Down-regulation of thyrotropin-releasing hormone (TRH) receptors in spinal cord after transection as revealed by quantitative autoradiography.

The autoradiographic localization of thyrotropin-releasing hormone (TRH) receptors was investigated in the rat spinal cord after transection at the level of T8-T9. The discrete distribution of [3H]-MeTRH binding was measured with a computerized image analyzer at the cervical (C6-C7) and lumbar (L2-L3) level, one week and three weeks after injury. The TRH receptor density was expressed in fmol/mg protein. There was no significant change in the density of TRH receptors below the injury site. In the cervical region, TRH receptor concentration in the dorsal gray matter did not differ from normal controls; in contrast we found a time dependent change in lamina 10 and in the ventral gray, with a significant decrease (25% and 19%, respectively) of TRH receptor binding sites one week after transection and a return to control levels by three weeks. From these data and the known increase of TRH immunoreactivity above a spinal injury, a down-regulation of spinal cord TRH receptors in response to elevated levels of TRH is suggested.

Solid scintillators for receptor assays: an environmentally safe alternative to liquid scintillation cocktails.

Scintillation counting of tritiated ligands is widespread in receptor assays and has necessitated the use of scintillation cocktails containing environmentally damaging solvents that pose health hazards to their users. A safer mode of dry scintillation counting, based on the solid scintillator Xtalscint, was evaluated in whole-cell and membrane receptor assays. The results compared favorably with those obtained with glass-fiber filters and conventional liquid counting methods. It is concluded that solid scintillators may be used as an environmentally safer alternative to liquid scintillation in these assays.

Effects of experimental spinal cord transection on substance P receptors: a quantitative autoradiography study.

The autoradiographic localisation of Substance P (SP) receptors was investigated in the rat spinal cord following cordotomy at the thoracic 8-9 level. The binding of [125I]-BH-SP was measured in discrete gray matter structures above (C6-C7 level) and below (L2-L3 level) the injury site, and expressed in fmol/mg protein. There was a statistically significant increase in SP receptor density 1 week after cordotomy in the dorsal horn and in the central canal of lumbar region, in laminae 3-4-5 and also in the ventral horn of cervical spinal cord. Elevated SP receptor density was also noted in cervical ventral horn at 3 weeks after thoracic cordotomy, whereas the increase seen at 1 week was normalized at 3 weeks everywhere else. Since SP concentration has been reported to show a similar localized increase below a spinal transection, the present results are consistent with an up-regulation of SP receptors. A similar direct relationship between SP receptors and SP content appears to occur also in the ventral horn above the injury site.

Glucocorticoid receptor from lactating goat mammary tissue comparison of native and activated forms in a cell free system.

Physicochemical properties of native and activated (DNA-binding) forms of the glucocorticoid receptor in cytosol prepared from lactating goat mammary tissue have been examined. Under hypotonic conditions the cytosolic receptor sediments at 8.4 S or 9.9 S in the absence or presence of 10 mM molybdate, respectively. The receptor in cytosol, either with or without molybdate elutes from DEAE-cellulose at approximately 200 mM potassium phosphate concentration. Isoelectric focusing reveals that this form of the receptor focuses at pH 5.5. Further, the cytosolic form of the receptor exhibits minimal binding affinity for polyanions such as DNA-cellulose. Its Stokes radius is 77 A and the mol. wt is approximately 331,000. Following exposure to in vitro activating conditions (including elevated ionic strength or temperature), the liganded receptor exhibits much lower affinity for DEAE-cellulose (elution at 35-55 mM potassium phosphate concentration). Other alterations in properties of the activated receptor, after partial purification, include sedimentation at 3.9 S in hypotonic sucrose gradients, binding to polyanions (DNA-cellulose), and an isoelectric point at pH 7.2. This receptor has a Stokes radius of 58 A and a mol wt of 98,000. A degraded form, with a mol. wt of approximately 57,000 and high affinity for polyanions, was the major form of the receptor obtained if appropriate precautions to prevent or remove proteolytic activity were not observed during purification and/or characterization of the activated receptor.

Autoradiographic localization of substance P receptors in rat spinal cord: effects of experimental spinal transection.

The autoradiographic localization of Substance P (SP) receptors was studied in the rat spinal cord following thoracic cordotomy. The binding of [125I]-BH-SP was measured in discrete gray matter structures above and below the injury site. There was a significant increase in SP receptor density 1 week after cordotomy in the dorsal horn and in the central canal of lumbar region, and in laminae 3-4-5 and also in the ventral horn of cervical spinal cord. Elevated SP receptor density was noted only in cervical ventral horn at 3 weeks after cordotomy. Since SP concentration has been reported to show similar localized increases, the present results are consistent with an up-regulation of SP receptors.

Kinetic analysis of thyrotropin-releasing hormone binding in the central nervous system: evidence for receptor desensitization.

To investigate the mechanism(s) of experimentally and clinically observed refractoriness of spinal lower motor neurons (LMNs) to the excitatory effects of high-dose TRH, we examined the kinetics of dissociation of [3H]TRH from its CNS-receptor. At 23 degrees C, the receptor was rapidly (40 min) and completely converted from a form with fast dissociation kinetics (complex I; t1/2 20-30 min) to one from which the peptide dissociated much more slowly (complex II; t1/2 greater than 120 min). This conversion required the presence of added agonist ([3H]TRH) and was not prevented by the GTP-analog Gpp(NH)p. We suggest that complexes I and II may respectively represent active and inactive (desensitized) forms of the TRH-receptor and that TRH-induced I to II conversion of the receptor is responsible for refractoriness of LMNs to the drug.

De novo neuromuscular junction formation on human muscle fibres cultured in monolayer and innervated by foetal rat spinal cord: ultrastructural and ultrastructural--cytochemical studies.

Ultrastructural features of neuromuscular junction formation and transverse tubule development were studied utilizing a newly developed model in which human muscle fibres cultured in monolayer are innervated by foetal rat spinal cord with dorsal root ganglia attached. At early innervation (7-10 days), when distinct 'boutons' are contacting muscle fibres, the contacts of nerve terminals with the muscle fibres are, ultrastructurally, superficial and unorganized, and there is no basal lamina-like material between nerve terminals and muscle fibres. A bouton consists, ultrastructurally, of a cluster of small nerve terminals contacting the muscle fibre. At 2-3 weeks of innervation, shallow 'beds' are formed on the muscle fibre just beneath nerve terminals, and occasionally there are irregular and miniscule fragments of basal lamina-like material in the cleft. There is no Schwann cell apposing the nerve terminal at this stage of innervation. After 4-5 weeks of innervation there is more definite basal lamina material in the cleft and suggestive postsynaptic plasmalemmal densities and invaginations. However, there is no Schwann cell apposing the nerve terminal at this stage. At 6-8 weeks of innervation, deep postsynaptic folds are present, a Schwann cell apposes the nerve terminal, and basal lamina surrounds the entire muscle fibre. At all four stages of innervation examined, ultrastructural cytochemistry of alpha-bungarotoxin binding reveals that nicotinic ACh receptors are located exclusively at the neuromuscular junctions. After 1-2 weeks of innervation, very few lanthanum-positive transverse tubules are observed and only in close proximity to the surface membrane. After 3 weeks of innervation, more lanthanum-positive tubules are present, and they are located deeper within the muscle fibre. Five weeks after innervation, somewhat more elaborated tubules (but no lateral sacs) appear, and honeycomb structures are often present. After 6-7 weeks of innervation the tubular system is very elaborate and lateral sacs are present. Hence, this study describes consecutive stages of the formation of neuromuscular junctions and transverse tubules in innervated cultured human muscle, and provides an important basis to which similar studies related to the diseased human muscle can be compared.

Lack of usefulness of DN-1417 for characterization of a CNS receptor for thyrotropin-releasing hormone.

CNS receptors for thyrotropin-releasing hormone (TRH) and its analogs are likely to mediate the experimentally and clinically observed net excitatory effect of these peptides on lower motor neurons. Previous findings suggest that several types of TRH receptors with distinct TRH analog specificities may be present in rat CNS. In particular, based on competition isotherm assays with unlabeled analog gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-prolineamide (DN-1417). Funatsu et al. claim the existence of a limbic forebrain site that binds this peptide and TRH with high affinity but that does not bind [3-methyl-histidyl2]-TRH (MeTRH). Using saturation and competition isotherm experiments, we have examined the binding of [3H]TRH and [3H]DN-1417 in three regions of rat CNS: pyriform cortex/amygdala, limbic forebrain, and lumbosacral spinal cord. In all three regions, saturation assays with [3H]TRH (0.4-100 nM) resolved only a single, saturable receptor with high affinity (KD = 12-14 nM) for TRH; in no case could more than one saturable site be identified. When [3H]DN-1417 was substituted as the assay ligand, no high-affinity binding component for this analog could be detected in the three regions. Competition curves for the binding of unlabeled DN-1417 to limbic forebrain and lumbosacral spinal cord ([3H]TRH as assay ligand) were monophasic (not biphasic like those of Funatsu et al.) and indicative of low-affinity binding of DN-1417 in these regions (Ki values = 2-3 microM; in agreement with values obtained in similar assays with [3H]MeTRH).(ABSTRACT TRUNCATED AT 250 WORDS)

Analogs of thyrotropin-releasing hormone: hypotheses relating receptor binding to net excitation of spinal lower motor neurons.

Experimentally and clinically, treatment with high-doses of TRH produces a net excitation of spinal lower motor neurons (LMNs) that is subsequently reduced or completely lost through continuous or repeated exposure to the peptide. This is operationally termed "autorefractoriness" (AR). We have performed biochemical and in vivo pharmacologic experiments to investigate the mechanism(s) of AR. Biochemically, we classified TRH and several analogs into three groups based on their binding by spinal-cord TRH-receptors (TRH-Rs): high-affinity, (low nanomolar range; MeTRH, TRH); intermediate-affinity (mid-nanomolar range; MK-771, RX77368) or low-affinity (micromolar range; DN-1417, PNP). When tested in vivo for LMN excitatory activity in cordotomized (T8) rats, TRH and MK-771 produced rapid-onset excitation followed AR. In contrast, sustained excitation with much less AR was produced by the low affinity analog DN-1417. Based on these results, we have formulated two receptor-based hypotheses to explain AR: a) rapid TRH-R desensitization (conversion to an inactive form) by high- but not low-affinity TRH-analogs; and b) a slower down-regulation (cellular internalization) of the agonist-receptor complex, most evident with high-affinity agonists. Thus, low-rather than high-affinity TRH-analogs may be superior to TRH for providing sustained LMN excitation (increase of strength) in motor neuron degenerative disorders.

Cells of neural crest origin as possible models to investigate thyrotropin releasing hormone action in the central nervous system.

Since TRH has been linked to trophic events in the motor neuron and is being used for symptomatic treatment of motor neuron disorders, we have examined seven neural crest cell lines for TRH-receptors (TRH-Rs) and for the enzymes choline acetyltransferase (ChAT) and creatine kinase (CK), with the intent to identify one that can be used for studies of TRH-R mediated cellular events in the CNS. Three of the seven (neuroblastomas) were ChAT-positive but were without detectable TRH-Rs and showed no major alterations in ChAT/CK-activities following acute (24 hr) treatment with 0.01-10 microM TRH. In contrast, high affinity TRH receptor sites were detected in a melanoma (rpmI 3460) cell line. Linking cellular events with these receptors could lead to the use of this established cell line as a tool for the biochemical investigation of the pathways and mechanisms of TRH action in the CNS.

Analog specificity of the thyrotropin-releasing hormone receptor in the central nervous system: possible clinical implications.

TRH has rapid-onset (30 sec), slow-offset (1-12 days) clinical benefit in patients with amyotrophic lateral sclerosis and other motor neuron disorders. This benefit is probably receptor-mediated and may have at least 2 components. To obtain a better understanding of the various responses to TRH of the spinal lower motor neurons (LMNs) in patients, and possibly to help guide selection of additional therapeutic agents, we utilized rat CNS (spinal-cord and brain membranes) to analyze the ability of certain molecules to inhibit specific binding of [3H]methyl TRH [( 3H]MeTRH) to the TRH receptor. We found: a) lack of high-affinity binding of the TRH-analog DN-1417 by spinal-cord and brain TRH receptor, despite its known strong TRH-like action physiologically on LMNs; b) lack of high-affinity binding of the TRH-product cyclo(His-Pro) by spinal-cord and brain TRH receptor despite its having some strong TRH-like physiologic actions on the CNS; and c) lack of any identifiable high-affinity receptor for cyclo(His-Pro) in spinal cord and brain. From these data we hypothesize that the acute transmitter-like action of DN-1417, TRH, and possibly other TRH-analogs and products on LMNs is via a non-TRH receptor, such as an amine or amino acid neurotransmitter receptor, e.g. a 5-hydroxytryptamine receptor. We further postulate that the CNS TRH-receptor may modulate a trophic-like influence of TRH on LMNs.

Identification of transformed glucocorticoid receptor from dexamethasone resistant melanoma.

We have examined properties of glucocorticoid-receptor complexes which might account for dexamethasone induced alterations in the growth and morphology of melanoma target cells. We have used cultured cells and solid tumors derived from the dexamethasone-sensitive RPMI 3460 Syrian hamster melanoma cell line together with clonal variants which are either more sensitive (clone 6) or resistant (clone 5) to the growth inhibiting effects of dexamethasone. Although differing markedly in their response to corticoids, each of the cell lines contains significant quantities of glucocorticoid receptor. The present studies were designed to determine if differences in the transformability (nuclear binding ability) of the glucocorticoid receptors from these target cells could account for their sensitivity or resistance to glucocorticoids. Cytosolic glucocorticoid-receptor complexes were analyzed by DEAE-cellulose chromatography to identify and quantitate the relative amounts of native (unactivated) and transformed (activated-nuclear binding) receptors present. We could readily separate these two major forms of the glucocorticoid-receptor complex in cytosols from each of the melanoma cell lines and solid tumors examined. The identity of these receptor complexes as native or transformed was confirmed using both ATP-agarose and isolated nuclei; only transformed receptor complexes are bound. When cytosol is prepared in the presence of 10 mM sodium molybdate, only native receptor is present. After molybdate is removed, native glucocorticoid receptor is readily transformed by increased ionic strength. We conclude that resistance to dexamethasone-induced changes in growth observed in resistant clone 5 cells and solid tumors cannot be attributed to an inability of the receptor they contain to exist as a stable, transformed complex.

Glucocorticoids and melanoma: receptor properties of dexamethasone sensitive and resistant tumors.

We have grown solid tumors using dexamethasone-sensitive (clone 6) and -resistant (clone 5) cells cloned from RPMI 3460 Syrian hamster melanoma. Clone 6 but not clone 5 tumor growth was retarded by dexamethasone, indicating that these tumors retain the growth sensitivity to the hormone characteristic of the cells from which they are derived. Both tumor types contain cytosolic glucocorticoid receptors (significantly higher levels in clone 5 tumors) with similar affinity and steroid specificity characteristics and these can exist as stable, activated (nuclear binding) complexes. Despite these similarities the receptor in the two tumor types differ by some physiochemical criteria. By sucrose gradient analysis, cytosols from both tumors contain 7S receptor complexes but clone 6 contains an additional 13S form. Activated receptors isolated by DEAE-cellulose chromatography from both clone 5 and 6 tumor cytosols sediment as a single peak at 4-5S. However, the DEAE-cellulose profiles indicate that clone 6 but not clone 5 activated complexes (Peak I) appear heterogeneous with respect to charge. Interestingly, DNA-cellulose chromatography indicates that activated receptors from clone 5 tumor cytosols may bind more tightly to DNA than those from clone 6. We are investigating these receptor differences in more detail to determine more precisely the role and pathways of action of glucocorticoid hormones in melanoma.

Molybdate and the measurements of androgen receptors in prostate cancer.

Using the sucrose density gradient technique, we have compared androgen receptor values obtained in the presence and absence of sodium molybdate from cytosols of surgically obtained human prostate cancer tissue. In all nine prostates examined, inclusion of molybdate in the buffers resulted in an increase in the amount of androgen receptor detected, in some cases dramatically. Since accurate androgen receptor assays could be valuable for therapeutic decision making in prostate cancer, we advocate the inclusion of sodium molybdate in assay buffers to guard against the possibility of false negative or artificially low values.