Comparative Passivation of Si(100) by H2 and D2 Atmospheres under Simultaneous Xe+ Bombardment: An X-ray Photoelectron Spectroscopy Analysis.

This study presents a comparison of H2 and D2 passivation on Si(100) under simultaneous Xe+ ion bombardment. The impact of Xe+ ions causes significant damage to the substrate surface, leading to an increase in H2 (D2) retention as Si-H (Si-D) bonds. The ion bombardment conditions are precisely controlled using a Kaufman ion gun. The atomic concentrations on the surface of the sample were investigated by quasi-in situ X-ray photoelectron spectroscopy. A simple methodology is employed to estimate the H (D) chemical concentration and the cover ratio of the sample, with regard to the oxygen concentration through residual water chemisorption present in the vacuum vessel. Differences in passivation are expected when using H2 or D2 atmospheres because their retained scission energies and physisorption properties differ. The results indicate an increase of the sticking coefficient for D2 and H2 under the ion bombardment. It is also found that the flux of H2 (D2) impinging on the surface contributes to play an important role in the whole process. Finally, a model is proposed to describe the phenomenon of the passivation of Si under Xe+ ion bombardment in the presence of H2 (D2).

Purinergic P2 and glutamate NMDA receptor coupling contributes to osmotically driven excitability in hypothalamic magnocellular neurosecretory neurons.

KEY POINTS: Purinergic and glutamatergic signalling pathways play a key role in regulating the activity of hypothalamic magnocellular neurosecretory neurons (MNNs). However, the precise cellular mechanisms by which ATP and glutamate act in concert to regulate osmotically driven MNN neuronal excitability remains unknown. Here, we report that ATP acts on purinergic P2 receptors in MNNs to potentiate in a Ca2+ -dependent manner extrasynaptic NMDAR function. The P2-NMDAR coupling is engaged in response to an acute hyperosmotic stimulation, contributing to osmotically driven firing activity in MNNs. These results help us to better understand the precise mechanisms contributing to the osmotic regulation of firing activity and hormone release from MNNs. ABSTRACT: The firing activity of hypothalamic magnocellular neurosecretory neurons (MNNs) located in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) is coordinated by the combined, fine-tuned action of intrinsic membrane properties, synaptic and extrasynaptic signalling. Among these, purinergic and glutamatergic signalling pathways have been shown to play a key role regulating the activity of MNNs. However, the precise cellular mechanisms by which ATP and glutamate act in concert to regulate osmotically driven MNN neuronal excitability remains unknown. Whole-cell patch-clamp recordings obtained from MNNs showed that ATP (100 µM) induced an increase in firing rate, an effect that was blocked by either 4-[[4-formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]2-pyridinyl]azo]1,3-benzenedisulfonic acid tetrasodium salt (PPADS) (10 µM) or kynurenic acid (1 mm). While ATP did not affect the frequency or magnitude of glutamatergic excitatory postsynaptic currents (EPSCs), it induced an inward shift in the holding current that was prevented by PPADS or kynurenic acid treatment, suggesting that ATP enhances a tonic extrasynaptic glutamatergic excitatory current. We observed that ATP-potentiated glutamatergic receptor-mediated currents were evoked by focal application of L-glu (1 mm) and NMDA (50 µM), but not AMPA (50 µM). ATP potentiation of NMDA-evoked currents was blocked by PPADS (10 µM) and by chelation of intracellular Ca2+ with BAPTA (10 mm). Finally, we report that a hyperosmotic stimulus (mannitol 1%, +55 mOsm/kgH2 O) potentiated NMDA-evoked currents and increased MNN firing activity, effects that were blocked by PPADS. Taken together, our data support a functional excitatory coupling between P2 and extrasynaptic NMDA receptors in MNNs, which is engaged in response to an acute hyperosmotic stimulus.

Small low-risk pulmonary nodules on chest digital radiography: can we predict whether the nodule is benign?

AIM: To evaluate digital chest radiography (CR) performance compared to computed tomography (CT) for characterising small low-risk pulmonary nodules detected incidentally in non-oncological patients. A second aim was to assess the prevalence of calcification and possible false-positive findings mimicking nodules. MATERIALS AND METHODS: Two hundred and seven patients who presented with a pulmonary nodule on CR and underwent CT were included prospectively. Nine radiologists blinded to the CT images reviewed the CRs assessing for the presence of nodules. Afterwards, the same radiologists evaluated the corresponding CT for the presence of nodules, dimensions, and calcification. If the nodule was not present on CT, it was considered a false-positive finding, and possible confounding factors on CR were investigated. RESULTS: Among all 213 nodules seen on CR, 32.4% were revealed to be false-positive findings on CT, mostly due to images formed by vessels (53.6%), osseous aetiologies (30.4%), and skin lesions (13%). Most nodules <6 mm detected on radiographies had benign calcification on CT (n=90; 67.7%). Comparatively, only 41.2% of nodules ≥6 mm on the CR had benign calcification. Among all nodules <6 mm detected on CR, 95.5% were calcified or not present at CT against 81.2% for those ≥6 mm (p<0.001). CONCLUSION: The present study demonstrated that 95.5% of pulmonary nodules smaller than 6 mm on CRs are either calcified (benign) or represent a false-positive finding on CT. These results suggest that nodule measures on CR smaller than 6 mm most likely represent a benign finding.

Purinergic P2 receptors in the paraventricular nucleus of the hypothalamus are involved in hyperosmotic-induced sympathoexcitation.

Increases in plasma osmolality activates the paraventricular nucleus of the hypothalamus (PVN) which in turn mounts a physiological response by increasing the release of arginine vasopressin and sympathetic nerve activity to end organs such as the kidney. The PVN expresses an abundance of purinergic receptors including P2X2 receptors. In the present study, we sought to determine (1) whether P2X2-expressing PVN neurons are activated by hypertonic saline or hypertonic mannitol and (2) what effects P2X receptor blockade has on sympathetic nerve activation mediated by a hyperosmotic stimulus. Male Wistar rats were randomly assigned to three groups and intravenously infused with either isotonic saline (0.154M, 0.5mL), hypertonic saline (3M, 0.5mL) or hypertonic mannitol (10% w/v, 0.5mL). Significantly greater numbers of Fos-positive cells were observed in the hypertonic saline (393±29)- and hypertonic mannitol (141±11)-infused rats compared with control, saline-treated, rats (47±2 neurons/PVN section). Furthermore, there was a significant increase in the number of activated (Fos-positive) P2X2 expressing PVN neurons in the hypertonic saline (65±7) and hypertonic mannitol (37±7)-treated rats compared with controls (16±2). Microinjection of a P2X receptor antagonist, PPADS, within the PVN significantly attenuated sympathetic nerve activation driven by a hyperosmotic stimulus. The hyperosmotically induced increase in lumbar sympathetic nerve activity was significantly blunted after PPADS pre-treatment. Collectively, our findings indicate that hyperosmotic stimulation activates a subset of P2X2 expressing PVN neurons that might facilitate increased sympathetic drive.

Angiotensin II acting on PVN induces sympathoexcitation and pressor responses via the PI3K-dependent pathway.

In vitro studies have shown that angiotensin II (ANG II), via activation of ANG II type 1 (AT1) receptor plays an important role on the neural control of the blood pressure (BP), through an intracellular signalling pathway involving PI3K in the paraventricular nucleus of the hypothalamus (PVN). However, to the best of our knowledge, no in vivo study has been performed yet to unravel the functional role of ANG II and its interaction with PI3K pathways in the neural control of circulation of non-anesthetized animals. Here, we demonstrate that exogenous ANG II microinjected into the PVN in anaesthetic-free animals evokes an increase in sympathetic nerve activity and BP in a PI3K-dependent manner.

Thyroid hormone reduces inflammatory cytokines improving glycaemia control in alloxan-induced diabetic wistar rats.

AIM: This study aimed at evaluating whether thyroid hormone treatment could improve glycaemia and insulin response in alloxan-induced diabetic rats by altering cytokine expression in the skeletal muscle and epididymal white adipose tissue (eWAT) as well as altering inflammatory cell infiltration in eWAT. METHODS: Diabetes mellitus (DM) was induced in male Wistar rats by alloxan injection, and a subset of the diabetic rats was treated with T3 (1.5 µg per 100 g body weight) for a 28-day period (DT3 ). Cytokines were measured in serum (MILIplex assay kit) as well as in soleus and EDL skeletal muscles and eWAT by Western blotting. Thyroid function was evaluated by morphological, molecular and biochemical parameters. Cardiac function was assessed by measuring heart rate, blood pressure, maximal rate of pressure development (dp/dtmax ) and decline (dp/dtmin ) as well as the contractility index (CI). Sixty rats were used in the study. RESULTS: Diabetic rats exhibited decreased thyroid function and increased inflammatory cytokines in serum, soleus muscle and eWAT. T3 treatment decreased glycaemia and improved insulin sensitivity in diabetic animals. These alterations were accompanied by decreased TNF-alpha and IL-6 content in soleus muscle and eWAT, and inflammatory cell infiltration in eWAT. T3 treatment did not affect cardiac function of diabetic rats. CONCLUSIONS: The present data provide evidence that T3 treatment reduces glycaemia and improves insulin sensitivity in diabetic rats, and that at least part of this effect could result from its negative modulation of inflammatory cytokine expression.

The cardiovascular actions of fractalkine/CX3CL1 in the hypothalamic paraventricular nucleus are attenuated in rats with heart failure.

The paraventricular nucleus (PVN) of the hypothalamus plays an important role in the regulation of sympathetic nerve activity, which is significantly elevated in chronic heart failure (CHF). Fractalkine (FKN) and its cognate receptor, CX3CR1, are constitutively expressed in the central nervous system, but their role and physiological significance are not well known. The aims of the present study were to determine whether FKN plays a cardiovascular role within the PVN and to investigate how the actions of FKN might be altered in CHF. We show that both FKN and CX3CR1 are expressed on neurons in the PVN of rats, suggesting that they may have a physiological function in this brain nucleus. Unilateral microinjection of FKN directly into the PVN of anaesthetized rats elicited a significant dose-related decrease in blood pressure (1.0 nmol, -5 ± 3 mmHg; 2.5 nmol, -13 ± 2 mmHg; 5.0 nmol, -22 ± 3 mmHg; and 7.5 nmol, -32 ± 3 mmHg) and a concomitant increase in heart rate (1.0 nmol, 6 ± 3 beats min(-1); 2.5 nmol, 11 ± 3 beats min(-1); 5 nmol, 18 ± 4 beats min(-1); and 7.5 nmol, 27 ± 5 beats min(-1)) compared with control saline microinjections. In order to determine whether FKN signalling is altered in rats with CHF, we first performed quantitative RT-PCR and Western blot analysis and followed these experiments with functional studies in rats with CHF and sham-operated control rats. We found a significant increase in CX3CR1 mRNA and protein expression, as determined by quantitative RT-PCR and Western blot analysis, respectively, in the PVN of rats with CHF compared with sham-operated control rats. We also found that the blood pressure effects of FKN (2.5 nmol in 50 nl) were significantly attenuated in rats with CHF (change in mean arterial pressure, -6 ± 3 mmHg) compared with sham-operated control rats (change in mean arterial pressure, -16 ± 6 mmHg). These data suggest that FKN and its receptor, CX3CR1, modulate cardiovascular function at the level of the PVN and that the actions of FKN within this nucleus are altered in heart failure.

Commissural nucleus of the solitary tract regulates the antihypertensive effects elicited by moxonidine.

The rostral ventrolateral medulla (RVLM) contains the presympathetic neurons involved in cardiovascular regulation that has been implicated as one of the most important central sites for the antihypertensive action of moxonidine (an α2-adrenergic and imidazoline agonist). Here, we sought to evaluate the cardiovascular effects produced by moxonidine injected into another important brainstem site, the commissural nucleus of the solitary tract (commNTS). Mean arterial pressure (MAP), heart rate (HR), splanchnic sympathetic nerve activity (sSNA) and activity of putative sympathoexcitatory vasomotor neurons of the RVLM were recorded in conscious or urethane-anesthetized, and artificial ventilated male Wistar rats. In conscious or anesthetized rats, moxonidine (2.5 and 5 nmol/50 nl) injected into the commNTS reduced MAP, HR and sSNA. The injection of moxonidine into the commNTS also elicited a reduction of 28% in the activity of sympathoexcitatory vasomotor neurons of the RVLM. To further assess the notion that moxonidine could act in another brainstem area to elicit the antihypertensive effects, a group with electrolytic lesions of the commNTS or sham and with stainless steel guide-cannulas implanted into the 4th V were used. In the sham group, moxonidine (20 nmol/1 µl) injected into 4th V decreased MAP and HR. The hypotension but not the bradycardia produced by moxonidine into the 4th V was reduced in acute (1 day) commNTS-lesioned rats. These data suggest that moxonidine can certainly act in other brainstem regions, such as commNTS to produce its beneficial therapeutic effects, such as hypotension and reduction in sympathetic nerve activity.

Purinergic and glutamatergic interactions in the hypothalamic paraventricular nucleus modulate sympathetic outflow.

P2X receptors are expressed on ventrolateral medulla projecting paraventricular nucleus (PVN) neurons. Here, we investigate the role of adenosine 5'-triphosphate (ATP) in modulating sympathetic nerve activity (SNA) at the level of the PVN. We used an in situ arterially perfused rat preparation to determine the effect of P2 receptor activation and the putative interaction between purinergic and glutamatergic neurotransmitter systems within the PVN on lumbar SNA (LSNA). Unilateral microinjection of ATP into the PVN induced a dose-related increase in the LSNA (1 nmol: 38 ± 6 %, 2.5 nmol: 72 ± 7 %, 5 nmol: 96 ±13 %). This increase was significantly attenuated by blockade of P2 receptors (pyridoxalphosphate-6-azophenyl-20,40-disulphonic acid, PPADS) and glutamate receptors (kynurenic acid, KYN) or a combination of both. The increase in LSNA elicited by L-glutamate microinjection into the PVN was not affected by a previous injection of PPADS. Selective blockade of non-N-methyl-D-aspartate receptors (6-cyano-7-nitroquinoxaline-2,3-dione disodium salt, CNQX), but not N-methyl-D-aspartate receptors (NMDA) receptors (DL-2-amino-5-phosphonopentanoic acid, AP5), attenuated the ATP-induced sympathoexcitatory effects at the PVN level. Taken together, our data show that purinergic neurotransmission within the PVN is involved in the control of SNA via P2 receptor activation. Moreover, we show an interaction between P2 receptors and non-NMDA glutamate receptors in the PVN suggesting that these functional interactions might be important in the regulation of sympathetic outflow.

Comparação entre imunofluorescência indireta e aglutinação direta para o diagnóstico sorológico da leishmaniose tegumentar americana em cães errantes / Comparison between indirect immunofluorescence and direct agglutination for the serologic diagnosis of American cutaneous leishmaniasis in stray dogs

Estudaram-se 92 cães errantes com o objetivo de comparar a imunofluorescência indireta (IFI) e a aglutinação direta (AD) na investigação da leishmaniose tegumentar americana (LTA) canina, no período de outubro de 1999 a novembro de 2001. Os animais foram examinados quanto à presença de lesões e submetidos à intradermorreação de Montenegro (IDRM) e à pesquisa de anticorpos anti-Leishmania por IFI e AD. A IFI apresentou sensibilidade de 78,9 por cento e especificidade de 93,8 por cento (título >40), e a AD sensibilidade de 57,9 por cento e especificidade de 68,8 por cento (título >80). Dois cães apresentavam lesão, mas a pesquisa do parasito foi negativa. A IDRM, realizada em 89 cães, foi positiva em três (3,4 por cento). Trinta e quatro cães (37,0 por cento) foram positivos para a IFI, 61 (66,3 por cento) para a AD e 69 (75,0 por cento) para a IFI e/ou a AD. Dos cães com a IFI positiva, sete eram de Floresta, 13 de Maringá, 12 de Leópolis, um de São Jorge do Ivaí e um (50,0 por cento) de Campo Mourão. Os resultados sugerem o contato prévio desses animais com o parasito.

Ninety-two stray dogs were studied in order to compare indirect immunofluorescence (IIF) and direct agglutination (DA) in the investigation of American cutaneous leishmaniasis (ACL), from October 1999 to November 2001. The dogs were evaluated for the presence of lesions, and submitted to the Montenegro skin test (MST) and the search for anti-Leishmania antibodies by IIF and DA. IIF showed 78.9 percent sensitivity and 93.8 percent specificity (titers >40), and DA presented 57.9 percent sensitivity and 68.8 percent specificity (titers >80). In two dogs there were lesions, but the parasite was not detected. The MST was positive in 3 (3.4 percent) out of 89 dogs. Thirty-four (37.0 percent) dogs showed anti-Leishmania antibodies through IIF, 61 (66.3 percent) through DA, and 69 (75.0 percent) through IIF and/or DA. Of the dogs with positive IIF, 7 came from the municipal areas of Floresta, 13 of Maringá, 12 of Leópolis, 1 of São Jorge do Ivaí and 1 (50.0 percent) of Campo Mourão. These data suggest previous contact of these animals with the parasite.

A spinal vasopressinergic mechanism mediates hyperosmolality-induced sympathoexcitation.

An elevation in plasma osmolality elicits a complex neurohumoral response, including an activation of the sympathetic nervous system and an increase in arterial pressure. Using a combination of in vivo and in situ rat preparations, we sought to investigate whether hypothalamic vasopressinergic spinally projecting neurones are activated during increases in plasma osmolality to elicit sympathoexcitation. Hypertonic saline (HS, i.v. bolus), which produced a physiological increase in plasma osmolality to 299 +/- 1 mosmol (kg water)(-1), elicited an immediate increase in mean arterial pressure (MAP) (from 101 +/- 1 to 121 +/- 3 mmHg) in vivo. Pre-treatment with prazosin reversed the HS-induced pressor response to a hypotensive response (from 121 +/- 3 to 68 +/- 2 mmHg), indicating significant activation of the sympathetic nervous system. In an in situ arterially perfused decorticate rat preparation, hyperosmotic perfusate consisted of either 135 mm NaCl, or a non-NaCl osmolyte, mannitol (0.5%); both increased lumbar sympathetic nerve activity (LSNA) by 32 +/- 5% (NaCl) and 21 +/- 1% (mannitol), which was attenuated after precollicular transection (7 +/- 3% and 1 +/- 1%, respectively). Remaining experiments used the NaCl hyperosmotic stimulus. In separate preparations the hyperosmotic-induced sympathoexcitation (21 +/- 2%) was also significantly attenuated after transection of the circumventricular organs (2 +/- 1%). Either isoguvacine (a GABA(A) receptor agonist) or kynurenic acid (a non-selective ionotropic glutamate receptor antagonist) microinjected bilaterally into the paraventricular nucleus (PVN) attenuated the increase in LSNA induced by the hyperosmotic stimulus (control: 25 +/- 2%; after isoguvacine: 7 +/- 2%; after kynurenic: 8 +/- 3%). Intrathecal injection of a V(1a) receptor antagonist also reduced the increase in LSNA elicited by the hyperosmotic stimulus (control: 29 +/- 6%; after blocker: 4 +/- 1%). These results suggest that a physiological hyperosmotic stimulus produces sympathetically mediated hypertension in conscious rats. These data are substantiated by the in situ decorticate preparation in which sympathoexcitation was also evoked by comparable hyperosmotic stimulation. Our findings demonstrate the importance of vasopressin acting on spinal V(1a) receptors for mediating sympathoexcitatory response to acute salt loading.

Orexins/hypocretins excite rat sympathetic preganglionic neurons in vivo and in vitro.

The two recently isolated hypothalamic peptides orexin A and orexin B, also known as hypocretin 1 and 2, are reported to be important signaling molecules in feeding and sleep/wakefulness. Orexin-containing neurons in the lateral hypothalamus project to numerous areas of the rat brain and spinal cord including the intermediolateral cell column (IML) of the thoracolumbar spinal cord. An in vivo and in vitro study was undertaken to evaluate the hypothesis that orexins, acting on sympathetic preganglionic neurons (SPNs) in the rat spinal cord, increase sympathetic outflow. First, orexin A (0.3, 1, and 10 nmol) by intrathecal injection increased mean arterial pressure (MAP) and heart rate (HR) by an average of 5, 18, and 30 mmHg and 10, 42, and 85 beats/min in urethane-anesthetized rats. Intrathecal injection of saline had no significant effects. Orexin B (3 nmol) by intrathecal administration increased MAP and HR by an average of 11 mmHg and 40 beats/min. The pressor effects of orexin A were attenuated by prior intrathecal injection of orexin A antibodies (1:500 dilution) but not by normal serum albumin. Intravenous administration of the alpha(1)-adrenergic receptor antagonist prazosin (0.5 mg/kg) or the beta-adrenergic receptor antagonist propranolol (0.5 mg/kg) markedly diminished, respectively, the orexin A-induced increase of MAP and HR. Second, whole cell patch recordings were made from antidromically identified SPNs of spinal cord slices from 12- to 16-day-old rats. Superfusion of orexin A or orexin B (100 or 300 nM) excited 12 of 17 SPNs, as evidenced by a membrane depolarization and/or increase of neuronal discharges. Orexin A- or B-induced depolarizations persisted in TTX (0.5 microM)-containing Krebs solution, indicating that the peptide acted directly on SPNs. Results from our in vivo and in vitro studies together with the previous observation of the presence of orexin A-immunoreactive fibers in the IML suggest that orexins, when released within the IML, augment sympathetic outflow by acting directly on SPNs.

Effects of the alpha antagonists and agonists injected into the lateral hypothalamus on the water and sodium intake induced by angiotensin II injection into the subfornical organ.

The subfornical organ (SFO) and the lateral hypothalamus (LH) have been shown to be important for the central action of angiotensin II (ANG II) on water and salt regulation. Several anatomical findings have demonstrated neural connections between the SFO and the LH. The present experiments were conducted to investigate the role of the alpha-adrenergic antagonists and agonists injected into the LH on the water and salt intake elicited by injections of ANG II into the SFO. Prazosin (an alpha1-adrenergic antagonist) injected into the LH increased the salt ingestion, whereas yohimbine (an alpha2-adrenergic antagonist) and propranolol (a beta-adrenergic antagonist) antagonized the salt ingestion induced by administration of ANG II into the SFO. Previous administration of clonidine (an alpha2-adrenergic agonist) or noradrenaline into the LH increased, whereas pretreatment with phenylephrine decreased the sodium intake induced by injection of ANG II into the SFO. Previous treatment with prazosin and propranolol reduced the water intake induced by ANG II. Phenylephrine increased the dipsogenic responses produced by ANG II, whereas previous treatment with clonidine injected into the LH reduced the water intake induced by ANG II administration into the SFO. The LH involvement with SFO on the excitatory and inhibitory mechanisms related to water and sodium intake is suggested.

Role of angiotensin II and vasopressin receptors within the supraoptic nucleus in water and sodium intake induced by the injection of angiotensin II into the medial septal area

In this study we investigated the effects of the injection into the supraoptic nucleus (SON) of non-peptide AT1- and AT2-angiotensin II (ANG II) receptor antagonists, DuP753 and PD123319, as well as of the arginine-vasopressin (AVP) receptor antagonist d(CH2)5-Tyr(Me)-AVP, on water and 3 percent NaCl intake induced by the injection of ANG II into the medial septal area (MSA). The effects on water or 3 percent NaCl intake were assessed in 30-h water-deprived or in 20-h water-deprived furosemide-treated adult male rats, respectively. The drugs were injected in 0.5 µl over 30-60 s. Controls were injected with a similar volume of 0.15 M NaCl. Antagonists were injected at doses of 20, 80 and 180 nmol. Water and sodium intake was measured over a 2-h period. Previous administration of the AT1 receptor antagonist DuP753 into the SON decreased water (65 percent, N = 10, P<0.01) and sodium intake (81 percent, N = 8, P<0.01) induced by the injection of ANG II (10 nmol) into the MSA. Neither of these responses was significantly changed by injection of the AT2-receptor antagonist PD123319 into the SON. On the other hand, while there was a decrease in water intake (45 percent, N = 9, P<0.01), ANG II-induced sodium intake was significantly increased (70 percent, N = 8, P<0.01) following injection of the V1-type vasopressin antagonist d(CH2)5-Tyr(Me)-AVP into the SON. These results suggest that both AT1 and V1 receptors within the SON may be involved in water and sodium intake induced by the activation of ANG II receptors within the MSA. Furthermore, they do not support the involvement of MSA AT2 receptors in the mediation of these responses

Characterisation of L-[3H]glutamate binding to fresh and frozen crude plasma membranes isolated from cerebral cortex of adult rats.

Specific [3H]glutamate binding to fresh crude plasma membranes (CPMs) was compared with binding to frozen CPMs and the optimal conditions for the binding to frozen CPMs isolated from cerebral cortex of adult rats were determined. Freezing reduced [3H]glutamate binding (3.5-fold), and pre-incubation of previously frozen membranes followed by three washes increased binding (4.5-fold) when compared to fresh samples. CPMs washed once, pre-incubated at 37 degrees C and washed 3 times was adopted as the most adequate condition for the binding assay of frozen membranes. In a Cl(-)-containing medium, [3H]glutamate binding (Bmax=97.9 pmol/mg, Kd=349.68 nM) to this frozen CPM preparation was significantly displaced by excess quisqualic acid (QA) (65%), L-2-amino-4-phosphonobutyric acid (L-AP4) (35%), trans-1-aminocyclopentane-1,3-dicarboxylate (1S,3R-ACPD) (25%) and alfa-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) (25%). In a Cl(-)-free medium, binding (Bmax=44.14 pmol/mg, 311 nM) was significantly displaced by QA (45%), L-AP4 (25%), ACPD (25%), AMPA (25%), kainic acid (20%) and N-methyl-D-aspartate (15%).

Role of angiotensin II and vasopressin receptors within the supraoptic nucleus in water and sodium intake induced by the injection of angiotensin II into the medial septal area.

In this study we investigated the effects of the injection into the supraoptic nucleus (SON) of non-peptide AT1- and AT2-angiotensin II (ANG II) receptor antagonists, DuP753 and PD123319, as well as of the arginine-vasopressin (AVP) receptor antagonist d(CH2)5-Tyr(Me)-AVP, on water and 3% NaCl intake induced by the injection of ANG II into the medial septal area (MSA). The effects on water or 3% NaCl intake were assessed in 30-h water-deprived or in 20-h water-deprived furosemide-treated adult male rats, respectively. The drugs were injected in 0.5 microliter over 30-60 s. Controls were injected with a similar volume of 0.15 M NaCl. Antagonists were injected at doses of 20, 80 and 180 nmol. Water and sodium intake was measured over a 2-h period. Previous administration of the AT1 receptor antagonist DuP753 into the SON decreased water (65%, N = 10, P < 0.01) and sodium intake (81%, N = 8, P < 0.01) induced by the injection of ANG II (10 nmol) into the MSA. Neither of these responses was significantly changed by injection of the AT2-receptor antagonist PD123319 into the SON. On the other hand, while there was a decrease in water intake (45%, N = 9, P < 0.01), ANG II-induced sodium intake was significantly increased (70%, N = 8, P < 0.01) following injection of the V1-type vasopressin antagonist d(CH2)5-Tyr(Me)-AVP into the SON. These results suggest that both AT1 and V1 receptors within the SON may be involved in water and sodium intake induced by the activation of ANG II receptors within the MSA. Furthermore, they do not support the involvement of MSA AT2 receptors in the mediation of these responses.

Central interaction between atrial natriuretic peptide and angiotensin II in the control of sodium intake and excretion in female rats.

We investigated the effects of estrogen on sodium intake and excretion induced by angiotensin II (ANG II), atrial natriuretic peptide (ANP) or ANG II plus ANP injected into the median nucleus (MnPO). Female Holtzman rats weighing 250-300 g were used. Sodium ingestion and excretion 120 min after the injection of 0.5 microliters of 0.15 M NaCl into the MnPO were 0.3 +/- 0.1 ml (N = 12) and 29 +/- 7 microEq in intact rats, 0.5 +/- 0.2 ml (N = 10) and 27 +/- 6 microEq in ovariectomized rats, and 0.2 +/- 0.08 (N = 11) and 36 +/- 8 microEq in estrogen-treated ovariectomized (50 micrograms/day for 21 days) rats, respectively. ANG II (21 microM) injection in intact, ovariectomized, and estrogen-treated ovariectomized rats increased sodium intake (3.8 +/- 0.4, 1.8 +/- 0.3 and 1.2 +/- 0.2 ml/120 min, respectively) (N = 11) and increased sodium excretion (166 +/- 18, 82 +/- 22 and 86 +/- 12 microEq/120 min, respectively) (N = 11). ANP (65 microM) injection in intact (N = 11), ovariectomized (N = 10) and estrogen-treated ovariectomized (N = 10) rats increased sodium intake (1.4 +/- 0.2, 1.8 +/- 0.3, and 1.7 +/- 0.3 ml/120 min, respectively) and sodium excretion (178 +/- 19, 187 +/- 9, and 232 +/- 29 microEq/120 min, respectively). Concomitant injection of ANG II and ANP into the MnPO of intact (N = 12), ovariectomized (N = 10) and estrogen-treated ovariectomized (N = 10) rats caused smaller effects than those produced by each peptide given alone: 1.3 +/- 0.2, 0.9 +/- 0.2 and 0.3 +/- 0.1 ml/120 min for sodium intake, respectively, and 86 +/- 9, 58 +/- 7, and 22 +/- 4 microEq/120 min for sodium excretion, respectively. Taken together, these results demonstrate that there is an antagonistic interaction of ANP and ANG II on sodium intake and excretion, and that reproductive hormones affect this interaction.

Central interaction between atrial natriuretic peptide and angiotensin II in the control of sodium intake and excretion in female rats

We investigated the effects of estrogen on sodium intake and excretion induced by angiotestin II (ANG II), atrial natriuretic peptide (ANP) or ANG II plus ANP injected into median preoptic nucleus MnPO). Female Holtzman rats weighing 250-300 g were used. Sodium ingestion and excretion 120 min after the injection of 0.5 mul of 0.15 M NaCl into the MnPO were 0.3 + 7 muEq in intact rats, 0.5 + 0.2 ml (N = 10) and 27 + 6 muEq in ovariectmized rats, and 0.2 + 0.08 (N = 11) and 36 + 8 muEq in estrogen-treated ovariectomized (50 mug/day for 21 days) rats, respectively. ANG II (21 muM) injection in intact, ovariectomized, and estrogen-treated ovariectomized rats increased sodium intake (3.8 + 0.4, 1.8 + 0.3 and 1.2 + 0.2 ml/120 min, respectively) (N = 11) and increased sodium excretion (166 + 18,82 + 22 and 86 + 22 and 86 + 12 muEq/120 min, respectively (N = 11). ANP (65 muM) injection in intact (N = 11), ovariectomized (N = 10) and estrogen-treated ovariectomized (N = 10) rats increased sodium intake (1.4 + 0.2, 1.8 + 0.3, and 1.7 + 0.3 ml/120 min, respectively) and sodium excretion (178 + 19, 187 + 9, and 232 + 29 muEq/120 min, respectively). Concomitant injection of ANG II and ANP into the MnPO of intact (N = 12), ovariectomized (N = 10) and estrogentreated ovariectomized (N = 10) rats caused smaller effects than those produced by each peptide given alone: 1.3 + 0.2, 0.9 + 0.2 and 0.3 + 0.1 ml/120 min for sodium intake, respectively, and 86 + 9,58 + 7, and 22 + 4 muEq/120 min for sodium excretion, respectively. Taken together, these results demonstrate that there is an antagonistic interaction of ANP and ANG II on sodium intake and excretion, and that reproductive hormones affect this interaction.