Hormones and neurotransmitters in the mammalian olfactory system.
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Animals↗
Biomedical subjects
R M Kream
Publications and source records attributed to R M Kream.
Substance P and luteinizing hormone-releasing hormone levels in the brain of the male golden hamster are both altered by castration and testosterone replacement.
The effects of castration and testosterone (T) replacement on levels of substance P (SP) and luteinizing hormone-releasing hormone (LHRH) were assessed in discrete areas of the male hamster brain. The animals were either castrated, castrated and given a chronically low or high dose of T by Silastic implant, or sham-operated. Brain tissues and trunk blood were collected 3 weeks after surgery. Plasma T levels were maintained within the normal range by the implants but at significantly lower or higher levels than the mean for sham-operated males. Levels of SP and LHRH were quantified in the olfactory bulbs, rostral basal forebrain, anterior hypothalamic and preoptic area, medial basal hypothalamic area, medial basal hypothalamic area and median eminence, and brain stem. In general, castration and T replacement effected opposite changes in levels of SP and LHRH. In the medial basal hypothalamic area and median eminence SP levels were found to be inversely related to the chronic T levels, whereas the LHRH levels were directly correlated. In the anterior hypothalamic and preoptic area, castration reduced levels of SP. Conversely, castration elevated levels of LHRH in this area. This inverse dynamic relationship between changing peptide levels was also observed in the rostral basal forebrain but not in the olfactory bulbs. In most of these forebrain regions, the dose-response curves for the experimental groups could not incorporate the peptide levels in the sham-operated control group. SP levels in the brain stem showed a monotonic inverse relationship to circulating T levels which did include the control group values.(ABSTRACT TRUNCATED AT 250 WORDS)
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Leukotriene D4 acts in part to contract guinea pig ileum smooth muscle by releasing substance P.
The role of neurotransmitter release in generation of the contractile response to leukotriene D4 (LTD4) by guinea pig ileum longitudinal smooth muscle preparations was studied. The contractile response to LTD4 was inhibited partially by tetrodotoxin (TTx) with early portions of the contraction being more sensitive to inhibition by TTx. This finding suggests that part of the LTD4 contraction is mediated by release of a neurotransmitter as a result of activation of Na+-dependent action potentials. Three approaches, study of the effects of substance P (SP) desensitization and of the effects of a SP antagonist on the LTD4 response and direct measurement of SP release were used to evaluate the possibility that SP is released by LTD4. Partial inhibition of the LTD4 contraction by SP desensitization, and by the SP antagonist, as well as direct measurement of TTx sensitive SP release in the presence of LTD4, indicates that SP is released from neurons by LTD4 in guinea pig ileum.
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Early exposure to delta 9-tetrahydrocannabinol influences neuroendocrine and reproductive functions in female rats.
Sexual differentiation of the rat brain is affected by certain compounds administered during the neonatal period. We evaluated the effects of exposure to THC during the critical period of sexual differentiation of the female rat brain on postpubertal estrous cycles and brain neurotransmitter levels. Newborn female rats were injected either with vehicle (oil) or with different doses of THC (0.38; 1.9 or 3.8 mg/100 g) subcutaneously during the first 5 days after birth. The rats were examined daily by vaginal lavage smears from 3 to 10 months of life for phases of estrous cyclicity. The animals were then sacrificed and the anterior hypothalamus preoptic area (AHPOA) and medial basal hypothalami (MBH) were collected, processed and the methionine-enkephalin (met-enkephalin), beta-endorphin-like immunoreactivity (beta-end LI), LHRH and substance-P were measured by radioimmunoassays. In addition, serum LH and prolactin levels were measured by radioimmunoassay. Compared with the control rats, the rats perinatally exposed to THC exhibited either constant metestrus diestrus type vaginal smears or irregular estrous cycles. In the THC-treated animals, the met-enkephalin and beta-end LI levels were lower in the AHPOA and higher in the MBH. The LHRH levels of THC-treated rats were significantly lower in the MBH. The substance-P levels were significantly lower in the AHPOA of THC treated animals. In the THC-treated rats, serum LH was low but, the prolactin levels were not significantly different from the control animals.(ABSTRACT TRUNCATED AT 250 WORDS)
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Precursor forms of substance P (SP) in nervous tissue: detection with antisera to SP, SP-Gly, and SP-Gly-Lys.
Antisera generated to substance P-Gly (SP-G) and substance P-Gly-Lys (SP-G-K), the likely unamidated COOH-terminally extended forms of substance P, were used to quantify and localize substance P precursor forms in hamster brain stem and spinal cord. The precursor determinant SP-G-K was liberated from larger heterogeneous forms by mild trypsinization of tissue extracts and was converted into the second precursor determinant, SP-G, by subsequent treatment with carboxypeptidase B. The basal levels of SP-G-K in brain stem and spinal cord were approximately equal to 0.5 pg/mg of tissue and rose 43- to 64-fold after trypsinization. Basal levels of SP-G were comparable to those of SP-G-K and rose 10- to 29-fold after combined enzyme treatments. Immunohistochemical labeling of axons and somata with anti-SP-G-K increased dramatically after trypsinization. This labeling was eliminated by preadsorption with authentic SP-G-K but not substance P or SP-G. Gel-permeation chromatography revealed SP-G-K-like immunoreactivity in fractions corresponding to considerably higher molecular weight than mature substance P. Collectively, these results support the hypothesis that substance P is synthesized from larger precursors and demonstrate that extended precursor forms are normally present in the axons and somata of neural systems that synthesize substance P.
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Substance P and catecholaminergic expression in neurons of the hamster main olfactory bulb.
A coordinated series of immunohistochemical and biochemical analyses have been conducted in the hamster to examine the dependence of substance P and tyrosine hydroxylase (TH) expression by second-order olfactory neurons, and the level of dopamine in the main olfactory bulb (MOB), on the integrity of carnosine- and olfactory marker protein (OMP)-containing primary afferent neurons. Substance P-like immunoreactivity (SPLI) is localized in external tufted cells and centrifugal afferents of the MOB; TH immunoreactivity has a wider distribution, in external tufted, middle tufted, periglomerular, and deep short-axon cells as well as in centrifugal afferents. To characterize the SPLI, this material was isolated by guanidine-HCl extraction and passage over a C18 SEP-PAK. The SPLI coelutes on HPLC with authentic substance P and, following oxidation, coelutes with substance P sulfoxide. It is sensitive to alpha-chymotrypsin and is resistant to trypsin. Thus, the SPLI in the MOB behaves as authentic substance P. Intranasal irrigation with 0.17 M ZnSO4 results in peripheral deafferentiation of the MOB for up to 8 months as evidenced by a persistent loss of OMP immunoreactivity and shrinkage of the olfactory nerve layer and glomeruli. By these criteria, the vomeronasal inputs to the accessory olfactory bulb are not destroyed and the spared vomeronasal receptor neurons do not innervate the vacated peripheral projection field in the MOB. The loss of peripheral inputs to the MOB is accompanied by marked and parallel reductions in the incidences of SPLI- and TH-positive second-order neurons despite an increase in the density of neuronal somata in the glomerular layer. Biochemical quantifications following peripheral deafferentation also demonstrate significant decreases of both substance P and dopamine, together with the expected decrease of carnosine. In contrast, the SPLI and the TH and serotoninlike immunoreactivities in centrifugal afferents as well as the TH immunoreactivity in deep interneurons do not appear to be reduced, and the MOB content of norepinephrine in centrifugal afferents is unaffected. These results collectively indicate that the loss of inputs from the primary olfactory receptor neurons can reduce the levels of at least two different, putatively neuroactive compounds (substance P and dopamine) in at least three classes of second-order neurons (external tufted, middle tufted, and periglomerular cells). The control of central neuron phenotype by the peripheral olfactory neurons thus appears to be a phenomenon of broad influence. It may play a role in processing chemosensory information as well as offering a system in which to study neuronal plasticit
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Olfactory marker protein: turnover and transport in normal and regenerating neurons.
A 19,000-dalton acidic protein designated olfactory marker protein (OMP) is a cell-specific marker of mature olfactory chemosensory neurons. Intranasal irrigation of mouse olfactory epithelium with [35S]methionine labeled OMP to high specific activity. Turnover and transport characteristics of 35S-labeled OMP were compared to those of 35S-labeled global cytosol protein in groups of young, adult, and Triton-treated adult mice. The latter contained primarily large numbers of regenerating olfactory neurons. In olfactory epithelium of young and Triton-treated mice, the specific activity of OMP was three times that of global cytosol protein, whereas in adults the two measures were equal. In all three groups, however, the rate of degradation of OMP was roughly equal to that of cytosol protein (T1/2 = 5 to 6 days). By contrast, differences in T1/2 for OMP decline in the bulb of adult, young, and Triton-treated adult mice were highly significant (T1/2's of 9.3, 6.1, and 4 to 5 days, respectively; p = 0.001). The specific activity of [35S]methionine incorporated in OMP exceeded that of the free amino acid 5-fold, indicating minimal precursor reutilization during the course of our experiments. Turnover data indicate that increased isotope incorporation into OMP in the epithelium is matched by an accelerated rate of degradation in the bulb. This may be correlated with the physiological state or developmental age of the primary neurons since in young and Triton-treated adult mice, rapidly maturing "young" olfactory neurons represent a larger proportion of the total population than in adults. Thus, OMP behaves as a typical, relatively slowly transported soluble protein (v = 2 to 4 mm/day, slow component b).
Aging↗
Evidence for the presynaptic localization of a high affinity opiate binding site on dopamine neurons in the pedal ganglia of Mytilus edulis (Bivalvia).
Considerable evidence has demonstrated an interrelationship between the enkephalinergic and dopaminergic systems in both the mammalian and invertebrate nervous systems. We have described recently the presence of two classes of high affinity opiate binding sites in the nervous tissue of the marine mollusc Mytilus edulis. In order to examine the physiological role of these high affinity opiate sites, M. edulis pedal ganglia (Pg) were treated with the selective neurotoxin 6-hydroxydopamine (1 micrograms/animal, applied topically to the intact Pg); animals were sacrificed 5 days after treatment. The dopamine content of the Pg from lesioned animals was reduced to 33% relative to that of Pg from control animals. Neither serotonin nor norepinephrine levels were reduced. Fluorescent micrographs of formaldehyde-treated Pg from both lesioned and control animals revealed that the neurotoxic substance accumulates in the synaptically rich neuropil and not in the cortex of the Pg. Thus, the partial reduction in dopamine levels may reflect nearly total loss of dopamine in terminals with essentially no change in the nerve cell bodies. High affinity binding of the potent opioid peptide 125I-labeled FK 33-824 (2 nM) was reduced by 81% and low affinity binding (10 nM peptide) by 43% in Pg from lesioned animals relative to that in control tissue. In addition, D-Ala2-Met5-enkephalin, beta-endorphin and etorphine failed to change dopamine levels in lesioned animals. Together, these results suggest that the high affinity opiate binding sites that mediate alteration in dopamine levels are on dopaminergic presynaptic terminals.
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Characterization of the dopamine stimulated adenylate cyclase in the pedal ganglia of Mytilus edulis: interactions with etorphine, beta-endorphin, DALA, and methionine enkephalin.
The dopamine-stimulated adenylate cyclase activity was studied both in vivo and in vitro in the central nervous system of the bivalve mollusc Mytilus edulis. Dopamine, epinine, and apomorphine stimulated the enzyme system. Fluphenazine, haloperidol, chlorpromaxine, and to a lesser extent BOL inhibited the dopamine-stimulated adenylate cyclase. Etorphine, beta-endorphine, DALA, and methionine enkephalin depressed cyclic AMP levels. This phenomena was naloxone reversible. In addition, the opioids inhibited the stimulation of adenylate cyclase by dopamine. This phenomena was also naloxone reversible. The study demonstrates an interaction among dopamine, the opioids, and cyclic AMP.
Adenylyl Cyclases↗
Demonstration of two classes of opiate binding sites in the nervous tissue of the marine mollusc Mytilus edulis. Positive homotropic cooperativity of lower affinity binding sites.
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Demonstration of stereospecific opiate binding in the nervous tissue of the marine mollusc Mytilus edulis.
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Animals↗
Binding characteristics of a potent enkephalin analog.
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Animals↗
Chemical crosslinking of a solubilized enkephalin macromolecular complex.
Covalently bound [3H]D-Ala2,Met5-enkephalinamide- and 125I-labeled D-Ala2,N-Me-Phe4,Met-(O)5-ol-enkephalin-macromolecule complexes have been prepared by crosslinking the solubilized noncovalent complexes from rat brain. Gel electrophoresis of the partially purified 125I-labeled enkephalin-macromolecule complex under nondenaturing conditions results in a single major/radioactive peak. The complex has a Stokes radius of approximately 48 A as determined by molecular exclusion chromatography; this radius corresponds to a molecular weight of 380,000 for a spherical molecule. In preliminary experiments, sodium dodecyl sulfate electrophoresis of the complex shows a major radioactive peak corresponding to a molecular weight of 35,000. The preparation of these specific covalent enkephalin-macromolecule complexes should be useful in purification of the receptor and in probing the molecular mechanism of opiate action.
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Release of substance P from guinea pig trachea leukotriene D4.
Coordinated studies of leukotriene D4 (LTD4)-mediated contractile responses and LTD4-evoked release of the tachykinin substance P (SP) in both intact and epithelium abraded guinea pig tracheal smooth muscle preparations were performed. A partial contribution by axon reflex mechanisms to the magnitude of LTD4-induced tracheal contractions was suggested by a maximum inhibition of 21% and 28% by 5 x 10(-6) M tetrodotoxin (TTX) in abraded and intact preparations, respectively. SP-induced contractions were antagonized by the SP analog [DPro4DTrp7,9]-SP 4-11 in both types of preparation. The SP analog produced 58% and 72% inhibition of contractile responses to 10(-8) M LTD4 in abraded and intact preparations, respectively. Direct measurement of SP release by radioimmunoassay of the bathing medium showed TTX-sensitive LTD4-evoked release of SP. Inhibition by 5 x 10(-6) M TTX of LTD4-evoked SP release was 77%. The SP antagonist produced greater inhibition of LTD4-evoked contractions (58% in abraded, and 72% in intact preparations) than maximum TTX inhibition of LTD4-evoked contractions (21% in abraded, and 28% in intact). However, LTD4 (10(-8) M)-evoked SP release was at least 77% blocked by maximum doses of TTX. We therefore suggest that an additional agent, released by TTX-insensitive mechanisms, but whose contractile effects are also antagonized by [DPro4DTrp7,9]-SP 4-11, may participate in the LTD4 response.
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