Impact of dry-off and lyophilized Aloe arborescens supplementation on plasma metabolome of dairy cows.

Positive effects have been observed as a result of Aloe arborescens supplementation in the dry-off phase in dairy cows. Metabolomic approaches can provide additional information about animal physiology. Thus, we characterized plasma metabolome around dry-off in 12 cows supplemented (AL) or not (CTR) with 10 g/d of lyophilized A. arborescens with an untargeted metabolomic approach. Overall, 1658 mass features were annotated. Regardless of treatment, multivariate statistics discriminated samples taken before and after dry-off. Overall, 490 metabolites were different between late lactation and early dry period, of which 237 were shared between AL and CTR. The most discriminant compounds (pentosidine and luteolin 7-O-glucoside) were related to the more fibrous diet. Pathway analysis indicated that pyrimidine and glycerophospholipid metabolisms were down-accumulated, suggesting reduced rumen microbial activity and liver load. Samples from AL were discriminated from CTR either the day of dry-off or 7 days after. At dry-off, aloin and emodin were the most discriminant metabolites, indicating that Aloe's bioactive compounds were absorbed. Seven days later, 534 compounds were different between groups, and emodin was among the most impacted. Pathway analysis highlighted that glycerophospholipid, pyrimidine, and folate metabolisms were affected. These results might indicate that Aloe has positive effects on liver function and a modulatory effect on rumen fermentation.

Regional distribution of sulfonylurea receptors in the brain of rodent and primate.

Glibenclamide, one of the most potent antidiabetic sulfonylureas, inhibits the activity of ATP-sensitive K+ channels in the pancreas as well as in the brain through its binding to specific receptors. Quantitative autoradiography was used to localize such receptors in the brain of rat, mouse, guinea-pig and marmoset, using [3H]glibenclamide as radioligand. In all four species, specific glibenclamide binding sites were found to be heterogeneously distributed. The highest densities were in the cerebral cortex, the molecular layer of the cerebellar cortex, the thalamus and the caudate-putamen. The globus pallidus and the substantia nigra were highly labelled in rat and mouse but poorly labelled in guinea-pig and marmoset. The distribution of glibenclamide binding sites in the hippocampus was different between the rodents and marmoset; in rodents, most binding sites were distributed in the fascia dentata and the CA3-CA4 fields of Ammon's horn, contrasting with a very homogeneous distribution in all subfields of the marmoset hippocampus. In conclusion, we demonstrate that primate brain contains specific binding sites for [3H]glibenclamide with a distribution not exactly similar to that in rodent brain.

Aminopeptidase A: distribution in rat brain nuclei and increased activity in spontaneously hypertensive rats.

Aminopeptidase A is a membrane-bound zinc metalloprotease which cleaves angiotensin II into angiotensin III. Using a new specific aminopeptidase A inhibitor, EC33, we evaluated its enzymatic activity in several microdissected brain nuclei involved in the control of cardiovascular functions and in the pituitary. We compared this distribution with that of the angiotensin I-converting enzyme which converts angiotensin I to angiotensin II. Aminopeptidase A activity was heterogenously distributed with a 150-fold difference between the lowest and the highest levels. The pituitary and the circumventricular organs were the richest source of enzyme, followed by the median eminence, the arcuate nucleus, the area postrema, the choroid plexus and the supraotic and paraventricular nuclei. We did not find any close parallel between aminopeptidase A and angiotensin I-converting enzyme distributions. We examined both enzymatic activities in brain nuclei of spontaneously hypertensive rats. Aminopeptidase A activity was higher in the spontaneously hypertensive rats than in age-matched Wistar Kyoto control rats. The difference was up to 2.5-fold in several brain nuclei involved in the blood pressure regulation; in contrast, no differences in angiotensin I-converting enzyme activity were found in the same regions. The close correspondence between the distribution of aminopeptidase A activity and angiotensin receptors and nerve terminals in the brain associated with the observation that aminopeptidase A activity was overactivated in the spontaneously hypertensive rats suggest that this enzyme may contribute, at least in part, to the regulation of cardiovascular functions by its ability to convert angiotensin II to angiotensin III.

Inhibition of vasopressinergic neurons by central injection of a specific aminopeptidase A inhibitor.

The brain angiotensin (Ang) system plays an important role in the central control of vasopressin release. Using EC33, a selective aminopeptidase A inhibitor which blocks the metabolism of Ang II in Ang III, we previously reported that vasopressin release was under the control of Ang III and not Ang II. To determine accurately the action of EC33, the effects of intracerebroventricular injection of Ang peptides or EC33 on extracellular unit activity of vasopressinergic neurons in the supraoptic nucleus of urethane-anaesthetized rats were examined. Angiotensin II (15-30 ng) or Ang III (15 ng) increased the firing rate of all neurons tested. Conversely, EC33 (10 microg) reduced or completely abolished (30-60 microg) the basal firing rate for 4-6 min in all eight neurons tested. EC33 (30 microg) also inhibited the activity induced by 30 ng Ang II. It was concluded that the observed activity of Ang II required its conversion to Ang III and that endogenous Ang III may exert a tonic control on the basal firing level of vasopressinergic neurons.

Two binding sites for [3H]glibenclamide in the rat brain.

The binding of [3H]glibenclamide, a potent sulfonylurea which blocks ATP-sensitive potassium channels, was studied in the rat brain. A Scatchard plot of saturation isotherms suggests that [3H]glibenclamide binds in various brain regions to a high- and a low-affinity binding site (Kd values of 0.21 nM and 111 nM and Bmax values of 41 and 1060 fmol/mg protein, respectively). Competitive binding assays with various unlabelled sulfonylureas showed biphasic displacements of [3H]glibenclamide with pseudo-Hill coefficients significantly different from unity. These data indicate the existence of a heterogeneity of binding sites to [3H]glibenclamide in the rat brain; this may correlate with the variability of effects of sulfonylureas observed from physiological experiments.

Galanin reduces release of endogenous excitatory amino acids in the rat hippocampus.

Galanin is a 29-amino acid peptide widely distributed in the central nervous system where it modulates the release of several neurotransmitters and neurohormones. Because previous data had postulated that galanin could inhibit the release of excitatory amino acids and protect hippocampal neurons against anoxia, we have investigated the effect of galanin on the release of endogenous glutamate and aspartate evoked by potassium depolarization in rat hippocampal slices. Galanin, added to a concentration of 0.3 microM, produced a 50-60% reduction in the release of endogenous glutamate and aspartate as evoked by 40 mM K+. This effect was concentration-dependent with half-maximal effective galanin concentrations (EC50) of 1.7 and 5.9 nM for glutamate and aspartate, respectively. Such an effect was found to occur preferentially in the ventral rather than in the dorsal region of the hippocampus. The inhibitory effect of galanin on the K(+)-evoked release of excitatory amino acids was reversed by the specific galanin antagonist M-15 (0.3 microM), and by the ATP-sensitive potassium channel blocker glibenclamide (10 microM). Furthermore, M-15 alone increased the basal and the K(+)-evoked release of glutamate and aspartate from hippocampal slices. It is concluded that galanin exerts a tonic inhibition of excitatory glutamate/aspartate neurotransmission in the rat ventral hippocampus. The efficacy of glibenclamide in antagonizing the effect of galanin suggests the involvement of ATP-sensitive or -insensitive potassium channels in such a regulation.

Autoradiographic study of the cellular localization of [3H]glibenclamide binding sites in the rat hippocampus.

[3H]Glibenclamide, a potent ATP-sensitive K+ channel blocker, is a specific ligand for the identification of the sulfonylureas receptors which are closely related to ATP-sensitive K+ channels. In order to determine the pre- or postsynaptic localization of these receptors in the rat hippocampus, we studied the effects of selective kainate or colchicine-induced lesions on the regional distribution of [3H]glibenclamide binding. A decreased binding was found in the following conditions: in CA3, after destruction of the mossy fiber terminals but not of the CA3 cells; in CA1, after destruction of the CA1 cells but not of the Schaffer collaterals; in the fascia dentata, after destruction of the granule cells. These results suggest that glibenclamide binding sites are mainly localized on the mossy fibers in CA3, and on the granular and pyramidal neurons in the fascia dentata and CA1, respectively.

Effect of potassium channel modulators on the release of glutamate induced by ischaemic-like conditions in rat hippocampal slices.

The effects of the potassium channel openers lemakalim, RP 52891 and galanin and the potassium channel blockers glibenclamide and gliquidone were evaluated by the release of endogenous glutamate from rat hippocampal slices subjected to a brief period of ischaemia (2-10 min). Ischaemia was mimicked by incubating slices in a glucose free medium equilibrated with 95% N2/5% CO2. These conditions evoked a release of glutamate which was insensitive to tetrodotoxin and Ca2+ indicating a non-vesicular origin. The release of glutamate evoked by a 6- or 8-min period of ischaemia was reduced by 25-40% in the presence of lemakalim (10 microM), RP 52891 (10 microM) or galanin (0.3 microM), whereas it was enhanced by 60 to 100% in the presence of glibenclamide (1 microM) and gliquidone (2 microM). These observations suggest that cellular damage resulting from ischaemia induced excessive release of glutamate in the hippocampus may be partly reduced by potassium channel openers, and conversely increased by sulfonylureas.

Bacterial-based additives for the production of artificial snow: what are the risks to human health?

For around two decades, artificial snow has been used by numerous winter sports resorts to ensure good snow cover at low altitude areas or more generally, to lengthen the skiing season. Biological additives derived from certain bacteria are regularly used to make artificial snow. However, the use of these additives has raised doubts concerning the potential impact on human health and the environment. In this context, the French health authorities have requested the French Agency for Environmental and Occupational Health Safety (Afsset) to assess the health risks resulting from the use of such additives. The health risk assessment was based on a review of the scientific literature, supplemented by professional consultations and expertise. Biological or chemical hazards from additives derived from the ice nucleation active bacterium Pseudomonas syringae were characterised. Potential health hazards to humans were considered in terms of infectious, toxic and allergenic capacities with respect to human populations liable to be exposed and the means of possible exposure. Taking into account these data, a qualitative risk assessment was carried out, according to four exposure scenarios, involving the different populations exposed, and the conditions and routes of exposure. It was concluded that certain health risks can exist for specific categories of professional workers (mainly snowmakers during additive mixing and dilution tank cleaning steps, with risks estimated to be negligible to low if workers comply with safety precautions). P. syringae does not present any pathogenic capacity to humans and that the level of its endotoxins found in artificial snow do not represent a danger beyond that of exposure to P. syringae endotoxins naturally present in snow. However, the risk of possible allergy in some particularly sensitive individuals cannot be excluded. Another important conclusion of this study concerns use of poor microbiological water quality to make artificial snow.

Characterization of sulfonylurea receptors and the action of potassium channel openers on cholinergic neurotransmission in guinea pig isolated small intestine.

Specific binding sites for [3H]glibenclamide, a potent ATP-sensitive K+ channel blocker, have been characterized in the isolated guinea pig longitudinal muscle-myenteric plexus preparation. The Scatchard plot of the saturation isotherm was curvilinear and revealed two binding sites, one of high affinity (Kd = 0.42 nM; maximum binding site = 156 fmol/mg of protein), the other of low affinity (Kd = 83 nM; maximum binding site = 3100 fmol/mg of protein). Displacement experiments in the presence of various sulfonylureas showed the same order of potency for the two binding sites (glibenclamide greater than gliquidone greater than glipizide glibornuride greater than chlorpropamide greater than tolbutamide). The K+ channel opener RP 49356 (but not diazoxide or cromakalim) displaced the [3H] glibenclamide with an IC50 of 4.8 microM. The effects of the K+ channel openers diazoxide, RP 49356, cromakalim and its two optical isomers BRL 38226 and BRL 38227 were also studied on the electrically induced contractions of isolated guinea pig small intestine. These compounds produce an inhibition of neurally evoked twitch height with pD2 values of 4.5 to 6.2 (BRL 38227 greater than cromakalim greater than RP 49356 greater than diazoxide, whereas BRL 38226 was practically ineffective). The effects of cromakalim and RP 49356 were antagonized competitively by glibenclamide (pA2 = 7.2) and other sulfonylureas, suggesting they act on ATP-sensitive K+ channels. Affinities of the sulfonylureas obtained from concentration-response curves to cromakalim on electrically induced contractions are better correlated with the IC50 value corresponding to the low affinity binding site than to the high affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleotides modulate the low affinity binding sites for [3H]glibenclamide in the rat brain.

Receptors for hypoglycemic sulfonylureas, such as glibenclamide, are commonly linked to the activity of ATP-sensitive K+ channels (K-ATP). High and low affinity binding sites for glibenclamide were described previously in numerous tissues. High affinity binding sites have been thought to be responsible of the modulation of K-ATP, but new evidences suggest that low affinity ones could also regulate these channels. In order to clarify the properties of the two binding sites, with respect to their interaction with K-ATP, we characterized biochemically and pharmacologically [3H]glibenclamide binding in the rat brain cortex. Competitive inhibition plots with [3H]glibenclamide performed on membranes of adult and neonatal rat brain cortex exhibited a biphasic pattern with similar binding parameters, indicating the presence of two similar binding sites in adult as well as in neonatal animals. Membranes of adult rat cortex treated with thiol groups modifying agents, N-ethylmaleimide or 1,4-dithiothreitol, increased the inhibition constant of glibenclamide for the low affinity binding sites (K(i)L) by about 4-fold. The divalent cations Mg++ and Ca++ also increased K(i)L by 3- to 6-fold and enhanced the low affinity binding capacity (BmaxL) by 55 and 103%, respectively, both cations increasing BmaxL by 144%. Among the numerous nucleotides studied, adenine and guanidine triphosphate nucleotides were the most potent to affect the low affinity binding sites. ATP, ADP, GTP and respective nonhydrolysable nucleotides increased K(i)L by 7- to 12-fold and decreased BmaxL by 10 to 30%. The effects of nucleotides were not Mg++ dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Dopaminergic regulation of enkephalin release.

The effects of dopamine receptor stimulation on enkephalin release were evaluated in vitro and in vivo by measuring the changes in the levels of [Met5]enkephalin (YGGFM) and Tyr-Gly-Gly (YGG), a characteristic extracellular enkephalin metabolite produced under the action of enkephalinase. In rat striatal slices, D1-receptor agonists or antagonists did not modify enkephalin release. By contrast, D2-receptor agonists enhanced the potassium-induced release of YGGFM and YGG without affecting spontaneous release from nondepolarized slices. This response was prevented by the D2-receptor antagonists haloperidol and RIV 2093, the latter compound being more potent, which suggested the involvement of a putative D2-receptor subtype. Acute administration of apomorphine or selective D2-receptor agonists, but not that of a D1-receptor agonist, enhanced the steady-state level of YGG without affecting the YGGFM level in rat striatum. The effect was blocked selectively by D2-receptor antagonists which, administered alone, had no effect. These observations indicate that D2-receptor stimulation in vitro or in vivo facilitates enkephalin release from striatal neurons, but that endogenous dopamine does not exert any tonic influence upon the opioid peptide neuron activity under basal conditions. However, chronic administration of haloperidol resulted in increases in striatal YGGFM and YGG, an effect presumably reflecting a long-term adaptive process.

Subcellular fractionation on Percoll gradient of mossy fiber synaptosomes: morphological and biochemical characterization in control and degranulated rat hippocampus.

A method for preparation of hippocampal mossy fiber synaptosomes directly from the postnuclear pellet is presented. This method represents an adaptation of that previously described for the isolation of synaptosomes by centrifugation through Percoll gradients directly from the supernatant fraction. We have characterized by electron microscopy two fractions, PII and PIII, enriched in mossy fiber synaptosomes; fraction PIII had 75% mossy fiber synaptosomes with well-preserved morphology (large size 3 microns, complex morphology, high synaptic vesicle density, multisynapses), whereas fraction PII contained 12%. These fractions were enriched in lactate dehydrogenase activity indicating that the integrity of synaptosomes was preserved. Compared with the other synaptosomal fractions, these fractions showed greater levels of dynorphin A (1-8) immunoreactivity and endogenous zinc, which are particularly concentrated in hippocampal mossy fiber terminals. Furthermore, we prepared synaptosomes from adult hippocampus after neonatal irradiation, which destroys the majority of granule cells and associated mossy fibers. The levels of dynorphin and zinc decreased by 88 and 70% in fraction PII and by 95 and 90%, respectively, in PIII. These results suggest that the rapid Percoll procedure is convenient for the purification of mossy fiber synaptosomes.

Identification of metabolic pathways of brain angiotensin II and III using specific aminopeptidase inhibitors: predominant role of angiotensin III in the control of vasopressin release.

Angiotensin (Ang) II and Ang III are two peptide effectors of the brain renin-angiotensin system that participate in the control of blood pressure and increase water consumption and vasopressin release. In an attempt to delineate the respective roles of these peptides in the regulation of vasopressin secretion, their metabolic pathways and their effects on vasopressin release were identified in vivo. For this purpose, we used recently developed selective inhibitors of aminopeptidase A (APA) and aminopeptidase N (APN), two enzymes that are believed to be responsible for the N-terminal cleavage of Ang II and Ang III, respectively. Mice received [3H]Ang II intracerebroventricularly (i.c.v.) in the presence or absence of the APN inhibitor, EC33 (3-amino-4-thio-butyl sulfonate) of the APN inhibitor, EC27 (2-amino-pentan-1,5-dithiol). [3H]Ang II and [3H]Ang III levels were evaluated from hypothalamus homogenates by HPLC. EC33 increased the half-life of [3H]Ang II 2.6-fold and completely blocked the formation of [3H]Ang III, whereas EC27 increased the half-life of [3H]Ang III 2.3-fold. In addition, the effects of EC33 and EC27 on Ang-induced vasopressin release were studied in mice. Ang II was injected i.c.v. in the presence or absence of EC33, and plasma vasopressin levels were estimated by RIA. While vasopressin levels were increased 2-fold by Ang II (5 ng), EC33 inhibited Ang II-induced vasopressin release in a dose-dependent manner. In contrast, EC27 injected alone increased in a dose-dependent manner vasopressin levels. The EC27-induced vasopressin release was completely blocked by the coadministration of the Ang receptor antagonist (Sar1-Ala8) Ang II. These results demonstrate for the first time that (i) APA and APN are involved in vivo in the metabolism of brain Ang II and Ang III, respectively, and that (ii) the action of Ang II on vasopressin release depends upon the prior conversion of Ang II to Ang III. This shows that Ang III behaves as one of the main effector peptides of the brain renin-angiotensin system in the control of vasopressin release.

Aminopeptidase A inhibitors as potential central antihypertensive agents.

Overactivity of the brain renin-angiotensin system (RAS) has been implicated in the development and maintenance of hypertension in several experimental models, such as spontaneously hypertensive rats and transgenic mice expressing both human renin and human angiotensinogen transgenes. We recently reported that, in the murine brain, angiotensin II (AngII) is converted to angiotensin III (AngIII) by aminopeptidase A (APA), whereas AngIII is inactivated by aminopeptidase N (APN). If injected into cerebral ventricles (ICV), AngII and AngIII cause similar pressor responses. Because AngII is metabolized in vivo into AngIII, the exact nature of the active peptide is not precisely determined. Here we report that, in rats, ICV injection of the selective APA inhibitor EC33 [(S)-3-amino-4-mercaptobutyl sulfonic acid] blocked the pressor response of exogenous AngII, suggesting that the conversion of AngII to AngIII is required to increase blood pressure (BP). Furthermore, ICV injection, but not i.v. injection, of EC33 alone caused a dose-dependent decrease in BP by blocking the formation of brain but not systemic AngIII. This is corroborated by the fact that the selective APN inhibitor, PC18 (2-amino-4-methylsulfonyl butane thiol), administered alone via the ICV route, increases BP. This pressor response was blocked by prior treatment with the angiotensin type 1 (AT(1)) receptor antagonist, losartan, showing that blocking the action of APN on AngIII metabolism leads to an increase in endogenous AngIII levels, resulting in BP increase, through interaction with AT(1) receptors. These data demonstrate that AngIII is a major effector peptide of the brain RAS, exerting tonic stimulatory control over BP. Thus, APA, the enzyme responsible for the formation of brain AngIII, represents a potential central therapeutic target that justifies the development of APA inhibitors as central antihypertensive agents.

Aminopeptidase A activity and angiotensin III effects on [Ca2+]i along the rat nephron.

BACKGROUND: This study examined the specific effects of angiotensin III (Ang III) along the nephron. METHODS: We examined the distribution of aminopeptidase A (APA) activity by using a specific APA inhibitor and by immunostaining with an antirat kidney APA antibody, the Ang III-induced variations of [Ca2+]i by using fura-2 and the characterization of the receptor subtype involved in the response to Ang III in cortical thick ascending limb (CTAL). RESULTS: APA activity was found all along the nephron but was higher in the cortex than in the medulla. This was confirmed by immunostaining. Increases in [Ca2+]i elicited by 10(-7) mol/liter Ang III were observed all along the nephron. The characterization of the receptor subtype involved in the [Ca2+]i response to Ang III in CTAL indicated that EC50 values for Ang III and Ang II were similar (13.5 and 10.3 nmol/liter, respectively), and Ang III-induced responses were totally abolished by AT1 receptor but not by AT2 receptor antagonists. There was a cross-desensitization of [Ca2+]i responses to 10(-7) mol/liter Ang III and Ang II, and the [Ca2+]i responses to 10(-7) mol/liter Ang II and Ang III were not additive. CONCLUSION: These results show that in CTAL, the [Ca2+]i responses to Ang II and Ang III occur through the same AT1a receptor because this subtype is predominant in this segment. Taken together, these data suggest that APA could be a key enzyme to generate Ang III from Ang II in the kidney.

PC18, a specific aminopeptidase N inhibitor, induces vasopressin release by increasing the half-life of brain angiotensin III.

Angiotensin III (AngIII), which is metabolized in vivo by aminopeptidase N (APN), was previously shown to be one of the main effector peptides of the brain renin-angiotensin system (RAS) in the control of vasopressin release. Recently, a potent APN inhibitor, PC18 (2-amino-4-methylsulfonyl butane thiol, methionine thiol), has been developed. In this study, we first checked the in vitro selectivity of PC18 towards APN, aminopeptidase A (APA) and aminopeptidase B (APB), three zinc metalloproteases with significant identity between their amino acid sequences. The Ki values of this compound on APN were found to be in the nanomolar range (Ki = 8.0 +/- 1.7 nM) but it was 2,150 and 125 times less active on APA and APB, respectively. Secondly, we evaluated in vivo the effect of brain APN inhibition with PC18 on the inactivation of brain AngIII and on vasopressin secretion in mice. For this purpose, mice received [3H]AngII intracerebroventricularly in the presence or absence of the APN inhibitor PC18 (30 microg). At different times after the injection, [3H]AngIII levels were evaluated from hypothalamus homogenates after separation by cation-exchange chromatography. PC18 induced an accumulation of [3H]AngIII, increasing its half-life 3.9 times as compared with control values. In addition, the effect of PC18 on vasopressin release was studied in mice. PC18 (10-100 microgram) was injected intracerebroventricularly, and plasma vasopressin levels were estimated by radioimmunoassay. PC18 increased vasopressin levels in a dose-dependent manner. The maximal increase in vasopressin release (+220%) is observed for a dose of PC18 of 100 microgram and was inhibited 75% by the coadministration of the AngII receptor antagonist (Sar1-Ala8)-AngII (0.5 microgram). These results indicate that in vivo, in the mouse brain, APN inhibition by PC18 increases the half-life of endogenous AngIII, resulting in an enhanced vasopressin release.