Abstract Although it is becoming increasingly evident that nitric oxide (NO) mediates some of estrogen’s actions in the brain, the effects of estrogen on NO production through NO synthases (NOS) in neuronal cells have not yet been identified. Here we assessed changes in NO production induced by 17β-estradiol (E2) in cells of neuronal origin using human SK-N-SH neuroblastoma cells, which we show express all three isoforms of NOS. Involvement of NOS isoforms in E2-induced NO production was examined using isoform-specific NOS inhibitors. E2 (10−10–10−6m) induced rapid increases in NO release and changes in endothelial NOS (eNOS) expression, which were blocked by ICI 182,780, an antagonist of estrogen receptors. Increased levels of NO release and NOS activity induced by E2 were blocked by N5-(1-Imino-3-butenyl)-l-ornithine, a neuronal NOS inhibitor, and N5-(1-Iminoethyl)-l-ornithine, an eNOS inhibitor, but not by 1400W, an inducible NOS inhibitor. These results demonstrate that E2-stimulated NO production occurs via estrogen receptor-mediated activation of the constitutive NOSs, neuronal NOS and eNOS. The E2-induced NO increase was abolished when extracellular Ca2+ was removed from the medium or after the addition of nifedipine, an L-type channel blocker, and was partially inhibited using 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester, an intracellular Ca2+ chelator. However, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester itself also caused an increase in NO release that was blocked by 1400W, suggesting that inducible NOS mediates this response. Together these data reveal that constitutive NOS activities are responsible for E2- induced NO production in neuroblastoma cells and that differential activation of NOS isoforms in these cells occurs in response to different treatments.
Abstract The distribution of leucine‐enkephalin, methionine‐enkephalin, neurotensin, somatostatin, substance P. oxytocin, vasopressin, and neurophysin II in cell bodies of sympathetic autonomic nuclei of the thoracolumbar (T‐L) spinal cord was studied immunohistochemically in cats after intrathecal administration of colchicine. Neurons containing only enkephalin‐, neurotensin‐, somatostatin‐, and substance P‐like immunoreactivity (ENK, NT, SS, SP, respectively) were found in the intermediolateral nucleus pars principalis (IMLp) and pars funicularis (IMLf), the nucleus intercalatus (IC), and the central autonomic area (CA). The size, shape, location, and numbers of the peptide‐positive neurons in the IMLp, IMLf, and IC suggested that they were sympathetic preganglionic neurons (SPN). This was confirmed by a combined retrograde tracing/immunohistochemical study showing that most of these neurons at the levels of the T‐L cord known to provide preganglionic fibers to the stellate ganglion were SPN. On the other hand, the functional identification of the neurons in the CA is uncertain as neurons were not observed which were both retrogradely labelled and contained ENK, NT, SS, or SP. Immunoreactive neurons in each area were counted in ten sections from each segment from C8 to L4. In the IMLp, the SPN with ENK were greatest in number (up to 25) in segments T4–T7 and L2–L3. The maximum number of SPN containing NT was found in segments T4–T7 (45 neurons). Of the four peptides, neurons containing SS were found in the greatest number (up to 48 in segments T2–T6); neurons containing SP were found in the smallest number (15 or fewer per segment). Few SPN containing each of the four peptides were found in the IC; CA neurons with ENK and NT were also few in number. A comparison of the numbers of immunoreactive neurons in the IML with earlier estimates for the total numbers of SPN in the IML at each level showed that the proportions of IML neurons containing each of the four peptides were fairly consistent throughout the T‐L cord, with some exceptions. These results suggest that the innervation of visceral organs is not obviously peptide‐specific, although some organs may be innervated by a greater proportion of SPN containing one of these peptides. Finally, the presence of ENK, NT, SS, and SP in SPN suggests that these four peptides act as neurotransmitters in preganglionic pathways to sympathetic ganglia.
Metabolic activity was assessed in the brains of spontaneously hypertensive rats (SHR) using the histochemical hexokinase (HK) technique and photodensitometric analysis. Of eight regions known to play a role in cardiovascular regulation, only the paraventricular nucleus of the hypothalamus (PVH) exhibited alterations in HK activity. Significantly lower levels of HK activity in SHR than in control Sprague-Dawley and Wistar-Kyoto rats were measured in both the parvo- and magnocellular divisions of the PVH. No differences in HK activity were found in the anterior hypothalamic nucleus, posterior hypothalamic nucleus, supraoptic nucleus, subfornical organ, central nucleus of the amygdala, or the medial nucleus of the tractus solitarius of SHR. Similar results were obtained in renal hypertensive rats; furthermore, a positive correlation was found between levels of arterial pressure and densitometric readings. These latter results strongly suggest that metabolic alterations in the PVH of SHR are directly related to the increases in arterial pressure and are not due to the genetic makeup of SHR. In light of studies by others, the data from the present study have been interpreted to suggest that the decreases in metabolic activity in the PVH of the adult SHR are the result of a central attempt to bring the level of the arterial pressure down to normal levels and not to the altered activity of a region that might be acting to keep arterial pressure elevated.
Abstract Retrograde tracer injections of fluorescein‐ and rhodamine‐labelled latex microspheres centered in the parvicellular zone of the hypothalamic/ paraventricular nucleus and pontine lateral parabrachial nucleus revealed that 36% of the labelled neurons in the dorsal raphe nucleus send collaterals to both structures. These cells were organized in a wellx‐distinguishable cluster within the dorsal raphe nucleus. By combining retrograde tracing with immunocytochemistry, it was found that less than 8% of the doublex‐labelled cells stained positively for serotonin. Of the remaining raphe nuclei that were examined, only the median raphe nucleus contributed a minor nonserotoninergic projection to the paraventricular or lateral parabrachial nuclei. Few of the retrogradely labelled cells in the median raphe nucleus contained both tracers. These results suggest that nonserotoninergic and serotoninergic neurons in the dorsal raphe nucleus, via collateral branching, may simultaneously influence the activity of two central nervous system nuclei involved in autonomic control.
Using the immunohistochemical localization of the protein product of the immediate early gene, c‐ fos , to localize activated neurons in the paraventricular nucleus of the hypothalamus (PVN), we studied the chemical phenotypes of neurons activated by circulating angiotensin II (AII). We determined the proportions of activated PVN neurons that expressed AII type I receptor‐like immunoreactivity (AT 1 ‐L) or the neurohormones vasopressin (VP) and oxytocin (OXY). In addition, we identified activated PVN neurons that putatively produce nitric oxide (NO) on the basis of histochemical staining for nicotinamide adenine dinucleotide phosphate diaphorase (NADPH‐d). Conscious rats received intravenous AII infusions at a rate sufficient to elevate mean arterial pressure by 40–60 mmHg for 90 min; control rats received infusions of vehicle. Brains were prepared for double immunohistochemistry [Fos‐like immunoreactivity (FLI)/AT 1 ‐L, FLI/VP or FLI/OXY] or FLI/NADPH‐d histochemistry. Systemic AII infusions led to activation of 149±14 PVN neurons per section. In contrast, control animals showed activation of 21±6 PVN neurons per section. AII infusions elicited the activation of the following numbers of chemically identified PVN neurons per section: AT 1 ‐L, 24±5; VP, 26±5; OXY, 11±2; NADPH‐d, 22±4. Control animals had few activated PVN neurons per section. For each of the chemically identified populations of PVN neurons, the following proportions were activated: AT 1 ‐L, 12.5%; VP, 15.2%; OXY, 7.2%; NADPH‐d, 17.3%. The results suggest that PVN neurons producing the AT 1 receptor, VP, OXY, and NO, participate in the mediation of the central responses to circulating AII.
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