Ribosomal DNA in spores of Physarum polycephalum.

DNA was isolated from plasmodia, spores and newly hatched amoebae of the slime mould Physarum polycephalum. The DNA preparations were fractionated in CsCl gradients and each fraction hybridised to combined 19 S + 26 S rRNA. In all three DNA preparations hybridisation was found to be limited to satellite DNA (rho = 1.714 g/cm3) and at saturation was found to reach a level of 0.16--0.18 % of total DNA. The main band of nuclear DNA (rho = 1.702 g/cm3) did not hybridise appreciably. Further experiments using analytical CsCl gradients revealed that the ratio of satellite to main band DNA was similar in all three preparations. It is concluded that the genes for ribosomal RNA are equally reiterated in spores, hatching amoebae and in plasmodia. They appear to be similarly organised in all stages of the life cycle examined so far.

Identification of zymogen and mature forms of human carboxypeptidase H. A processing enzyme for the synthesis of peptide hormones.

Carboxypeptidase H (CPH) is one of several processing enzymes required for the conversion of peptide hormone precursors into their smaller active forms. In this study, high levels of CPH activity was found in a liver metastasis of a human ileal carcinoid which expresses beta-preprotachykinin mRNA and the tachykinin neuropeptides, substance P and substance K. This human CPH showed properties of a zinc-metallopeptidase that is structurally similar to bovine and rat CPH. Immunoblots of the human ileal carcinoma with anti-bovine CPH showed that CPH activity is represented by two proteins of apparent molecular masses 57 and 55 kDa. Cell-free translation of poly(A)+ RNA followed by immunoprecipitation with anti-bovine CPH showed that human CPH mRNA encodes a precursor protein of apparent molecular mass 75 kDa. These data demonstrate that human CPH is synthesized as a zymogen, prepro-CPH, which must be cleaved to form catalytically active CPH.

Comparative distribution of mRNAs for glutamic acid decarboxylase, tyrosine hydroxylase, and tachykinins in the basal ganglia: an in situ hybridization study in the rodent brain.

Neurotransmitter-related messenger RNAs were detected by in situ hybridization in sections of rat and mouse brains by using 35S-radiolabelled RNA probes transcribed from cDNAs cloned in SP6 promoter-containing vectors. The distribution of messenger RNAs for glutamic acid decarboxylase, tachykinins (substance P and K), and tyrosine hydroxylase was examined in the striatum, pallidum, and substantia nigra. Dense clusters of silver grains were observed with the RNA probe complementary of the cellular messenger RNA for glutamic acid decarboxylase (antisense RNA) over most large neurons in the substantia nigra pars reticulata and medium-sized to large neurons in all pallidal subdivisions. A few very densely and numerous lightly labelled medium-sized neurons were present in the striatum. Among the areas examined, only the striatum contained neurons labelled with the antisense tachykinin RNA. Most of these neurons were of medium size, and a few were large. With the antisense tyrosine hydroxylase RNA, silver grains were found over neurons of the substantia nigra pars compacta and adjacent A10 and A8 dopaminergic cell groups. No signal was observed with RNAs identical to the cellular messenger RNA for glutamic acid decarboxylase or tachykinin (sense RNA). These results show a good correlation with immunohistochemical studies, suggesting that documented differences in the distribution and the level of glutamic acid decarboxylase, tyrosine hydroxylase, and substance P immunoreactivities in neurons of the basal ganglia are related to differences in the level of expression of the corresponding genes rather than to translation accessibility, stability, or transport of the gene products.

Hormone receptor regulated proopiomelanocortin gene expression.

The activity of the POMC gene is regulated by both stimulatory and inhibitory hormones. Presumably, some balance exists in the influence of these hormones in order to maintain a steady-state level of activity of this gene. Physiological insults, such as stress, may upset this balance and change the rate of production of POMC and its biologically active peptides. The relative strength of these different hormones may therefore determine the long-term expression of this gene. Chronic administration of CRF to rats, primates and humans produces prolonged increases in plasma ACTH levels. This long-term effect is most likely due to an activation of the POMC gene. Interestingly, chronic treatment of anterior pituitary cells with CRF desensitizes CRF receptors. Thus, despite the corticotrophs becoming refractory to the acute stimulatory actions of CRF, the POMC gene remains stimulated. These findings suggest that corticotrophs do not have to be continuously activated by CRF to maintain a long-term increase in POMC gene expression. This contrasts with the actions of glucocorticoids whose effects are abruptly terminated following their removal from the target tissue. The molecular basis of this form of cellular memory to the actions of CRF may involve cAMP regulatory phosphoproteins binding to and activating the POMC gene. If this phenomenon is shown to occur and the phosphorylation state of these nuclear proteins is found to govern their level of interaction with POMC gene, then it would represent a novel mechanism of gene regulation. Proof for this mechanism and the elucidation of how other second messengers such as protein kinase C and calcium regulate the POMC gene will greatly aid our understanding of the precise molecular mechanisms controlling opioid peptide expression.

Expression of substance P and preprotachykinin mRNA by primary sensory neurons in culture: regulation by factors present in peripheral and central target tissues.

Primary sensory neurons display various neuronal phenotypes which may be influenced by factors present in central or peripheral targets. In the case of DRG cells expressing substance P (SP), the influence of peripheral or central targets was tested on the neuronal expression of this neuropeptide. DRG cells were cultured from chick embryo at E6 or E10 (before or after establishment of functional connections with targets). Preprotachykinin mRNA was visualized in DRG cell cultures by either Northern blot or in situ hybridization using an antisense labeled riboprobe, while the neuropeptide SP was detected by immunostaining with a monoclonal antibody. In DRG cell cultures from E10, only 60% of neurons expressed SP. In contrast, DRG cell cultures performed at E6 showed a significant hybridization signal and SP-like immunoreactivity in virtually all the neurons (98%). The addition of extracts from muscle, skin, brain or spinal cord to DRG cells cultured at E6 reduced by 20% the percentage of neurons which express preprotachykinin mRNA and SP-like immunoreactivity. Our results indicate that factors issued from targets inhibit SP-expression by a subset of primary sensory neurons and act on the transcriptional control of preprotachykinin gene.

Nerve growth factor (NGF) regulates tachykinin gene expression and biosynthesis in rat sensory neurons during early postnatal development.

The regulatory effects of nerve growth factor (NGF) on tachykinin biosynthesis in rat primary sensory neurons during the period of postnatal development were examined under in vivo and in vitro conditions. Administration of NGF to neonatal rats led to a significant increase in protein levels of substance P (SP) and neurokinin A (NKA) in trigeminal and dorsal root ganglia (DRG). In addition, Northern blot analysis revealed that preprotachykinin mRNA was upregulated in sensory ganglia of neonatal animals after treatment with NGF. Using a well-defined in vitro system for neonatal rat DRG and trigeminal ganglia neurons, we found that addition of NGF induced SP and NKA protein levels in a dose-dependent manner. Furthermore, preprotachykinin mRNA was markedly increased in cultured DRG and trigeminal ganglia neurons in the presence of NGF. Thus, our results clearly demonstrate that NGF regulates tachykinin gene expression and biosynthesis both in vivo and in vitro during the developmental period of rat sensory neurons.

Elevated potassium stimulates enkephalin biosynthesis in bovine chromaffin cells.

Exposure of bovine adrenal medullary cells in culture to a depolarizing concentration of potassium (50 mM), causes a rapid rise in both cellular and secreted Met-enkephalin peptide. The induction of peptide is preceded by the appearance of a nuclear preproenkephalin transcript and subsequent increases in cytoplasmic preproenkephalin mRNA. These data suggest that the depolarizing medium acts by enhancing enkephalin gene transcription. Potassium stimulation of Met-enkephalin biosynthesis requires the presence of extracellular Ca2+ and is not observed in either low Ca2+ medium or in the presence of D600, a Ca2+ channel blocker. As similar depolarizing stimuli inhibit enkephalin biosynthesis in the rat adrenal gland, these findings suggest that the regulation of enkephalin peptide content in neuroendocrine cells is highly species specific.

Preprotachykinin messenger RNA detected by in situ hybridization in striatal neurons of the human brain.

Sections from postmortem human brain were processed for in situ hybridization histochemistry using a 35S-labelled RNA probe transcribed from a cDNA coding for the human preprotachykinin which contains both substance P and K. Labelled neurons were observed in the caudate nucleus and the putamen but not the cerebellum. The labelled cells were of medium size and their distribution and morphology were compatible with previous data on substance P-like immunoreactivity in the human brain. The results confirm the presence of preprotachykinin mRNA in a subpopulation of striatal neurons in the human and show that in situ hybridization can be used to detect specific neurotransmitter-related mRNAs in postmortem tissue from normal and diseased humans.

A cDNA encoding the precursor of the rat neuropeptide, neurokinin B.

We have isolated a cDNA clone from a rat cerebral cortex library which encodes the 116 amino acid precursor of the neuropeptide, neurokinin B. The precursor has 68% amino acid homology to the bovine precursor and encodes a single peptide of the tachykinin family. Except for possible small variations at both ends of the message, there appears to be only a single species of neurokinin B mRNA in rat cerebral cortex. In situ hybridization histochemistry indicates that the message is widely distributed in the rat brain in a pattern distinct from that of substance P message.

[Regulation of enkephalin biosynthesis in chromaffin cells]. / Régulation de la biosynthèse des enképhalines dans les cellules chromaffines.

Enkephalin peptides (ENK) are co-released with catecholamines from bovine chromaffin cells in culture. Drugs mimicking the effects of c-AMP increase ENK biosynthesis by increasing ENK mRNA, but are uneffective on ENK secretion. Nicotine, which causes a rapid release of ENK from these cells, also induces an increase in ENK biosynthesis and ENK mRNA. Reserpine enhance ENK precursor processing. The actions of these different pharmacological agents show that ENK biosynthesis is regulated at different levels in chromaffin cells.

Corticotropin releasing factor increases proopiomelanocortin messenger RNA in mouse anterior pituitary tumor cells.

The ability of corticotropin releasing factor (CRF) to stimulate adrenocorticotropin (ACTH) synthesis in corticotrophs was assessed by measuring total cell content of ACTH and the levels of proopiomelanocortin (POMC) mRNA in a cloned tumor cell line of the mouse anterior pituitary (AtT-20/D16-16). CRF treatment caused a time-dependent increase in POMC mRNA levels (measured using a hybridization technique) as well as elevating total ACTH content in AtT-20 cells. The increase in POMC mRNA levels preceded changes in ACTH content and slowly returned toward control levels after CRF withdrawal. The rise in POMC mRNA levels following CRF stimulation appeared to be specific since beta-actin mRNA levels were not affected by CRF treatment. Both 8-bromo-cAMP and phorbol ester increased POMC mRNA levels in AtT-20 cells, suggesting that CRF may act through different protein kinases to regulate the POMC gene. CRF appears to activate the POMC gene since treatment of the AtT-20 cells with the peptide increased the levels of an RNA species in the nuclei having the expected molecular weight of the transcript of the POMC gene. The results indicate that continued exposure of corticotrophs to CRF induces long term increases in the ACTH synthetic capacity of those cells.

Spiritual dimensions of a mind-body group for people with severe mental illness.

In a mind-body group designed to address issues of well-being for people with severe mental disorders, experiences with spiritual themes of optimal functioning and ultimate meaning emerged with surprising clarity.

Corticotropin-releasing factor-induced adrenocorticotropin hormone release and synthesis is blocked by incorporation of the inhibitor of cyclic AMP-dependent protein kinase into anterior pituitary tumor cells by liposomes.

Corticotropin-releasing factor (CRF) is the most potent and effective natural stimulant of corticotropin (ACTH) secretion. In a tumor cell line of the mouse anterior pituitary (AtT-20/D16-16) consisting of a homogeneous population of corticotrophs, CRF is known to increase adenylate cyclase and cAMP-dependent protein kinase activities as well as to release ACTH. To determine whether activation of cAMP-dependent protein kinase is essential for CRF to evoke the secretion of ACTH, an inhibitor (PKI) of this kinase was inserted into AtT-20 cells. This was accomplished by first encapsulating PKI into liposomes and then covalently coupling them to protein A for binding to antibodies directed against an AtT-20 cell surface antigen, N-CAM (neural cell adhesion molecule). The binding of the liposomes to the anti-N-CAM antibodies led to the internalization of the PKI into the tumor cells. The PKI treatment greatly attenuated CRF-stimulated ACTH release as well as the secretory response to beta-adrenergic agonists. However, ACTH release in response to caerulein, an agonist of cholecystokinin 8 receptors, was not altered by the PKI treatment. CRF treatment also increased the levels of mRNA for proopiomelanocortin (POMC), the precursor for ACTH in AtT-20 cells. Application of liposomes containing PKI to AtT-20 cells blocked the ability of CRF and 8-bromo-cAMP, but not phorbol ester, to increase POMC mRNA levels. The results revealed an essential role for cAMP in mediating the effect of CRF on ACTH release and POMC gene expression.

Cleavage of recombinant enkephalin precursor by endoproteolytic activity in bovine chromaffin granules.

To identify endoproteolytic activity that processes the enkephalin precursor, a novel approach was undertaken for the production of model substrate in the form of recombinant 35S-(Met)-preproenkephalin (35S-(Met)-PPE), generated by in vitro transcription and translation of the rat PPE cDNA. Endoproteolytic activity in bovine chromaffin granules cleaved 35S-(Met)-PPE with a pH optimum of 4.5 and generated multiple products containing the NH2-terminal segment of the precursor. Processing of 35S-(Met)-PPE, as well as endogenous enkephalin intermediates, was inhibited by pepstatin A and stimulated by DTT. These results suggest that aspartyl and thiol proteolytic activity(ies) are involved in cleaving the enkephalin precursor.

Carboxypeptidase H in the hypothalamo-neurohypophysial system: evidence for processing and activation of a prohormone-processing enzyme during axonal transport.

Investigations of peptide precursor processing in nerve cells, including studies on prooxytocin and provasopressin processing in the rat hypothalamo-neurohypophysial system, show that prohormone processing occurs during axonal transport of maturing secretory vesicles. Recent studies (Fricker et al., 1989; Rodriguez et al., 1989) show that carboxypeptidase H (CPH), one of several proteases required for prohormone processing, is synthesized as a proenzyme that presumably requires activation. To determine if pro-CPH, like prohormone precursors, is processed and activated during axonal transport, we have analyzed the molecular forms of CPH present at several levels in the rat hypothalamo-neurohypophysial system. These biochemical and immunochemical studies showed that the supraoptic nucleus (SON), a region enriched in neuronal cell bodies, possesses primarily an inactive 65-kDa species of CPH. The median eminence and pituitary stalk regions that are enriched in axons possess both the inactive 65-kDa and the active 55-kDa forms of CPH, and nerve terminals of the posterior pituitary contain primarily the active 55-kDa CPH. These results support the hypothesis that pro-CPH is processed and activated during axonal transport from neuronal perikarya of SON to nerve terminals of the posterior pituitary. Furthermore, analysis of immunoreactive CPH in the rat and bovine pituitary showed that each tissue possessed different relative amounts of zymogen compared to mature forms of CPH, suggesting that tissue-specific processing of pro-CPH occurs. Thus, the biosynthesis of active peptide hormones requires the simultaneous processing of proenzyme and prohormone.

Bovine chromogranin A sequence and distribution of its messenger RNA in endocrine tissues.

Chromogranin A is contained in storage vesicles of chromaffin cells of the adrenal medulla and released with catecholamines when the splanchnic nerve is stimulated. Chromogranin A is similar to secretory protein I (SP-I), a major secreted protein of the parathyroid. Chromogranin A/SP-I immunoreactivity is abundant in endocrine cells that secrete peptide hormones from storage vesicles. Chromogranins may act in neuroendocrine secretion by binding intravesicular calcium. Serum levels of chromogranin are raised in hypertension and endocrine neoplasia. We report here the isolation and sequencing of a cDNA encoding bovine chromogranin A, providing the first complete primary structure of a chromogranin protein. Chromogranin A is a highly acidic protein with an apparent relative molecular mass (Mr) of 75,000 on SDS-PAGE, but an actual Mr of 48,000. Adrenal medulla, brain, pituitary and parathyroid are all sites of synthesis of chromogranin A. The primary structure of chromogranin A, and the presence of chromogranin mRNA in the parathyroid, indicate that chromogranin A and SP-I are identical.

Nicotinic receptor stimulation activates enkephalin release and biosynthesis in adrenal chromaffin cells.

Neuroendocrine cells release a portion of their stored secretory hormone content when exposed to tissue-specific secretagogues. In the case of the adrenal medulla, catecholamines and enkephalin peptides, as well as other secretory proteins, are secreted in response to acetylcholine, which is released onto cholinergic receptors on chromaffin cells upon splanchnic nerve stimulation in vivo. Secretagogue stimulation thus depletes intracellular stores of exportable hormone. We were interested to know whether the signal for exportable hormone release might also function as a signal for compensatory hormone repletion by enhancing the biosynthesis of the released hormone(s). Accordingly, we have investigated the effect of nicotinic receptor stimulation on Met-enkephalin peptide biosynthesis and expression of proenkephalin messenger RNA in primary cultures of bovine chromaffin cells. Our results, reported here, suggest a model for stimulus-secretion-synthesis coupling in which nicotinic receptor occupancy activates two pathways. One pathway, dependent on calcium and not mimicked by increased intracellular cyclic AMP, leads to exocytotic hormone release; the other, probably via a calcium-dependent increase in intracellular cyclic AMP, leads to a compensatory increase in intracellular enkephalin through activation of transcription of the proenkephalin structural gene.

Alternative modes of enkephalin biosynthesis regulation by reserpine and cyclic AMP in cultured chromaffin cells.

Exposure of bovine chromaffin cells in primary culture to 5 microM reserpine or 25 microM forskolin results in an increase in enkephalin peptide levels within 24-48 hr; 25 microM forskolin (or cholera toxin at 50 micrograms/ml) causes a 1.5- to 2-fold increase in enkephalin peptide levels, which is maximal after 48 hr of exposure and is totally blocked by addition of cycloheximide (0.5 microgram/ml). Reserpine (5 microM) elicits a 1.5- to 2-fold increase in enkephalin peptide levels within 24 hr, which is only partially blocked by cycloheximide. Chromatographic analysis of cellular extracts shows that forskolin increases levels of both [Met]enkephalin pentapeptide and high molecular weight enkephalin-containing peptides, while reserpine causes an increase in [Met]enkephalin pentapeptide and a concomitant decrease in high molecular weight enkephalin-containing peptides, suggesting enhanced conversion of enkephalin precursor(s) to the mature polypeptide hormone. Measurement of preproenkephalin messenger RNA (mRNAenk) by RNA blot hybridization with a cDNA probe for mRNAenk reveals that forskolin and cholera toxin cause a relatively rapid (less than 17 hr) 3- to 5-fold increase in mRNAenk, while exposure to reserpine elicits a gradual decrease in enkephalin mRNA (a 50%-80% decline) beginning within 24 hr and continuing over a 72-hr period. These results suggest that forskolin and reserpine differentially regulate enkephalin biosynthesis in cultured chromaffin cells, the former by increasing, presumably via a cAMP-dependent mechanism, cellular mRNA coding for preproenkephalin and the latter by a post-translational increase in proenkephalin processing.

Dopamine antagonist haloperidol decreases substance P, substance K, and preprotachykinin mRNAs in rat striatonigral neurons.

Rat genomic clones were used to quantitate preprotachykinin mRNAs in the rat basal ganglia, while the tachykinin peptide products substance P and substance K were measured by radioimmunoassay. Administration of the dopamine antagonist (antipsychotic) drug haloperidol significantly decreased substance P, substance K, and both alpha (substance P encoding) and beta (substance P/substance K encoding) preprotachykinin mRNAs, suggesting a drug-induced decrease in striatonigral tachykinin biosynthesis. The time course for decreased preprotachykinin mRNAs and tachykinins apparently parallels the period of maximum risk for the development of certain antipsychotic drug-induced extrapyramidal side effects seen clinically. Tachykinin interaction with dopamine neurons may play an important role in the modulation of basal ganglia function.