones and comparison with the previously determined canine sequence has revealed that 76% of the amino acid residues are conserved in all three species. The protein precursor sequences predicted from bovine hypothalamus and canine intestine cDNA clones vary at only 9 of 170 amino acid residues suggesting that within a species identical precursors are synthesized in both the central nervous system and intestine. The rat gene spans approximately 10.2 kilobases (kb) and is divided into four exons by three introns. The neurotensin and neuromedin N coding domains are tandemly positioned on exon 4. RNA blot analysis has revealed that the rat gene is transcribed to yield two distinct mRNAs, 1.0 and 1.5 kb in size, in all gastrointestinal and all neural tissues examined except the cerebellum. There is a striking variation in the relative levels of these two mRNAs between brain and intestine. The smaller 1.0-kb mRNA greatly predominates in intestine while both mRNA species are nearly equally abundant in hypothalamus, brain stem, and cortex. Sequence comparisons and RNA blot analysis indicate that these two mRNAs result from the differential utilization of two consensus poly(A) addition signals and differ in the extent of their 3' untranslated regions. The relative combined levels of the mRNAs in various brain and intestine regions correspond roughly with the relative levels of immunologically detectable neurotensin except in the cerebral cortex where mRNA levels are 6 times higher than anticipated.     

Evidence for a novel pituitary protein (7B2) in human brain, cerebrospinal fluid and plasma: brain concentrations in controls and patients with Alzheimer's disease

A novel pituitary protein, designated as 7B2, recently purified in our laboratory was measured using a specific radioimmunoassay in conjunction with immuno-affinity extraction, in cerebrospinal fluid (CSF) and in plasma obtained from normal volunteers. The mean concentrations of immunoreactive (IR)-7B2 were 2154 pg/ml in CSF and 29 pg/ml in plasma. Studies by SDS-poly-acrylamide gel electrophoresis revealed that both CSF IR-7B2 and plasma IR-7B2 have an apparent molecular weight of around 20,000-21,000 as previously observed in various rat tissues. IR-7B2 was also measured in various brain regions obtained from control subjects and patients with Alzheimer's disease. IR-7B2 was widely distributed in the human brain, with the highest concentrations in substantia nigra and caudate. IR-7B2 brain concentrations were found to be similar between control subjects and patients with Alzheimer's disease. Gel permeation chromatography of extracts of various brain regions revealed two major peaks with apparent molecular weights of 45,000-50,000 and 11,000-16,000 in hypothalamus, caudate, frontal cortex, hippocampus, putamen and locus coeruleus, and only one peak with an apparent molecular weight of 14,000-16,000 in substantia nigra and globus pallidus. These data suggest that this novel pituitary protein may play a role of consequence perhaps as a neurotransmitter or as a neuromodulator in the human central nervous system.     

Quantitation of Vasopressin mRNA and Oxytocin mRNA in Hypothalamic Nuclei by Solution Hybridization Assays

Concentrations of vasopressin (VP) precursor and oxytocin (OT) precursor mRNA were measured in magnocellular cell groups of the rat hypothalamus by newly developed solution hybridization assays. The assays employed single-stranded 35S-labeled VP-specific and OT-specific DNA probes that were prepared by primer extension on recombinant M13 DNA templates. Solution hybridization assays were standardized by known amounts of cloned DNA. The detection limit was less than 1 pg DNA equivalent of the respective mRNA. In total RNA preparations of microdissected supraoptic nucleus (SON) mean (+/- SEM) basal levels of 1.37 +/- 0.18 pg VP mRNA and 1.95 +/- 0.14 pg OT mRNA were measured. RNA of the microdissected paraventricular nucleus (PVN) contained 0.35 +/- 0.02 pg VP mRNA and 1.77 +/- 0.15 pg OT mRNA. Elevation of plasma osmolality induced by drinking of 2% saline for 25 days resulted in a 1.85-fold increase in VP mRNA levels of the SON and a 1.6-fold increase in VP mRNA levels of the PVN. The solution hybridization assays are suitable tools to study the regulation of VP and OT mRNAs in magnocellular neurons of the brain.     

Xenopus fibrinogen: characterization of the mRNAs for the three subunits.

We purified and characterized the mRNAs coding for each of the three subunits of Xenopus fibrinogen. Purification was accomplished by electrophoretic separation of liver polyadenylated RNA in a fully denaturing gel, followed by recovery of the RNA from the gel via transfer to an ion-exchange membrane. This procedure yielded fractions which were highly enriched for the mRNAs for each of the fibrinogen chains. The fibrinogen mRNAs were identified by two methods: (i) in vitro translation followed by subunit-specific cleavage with the proteases thrombin and batroxobin; and (ii) cross-hybridization with cDNA clones for individual subunits of rat fibrinogen. The results demonstrate that the A alpha and gamma chains of frog fibrinogen are each coded by a single mRNA species. The A alpha mRNA is ca. 3,100 nucleotides in length, which is nearly twice the minimum size required to code for the A alpha precursor polypeptide. The gamma chain mRNA comprises about 1,600 bases and includes only a small untranslated region. In contrast, the B beta subunit is synthesized from two mRNAs, one of which is 2,500 and the other 1,800 nucleotides long. The 2,500-base mRNA includes a large noncoding region, whereas the smaller one is near the minimum required size. The larger B beta mRNA is ca, fivefold more abundant that the smaller species.     

Topographical localisation of endothelin mRNA and peptide immunoreactivity in neurones of the human brain

The distribution of endothelin mRNA and immunoreactivity in the human brain was investigated using the technique of in situ hybridization and immunocytochemistry. Cryostat sections from 22 cases of neurologically normal adult human brain, collected 3-7 h post-mortem were hybridized with 35S-labelled complementary (c)RNA probes prepared from the 3' non-coding region of endothelin-1 cDNA, and the chromosomal genes encoding endothelin-2 and -3. In situ hybridization with all three cRNA probes revealed labelled neuronal cell bodies in laminae III-VI of the parietal, temporal and frontal cortices. Labelled cells were also seen, scattered throughout the para- and periventricular, supraoptic and lateral hypothalamic nuclei, the caudate nucleus, amygdala, hippocampus, basal nucleus of Meynert, substantia nigra, raphe nuclei, Purkinje cell layer of the cerebellum and in the dorsal motor nuclei of the vagus of the medulla oblongata. The distribution of neurones immunoreactive to endothelin was similar to that of endothelin mRNA, although fewer immunoreactive cells throughout the brain, were noted. Immunoreactive fibres were present mainly in the cortex and hypothalamus, and to a lesser extent in the brain stem. Combined in situ hybridization and immunocytochemistry on the same section revealed the presence of endothelin-1 mRNA and immunoreactivity in the same cortical neuronal cell. Colocalisation studies in the cortex revealed endothelin-1 mRNA and immunoreactivity in a number of cells which also expressed neuropeptide Y mRNA and immunoreactivity. In the hypothalamus and basal nucleus of Meynert endothelin immunoreactivity was colocalised to a subset of neurophysin- and galanin-immunoreactive cell bodies respectively. Endothelin mRNA and immunoreactivity was also seen in some blood vessel endothelial cells. The findings of endothelin mRNAs and immunoreactivity in heterogenous neuronal populations further emphasises the potential role of endothelin as a neuropeptide, probably having diverse actions in the nervous system of man.     

SUBCELLULAR DISTRIBUTION OF DOPAMINE-β-HYDROXYLASE AND INHIBITORS IN THE HIPPOCAMPUS AND CAUDATE NUCLEUS IN SHEEP BRAIN

—The enzyme dopamine-β-hydroxylase (EC 1.14.17.1) which converts dopamine to noradrenaline was found to be present in substantial amounts in sheep brain hypothalamus and caudate nucleus and was located to the synaptic vesicle fractions in these two brain regions by subcellular fractionation. This dopamine-β-hydroxylase was associated with paniculate matter in these two brain regions since it was resistant to solubilization with butan-1-ol and 0.1% Triton X-100. As highly significant levels of dopamine-β-hydroxylase were present in the caudate nucleus, factors other than a simple lack of this enzyme must operate to maintain the low levels of noradrenaline and high levels of dopamine in the caudate nucleus. Purified adrenal dopamine-β-hydroxylase was substantially inhibited by two factors prepared from sheep brain hypothalamus and caudate nucleus. These were found to be cupric ions and a sulphydryl inhibitor. High levels of the sulphydryl inhibitor of dopamine-β-hydroxylase were found in synaptosomal fractions from sheep brain hypothalamus and caudate nucleus and the levels were comparable in both regions. Upon subfractionation of a synaptosome-containing fraction from the hypothalamus, the inhibitor was located predominantly in the soluble fraction, although there were significant levels in the synaptic vesicle fraction. Therefore, the sulphydryl inhibitor must be considered as a possible regulator of dopamine-β-hydroxylase activity. Free cupric ion concentrations as low as 2·5 μM were found to inhibit purified adrenal dopamine-β-hydroxylase in vitro and the concentration of copper in the soluble tissue component of hypothalamus and caudate nucleus was well above this minimal copper concentration. The percentage content of soluble copper in the caudate nucleus was significantly higher than in the hypothalamus. The importance of the soluble to particulate-bound ratio of copper in brain was shown in studies of the developing rat brain. A rapid increase in the level of copper in brain was found in the first 4 weeks but the level was constant by 2 months of age. The percentage of soluble copper, however, was maximal soon after birth and had declined to a constant figure by 2 months of age. A scheme for the regulation of dopamine-β-hydroxylase activity involving these factors is proposed.     

Characterization of myelin proteolipid mRNAs in normal and jimpy mice.

A clone specific for the rat myelin proteolipid protein (PLP) was isolated from a cDNA library made in pUC18 from 17-day-old rat brain stem mRNA. This clone corresponded to the carboxyl-terminal third of the PLP-coding region. The clone was used to identify PLP-specific mRNAs in mouse brain and to establish the time course of PLP mRNA expression during mouse brain development. Three PLP-specific mRNAs were seen, approximately 1,500, 2,400, and 3,200 bases in length, of which the largest was the most abundant. During brain development, the maximal period of PLP mRNA expression was from 14 to 25 days of age, and this was a similar time course to that for myelin basic protein mRNA expression. When the jimpy mouse, an X-linked dysmyelination mutant, was studied for PLP mRNA expression, low levels of PLP mRNA were seen which were approximately 5% of wild-type levels at 20 days of age. When jimpy brain RNA was analyzed by Northern blotting, the PLP-specific mRNA was shown to be 100 to 200 bases shorter than the wild-type PLP-specific mRNA. This size difference was seen in the two major PLP mRNAs, and it did not result from a loss of polyadenylation of these mRNAs.     

Differential thyroid hormone-regulated rat thyrotropin beta gene expression detected by blot hybridization

In the rat, there is a single TSH beta-subunit gene represented by three exons interrupted by two introns. This gene contains two promoters which determines the synthesis of two mRNAs with 5'-untranslated regions that differ by 43 base pairs. This study evaluates the steady state levels of these TSH beta mRNAs in various thyroidal states. Blot hybridization analyses of pituitary mRNA with synthetic probes designed to detect either one or both TSH beta mRNAs were performed. One probe corresponds to 24 bases in the 5'-untranslated region of mRNA1 and a second corresponds to 25 nucleotides in the coding region and detects both mRNA1 and mRNA2. These studies indicate the presence of TSH beta mRNA species of indistinguishable size consistent with the presence of two TSH beta mRNAs that contain slightly different 5'-untranslated regions. Comparison of pituitary RNA obtained from normal and hypothyroid rats reveals that the shorter mRNA (mRNA2) is increased approximately 6- to 8-fold with hypothyroidism while the abundance of the longer mRNA (mRNA1) is relatively unchanged. Treatment of either normal or hypothyroid animals with T3 decreases the abundance of mRNA2 while again mRNA1 is relatively unaffected. Thus, although both mRNAs are detected, only one mRNA is dramatically altered by thyroidal status. Therefore, the single rat TSH beta gene is transcribed into two mRNAs via the use of alternative promoters of which only one is markedly regulated by thyroid hormones.     

Membrane-associated peptidylglycine alpha-amidating monooxygenase in the heart

Recent investigations have shown that the heart atrium is an endocrine tissue. In the present studies, high levels of peptidylglycine alpha-amidating monooxygenase (PAM), which catalyzes the formation of bioactive alpha-amidated peptides from their glycine-extended precursors, have been found in particulate fractions from bovine and rat heart atrium; only low levels of PAM activity were present in soluble fractions. Corresponding fractions from the ventricles contained 20-fold less activity. Immunocytochemical studies demonstrated that PAM was localized primarily to atrial cardiocytes, with a distribution resembling that of atriopeptin. Following differential centrifugation of rat atrial homogenates, most of the PAM activity was associated with crude granule fractions, with lesser amounts of activity associated with crude microsomal fractions. Upon further subcellular fractionation, PAM activity in the rat atrium was found primarily with immunoactive atriopeptin in fractions enriched in secretory granules. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, antisera to purified bovine pituitary PAM identified a 113,000-dalton protein in bovine atrial microsomes and secretory granules; the protein predicted from the sequence of the cDNA encoding bovine pituitary PAM is of similar size (Eipper, B. A., Park, L. P., Dickerson, I. M., Keutmann, H. T., Thiele, E. A., Rodriguez, H., Schofield, P. R., and Mains, R. E. (1987) Mol. Endocrinol. 1, 777-790). Northern blot analysis using cDNA probes encoding bovine pituitary PAM demonstrated higher levels of PAM mRNA in heart atrium than in anterior pituitary. Rat heart contains PAM mRNA species of 3.6 and 3.8 kilobases, the smaller mRNA species corresponding in size to the PAM mRNA expressed in rat anterior pituitary.     

Localization of somatostatin mRNAs in the brain and pancreas of rainbow trout (Oncorhynchus mykiss)

Rainbow trout possess three distinct mRNAs, each encoding a separate precursor: PPSS I, which contains a 14-amino acid sequence at its C-terminus (somatostatin-14) that is highly conserved among vertebrates, as well as two others, PPSS II' and PPSS II", both containing [Tyr(7), Gly(10)]-somatostatin-14 at their C-terminus. In this study, we used RNA template-specific PCR and in situ hybridization to determine the distribution and cellular localization of PPSS mRNAs in the brain and Brockmann body of rainbow trout. PPSS I, PPSS II' and PPSS II" were expressed in the Brockmann body and pituitary; the expression of PPSS mRNAs in the brain was region specific. PPSS I mRNA was expressed in the Brockmann body predominantly by cells other than those that expressed PPSS IIs; however, there were several instances where PPSS I and PPSS IIs were co-expressed within the same cell. Of the PPSS II-expressing cells, many were observed to express both PPSS II' and PPSS II" mRNA; however, some cells expressed only PPSS II' mRNA, while other cells expressed only PPSS II" mRNA. In the brain, PPSS I mRNA was expressed in the optic tectum (OT) and in many hypothalamic nuclei, including the nucleus rotundus (NR), nucleus anterioris hypothalami (NAH), nucleus anterior tuberis (NAT), nucleus lateral tuberis (NLT), as well as in the pituitary (adenohypophysis). PPSS II" mRNA was present in the same regions as PPSS I mRNA; however, PPSS II' mRNA was present primarily in OT, NAT, NLT and adenohypohysis. These results indicate that PPSS mRNAs are expressed differently by different cells, suggesting that cell-specific mechanisms are involved with the control of PPSS expression and that particular biological responses may be associated with a specific SS isoform.     

Topographical localisation of endothelin mRNA and peptide immunoreactivity in neurones of the human brain

Summary The distribution of endothelin mRNA and immunoreactivity in the human brain was investigated using the technique of in situ hybridization and immunocytochemistry. Cryostat sections from 22 cases of neurologically normal adult human brain, collected 3–7 h post-mortem were hybridized with³⁵S-labelled complementary (c)RNA probes prepared from the 3 non-coding region of endothelin-1 cDNA, and the chromosomal genes encoding endothelin-2 and -3. In situ hybridization with all three cRNA probes revealed labelled neuronal cell bodies in laminae III–VI of the parietal, temporal and frontal cortices. Labelled cells were also seen, scattered throughout the para- and periventricular; supraoptic and lateral hypothalamic nuclei, the caudate nucleus, amygdala, hippocampus, basal nucleus of Meynert, substantia nigra, raphe nuclei, Purkinje cell layer of the cerebellum and in the dorsal motor nuclei of the vagus of the medulla oblongata. The distribution of neurones immunoreactive to endothelin was similar to that of endothelin mRNA, although fewer immunoreactive cells throughout the brain, were noted. Immunoreactive fibres were present mainly in the cortex and hypothalamus, and to a lesser extent in the brain stem. Combined in situ hybridization and immunocytochemistry on the same section revealed the presence of endothelin-1 mRNA and immunoreactivity in the same cortical neuronal cell. Colocalisation studies in the cortex revealed endothelin-1 mRNA and immunoreactivity in a number of cells which also expressed neuropeptide Y mRNA and immunoreactivity. In the hypothalamus and basal nucleus of Meynert endothelin immunoreactivity was colocalised to a subset of neurophysin- and galanin-immunoreactive cell bodies respectively. Endothelin mRNA and immunoreactivity was also seen in some blood vessel endothelial cells. The findings of endothelin mRNAs and immunoreactivity in heterogenous neuronal populations further emphasises the potential role of endothelin as a neuropeptide, probably having diverse actions in the nervous system of man.     

Distribution and characterization of synenkephalin immunoreactivity in the bovine brain and pituitary

The distribution of synenkephalin, the N-terminal fragment of proenkephalin, was studied in various parts of the bovine brain (globus pallidus, caudate nucleus, hypothalamus) and in the posterior pituitary by the use of a radioimmunoassay. The distribution of synenkephalin-immunoreactivity (IR) was compared to the distribution of Met-enkephalin-IR. Gel exclusion chromatography was used to examine the molecular forms of the immunoreactivities present in the tissues. The distribution of synenkephalin-IR was similar to the distribution of Met-enkephalin-IR, with a molar ratio of Met-enkephalin/synenkephalin ranging between 2.7 and 5.9. In all regions tested except the hypothalamus the synenkephalin-IR was present as a single species. However, in the hypothalamus a small amount of IR material (3% of the total synenkephalin-IR) was detected in fractions where larger Met-enkephalin-containing peptides eluted. Based on the concordance between the molar ratio of Met-enkephalin to synenkephalin found in the tissues and the molar ratio present in the sequence of adrenal proenkephalin, it is concluded that the brain and adrenal glands utilize a similar precursor for enkephalin biosynthesis.     

In situ hybridization of corticotropin-releasing factor-encoding messenger RNA in the hypothalamus of the white sucker,Catostomus commersoni

Summary In situ hybridization procedure with a ³²P-labelled synthetic oligonucleotide probe was used to detect corticotropin-releasing factor-encoding messenger RNA (CRF mRNA) in the hypothalamus of the white sucker, Catostomus commersoni. Adjacent sections were immunostained by a sucker CRF-specific antiserum. CRF mRNA-containing neurons were identified by autoradiography in the magnocellular and parvocellular subdivisions of the preoptic nucleus (PON). Many of these neurons were also immunostained by sucker antiserum, showing the same distribution patterns. These results confirm the presence of CRF mRNA and CRF peptide in the white sucker hypothalamus and support the view that the magnocellular and parvocellular neurons of the PON may be involved in the control of adrenocorticotropic hormone secretion from the pituitary in the white sucker.