Biomedical subjects
E M Hol
Publications and source records attributed to E M Hol.
The role of calcium and cAMP in the mechanism of action of two melanocortins: alpha MSH and the ACTH4-9 analogue Org 2766.
Melanocortins accelerate functional recovery after nerve crush and enhance neurite outgrowth in vitro. To get more insight in the mechanism of action of melanocortins, we studied the effects of two neurotrophic peptides: alpha-melanocyte-stimulating hormone (alpha MSH) and an adrenocorticotropin4-9 analogue Org 2766 on second messengers in cultures of spinal cord (SC), dorsal root ganglion (DRG) and Schwann cells. alpha MSH (10 microM) enhanced the forskolin-induced cAMP production in SC- (45%) and in DRG-cells (35%). Org 2766 (1 microM) induced an increase in cAMP only in SC-cells (39%). The peptides did not affect the cAMP levels in Schwann cells. Neither peptide evoked significant changes in the intracellular free calcium concentration ([Ca2+]i) in batch-measurements of all cell types, however, Ca(2+)-imaging revealed an infrequent occurrence of large [Ca2+]i-elevations in individual SC-neurons. The results indicate that SC- and DRG-cells are targets for both peptides, while Schwann cells are not or exploit different pathways. We observed for alpha MSH that cAMP production always coincides with outgrowth stimulation, whereas for Org 2766 cAMP production and outgrowth stimulation appear not causally related. These differences in second messenger stimulation could be explained by receptor heterogeneity. We suggest that alpha MSH and Org 2766 act through different receptors, each with its own signalling pathways.
Dorsal root ganglia as an in vitro model for melanocortin-induced neuritogenesis. Pharmacological and mechanistic aspects.
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Involvement of calcium and cAMP in the mechanism of action of two melanocortins: alpha MSH and an ACTH-(4-9) analogue.
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Protection by an ACTH4-9 analogue against the toxic effects of cisplatin and taxol on sensory neurons and glial cells in vitro.
Sensory neuropathy is a serious side effect of anti-tumour drugs such as cisplatin and taxol. There are indications that an analogue of the adrenocorticotrophic hormone 4-9 fragment (ACTH4-9: Met(O2)-Glu-His-Phe-D-Lys-Phe) can prevent these neurotoxic effects. We studied the potential protective effects of this analogue in cultures of chick dorsal root ganglia and rat Schwann cells treated with cisplatin or taxol to gain insight into the mode of action and characteristics of this neuroprotection. Neurite outgrowth of sensory neurons in vitro was dose-dependently inhibited by cisplatin and taxol; after 48 hr, 10 micrograms/ml cisplatin reduced outgrowth from 431 +/- 17 microns to 220 +/- 6 microns and 0.01 micrograms/ml taxol from 344 +/- 3 microns to 200 +/- 43 microns. Co-treatment of 10 micrograms/ml cisplatin with the ACTH4-9 analogue (0.1 nM-1 nM) resulted in about 35% more outgrowth than cisplatin alone. In contrast, the analogue could not prevent taxol neurotoxicity. Migration of neurons and satellite cells from the DRG-body is completely inhibited by 10 micrograms/ml cisplatin. Taxol had no effect on the migration of these cells. In addition, cisplatin was more toxic to Schwann cells than taxol; 3-10 micrograms/ml cisplatin significantly reduced their laminin content, total protein, 2',3'-cyclic nucleotide 3'-phosphodiesterase activity, and cell division. The ACTH4-9 analogue (0.01 nM-100 nM) had no effect on the migration of cells out of the DRGs and could not prevent the toxic effect on the Schwann cells. These data support our hypothesis that the neuroprotective effect of ACTH4-9 analogue is brought about by a direct action on neurons, possibly by replacing a Schwann-/satellite-cell derived trophic factor.
Trophic effects of melanocortins on neuronal cells in culture.
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alpha-MSH and ACTH4-9 analogue Org 2766 induce a cAMP increase in cultured rat spinal cord cells.
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The expression of B-50/GAP-43 in Schwann cells is upregulated in degenerating peripheral nerve stumps following nerve injury.
We have detected mRNA for B-50 (GAP-43, pp46, F1, neuromodulin), which was originally believed to be a neuron-specific protein, in non-neuronal cells in the rat sciatic nerve. In control rats, the level of B-50 mRNA in sciatic nerve tissue was much lower than in dorsal root ganglia. Following nerve crush or transection, the expression of B-50 mRNA in the distal nerve stump increased dramatically between 1 and 2 days post-injury. The B-50 mRNA levels in the distal stump of crushed nerves remained elevated for up to 4 weeks and subsequently returned to control levels after 7 weeks. In contrast, after nerve transection B-50 mRNA levels in the distal nerve portion continued to increase up to 7 weeks post-lesion. No changes in the levels of the B-50 transcript were observed in the proximal portion of either crush-lesioned or transected sciatic nerves. In situ hybridization demonstrated B-50 mRNA associated with Schwann cells in the distal nerve stump. The observation that Schwann cells are capable of producing B-50 mRNA was confirmed by Northern blot analysis of total RNA isolated from primary Schwann cell cultures. Taken together, these data show the expression of B-50 mRNA by Schwann cells and the up-regulation of B-50 mRNA in reactive Schwann cells upon loss of axonal contact.
Nimodipine protects cultured spinal cord neurones from depolarization-induced inhibition of neurite outgrowth.
In the nervous system calcium ions play a crucial role in the regulation of growth cone motility, cell migration and neurite outgrowth. High intracellular Ca2+ concentrations severely disturb Ca(2+)-regulated processes and may lead to neuronal death. We studied whether the Ca(2+)-antagonist nimodipine could prevent inhibition of neurite outgrowth which occurs in depolarized cultures of rat foetal spinal neurones. Spinal cord slices were depolarized in culture with 50 mM K+. Nimodipine (0.01-10 microM) was added before depolarization. After 5 and 7 days the effect of treatment was determined by: (a) blind scoring of neurite outgrowth under phase contrast; and (b) measuring neurofilament (NF) protein with an ELISA. Neurite outgrowth was markedly decreased after depolarization, but was restored to control values by nimodipine (0.1 microM). Depolarization also led to a decrease in total NF content (18%). The NF content of depolarized slices incubated with 0.1 microM nimodipine was the same as in the controls. Thus, depolarization-induced Ca2+ entry into spinal neurones inhibits neurite outgrowth from spinal neurones. Low concentrations of nimodipine prevented this inhibition. As nimodipine had no effect on neurite outgrowth in control cultures, we conclude that nimodipine does not act as a neurotrophic factor but rather as a neuroprotective agent.
alpha MSH but not Org 2766 induces expression of c-fos in cultured rat spinal cord cells.
Alpha-melanocyte stimulating hormone (alpha MSH) and Org 2766 (an adrenocorticotrophic hormone (ACTH) MSH4-9 analogue) stimulate sprouting after nerve damage in vivo. In cultures of rat spinal cord cells only alpha MSH enhances neurite outgrowth. In order to analyse the mode of action of alpha MSH and to get a clue why its action differs from that of Org 2766, we studied whether expression of c-fos was induced in spinal cord cells by treatment with alpha MSH or Org 2766. We found a rapid and transient induction of both c-fos mRNA and protein after treatment with 100 microM alpha MSH. Treatment with Org 2766 (100 pM-100 microM) had no effect on c-fos expression. These results suggest that c-fos is involved in the signal transduction system of alpha MSH stimulated neuronal outgrowth in vitro.
Endorphin-like immunoreactivities in uncultured and cultured human peripheral blood mononuclear cells.
It has been reported that cells of the immune system produce and release considerable amounts of pro-opiomelanocortin (POMC) -derived peptides in response to coculture with a variety of stimulatory agents. The present study investigated whether extracts of human peripheral blood mononuclear cells (PBMC) contain immunoreactivity for beta-endorphin (beta E) and related peptides. Using four endorphin RIA systems with different specificities, extracts of freshly isolated PBMC and PBMC cultured in the presence or absence of mitogens or of corticotropin releasing factor (CRF) and vasopressin (VP), were analyzed. With a radioimmunoassay (RIA) system directed to the midportion of beta E, immunoreactivity (MP beta E-IR) was readily detectable, although the concentration was extremely low (ca. 200 pg/10(7) cells). beta E immunoreactivity (beta E-IR) and alpha-endorphin immunoreactivity (alpha E-IR), as determined in C-terminally directed RIA systems, were present in even lower concentrations. gamma-Endorphin immunoreactivity (gamma E-IR) was hardly detectable. Of subsets enriched in T-cells, B-cells or monocytes, the highest concentration of MP beta E-IR was detected in extracts of monocytes. Coculture of PBMC with the mitogen Concanavalin A (Con A) or Phytohaemagglutinin (PHA) increased the amount of MP beta E-IR in extracts of the cells. No increase in alpha E-IR, however, was detected, whereas beta E-IR was only increased in extracts of cells cultured in the presence of Con A. No increase, in any of the immunoreactivities, was observed in extracts of PBMC cultured with bacterial lipopolysaccharide (LPS) or with the combination of CRF and VP, both stimuli that have been reported to induce POMC peptides in cultured PBMC. The present data show that human PBMC contain endorphin-like immunoreactivity, but in very small amounts. The extremely low concentrations and the ineffectiveness of LPS and the combination of CRF and VP to increase the endorphin-like immunoreactivity raise questions about the reported capacity of PBMC to synthesize POMC-derived peptides.
Characterization of dopamine D2 receptors in the pituitary of the African catfish, Clarias gariepinus.
Dopamine receptors in the pituitary of the African catfish, Clarias gariepinus, were characterized using [3H]spiperone as radioligand. Specific binding of [3H]spiperone to pituitary membranes reached equilibrium within 60 min of incubation. The binding of the radioligand was tissue specific since the amount of binding was linear with pituitary membrane content in the incubations. In addition, pituitary membranes were observed to bind considerably more [3H]spiperone, compared to membrane preparation of various other tissues. Saturation experiments revealed the presence of a single class of high affinity/low capacity binding sites. The binding characteristics, estimated by Scatchard analysis, were: Kd = 3.2 +/- 0.5 x 10(-9) M and Bmax = 105 +/- 5 fmol/mg protein. Specific binding was displaceable with dopamine and with various specific D2 agonists and antagonists. The nature of displacement curves resembles those observed in studies on mammalian dopamine receptors. Binding experiments with cell fractions, obtained after centrifugation of dispersed pituitary cells over a Percoll density gradient, showed that most [3H]spiperone binding was obtained in an enriched gonadotropic cell fraction. This observation indicates that the receptor characteristics, estimated with the [3H]spiperone assay, are representative for dopamine receptors on the gonadotropic cells.
Stimulation by melanocortins of neurite outgrowth from spinal and sensory neurons in vitro.
Using ELISAs for B-50/GAP43 and neurofilament (NF), we tested ACTH(1-24), alpha-MSH, ACTH(4-10), and an ACTH(4-9) analogue (ORG2766) for their ability to induce sprouting and neuritogenesis from spinal and sensory neurons. Dissociated fetal rat spinal cord neurons or neonatal rat dorsal root ganglion (DRG) cells were cultured with peptide and assayed after 24, 48, or 96 h. In spinal neurons, alpha-MSH and ACTH(1-24) induced the expression of B-50 dose dependently. After 24 h alpha-MSH had a stimulatory effect (from 10 nM onwards), with a maximum at 100 microM (36% increase). After 96 h the maximal effect of 100 microM alpha-MSH on B-50/GAP43 was lower (19%). ACTH(1-24) (100 microM) stimulated B-50/GAP43 by 19%. Neurofilament levels (96 h) were elevated maximally by 64% at 100 microM alpha-MSH. In DRG neurons a bell-shaped dose-response curve was found for alpha-MSH, the maximal effect being observed after 48 h at 100 nM: 54% for B-50/GAP43 and 22% for NF. In both culture systems neither ACTH(4-10) nor ORG2766 was effective. We conclude that alpha-MSH stimulates the expression of B-50/GAP43 (sprouting) and the formation of NF (neurite elongation) and may therefore be considered a neurotrophic factor.
Molecular misreading: a new type of transcript mutation expressed during aging.
Dinucleotide deletions (e.g. DeltaGA, DeltaGU) are created by molecular misreading in or adjacent to GAGAG motifs of neuronal mRNAs. As a result, the reading frame shifts to the +1 frame, and so-called "+1 proteins" are subsequently synthesized. +1 Proteins have a wild-type N-terminus, but an aberrant C-terminus downstream from the site of the dinucleotide deletion. Molecular misreading was discovered in the rat vasopressin gene associated with diabetes insipidus and subsequently in human genes linked to Alzheimer's disease (AD), e.g. beta amyloid precursor protein (betaAPP) and ubiquitin-B (UBB). Furthermore, betaAPP(+1) and UBB(+1) proteins accumulate in the neuropathological hallmarks (i.e. in the tangles, neuritic plaques, and neuropil threads) of AD. As these +1 proteins were also found in elderly nondemented controls, but not in younger ones (<51 years), molecular misreading in nondividing cells might act as a factor that only becomes manifest at an advanced age. Frameshift mutations (UBB(+1)) and pretangle staining (Alz-50 and MC1) seem to occur independently of each other during early stages of AD. We recently detected +1 proteins, not only in proliferating cells present in non-neuronal tissues such as the liver and epididymis, but also in neuroblastoma cell lines. These observations suggest that molecular misreading is a general source of transcript errors that are involved in cellular derangements in various age-related pathologies.