Effects of calcium and sodium on ATP-induced vasopressin release from rat isolated neurohypophysial terminals.

ATP-receptors (P2X2, P2X3, P2X4 & P2X7) are found in neurohypophysial terminals (NHT). These purinergic receptor subtypes are known to be cation selective. Here we confirm that both sodium (Na+ ) and calcium (Ca2+ ) are permeable through these NHT purinergic receptors, but to varying degrees (91% vs. 9%, respectively). Furthermore, extracellular calcium inhibits the ATP-current magnitude. Thus, the objective of this study was to determine the effects of extracellular Na+ vs. Ca2+ on ATP-induced vasopressin (AVP) release from populations of rat isolated NHT. ATP (200 µM) perfused exogenously for 2 minutes in Normal Locke's buffer caused an initial transient increase in AVP release followed by a sustained increase in AVP release which lasted for the duration of the ATP exposure. Replacing extracellular NaCl with NMDG-Cl had no apparent effect on the ATP-induced transient increase in AVP release but abolished the sustained AVP release induced by ATP. Furthermore, removal of extracellular calcium resulted in no ATP-induced transient increase in AVP release, but had no effect on the delayed, sustained increase in AVP release. The ATP-induced calcium-dependent transient increase in AVP release was >95% inhibited by 10 µM of the P2X purinergic receptor antagonist PPADS, a dose sufficient to block P2X2 and P2X3 receptors but not P2X4 or P2X7 receptors. Interestingly, the ATP-induced calcium-independent, sodium-dependent sustained increase in AVP release was not affected by 10 µM PPADS. The ATP-induced calcium-dependent transient increase in AVP release was not affected by the P2X7 receptor antagonist BBG (100 nM). However, the ATP-induced sodium-dependent sustained AVP release was inhibited by 50%. Therefore, these results show that rat isolated NHT exhibit a biphasic response to exogenous ATP that is differentially dependent on extracellular calcium and sodium. Furthermore, the initial transient release appears to be through P2X2 and/or P2X3 receptors and the sustained release is through a P2X7 receptor. This article is protected by copyright. All rights reserved.

Ecophysiological interactions and water-related physicochemical parameters among freshwater stingrays / Interações ecofisiológicas e dos parâmetros físico-químico da água em arraias de água doce

Abstract The objective of this study was to compare and correlate the ecology of neonates and young individuals of Potamotrygon wallacei, Potamotrygon motoro and Paratrygon aiereba with regard to their hematological profile and the physicochemical parameters of the water that they inhabit. Principal component analysis (PCA) on the complete blood count revealed total variation of 72.92%, thus demonstrating a differentiation system for oxygen demand. On the other hand, P. motoro was considered to be an intermediate species, given that its complete blood count characteristics interacted with both P. wallacei and with P. aiereba. The interaction among the biochemical variables was shown to total 64.67% of the factors. This allowed differentiation of P. wallacei from P. aiereba, while P. motoro maintained an intermediate position. These characteristics of differentiation within the preferred environment corroborate the PCA of the present study and confirm that these species can be differentiated through considering the complete blood count and biochemical parameters. The PCA on water properties showed 68.57% differentiation, mainly comprising the x axis (49.44%). It can be affirmed that P. motoro has the capacity to inhabit the preferential areas of P. wallacei and P. aiereba, as well as occupying localities in which other stingrays are not found. In conclusion, P. wallacei presents patterns differentiating it from P. aiereba, while P. motoro is a species that presents intermediate characteristics. The latter can be considered to be a more broadly distributed species regarding its ecophysiological characteristics.

Resumo Este trabalho tem por objetivo investigar o perfil hematológico e os parâmetros físico-químicos da água, comparando e correlacionando ecologicamente entre neonatos e jovens de Potamotrygon wallacei (arraia cururu), Potamotrygon motoro e Paratrygon aiereba. A análise de componentes principais (PCA) do hemograma revelou um total 72,92% de variação, constituindo-se em um sistema de diferenciação na demanda por oxigênio. P. wallacei apresenta diferenciação no eixo X quando comparada a P. aiereba, por outro lado P. motoro constitui-se como uma espécie intermediária que apresenta as características do hemograma interagindo tanto com P. wallacei quanto com P. aiereba. A interação entre as variáveis bioquímica demonstram um total de 64,67% dos fatores, no qual foi possível diferenciar, a arraia P. wallacei de P. aiereba, tendo P. motoro uma aspecto de espécie intermediária entre as demais. Esses aspectos de diferenciação de ambiente de preferência corroboraram a PCA obtida no presente estudo e confirmam que essas espécies podem ser diferenciadas quando se considerar as variáveis referentes ao hemograma e a bioquímica. Nos íons, no trombograma e no leucograma, não foi possível diferenciar as espécies. O PCA das propriedades da água foi constituído por 68,57% de diferenciação que se constituiu principalmente no eixo x (49,44%). É possível confirmar que P. motoro tem a capacidade de habitar as áreas preferências de P. wallacei e P. aiereba, além do mais esta possui uma localidade que as demais arraias não são encontradas. Conclui-se que P. wallacei, apresenta padrões diferenciados de P. aiereba, além do mais P. motoro é uma espécie que apresenta características intermediárias entre as descritas, o qual pode ser considerado uma espécie com distribuição mais ampla em seus aspectos ecofisiológicos.

Ecophysiological interactions and water-related physicochemical parameters among freshwater stingrays.

The objective of this study was to compare and correlate the ecology of neonates and young individuals of Potamotrygon wallacei, Potamotrygon motoro and Paratrygon aiereba with regard to their hematological profile and the physicochemical parameters of the water that they inhabit. Principal component analysis (PCA) on the complete blood count revealed total variation of 72.92%, thus demonstrating a differentiation system for oxygen demand. On the other hand, P. motoro was considered to be an intermediate species, given that its complete blood count characteristics interacted with both P. wallacei and with P. aiereba. The interaction among the biochemical variables was shown to total 64.67% of the factors. This allowed differentiation of P. wallacei from P. aiereba, while P. motoro maintained an intermediate position. These characteristics of differentiation within the preferred environment corroborate the PCA of the present study and confirm that these species can be differentiated through considering the complete blood count and biochemical parameters. The PCA on water properties showed 68.57% differentiation, mainly comprising the x axis (49.44%). It can be affirmed that P. motoro has the capacity to inhabit the preferential areas of P. wallacei and P. aiereba, as well as occupying localities in which other stingrays are not found. In conclusion, P. wallacei presents patterns differentiating it from P. aiereba, while P. motoro is a species that presents intermediate characteristics. The latter can be considered to be a more broadly distributed species regarding its ecophysiological characteristics.

Plantas medicinais conhecidas na zona urbana de Cajueiro da Praia, Piauí, Nordeste do Brasil / Known medicinal plants found in the urban Cajueiro da Praia urban area, Piauí state, Northeast Brazil

RESUMO A utilização de plantas com fins medicinais para tratamento, cura e prevenção de doenças é considerada uma das mais antigas formas de prática medicinal da humanidade. O presente estudo teve como objetivo identificar as espécies de plantas medicinais usadas dentro da comunidade de Cajueiro da Praia, Piauí, bem como a parte da planta utilizada, o modo de preparo, a sua importância relativa, o valor de uso e o consenso em relação às propriedades terapêuticas das espécies citadas. Foram aplicadas 12 entrevistas semiestruturadas com especialistas locais indicados, utilizando o método de amostragem por “bola-de-neve” e a técnica de “turnê-guiada” para coleta das espécies citadas. Análises quantitativas tais como Valor de Uso, Importância Relativa e Fator de Consenso dos Informantes foram associadas à análises qualitativas. As coletas botânicas seguiram a metodologia usual. Foram citadas 43 espécies, distribuídas em 24 famílias botânicas, sendo Lamiaceae a mais representada em número de espécies (oito espécies), seguida de Euphorbiaceae, Fabaceae, Myrtaceae e Rutaceae (todas com três). Chenopodium ambrosioides L. (mastruz) obteve o maior valor de uso (VU=1,58), sendo portanto, a planta com elevado potencial de uso para a comunidade. Quanto ao uso terapêutico, observou-se que um maior número de espécies foi indicado no combate à gripe, seguido por má digestão. O IR demonstrou que planta mais versátil foi Chenopodium ambrosioides L. (mastruz), com IR=2, referido em cinco categorias de uso e o FCI apontou que os sistemas corporais que apresentam maior importância local são: sistema circulatório (FCI=0,57), seguido por lesões e consequências de causas externas (FCI=0,50), sistema endócrino (FCI=0,50) e respiratório (FCI=0,40). No âmbito geral, verificou-se que a diversidade de plantas medicinais conhecida e a obtenção das plantas na comunidade estudada sugerem uma correlação entre uso/conhecimento de plantas medicinais e disponibilidade das mesmas; que a flora medicinal é representada, em boa parte, por plantas exóticas cultivadas nos quintais e que a transmissão do conhecimento tradicional feito localmente e por via oral demonstra uma herança cultural na cidade.

ABSTRACT The use of medicinal plants for the purposes of treating, curing and preventing diseases is considered one of the oldest forms of humanity’s medical practices. This study aimed to identify the species of medicinal plants used in the Cajueiro da Praia community, Piauí state, the part of the plant used, the method of preparation, their relative importance, the use value and the consensus on the therapeutic properties of the species mentioned. 12 semi-structured interviews with indicated local experts were performed, using the “snow-ball” sampling method and the “guided tour” technique to collect the aforementioned species by a standardized method. Quantitative analyzes such as Use value, relative importance and informant consensus factor were performed alongside qualitative analyzes. 43 species belonging to 24 botanical families were cited, Lamiaceae being the most represented in number of species (eight species) followed by Euphorbiaceae, Fabaceae, Myrtaceae and Rutaceae (with three each). Chenopodium ambrosioides L. (mastruz) had the highest use value (UV = 1.58), and therefore was the plant with the highest potential for use by the community. For therapeutic use, it was observed that a greater number of species have been shown to combat influenza, followed by maldigestion. The RI showed that the most versatile plant was Chenopodium ambrosioides L. (mastruz) with RI = 2, found in five usage categories and the ICF pointed out that the bodily systems that have greater local importance are: the circulatory system (ICF = 0.57), followed by lesions and consequences by external causes (ICF = 0.50), the endocrine (ICF = 0.50), and the respiratory system (ICF = 0.40). Within the general framework, it was found that the diversity of known medicinal plants and plants obtained from the population studied, suggested a correlation between the use/knowledge of medicinal plants and the availability there of; the medicinal flora is largely represented by exotic plants that are grown in backyards andby the transmission of traditional knowledge made locally and orally demonstrates a cultural heritage in the city.

Plantas medicinais conhecidas por especialistas locais de uma comunidade rural maranhense / Known medicinal plants by local experts in a rural community in Maranhão

RESUMO Este trabalho objetivou realizar o inventário das plantas medicinais de uma comunidade rural maranhense, analisando o valor de uso e a importância relativa das espécies, assim como o consenso dos informantes quanto às doenças tratadas. Foram realizadas 50 entrevistas semiestruturadas utilizando-se o método de amostragem “bola-de-neve” e usou-se a técnica “turnê-guiada” para coletar as espécies citadas. Os especialistas locais citaram 63 espécies pertencentes a 44 famílias, das quais Fabaceae, Apocynaceae, Lamiaceae e Myrtaceae apresentaram o maior número de espécies. A planta que obteve maior valor de uso foi Morinda citrifolia L.. Himatanthus drasticus (Müll.Arg.) Woodson foi a espécie que apresentou maior importância relativa. Dentre os sistemas corporais tratados com os recursos vegetais, os que obtiveram maior consenso entre os informantes foram os relacionados às doenças do estômago, esôfago e duodeno e infecções virais caracterizadas por lesões na pele (ambas com FCI = 0,83). Em virtude da riqueza de conhecimento sobre plantas medicinais em um Estado do Brasil onde o sistema de saúde é bastante precário e onde os estudos de cunho etnobotânico ainda são escassos, sugere-se que novos estudos etnobotânicos sejam realizados afim de evidenciar a dinâmica de conhecimento local e suas implicações ecológicas e sociais.

ABSTRACT This study aimed to conduct an inventory of medicinal plants in a rural community in Maranhão, analyzing the use value and the relative importance of species as well as the consensus of the informants about the treated diseases. Fifty semi-structured interviews were conducted using the sampling method “snow-ball” and the technique “guided tour” was employed in order to collect the species mentioned. Local experts mentioned 63 species belonging to 44 families, from which Fabaceae, Apocynaceae, Lamiaceae and Myrtaceae represented the highest number of species. The plant which had the highest use value was the Morinda citrifolia L.. The Himatanthus drasticus (Müll.Arg.) Woodson was the species with the highest relative importance. Among the body systems treated with plant resources, those who had the most consensus among the informants were related to stomach, esophagus and duodenum diseases and viral infections characterized by skin lesions (both ICF = 0.83). Because of the abundance of knowledge about medicinal plants in this state of Brazil, where the health system is quite precarious and where the nature of ethnobotanical studies are still scarce, it is suggested that new ethnobotanical studies are conducted in order to show the dynamics of local knowledge and its ecological and social implications.

THE INTAKE OF FIBER MESOCARP PASSIONFRUIT (PASSIFLORA EDULIS) LOWERS LEVELS OF TRIGLYCERIDE AND CHOLESTEROL DECREASING PRINCIPALLY INSULIN AND LEPTIN.

BACKGROUND: Diabetes mellitus (DM) is a major risk factor for coronary artery disease, renal failure, retinopathy, and neuropathy. Over the last years, there has been an increasing demand in folk medicine for natural sources that could help in the treatment of chronic diseases, including diabetes. The rind of passion fruit (Passiflora edulis f. Flavicarpa) is traditionally used as a functional food due to its high concentration of soluble and insoluble fiber. OBJECTIVE: The aim of this study was to determine the effect of high-fiber diet albedo of passion fruit on the metabolic and biochemical profile in diabetic rats induced by alloxan (2%). DESIGN: The passion fruit mesocarp fiber was dried in an oven with circulating air at 60°C and pulverized. We used 32 adult male rats, divided into 4 groups: Wistar group 1 control (GC), Wistar group 2, 15% fiber (GF15), Wistar group 3, 30% fiber (GF30), Wistar group 4, fiber disolved in water (GFH2O). The ratio of passion fruit was prepared according to the AIN 93M guidelines, varying only the source of dietary fiber. The corresponding diet for each group was offered to the animals for 60 days. RESULTS: There was a statically significant decrease in plasma glucose for GFH2O, GF15%, and GF30% groups with 27.0%, 37.4%, and 40.2%, respectively. CONCLUSION: The use of mesocarp fiber of passion fruit at concentrations of 15% and 30% are an important dietary supplement for the treatment of DM due to its potential hypoglycemic effect, and its ability to reduce triglycerides and VLDL-cholesterol levels with a principal reduction of insulin and leptin.

THE CONSUMPTION OF RED PUPUNHA (BACTRIS GASIPAES KUNTH) INCREASES HDL CHOLESTEROL AND REDUCES WEIGHT GAIN OF LACTATING AND POST-LACTATING WISTAR RATS.

INTRODUCTION: The lactating and post-lactating periods are marked by large metabolic change. Production of milk is 60% lipid dependent. We reported in a recent scientific meeting that Red pupunha palm tree fruit increases HDL cholesterol in lactating rats. This study evaluated if consumption of Red Pupunha by adult female rats has a beneficial impact on the lipid metabolism of lacting and post-lacting adult rats. OBJECTIVE: Evaluate if consumption of red pupunha has a beneficial effect in the lipid metabolism of lacting and post-lacting adult Wistar rats. RESEARCH METHODS: Four groups including two for control; (1) control adult lactating rats, (2) control adults post-lactating rats; and two experimental groups; (3) pupunha adults lactating rats and (4) pupunha adult post-lactating rats were evaluated and compared regarding: weight gain, food consumption, plasma total protein, glucose, total lipid, triglycerides, total cholesterol and HDL-cholesterol levels. The mean difference and its 95% confidence intervals were used for group comparisons. Group comparisons were evaluated by using analysis of variance (one-way ANOVA). The statistical significance of the pairwise differences among groups was assessed by using the two-sided Tukey test. RESULTS: There were no important differences in food consumption, plasma glucose, total lipids and triglycerides among groups. The red pupunha lactating group gain less weight showing lower body mass index (BMI) than controls (p < 0.05). Total cholesterol was lower in red pupunha lactating than in controls but not in the red pupunha post-lactating group as compared to controls. Triglycerides were lower in the post-lactating red pupunha group as compared to the control group (p = 0.039) but not for the lactating groups. Red pupunha lactating and post-lactating groups had higher HDL-cholesterol than their corresponding control groups (p ≤ 0.01). CONCLUSION: Original findings include the beneficial effect of red pupunha in post-lactating rats increasing the HDL-cholesterol and lowering the BMI. Red pupunha was confirmed to increase HDL-cholesterol in lactating rats. These results suggest that red pupunha is a healthy fruit to be consumed during lactating and post-lactating periods as it is related to better lipid profile and less body weight gain.

Adenosine trisphosphate appears to act via different receptors in terminals versus somata of the hypothalamic neurohypophysial system.

ATP-induced ionic currents were investigated in isolated terminals and somata of the hypothalamic neurohypophysial system (HNS). Both terminals and somata showed inward rectification of the ATP-induced currents and reversal near 0 mV. In terminals, ATP dose-dependently evoked an inactivating, inward current. However, in hypothalamic somata, ATP evoked a very slowly inactivating, inward current with a higher density, and different dose dependence (EC(50) of 50 µm in somata versus 9.6 µm in terminals). The ATP-induced currents, in both the HNS terminals and somata, were highly and reversibly inhibited by suramin, suggesting the involvement of a purinergic receptor (P2XR). However, the suramin inhibition was significantly different in the two HNS compartments (IC(50) of 3.6 µm in somata versus 11.6 µm in terminals). Also, both HNS compartments show significantly different responses to the purinergic receptor agonists: ATP-γ-S and benzoyl-benzoyl-ATP. Finally, there was an initial desensitisation to ATP upon successive stimulations in the terminals, which was not observed in the somata. These differences in EC(50) , inactivation, desensitisation and agonist sensitivity in terminals versus somata indicate that different P2X receptors mediate the responses in these two compartments of HNS neurones. Previous work has revealed mRNA transcripts for multiple purinergic receptors in micropunches of the hypothalamus. In the HNS terminals, the P2X purinergic receptor types P2X2, 3, 4 and 7 (but not 6) have been shown to exist in AVP terminals. Immonohistochemistry now indicates that P2X4R is only present in AVP terminals and that the P2X7R is found in both AVP and oxytocin terminals and somata. We speculate that these differences in receptor types reflects the specific function of endogenous ATP in the terminals versus somata of these central nervous system neurones.

P2X purinergic receptor knockout mice reveal endogenous ATP modulation of both vasopressin and oxytocin release from the intact neurohypophysis.

Bursts of action potentials are crucial for neuropeptide release from the hypothalamic neurohypophysial system (HNS). The biophysical properties of the ion channels involved in the release of these neuropeptides, however, cannot explain the efficacy of such bursting patterns on secretion. We have previously shown that ATP, acting via P2X receptors, potentiates only vasopressin (AVP) release from HNS terminals, whereas its metabolite adenosine, via A1 receptors acting on transient Ca(2+) currents, inhibits both AVP and oxytocin (OT) secretion. Thus, purinergic feedback-mechanisms have been proposed to explain bursting efficacy at HNS terminals. Therefore, in the present study, we have used specific P2X receptor knockout (rKO) mice and purportedly selective P2X receptor antagonists to determine the P2X receptor subtype responsible for endogenous ATP induced potentiation of electrically-stimulated neuropeptide release. Intact neurohypophyses (NH) from wild-type (WT), P2X3 rKO, P2X2/3 rKO and P2X7 rKO mice were electrically stimulated with four 25-s bursts (3 V at 39 Hz) separated by 21-s interburst intervals with or without the P2X2 and P2X3 receptor antagonists, suramin or pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). These frequencies, number of bursts, and voltages were determined to maximise both AVP and OT release by electrical stimulations. Treatment of WT mouse NH with suramin/PPADS significantly reduced electrically-stimulated AVP release. A similar inhibition by suramin was observed in electrically-stimulated NH from P2X3 and P2X7 rKO mice but not P2X2/3 rKO mice, indicating that endogenous ATP facilitation of electrically-stimulated AVP release is mediated primarily by the activation of the P2X2 receptor. Unexpectedly, electrically-stimulated OT release from WT, P2X3, P2X2/3 and P2X7 rKO mice was potentiated by suramin, indicating nonpurinergic effects by this 'selective' antagonist. Nevertheless, these results show that sufficient endogenous ATP is released by bursts of action potentials to act at P2X2 receptors in a positive-feedback mechanism to 'differentially' modulate neuropeptide release from central nervous system terminals.

Micro-opioid receptor preferentially inhibits oxytocin release from neurohypophysial terminals by blocking R-type Ca2+ channels.

Oxytocin release from neurophypophysial terminals is particularly sensitive to inhibition by the micro-opioid receptor agonist, DAMGO. Because the R-type component of the neurophypophysial terminal Ca2+ current (ICa) mediates exclusively oxytocin release, we hypothesised that micro-opioids could preferentially inhibit oxytocin release by blocking this channel subtype. Whole-terminal recordings showed that DAMGO and the R-type selective blocker SNX-482 inhibit a similar ICa component. Measurements of [Ca2+]i levels and oxytocin release confirmed that the effects of DAMGO and SNX-482 are not additive. Finally, isolation of the R-type component and its associated rise in [Ca2+]i and oxytocin release allowed us to demonstrate the selective inhibition by DAMGO of this channel subtype. Thus, micro-opioid agonists modulate specifically oxytocin release in neurophypophysial terminals by selectively targeting R-type Ca2+ channels. Modulation of Ca2+ channel subtypes could be a general mechanism for drugs of abuse to regulate the release of specific neurotransmitters at central nervous system synapses.

mu-Opioid receptor modulates peptide release from rat neurohypophysial terminals by inhibiting Ca(2+) influx.

The activation of opioid receptors in neurones of the central nervous system leads to a variety of effects including the modulation of diuresis and parturition, processes that are directly controlled by the hypothalamic-neurohypophysial system (HNS). The effects of mu-opioid receptor activation on peptide release, voltage-gated Ca2+ currents and intracellular calcium levels ([Ca2+]i) were studied in isolated nerve terminals of the HNS. The mu-receptor agonist, DAMGO ([d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin) inhibited high K+-induced peptide release in a dose-dependent manner, with oxytocin release being more sensitive to block than vasopressin release at all concentrations tested. The addition of the mu-receptor antagonist CTOP (d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr amide) was able to overcome the inhibitory effects of DAMGO. By contrast to previous results, voltage-gated Ca2+ currents were sensitive to blockage by DAMGO and this inhibition was also prevented by CTOP. Furthermore, [Ca2+]i measurements with Fura-2 corroborated the inhibition by DAMGO of calcium entry and its reversal by the micro -receptor antagonist in these nerve terminals. Thus, the decrease in neuropeptide release, particularly for oxytocin, induced by the activation of mu-opioid receptors in neurohypophysial terminals is mediated, at least in part, by a corresponding decrease in Ca2+ entry due to the inhibition of voltage-gated Ca2+ channels.

Trp2 regulates entry of Ca2+ into mouse sperm triggered by egg ZP3.

In many cells, receptor activation initiates sustained Ca2+ entry which is critical in signal transduction. Mammalian transient receptor potential (Trp) proteins, which are homologous to the Drosophila photoreceptor-cell Trp protein, have emerged as candidate subunits of the ion channels that mediate this influx. As a consequence of overexpression, these proteins produce cation currents that open either after depletion of internal Ca2+ stores or through receptor activation. However, determining the role of endogenous Trp proteins in signal transduction is complicated by the absence of selective antagonists. Here we examine Trp function during sperm-egg interaction. The sperm acrosome reaction is a Ca2+-dependent secretory event that must be completed before fertilization. In mammals, exocytosis is triggered during gamete contact by ZP3, a glycoprotein constituent of the egg's extracellular matrix, or zona pellucida (ZP). ZP3 activates trimeric G proteins and phospholipase C and causes a transient Ca2+ influx into sperm through T-type Ca2+ channels. These early responses promote a second Ca2+-entry pathway, thereby producing sustained increases in intracellular Ca2+ concentration ([Ca2+]i) that drive acrosome reactions. Our results show that Trp2 is essential for the activation of sustained Ca2+ influx into sperm by ZP3.

Tolerance to acute ethanol inhibition of peptide hormone release in the isolated neurohypophysis.

BACKGROUND: Acute ethanol (EtOH) exposure reduces the evoked release of vasopressin (AVP) and oxytocin (OT) from excised neurohypophyses and from dissociated neurohypophysial terminals of the rat. METHODS AND RESULTS: Rats placed on a diet that maintained blood levels of 30 mM EtOH for 20 to 40 days developed tolerance to acute EtOH inhibition of release. In the presence of 10 mM EtOH, high (50 mM) K+-induced release of AVP from isolated neurohypophysial terminals of EtOH-naive rats was reduced by 77.7+/-1.4%, whereas in the chronic EtOH group, release was reduced by only 9.4+/-8.7%. Similar tolerance was evident during acute challenge with 75 mM EtOH, as well as for release of OT from isolated terminals. Animals treated with an intraperitoneal injection of EtOH and sacrificed 90 min postinjection did not exhibit the reduced EtOH inhibition of release from dissociated terminals during a 75 mM EtOH acute challenge. CONCLUSIONS: The altered component responsible for the tolerance to inhibition of release resides in the isolated terminal, because tolerance measured in vitro from intact neurohypophyses was similar to that seen in isolated terminals. The failure of EtOH-injected animals to exhibit reduced inhibition of release in response to an acute EtOH challenge indicates that short-term elevated blood alcohol level does not induce this tolerance. The finding of tolerance to EtOH-induced inhibition of release from the intact neurohypophysis and isolated terminals provides a physiological preparation in which to examine the molecular targets of acute drug action modified after chronic exposure to the drug.

Excitatory versus inhibitory modulation by ATP of neurohypophysial terminal activity in the rat.

Much is now known about the electrophysiological properties of the magnocellular neurones of the hypothalamus. Oxytocin neurones are characterized by an intermittent high frequency discharge during suckling that leads to the pulsatile release of oxytocin into the blood and to subsequent milk ejection. Vasopressin neurones are characterized by their asynchronous phasic activity (bursting) during maintained vasopressin release and the subsequent regulation of water balance. In both cases, it is the clustering of spikes, albeit with different time courses for each peptide, that facilitates hormone release. The mechanism underlying this differential facilitation is one of the major unanswered questions in neuroendocrinology. This paper considers recent evidence that indicates that ATP, co-secreted with vasopressin and oxytocin, may play a key role in the regulation of stimulus-secretion coupling in the neurohypophysis. The activity of the type (II) Ca2+-activated K+ (K(Ca)) channel found in the nerve terminals was significantly increased in the presence of ATP on the cytoplasmic side of the channel. Extracellular ATP, in contrast, inhibited the type II K(Ca) current in a dose-dependent manner. Thus, intracellular and extracellular ATP exert opposite effects on the type II K(Ca) channel of neurohypophysial terminals. Furthermore, ATP opens P2X2 channels to increase intracellular [Ca2+] in the nerve terminals and subsequent arginine vasopressin (AVP) release. In contrast, adenosine, acting via A1 receptors, specifically inhibits only the N-type Ca2+ channel, thus decreasing neuropeptide release. These multiple, conflicting effects of ATP and its metabolite adenosine could explain the patterns of AVP release observed during physiological stimulation in vivo.

An R-type Ca(2+) current in neurohypophysial terminals preferentially regulates oxytocin secretion.

Multiple types of voltage-dependent Ca(2+) channels are involved in the regulation of neurotransmitter release (Tsien et al., 1991; Dunlap et al., 1995). In the nerve terminals of the neurohypophysis, the roles of L-, N-, and P/Q-type Ca(2+) channels in neuropeptide release have been identified previously (Wang et al., 1997a). Although the L- and N-type Ca(2+) currents play equivalent roles in both vasopressin and oxytocin release, the P/Q-type Ca(2+) current only regulates vasopressin release. An oxytocin-release and Ca(2+) current component is resistant to the L-, N-, and P/Q-type Ca(2+) channel blockers but is inhibited by Ni(2+). A new polypeptide toxin, SNX-482, which is a specific alpha(1E)-type Ca(2+) channel blocker (Newcomb et al., 1998), was used to characterize the biophysical properties of this resistant Ca(2+) current component and its role in neuropeptide release. This resistant component was dose dependently inhibited by SNX-482, with an IC(50) of 4.1 nM. Furthermore, SNX-482 did not affect the other Ca(2+) current types in these CNS terminals. Like the N- and P/Q-type Ca(2+) currents, this SNX-482-sensitive transient Ca(2+) current is high-threshold activated and shows moderate steady-state inactivation. At the same concentrations, SNX-482 blocked the component of oxytocin, but not of vasopressin, release that was resistant to the other channel blockers, indicating a preferential role for this type of Ca(2+) current in oxytocin release from neurohypophysial terminals. Our results suggest that an alpha(1E) or "R"-type Ca(2+) channel exists in oxytocinergic nerve terminals and, thus, functions in controlling only oxytocin release from the rat neurohypophysis.

Rat supraoptic magnocellular neurones show distinct large conductance, Ca2+-activated K+ channel subtypes in cell bodies versus nerve endings.

1. Large conductance, Ca2+-activated K+ (BK) channels were identified in freshly dissociated rat supraoptic neurones using patch clamp techniques. 2. The single channel conductance of cell body BK channels, recorded from inside-out patches in symmetric 145 mM K+, was 246.1 pS, compared with 213 pS in nerve ending BK channels (P<0.01). 3. At low open probability (Po), the reciprocal of the slope in the ln(NPo)-voltage relationship (N, number of available channels in the patch) for cell body and nerve ending channels were similar: 11 vs. 14 mV per e-fold change in NPo, respectively. 4. At 40 mV, the [Ca2+]i producing half-maximal activation was 273 nM, as opposed to >1.53 microM for the neurohypophysial channel, indicating the higher Ca2+ sensitivity of the cell body isochannel. 5. Cell body BK channels showed fast kinetics (open time constant, 8.5 ms; fast closed time constant, 1.6 and slow closed time constant, 12.7 ms), identifying them as 'type I' isochannels, as opposed to the slow gating (type II) of neurohypophysial BK channels. 6. Cell body BK activity was reduced by 10 nM charybdotoxin (NPo, 37% of control), or 10 nM iberiotoxin (NPo, 5% of control), whereas neurohypophysial BK channels are insensitive to charybdotoxin at concentrations as high as 360 nM. 7. Whilst blockade of nerve ending BK channels markedly slowed the repolarization of evoked single spikes, blockade of cell body channels was without effect on repolarization of evoked single spikes. 8. Ethanol reversibly increased neurohypophysial BK channel activity (EC50, 22 mM; maximal effect, 100 mM). In contrast, ethanol (up to 100 mM) failed to increase cell body BK channel activity. 9. In conclusion, we have characterized BK channels in supraoptic neuronal cell bodies, and demonstrated that they display different electrophysiological and pharmacological properties from their counterparts in the nerve endings.

Ethanol potentiation of calcium-activated potassium channels reconstituted into planar lipid bilayers.

We examined the actions of ethanol on the single channel properties of large conductance Ca2+-activated K+ (BK) channels isolated from skeletal muscle T-tubule membranes and incorporated into planar lipid bilayer membranes. We have taken advantage of this preparation, because it lacks most elements of cellular complexity, including cytoplasmic constituents and complex membrane lipid composition and architecture, to examine the minimum requirements for the effects of alcohol. Clinically relevant concentrations (25-200 mM) of ethanol increased the activity of BK channels incorporated into bilayers composed of phosphatidylethanolamine (PE) alone or PE and phosphatidylserine. The potentiation of channel activity by ethanol was attributable predominantly to a decrease in the average amount of time spent in closed states. Ethanol did not significantly affect the current amplitude-voltage relationship for BK channels, indicating that channel conductance for K+ was unaffected by the drug. Although base-line characteristics of BK channels incorporated into bilayers composed only of PE differed from those of channels in PE/ phosphatidylserine in a manner expected from the change in bilayer charges, the actions of ethanol on channel activity were qualitatively similar in the different lipid environments. The effects of ethanol on single channel properties of BK channels in the planar bilayer are very similar to those reported for the action of ethanol on neurohypophysial BK channels studied in native membrane, and for cloned BK channels expressed in Xenopus laevis oocytes, which suggests that ethanol's site and mechanism of action are preserved in this greatly simplified preparation.

Ethanol reduces the duration of single evoked spikes by a selective inhibition of voltage-gated calcium currents in acutely dissociated supraoptic neurons of the rat.

The effects of ethanol were studied on single evoked spikes recorded at 20 degrees C with the perforated-patch method in acutely dissociated rat supraoptic neurons. In seven out of eight neurons, ethanol (50 mM) significantly reduced the spike duration by selectively decreasing the decay time (82+/-2% of the control), leaving the amplitude and rise time unaffected. Resting potential and threshold did not change. Similarly, CdCl2 at a concentration of 100 microM, which blocks all voltage-activated calcium current in the supraoptic neurons, reduced the decay time of single evoked spikes (76+/-3% of the control, n=10) without modifying the other above-mentioned parameters. In addition, exposure to 100 microM CdCl2 prevented any subsequent effect of 50 mM ethanol (n = 5). Exposure to apamin (10 nM) and iberiotoxin (10 nM) did not have any effect on single evoked spikes. Because these concentrations are effective in blocking, respectively, small (SK) and large (BK) conductance calcium-dependent potassium channels in these neurons, this result shows that these currents are not involved in either the shaping of single evoked spikes or the actions of ethanol on spike shape. The sustained component of whole-cell recorded calcium current measured at -10 mV (hp -60 mV) was inhibited by ethanol in a dose-dependent manner, with a significant effect detectable at 25 mM. Exposure to 50 mM ethanol significantly reduced the sustained current to 70+/-5% of the control (n=12), without any apparent shift of the current-voltage relationship. Control exposure of the neurons to either 50 mM urea or 50 mM sucrose did not affect the voltage-gated calcium currents. We conclude that ethanol reduces the duration of single evoked spikes by a specific inhibition of voltage-activated calcium currents. The results suggest that, in addition to its direct effects on release of vasopressin and oxytocin from neurohypophysial terminals, ethanol could also affect hormonal release via changes in firing patterns arising in the cell bodies.