Gonadotropin-releasing hormone release by superfused hypothalami in response to norepinephrine

We have been studying the release of gonadotropin-releasing hormone (GnRH) from adult male rat medial basal hypothalamus (MBH) utilizing a continuous flow superfusion system. This model system allows for direct application of modifying substances into the superfusion chambers and for continuous collection of effluent for radioimmunoassay of GnRH levels. Gonadotropin-releasing hormone is rapidly released in response to specific chemical stimuli. As demonstrated by others, pulses of KCl or prostaglandin E2 (PGE2) result in sharp peaks of GnRH release followed by rapid return to baseline. Forty millimolar KCl increases [GnRH] 3- to 4-fold, consistent with a membrane-associated secretory process for GnRH release. A 50-micrograms bolus of PGE2 results in a 2-fold rise in GnRH. Norepinephrine stimulates the release of GnRH in a log-linear dose-dependent manner in the range of 10(-9) to 10(-5) M norepinephrine (NE). At 10(-11) M, NE does not increase GnRH release above baseline, whereas at 10(-9) M NE GnRH release is increased 2-fold. The alpha-receptor blocker phentolamine significantly inhibits the NE-induced rise in GnRH. Propranolol, a beta-adrenergic receptor blocker, does not inhibit the GnRH response to NE. This study demonstrates a direct, dose-dependent, alpha-mediated stimulatory effect of NE on GnRH release from superfused male rat MBH, and establishes the potential of this system for the investigation of the GnRH response to other aminergic agents and their extraneural modifiers, including steroid hormones.     

Choroidal readaptation to gravity in rats after spaceflight and head-down tilt

Davet, Julien, Benoit Clavel, Lucien Datas, LaurenceMani-Ponset, Daniel Maurel, Serge Herbuté, Michel Viso, WilliamHinds, Joellen Jarvi, and Jacqueline Gabrion.Choroidal readaptation to gravity in rats after spaceflight andhead-down tilt. J. Appl. Physiol.84(1): 19-29, 1998.To determine when choroidal structures wererestored after readaptation to Earth gravity or orthostatic position,fine structure and protein distribution were studied in rat choroidplexus dissected either 6 h [Space Life Sciences-2 (SLS-2)experiments] or 2 days [National Institutes ofHealth-Rodent 1 (NIH-R1) experiments] after a spaceflight, or 6 hafter head-down tilt (HDT) experiments. Apical alterations were notedin choroidal cells from SLS-2 and HDT animals, confirming thatweightlessness impaired choroidal structures and functions. However,the presence of small apical microvilli and kinocilia and the absenceof vesicle accumulations showed that the apical organization began tobe restored rapidly after landing. Very enlarged apical microvilli appeared after 2 days on Earth, suggesting increased choroidal activity. However, as distributions of ezrin and carbonic anhydrase IIremained altered in both flight and suspended animals after readaptation to Earth gravity, it was concluded that choroidal structures and functions were not completely restored, even after 2 days in Earth's gravity.

     

Desensitization to norepinephrine includes refractoriness of calcium release in myocardial cells

Localization of myoplasmic free calcium was measured in fura2-loaded single rat myocardial cells to determine whether the mechanism of norepinephrine desensitization includes redistribution of calcium. Fluorescence intensities at each pixel were quantitated by use of a photon-counting, microchannel plate camera. From these images, values of calcium-dependent fluorescence intensity averages in whole cells, areas of calcium release (as zones of high intracellular calcium concentrations), and ratios of fluorescence intensity in central vs. peripheral sites were determined. Stimulation by 1 nM norepinephrine caused an increase in total free intracellular calcium and an activation of intracellular calcium release sites from subsarcolemmal pools initially and later from centrally located calcium pools. Subsequent addition of 100 nM norepinephrine failed to cause significant intracellular calcium release from centrally located pools. In contrast, forskolin exposure still released high concentrations of calcium from these central pools. These results indicate that pretreatment with even a relatively small concentration of norepinephrine causes markedly decreased subsequent intracellular calcium release from centrally located sarcoplasmic reticulum because of a refractoriness of the link between receptor activation and calcium release.     

PTH-receptors regulate norepinephrine release in human heart and kidney

Recent data suggests that chronic renal failure and hyperparathyroidism are associated with sympathetic overactivity. Since peptide hormones are known to modulate norepinephrine (NE) release by activating prejunctional receptors, this study investigates whether parathyroid hormone fragment (1-34) (hPTH(1-34)) increases neuronal NE release in human heart and kidney. Using specific PTH-receptor agonists and antagonists, this study furthermore highlights functional differences between PTH1 and PTH2 receptors. Human atrial and renal tissues were incubated with [(3)H]-NE and superfused. Three electrical stimulations (5Hz, 1min) induced a stable [(3)H]-NE release which was taken as an index of endogenous NE release. RT-PCR with specific primers for PTH1- and PTH2-receptor was performed in heart and kidney. hPTH(1-34) (0.01-0.1μmol/L) and a stable analog of its second messenger cAMP (8-bromo-cAMP) increased [(3)H]-NE release in human atria. This facilitatory effect of PTH was also observed in human renal cortex. The PTH1-receptor antagonist (D-Trp(12), Tyr(34))-pTH-(7-34) (0.5μmol/L) abolished the effect of hPTH(1-34). This data was verified using isolated perfused mouse kidneys. Tuberoinfundibular peptide of 39 residues (TIP-39) (0.1nmol/L-0.1μmol/L) decreased [(3)H]-NE release in atria. PTH1- and PTH2-receptor expressions were demonstrated in human heart and kidney. Moreover, a splice variant of the PTH2-receptor was detected in human kidney. In conclusion, PTH is able to facilitate NE release in human atria and renal cortex by activation of PTH1-receptors. The highly increased PTH levels that can be observed in chronic renal failure might be one contributor for the elevated sympathetic nerve activity and the associated cardiovascular mortality in patients with end stage renal disease.     

Vagosympathetic interactions in ischemia-induced myocardial norepinephrine and acetylcholine release

To elucidate the pathophysiological roles of vagosympathetic interactions in ischemia-induced myocardial norepinephrine (NE) and acetylcholine (ACh) release, we measured myocardial interstitial NE and ACh levels in response to a left anterior descending coronary occlusion in the following groups of anesthetized cats: intact autonomic innervation (INT, n = 7); vagotomy (VX, n = 6); local administration of atropine (Atro, n = 6); transection of the stellate ganglia (TSG, n = 5); local administration of phentolamine (Phen, n = 6); and combined vagotomy and transection of the stellate ganglia (VX+TSG, n = 5). The maximum NE release was enhanced in the VX group (141 +/- 30 nmol/l, means +/- SE, P < 0.05) compared with the INT group (61 +/- 12 nmol/l). Neither the Atro (50 +/- 24 nmol/l) nor VX+TSG groups (84 +/- 25 nmol/l) showed enhanced NE release. The maximum ACh release was unaltered in the TSG and Phen groups compared with the INT group (19 +/- 4, 18 +/- 4, and 13 +/- 3 nmol/l, respectively). These findings indicate that the cardiac vagal afferent but not efferent activity reduced the ischemia-induced myocardial NE release. In contrast, the cardiac sympathetic afferent and efferent activities played little role in the ischemia-induced myocardial ACh release.     

Peripheral presynaptic adrenoreceptor regulation of norepinephrine release in humans

The concept of peripheral presynaptic regulation of neuronal norepinephrine (NE) release via alpha 2 adrenoreceptors has received extensive support from in vitro evidence. Despite this, the importance of such a system under physiological and pathophysiological conditions remains to be defined in humans. This largely reflects the limitations of using plasma NE as an index of neuronal amine release in vivo and the difficulties of interpreting the hemodynamic responses to adrenoreceptor agonists or antagonists administered in vivo. Efficient probes and sensitive indices of neuronal NE release are required to clarify the importance of peripheral presynaptic mechanisms in humans.     

PACAP enhances stimulation-induced norepinephrine release in canine pancreas in vivo

The present study was to investigate whether pituitary adenylate cyclase activating polypeptide (PACAP) can modify norepinephrine (NE) release in response to pancreatic nerve stimulation in anesthetized dogs. Plasma catecholamine concentrations in aortic and superior pancreaticoduodenal (SPD) venous blood were determined by a high performance liquid chromatography method. SPD venous blood flow was measured with an electromagnetic flowmeter. Pancreatic nerves were directly stimulated for 1 min (2 ms, 12 V) at various frequencies at the level of the SPD artery. Various doses of PACAP1-27 (PACAP27) were locally infused into the pancreas through the SPD artery. Nerve stimulation significantly increased both SPD venous NE concentration and its output from the pancreas in a frequency-dependent manner. With PACAP27 alone, neither SPD venous NE concentration nor its output changed significantly following the local administration of PACAP27 at any dose tested. In the presence of PACAP27, however, the net increases in NE concentration and its output in response to nerve stimulation at 2 Hz were significantly enhanced in a dose-dependent manner. The enhanced NE responses to nerve stimulation by PACAP27 were thus significantly greater than those obtained from the group receiving either PACAP27 or stimulation alone. Increases in NE concentration and its output induced by local administration of tyramine were virtually abolished by desipramine, a neural amine uptake inhibitor. However, the NE response to tyramine was not diminished by PACAP27. The results indicate that PACAP27 enhances the stimulation-induced NE release in the pancreas, and that this facilitatory effect of PACAP27 does not result from an inhibition of the neural amine uptake mechanism. The study suggests that PACAP receptor-mediated mechanisms may be involved either directly or indirectly in the local modulation of neural NE release in the canine pancreas in vivo.     

Chromosome aberrations in the blood lymphocytes of astronauts after space flight.

Cytogenetic analysis of the lymphocytes of astronauts provides a direct measurement of space radiation damage in vivo, which takes into account individual radiosensitivity and considers the influence of microgravity and other stress conditions. Chromosome exchanges were measured in the blood lymphocytes of eight crew members after their respective space missions, using fluorescence in situ hybridization (FISH) with chromosome painting probes. Significant increases in aberrations were observed after the long-duration missions. The in vivo dose was derived from the frequencies of translocations and total exchanges using calibration curves determined before flight, and the RBE was estimated by comparison with individually measured physical absorbed doses. The values for average RBE were compared to the average quality factor (Q) from direct measurements of the lineal energy spectra using a tissue-equivalent proportional counter (TEPC) and radiation transport codes. The ratio of aberrations identified as complex was slightly higher after flight, which is thought to be an indication of exposure to high-LET radiation. To determine whether the frequency of complex aberrations measured in metaphase spreads after exposure to high-LET radiation was influenced by a cell cycle delay, chromosome damage was analyzed in prematurely condensed chromosome samples collected from two crew members before and after a short-duration mission. The frequency of complex exchanges after flight was higher in prematurely condensed chromosomes than in metaphase cells for one crew member.     

Multiple factors regulating the release of norepinephrine consequent to nerve stimulation.

Whereas extracellular calcium is absolutely required for neurotransmitter release consequent to stimulation of adrenergic and other neurons, a large number of substances are known to modify the amount of norepinephrine released per nerve impulse. In general, cyclic nucleotides, phosphodiesterase inhibitors, beta-adrenoceptor agonists, cholinergic nicotinic agonists, and angiotensin are able to enhance neurally mediated norepinephrine release, whereas alpha-adrenoreceptor agonists, cholinergic muscarinic agonists, prostaglandins of the E series, opiates, enkephalins, dopamine, and adenosine inhibit neurally mediated norepinephrine release. Although it has been proposed that cyclic AMP may enhance, and endogenous cyclic GMP may inhibit, neurotransmitter release, no consistent relationship between the effects of the several modulators of neurally mediated norepinephrine release and their effects on adenylate and guanylate cyclase is as yet apparent. The demonstration of whether such a relationship exists must await the development of techniques that will allow the measurement of cyclic nucleotide levels in the presynaptic adrenergic nerve terminal after exposure to the putative modulators of release and consequent to nerve stimulation.     

Biochemical changes in rat liver after 18.5 days of spaceflight

The effect of "weightlessness" on liver metabolism was examined using tissue from rats flown in earth orbit for 18.5 days aboard the Soviet Cosmos 936 biosatellite. Changes in the activities of certain carbohydrate and lipid enzymes were noted. Of the 28 hepatic enzyme activities assayed, two, palmitoyl-CoA desaturase and lactate dehydrogenase, increased, whereas five, glycogen phosphorylase, 6-phosphogluconate dehydrogenase, both acyltransferases which act on alpha-glycerolphosphate and diglycerides, and aconitate hydratase decreased. The remaining enzyme activities measured were unchanged. In addition, increased levels of liver glycogen and palmitoleate were noted which probably resulted from the lowered glycogen phosphorylase and increased palmitoyl-CoA desaturase activities, respectively, in those animals that experienced weightlessness. These changes caused by weightlessness were transient since all of the aforementioned alterations returned to normal values when measured in the livers of other rats which had flown in the biosatellite 25 days after recovery.     

Determination of norepinephrine apparent release rate and clearance in humans

A method for estimating the rate of entry of norepinephrine into plasma (norepinephrine apparent release rate) and clearance of norepinephrine from plasma in humans is presented. The procedure involves the intravenous infusion of tritiated ?-norepinephrine, of sufficiently high specific activity to avoid elevating blood pressure, until plateau concentration is reached in plasma, and measurement of norepinephrine specific activity under steady state conditions. In ten normal subjects at rest, the apparent release rate of norepinephrine was 0.54 ± 0.20 μg/m²/min. (mean ± standard deviation). It was significantly lower in four patients with idiopathic peripheral autonomic insufficiency, 0.19 ± 0.12 μg/m²/min., but in the latter, despite reduced norepinephrine release, plasma norepinephrine concentration was near normal because of slowed clearance of norepinephrine from the circulation, 1.69 ± 0.44 ?/min. compared with 2.80 ± 0.73 ?/min. in normal subjects (p<0.05). In four normal subjects given the norepinephrine uptake inhibitor, desipramine, to slow removal of norepinephrine from the circulation, again the plasma concentration of neurotransmitter was higher than would be expected from the existing apparent release rate of norepinephrine. The findings suggest that methods which measure the dynamic processes of norepinephrine release and removal quantify sympathetic nervous activity better than steady state plasma norepinephrine measurements alone.     

A release of prostaglandins may be responsible for acute tolerance to norepinephrine infusions

A chick isolated rectum pretreated with atropine and indomethacin and superfused with the oxygenated mixed venous blood of anaesthetized cats, was selectively contracted by PGE₁ and PGE₂ at concentrations of <1 ng/ml. Intravenous infusion of norepinephrine (0.2 – 8.0 μg/kg/min) into the cats resulted in a contraction of the blood-bathed chick rectum. This was matched by contractions produced by PGE₂ (0.4 – 7 ng/ml) infused directly over the assay organ. The appearance of a chick rectum contracting substance in the venous blood was paralleled by a decline in the pressor response to norepinephrine. A single injection of indomethacin (3 – 10 mg/kg) prevented both the formation of the prostaglandin-like material and the acute tolerance to the pressor response to norepinephrine. Both effects could then be reproduced by an intra-arterial infusion of PGE₂ at a rate 0.125 – 0.5 μg/kg/min. β-Adrenoceptor blockade had no influence on the response of chick rectum and arterial blood pressure to an infusion of norepine phrine, but α-adrenoceptor blockade abolished both responses. It is postulated that the acute tolerance to norepinephrine infusions is the result of a release of PGE-like material from the contracting vascular bed.     

Up-regulation of SKIP relates to retinal ganglion cells apoptosis after optic nerve crush in vivo

Cell cycle re-entry is one of the key processes in neuronal apoptosis. Previous studies have shown that Ski-interacting protein (SKIP) played an important role in cell cycle re-entry. However, its expression and function in optic nerve injury are still with limited acquaintance. To investigate whether SKIP is involved in retinal ganglion cells (RGCs) death, we performed an optic nerve crush (ONC) model in adult rats. Western blot analysis revealed that up-regulation of SKIP was present in retina at 5 days after ONC. Immunofluorescent labeling indicated that up-regulated SKIP was found mainly in RGCs. We also investigated co-localization of SKIP with active-caspase-3 and TUNEL (apoptotic markers) -positive cells in the retina after ONC. In addition, the expression of SKIP was increased in parallel with P53 and P21 in retina after ONC. All these results suggested that up-regulation of SKIP in the retina was associated with RGCs death after ONC.     

Increase in rat soleus myotendinous interface after a 14-d spaceflight

Myotenclinous junctions (MTJs) transmit contractile force from skeletal muscles to tendons. The effects of a 14-d spaceflight on MTJ were studied in the soleus muscle of male adult Sprague Dawley rats by transmission electron microscopy and histomorphometric techniques. We showed that the length of the junctional membrane relative to the muscle fiber diameter increased by 58% after 14 d of spaceflight. This increase accompanies morphological changes at MTJs. The flight MTJs appeared more shredded. The ends of the muscle fibers exhibited T tubule dilatation, swollen mitochondria, Z-disk streaming, loss of myofilaments, a thinning down of subplasmalemmal densities, multivesicular bodies and signs of junctional membrane and basal lamina remodelling. The ultrastructural observations suggest that the increase in myotendinous interface could result from the extracellular matrix spreading into remodelling muscle fiber, whereas the constraints related to unloading were reduced by spaceflight conditions.