It is now widely accepted that ATP functions as a signalling substance in the nervous system. The presence of P2 receptors mediating the action of extracellular ATP in brain regions involved in ...hormonal regulation raises the possibility that a similar role for ATP might also exist in the neuroendocrine system. In this study, the release from the rat isolated neurohypophysis preparation of endogenous ATP, oxytocin and vasopressin (AVP) were measured simultaneously using luciferin-luciferase and RIA techniques. After 70 min preperfusion, electrical field stimulation caused a rapid increase in the amount of ATP in the effluent and the release of AVP and oxytocin also increased stimulation-dependently. Inhibition of voltage-dependent Na+ channels by tetrodotoxin (1 microM) reduced the stimulation-evoked release of AVP and oxytocin; however, the evoked release of ATP remained unaffected. The effect of endogenous ATP on the hormone secretion was tested by suramin (300 microM), the P2 receptor antagonist. Suramin significantly increased the release of AVP, and the release of oxytocin was also enhanced. ATP, when applied to the superfusing medium, decreased the release of AVP, but not that of oxytocin, and its effect was prevented by suramin. ATP (60 nmol), added to the tissues, was readily decomposed to ADP, AMP and adenosine measured by HPLC combined with ultraviolet light detection, and the kinetic parameters of the enzymes responsible for inactivation of ATP (ectoATPase and ecto5'-nucleotidase) were also determined (Km=264+/-2.7 and 334+/-165 microM and vmax=6.7+/-1.1 and 2.54+/-0.24 nmol/min per preparation (n=3) for ectoATPase and ecto5'-nucleotidase respectively). Taken together, our data demonstrate the stimulation-dependent release, P2 receptor-mediated action and extracellular metabolism of endogenous ATP in the posterior lobe of the hypophysis and indicate its role, as a paracrine regulator, in the local control of hormone secretion.
Background and purpose: 2,3,5-Triphenyltetrazolium chloride (TTC) staining measures tissue viability used to evaluate infarct size. The goal of this study was to compare viability of neuronal tissue ...during the early phases of ischemia–reperfusion assessed either by perfusion of the brain with TTC solution transcardially,
in vivo, or by staining brain slices,
in vitro.
Methods: The middle cerebral artery was occluded for 1 h in male SPRD rats and then reperfused for 0, 1, 4, 8, 16 and 24 h. Ischemic damage was evaluated by TTC staining,
in vivo and
in vitro, and by histology (Luxol Fast Blue and Fluoro-Jade staining, electron microscopy).
Results: Core volume of tissue injury measured
in vivo was large at 0 h and steadily decreased by 50% (
p
<
0.001) up to 16 h, but substantially increased from 16 to 24 h of reperfusion. In contrast, a significant core volume appeared at 4 h only,
in vitro, and gradually increased up to 24 h. Core volume was larger
in vivo than
in vitro at all times except at 16 h when the opposite was observed. Evans blue administered intracardially stained TTC-negative areas at 1 and 24 h. Histology covered the evolution of serious tissue injury but also demonstrated some morphologically preserved neurons in the infracted area at 24 h.
Conclusions: Formation of formazan from TTC can depend on both the staining method and the metabolic burden of the brain tissue causing uncertainties in the volume of ischemia-induced brain injury measured by TTC staining.
Using histochemical and immunocytochemical methods, cholinergic nerve fibres were demonstrated in the rat adrenal cortex, primarily in the capsule and zona glomerulosa, and in the medulla. Some ...terminated among the glomerulosa cells or around blood vessels. Occasional fibres were also seen in the fasciculata, ending in islets of chromaffin tissue without ramifications on cortical cells. To clarify the role of cholinergic innervation, a microvolume perifusion system was used to study steroid production by the rat adrenal capsule-glomerulosa. Acetylcholine (ACh) itself had no reproducible effects; however, since variable amounts of endogenous ACh were present, the actions of antagonists were also studied. The M1 muscarinic receptor antagonist pirenzepine (10 and 100 microM) stimulated aldosterone secretion. This stimulation was abolished by co-incubation with carbachol, the M1 agonist McN A-343 and by atropine. We found that the action of pirenzepine was blocked by nifedipine (Ca2+ channel blocker), suggesting that pirenzepine (through release of endogenous ACh) provides an acute stimulus by enhancing Ca2+ inflow. Hemicholine, a choline uptake blocker, reduced the stimulatory effect of pirenzepine on steroid secretion, confirming that stimulation was of neural origin. Neither the non-selective muscarinic receptor antagonist atropine, the selective M1-M3 muscarinic receptor antagonist 4-DAMP, nor the selective M2 muscarinic receptor antagonist methoctramine influenced aldosterone output. Receptor-binding studies revealed the existence of M3 receptors in capsule-glomerulosa homogenates. We conclude that pirenzepine acts on presynaptic M1 autoreceptors to increase spontaneous ACh release from varicose axon terminals that lie in close proximity to the glomerulosa cells. In turn ACh may thus stimulate steroidogenesis acutely through M3 receptors. These results support the concept of a direct cholinergic influence on zona glomerulosa function in the rat.
After aerosol deposition from the atmosphere, black carbon (BC) takes part
in the snow albedo feedback contributing to the modification of the Arctic
radiative budget. With the initial goal of ...quantifying the concentration of
BC in the Arctic snow and subsequent climatic impacts, snow samples were
collected during the research vessel (R/V) Polarstern expedition of PASCAL (Physical Feedbacks of Arctic Boundary Layer, Sea Ice,
Cloud and Aerosol; Polarstern cruise 106) in the sea-ice-covered Fram Strait in early summer 2017. The refractory BC (rBC) content was then measured in the laboratory of the Alfred Wegener Institute with the single particle soot photometer (SP2). Based on the strong observational correlations between both rBC concentration and rBC diameter with snow salinity, we hypothesize
a salt-induced matrix effect interfering with the SP2 analysis. This paper
evaluates the impact of sea salt, based on the measurement of electrical
conductivity (κ) in water samples, on rBC measurements made with a
SP2 nebulizer technique. Under realistic salinity conditions, laboratory
experiments indicated a dramatic six-fold reduction in observed rBC
concentration with increasing salinity. In the salinity conditions tested in the present work (salt concentration below 0.4 g L−1) the impact of salt on the nebulization of water droplets might be negligible. However, the SP2 mass detection efficiency systematically decreased with increasing
salinity, with the smaller rBC particles being preferentially undetected.
The high concentration of suspended salt particles and the formation of
thick salt coatings on rBC cores caused problems in the SP2 analog-to-digital conversion of the signal and incandescence quenching,
respectively. Changes to the signal acquisition parameters and the laser power of the SP2 improved the mass detection efficiency, which, nonetheless, stayed below unity. The present work provides evidence that a high concentration of sea salt undermines the quantification of rBC in snow performed with the SP2 nebulizer system described here. This interference has not been previously reported and might affect the future such analysis of rBC particles in snow collected, especially over sea ice or coastal regions strongly affected by sea salt deposition.
Midbrain slices containing the dorsal and medial raphe nuclei were prepared from rat brain, loaded with 3Hserotonin (3H5-HT), superfused, and the electrically induced efflux of radioactivity was ...determined. The nonselective 5-HT receptor agonist 5-carboxamido-tryptamine (5-CT; 0.001 to 1 microM) inhibited the electrically stimulated 3H5-HT overflow from raphe nuclei slices (IC50 of 3.34 +/- 0.37 nM). This effect of 5-CT on 3H5-HT overflow was antagonized by the 5-HT7 receptor antagonist SB-258719 (10 microM) and the 5-HT(1B/1D) antagonist SB-216641 (1 microM), the IC50 values for 5-CT in the presence of SB-258719 and SB-216641 were 94.23 +/- 4.84 and 47.81 +/- 4.66 nM. The apparent pA2 values for SB-258719 and SB-216641 against 5-CT were 6.43 and 7.12, respectively. The inhibitory effect of 5-CT on 3H5-HT overflow was weakly antagonized by 10 microM of WAY-100635, a 5-HT1A receptor antagonist (IC50 6.65 +/- 0.56 nM, apparent pA2 4.99). The antagonist effect of SB-258719 (10 microM) on 5-CT-evoked 3H5-HT overflow inhibition was also determined in the presence of 1 microM SB-216641 or 1 microM SB-216641 and 10 microM WAY-100635, and additive interactions were found between the antagonists of 5-HT7 and 5-HT1 receptor subtypes. Addition of the Na+ channel blocker tetrodotoxin (1 microM) in the presence of SB-216641 (1 microM) and WAY-100635 (10 microM) attenuated the inhibitory effect of 5-CT on KCl-induced 3H5-HT overflow. These findings indicate that 5-CT inhibits 3H5-HT overflow from raphe nuclei slices of the rat by stimulation of 5-HT7 and 5-HT(1B/1D receptors, whereas the role of 5-HT1A receptors in this inhibition is less pronounced. They also suggest that 5-HT7 receptors are probably not located on serotonergic neurons and thus may serve as heteroreceptors in regulation of 5-HT release in the raphe nuclei. 5-CT (0.1 microM) also inhibited 3Hglutamate release, and SB-258719 (10 microLM) suspended this effect. We therefore speculated that the axon terminals of the glutamatergic cortico-raphe neurons may possess 5-HT7 receptors that inhibit glutamate release, which consequently leads to decreased activity of serotonergic neurons. The postulated glutamatergic-serotonergic interaction in the raphe nuclei was further evidenced by the finding that N-methyl-D-aspartate and AMPA enhanced 3H5-HT release.
The release of endogenous ATP, measured by the luciferin-luciferase assay, and the release of
3Hacetylcholine from the isolated superior cervical ganglion of the rat loaded with
3Hcholine were ...studied simultaneously. Electrical field stimulation enhanced the release of endogenous ATP and acetylcholine in a Ca
2+
o-dependent manner. The Na
+ channel blocker, tetrodotoxin (1
μM) inhibited the stimulation-evoked release of endogenous ATP and of
3Hacetylcholine, but did not change the resting release. The release of ATP was dependent on the frequency of stimulation between 2 and 10
Hz, when the number of shocks was kept constant (360 shocks), while acetylcholine was not released in a frequency-dependent fashion. Ten days after cutting of the preganglionic nerve of the superior cervical ganglion the stimulation-evoked release of acetylcholine and ATP was abolished and the uptake of
3Hcholine was significantly reduced but not inhibited. Hexamethonium, (100
μM) a nicotinic acetylcholine receptor antagonist, significantly reduced the release of both acetylcholine and ATP, indicating a positive feedback modulation of ACh and ATP release. 8-Cyclopentyl-1,3-dipropylxanthine (10 nM), the selective A
1-adenosine receptor antagonist exhibited similar effect on the release of ATP and acetylcholine: both of them were augmented, showing that the stimulation-evoked release of ATP and acetylcholine are under the inhibitory control of A
1-adenosine receptors. When the temperature was reduced to 7°C to inhibit carrier-mediated processes, the resting and stimulated release of acetylcholine was not changed. Conversely, the release of ATP in response to stimulation was reduced by 79.9±5.6%, and the basal release was also almost completely blocked. Carbamylcholine by itself was able to release ATP, but not acetylcholine, in a hexamethonium-inhibitable manner, even from ganglia whose preganglionic nerve had been cut 10
days prior to experiments, suggesting that ATP release can occur in response to nicotinic receptor stimulation of postsynaptic cells. The breakdown of ATP or AMP by superior cervical ganglion was measured by high performance liquid chromatography combined with UV detection. ATP and AMP, added to the tissues, were readily decomposed: the K
m (apparent Michaelis constant) and V
max (apparent maximal velocity) were 475±24
μM and 3.50±0.18
nmol/min per mg for ectoATPase and 1550±120
μM and 14.5±0.9
nmol/min per mg tissue for 5′-nucleotidase. In addition, by using electron microscopic enzyme histochemistry, the presence of ectoATPase was also shown in the superior cervical ganglion. It is concluded that endogenous ATP and acetylcholine are released simultaneously in response to stimulation of preganglionic nerve terminals in the superior cervical ganglion in a Ca
2+
o-dependent, tetrodotoxin-sensitive manner and is metabolized by ectoenzymes present in the tissue.
The dissociation of the release of ATP and acetylcholine at different stimulation frequencies and temperatures shows that the release-ratio of acetylcholine and ATP can vary upon the condition of stimulation: this can reflect either the different composition of synaptic vesicles in the preganglionic nerve terminals or a significant contribution of non-exocytotic, carrier-mediated type of release of ATP to the bulk release.
In addition to hypophyseal control, steroid synthesis and secretion in the adrenal cortex is also under direct local neural modulation. We obtained morphological and neurochemical evidence that a ...substantial proportion of the noradrenergic nerve endings lie in close proximity to zona glomerulosa cells without making synaptic contact, thus providing evidence for a direct local modulatory role of catecholamines in steroid secretion. These noradrenergic neurones, like other noradrenergic neurones in the central nervous system, are able to take up dopamine (DA), convert it partly into noradrenaline (NA) and to release both NA and DA together with the co-transmitter ATP when neuronal activity drives them to do so. These catecholamines and ATP may reach zona glomerulosa cells via diffusion in a paracrine way and modulate the synthesis of aldosterone. The presence of ecto-Ca-ATPases, enzymes that may terminate the effect of ATP, was demonstrated around the nerve profiles indicating that not only ATP but its metabolites (ADP, AMP, adenosine) can also influence the production of aldosterone. These data strongly support the possibility of a paracrine, non-synaptic modulatory role of catecholamines and ATP in the regulation of adrenocortical steroid secretion.
The objective of this study was to study how the outflow of
3
H
purines is altered during a brief period of ischemic-like conditions in superfused hippocampal slices and to show whether it is ...regulated by P
2 purinoceptors and the nitric oxide (NO) pathway. The outflow of
3
H
purines increased in response to 5 min of combined hypoxia/hypoglycemia. High performance liquid chromatography analysis verified the efflux of
3
H
adenosine-triphosphate,
3
H
adenosine-diphosphate,
3
H
adenosine-monophosphate,
3
H
adenosine,
3
H
inosine, and
3
H
hypoxanthine in response to ischemic-like conditions. The P
2 receptor antagonists suramin and pyridoxal-phosphate-6-azophenyl-2′-4′-disulphonic-acid-tetrasodium (PPADS) reduced significantly the
3
H
purine efflux evoked by ischemic-like conditions, showing that P
2 purinoceptors are involved in the initiation of purine outflow. The NO synthase inhibitor
N-nitro-
l-arginine-methyl-ester (
l-NAME) attenuated significantly the
3
H
purine outflow, evoked by ischemic-like conditions, while 7-nitroindazole (7-NI) caused only a mild decrease in the outflow. The NO donor sodium nitroprusside increased significantly the basal efflux of
3
H
purines. In summary, a brief period of combined hypoxia/hypoglycemia induced the efflux of ATP in addition to the outflow of other purines. Since P
2 receptor antagonists decreased the
3
H
purine outflow evoked by ischemic-like conditions we propose that ATP, acting on P
2 purinoceptors, is responsible for further efflux of purines after ischemic-like period. It seems likely that NO is also involved in the regulation of purine outflow, since inhibition of NO production attenuated the
3
H
purine outflow, evoked by ischemic-like conditions, while exogenous NO facilitated the basal outflow.
Aerosol–cloud interaction is considered one of the
largest sources of uncertainty in radiative forcing estimations. To better
understand the role of black carbon (BC) aerosol as a cloud nucleus and ...the
impact of clouds on its vertical distribution in the Arctic, we report
airborne in situ measurements of BC particles in the European Arctic near
Svalbard during the “Arctic CLoud Observations Using airborne measurements
during polar Day” (ACLOUD) campaign held in the summer of 2017. BC was measured with
a single-particle soot photometer aboard the Polar 6 research aircraft from the lowest atmospheric layer up to approximately 3500 m a.s.l (metres above sea level). During
in-cloud flight transects, BC particles contained in liquid droplets (BC
residuals) were sampled through a counterflow virtual impactor (CVI) inlet.
Four flights, conducted in the presence of low-level, surface-coupled, inside-inversion, and mixed-phase clouds over sea ice, were selected to address
the variability in BC above, below, and within the cloud layer. First, the
increase in size and coating thickness of BC particles from the free
troposphere to the cloud-dominated boundary layer confirmed that ground
observations were not representative of upper atmospheric layers. Second,
although only 1 % of liquid droplets contained a BC particle, the higher
number concentration of BC residuals than BC particles sampled below cloud
indicated that the totality of below-cloud BC was activated by nucleation
scavenging but also that alternative scavenging processes such as the
activation of free-tropospheric BC at the cloud top might occur. Third, the
efficient exchange of aerosol particles at cloud bottom was confirmed by the
similarity of the size distribution of BC residuals and BC particles sampled
below cloud. Last, the increase in the BC residual number concentration
(+31 %) and geometric mean diameter (+38 %) from the cloud top to
the cloud bottom and the absolute enrichment in larger BC residuals compared
with outside of the cloud supported the hypothesis of concomitant scavenging
mechanisms but also suggested the formation of BC agglomerates caused by
cloud processing. The vertical evolution of BC properties from inside the cloud
and below the cloud indicated an efficient aerosol exchange at cloud bottom,
which might include activation, cloud processing, and sub-cloud release of
processed BC agglomerates. In the case of persistent low-level Arctic
clouds, this cycle may reiterate multiple times, adding an additional
degree of complexity to the understanding of cloud processing of BC particles in the Arctic.
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