Cerebrospinal Aß11-x and 17-x levels as indicators of mild cognitive impairment and patients' stratification in Alzheimer's disease.

In the present work, the concentrations of Aß11-x and Aß17-x peptides (x=40 or 42), which result from the combined cleavages of ß-amyloid precursor protein (AßPP) by ß'/α or α/γ-secretases, respectively, were assessed in cerebrospinal fluid (CSF) samples from patients with Alzheimer's disease (AD) or mild cognitive impairment (MCI). Specific multiplexed assays were set up using new anti-40 and anti-42 monoclonal antibodies (mAbs) for the capture of these N-truncated Aß peptides and anti-11 or anti-17 mAbs for their detection. The specificity, sensitivity and reproducibility of such assays were assessed using synthetic peptides and human cell models. Aß11-x and Aß17-x were then measured in CSF samples from patients with AD (n=23), MCI (n=23) and controls with normal cognition (n=21). Aß11-x levels were significantly lower in patients with MCI than in controls. Compared with the combined quantification of Aß1-42, total Tau (T-Tau) and phosphorylated Tau (P-Tau; AlzBio3, Innogenetics), the association of Aß11-40, Aß17-40 and T-Tau improved the discrimination between MCI and controls. Furthermore, when patients with MCI were classified into two subgroups (MCI ≤1.5 or ≥2 based on their CDR-SB (Cognitive Dementia Rating-Sum of Boxes) score), the CSF Aß17-40/Aß11-40 ratio was significantly higher in patients with CDR-SB ≤1.5 than in controls, whereas neither Aß1-42, T-Tau nor P-Tau allowed the detection of this subpopulation. These results need to be confirmed in a larger clinical prospective cohort.

Effect of ionizing radiation on thymidine uptake, differentiation, and VEGFR2 receptor expression in endothelial cells: the role of VEGF(165).

PURPOSE: Late thrombosis of irradiated vascular segments may be the consequence of endothelial cell (EC) dysfunction after radiation therapy. We investigated the effects of beta ionizing radiation on human EC viability, thymidine uptake, and differentiation. METHODS AND MATERIALS: Endothelial cells were exposed to (32)P-labeled DNA oligonucleotides in incremental doses of 2, 6, and 10 Gy. The modulation of the VEGFR2 receptor expression after irradiation and the overall potential radioprotective effect of VEGF(165) on these functions were assayed. RESULTS: A dose-dependent inhibitory effect of beta irradiation on ECs' thymidine uptake and differentiation was observed. EC viability, however, was not affected at levels of radiation up to 10 Gy. VEGF(165) proved to have a radioprotective effect as ECs' thymidine uptake, after radiation doses of 2, 6, and 10 Gy, was increased by 1.5-, 2-, and 4-fold, respectively, in the presence of 10 ng/ml of VEGF(165) (p < 0.05 vs. LacZ). This concentration of VEGF(165) also proved beneficial in maintaining cell differentiation at 16 h postirradiation when compared to controls. These biologic effects were in direct correlation with the upregulation of VEGFR2 receptor expression in irradiated ECs. CONCLUSIONS: beta irradiation interacts directly with EC functions by significantly reducing their ability to differentiate and proliferate, associated with upregulation of VEGFR2. These effects can be prevented in part by pretreating cells with VEGF(165), an effect potentially favored by the upregulation of VEGFR2 receptor expression after irradiation.

Altered cardiac endothelin receptors and protein kinase C in deoxycorticosterone-salt hypertensive rats.

The aim of the present study was to assess the status of ET-1 receptor subtypes (ET(A)and ET(B)) in ventricular myocytes and fibroblasts and to determine the role of PKC-dependent pathways in ET-1-stimulated cardiac cells in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Systolic blood pressure and relative heart to body weight were significantly increased in DOCA-salt rats. In unilaterally nephrectomized (Uni-Nx) control rats, more than 90% of cardiomyocyte ET receptors were of the ET(A)subtype, whereas in fibroblasts ET(A)and ET(B)receptors were present in a 1:3 ratio. In DOCA-salt rats, the density of the ET(A)receptor subtype was reduced by 31% in cardiomyocytes and in cardiac fibroblasts only ET(B)receptor density was decreased by 29%. Affinity was unchanged. The relative expression of immunoreactive PKC alpha, gamma and epsilon was significantly increased, whereas PKC delta was not altered in cardiac extracts of DOCA-salt rats. In cardiac fibroblasts from DOCA-salt rats PKC delta was significantly increased and PKC epsilon was not translocated after ET-1 stimulation. The hearts of DOCA-salt hypertensive rats are thus characterized by: (1) decreased density of cardiomyocyte ET(A)receptors and fibroblast ET(B)receptors; (2) cell-specific enhanced expression of some PKC isoenzymes (alpha, gamma, delta and epsilon); and (3) unresponsiveness of PKC epsilon to translocate in the presence of ET-1. Together with alterations of ET-1-induced Ca(2+)handling in cardiac myocytes and fibroblasts, which we previously reported, results from the present study indicate a marked modification of the cardiac ET-1 system of DOCA-salt hypertensive rats.

Cellular effects of beta-particle delivery on vascular smooth muscle cells and endothelial cells: a dose-response study.

BACKGROUND: Although endovascular radiotherapy inhibits neointimal hyperplasia, the exact cellular alterations induced by beta irradiation remain to be elucidated. METHODS AND RESULTS: We investigated in vitro the ability of 32P-labeled oligonucleotides to alter (1) proliferation of human and porcine vascular smooth muscle cells (VSMCs) and human coronary artery endothelial cells (ECs), (2) cell cycle progression, (3) cell viability and apoptosis, (4) cell migration, and (5) cell phenotype and morphological features. beta radiation significantly reduced proliferation of VSMCs (ED50 1.10 Gy) and ECs (ED50 2.15 Gy) in a dose-dependent manner. Exposure to beta emission interfered with cell cycle progression, with induction of G0/G1 arrest in VSMCs, without evidence of cell viability alteration, apoptosis, or ultrastructural changes. This strategy also proved to efficiently inhibit VSMC migration by 80% and induce contractile phenotype appearance, as shown by the predominance of alpha-actin immunostaining in beta-irradiated cells compared with control cells. CONCLUSIONS: 32P-labeled oligonucleotide was highly effective in inhibiting proliferation of both VSMCs and ECs in a dose-dependent fashion, with ECs showing a higher resistance to these effects. beta irradiation-induced G1 arrest was not associated with cytotoxicity and apoptosis, thus demonstrating a potent cytostatic effect of beta-based therapy. This effect, coupled to that on VSMC migration inhibition and the appearance of a contractile phenotype, reinforced the potential of ionizing radiation to prevent neointima formation after angioplasty.

Endothelin-1 signaling is altered in cardiac cells from deoxycorticosterone acetate-salt hypertensive rats.

The cardiac cellular effects of endothelin-1 (ET-1) on intracellular free Ca2+ concentration ([Ca2+]i) were investigated in deoxycorticosterone acetate (DOCA)-salt rats with severe cardiac hypertrophy. [Ca2+]i was measured by fura-2 methodology in ventricular cardiomyocytes and fibroblasts of DOCA-salt hypertensive and control unilaterally nephrectomized rats (Uni-Nx). Blood pressure and heart weight were increased (p < 0.01) in DOCA-salt rats compared to control rats. ET-1 (10(-12)-10(-6) M) increased [Ca2+]i in a dose-dependent manner in both cell types from control and hypertensive rats. However, ET-1-induced [Ca2+]i responses were significantly attenuated (p < 0.01) in cardiomyocytes and fibroblasts of DOCA-salt rats. Sarafotoxin S6c (S6c) increased [Ca2+]i in fibroblasts but not in cardiomyocytes. In conclusion, ET-1 dose-dependently increased [Ca2+]i in cardiomyocytes (primarily via ETA receptors) and fibroblasts (via ETA and ETB receptors). Cardiac cell ET-1 signaling pathways are blunted in DOCA-salt hypertensive rats. ET-1 may not play a critical role in the pathophysiology of the severe concentric cardiac hypertrophy present in DOCA-salt hypertensive rats.

Cardiac type-1 angiotensin II receptor status in deoxycorticosterone acetate-salt hypertension in rats.

The regulation of angiotensin II (Ang II) receptors and Ang II-induced modulation of intracellular Ca2+ concentration in cardiac cells from hearts of experimentally induced hypertensive deoxycorticosterone acetate (DOCA)-salt and control unilaterally nephrectomized (Uni-Nx) Sprague-Dawley rats was assessed. Ang II receptor density and intracellular Ca2+ concentration measurements were examined in adult ventricular myocytes and fibroblasts by radioligand binding assay and digital imaging using fura 2 methodology, respectively. Four-week DOCA-salt treatment induced hypertension associated with cardiac hypertrophy. Ang II binding studies demonstrated that adult ventricular myocytes and fibroblasts possess mainly the AT1 subtype receptor. Moreover, DOCA-salt hypertension was associated with a 1.8-fold increase in Ang II-specific binding compared with myocytes from Uni-Nx control rats. Intracellular Ca2+ responses induced by increasing Ang II concentrations (10[-12] to 10[-4] mol/L) were significantly enhanced in cardiomyocytes from DOCA-salt rats. The effects of Ang II on intracellular Ca2+ spike frequency were unaltered in cardiomyocytes from DOCA-salt-hypertensive rats. The density of AT1 subtype receptors was not modified in ventricular fibroblasts after DOCA-salt treatment. Ang II increased intracellular Ca2+ concentration similarly in ventricular fibroblasts from normal and hypertensive rats. In conclusion, DOCA-salt hypertension is characterized by an increased AT1 receptor density and intracellular calcium responses in ventricular myocytes, whereas in ventricular fibroblasts the AT1 receptor status is unaltered. These findings report for the first time the cardiac cell-specific implication of Ang II and the intracellular calcium signaling pathway stimulated by the AT1 receptor in cardiac hypertrophy in DOCA-salt-hypertensive rats.

Intracellular Ca2+ modulation by angiotensin II and endothelin-1 in cardiomyocytes and fibroblasts from hypertrophied hearts of spontaneously hypertensive rats.

The vasoactive peptides angiotensin II (Ang II) and endothelin-1 (ET-1) have been implicated in cardiac hypertrophy. This study investigates Ang II and ET-1 effects on intracellular free calcium concentration and the receptor subtype through which agonist-induced calcium responses are mediated in isolated cardiomyocytes and fibroblasts from hypertrophied hearts of spontaneously hypertensive rats (SHR). We measured intracellular free calcium concentration by fura 2 methodology and determined receptor status by radioligand binding assays. Ang II (10(-12) to 10(-7) mol/L) had no effect on cardiomyocyte calcium levels in control Wistar-Kyoto rats but significantly increased (P < .01) intracellular free calcium concentration in a dose-dependent manner in cardiomyocytes from SHR. Ang II total and specific binding were increased (P < .05) in SHR cardiomyocytes. Calcium responses elicited by 10(-7) to 10(-5) mol/L Ang II were significantly reduced (P < .01) in SHR fibroblasts despite no significant change in Ang II receptor density. The angiotensin type 1 receptor blocker losartan (1 mumol/L) blocked Ang II-stimulated calcium transients, whereas the angiotensin type 2 receptor blocker PD 123319 had no effect. ET-1- and sarafotoxin S6c-induced calcium responses in cardiomyocytes and fibroblasts were not different between hypertensive and control groups. In conclusion, Ang II and ET-1 elicit distinct and differential responses in a cell-specific manner in cardiomyocytes and fibroblasts from hypertrophied hearts of SHR. Whereas Ang II-mediated effects, which are elicited via angiotensin type 1 receptors, are detectable in cardiomyocytes from SHR, responses to Ang II are blunted in fibroblasts from SHR, and ET-1-related actions are similar in cells from both rat groups. Stimulation of cardiomyocytes by Ang II in hypertrophied hearts associated with pressure overload in genetic hypertension suggests that Ang II could modulate the function of cardiomyocytes of SHR but not those of Wistar-Kyoto rats, whereas cardiac actions of ET-1 do not change with the development of hypertension.

Changes in apical organization of choroidal cells in rats adapted to spaceflight or head-down tilt.

Structural changes observed in choroid plexuses from rats dissected aboard a space shuttle, on day 13 of an orbital flight (NASA STS-58 mission, SLS-2 Experiments) demonstrated that choroidal epithelial cells display a modified organization in a microgravitational environment. Results were compared with ultrastructural observations of choroid plexus from rats maintained under anti-orthostatic restraint (head-down tilt) for 14 days. In both experiment types, the main alterations observed by transmission electron microscopy, at the level of choroidal epithelial cells from the third and fourth ventricles, concerned the formation and the organization of apical microvilli, whereas pseudopod-like structures appeared. Immunocytochemical distribution of ezrin, a cytoskeletal protein involved in apical cell differentiation in choroid plexus, confirmed the structural alteration of microvilli in head-down tilted rats, Kinocilia tended to disappear from the apical surface, suggesting a partial loss of cell polarization. In addition, large amounts of clear vesicles were gathered in the apical cytoplasm of choroidal epithelial cells. Disorganization of apical microvilli accumulations of apical vesicles and partial loss of cell polarity showed that long-stays in weightlessness induced alterations in the fine structure of choroid plexus, consistent with a marked reduction of cerebrospinal fluid production.

Cytosolic calcium changes induced by angiotensin II in neonatal rat atrial and ventricular cardiomyocytes are mediated via angiotensin II subtype 1 receptors.

We determined the effects of angiotensin II (Ang II) on cytosolic free calcium concentrations ([Ca2+]i) in the absence and presence of the selective angiotensin subtype 1 (AT1) receptor antagonist losartan or the selective AT2 antagonist PD 123319 in cultured neonatal rat atrial and ventricular cardiomyocytes. We also Ang II receptor density, affinity, and mRNA expression. [Ca2+]i was measured in single cells microphotometrically and by fluorescent digital imaging with fura 2 methodology. Receptor parameters were assessed by competitive binding studies with 125I-[Sar1,Ile8]Ang II in the presence of increasing concentrations of [Sar1,Ile8]Ang II, losartan, and PD 123319. AT1 receptor (types AT1A and AT1B) mRNA abundance was measured by reverse transcription-polymerase chain reaction. Ang II produced concentration-dependent increases in [Ca2+]i values in atrial and ventricular cells were similar but Ang II (10-9 mol/L)-induced [Ca2+]i changes were significantly greater in atrial compared with ventricular cells Ang II responses were blocked by losartan (10-7 mol/L) but not PD 123319 (10-7 mol/L). Binding studies demonstrated a single class of high-affinity. Ang II binding sites on cardiomyocyte membranes (Kd = 0.71 +/- 0.11 mumol/L). 125I-[Sar1,Ile8]Ang II was displaced by losartan but not by PD 123319. AT1 receptor mRNA was detected by reverse transcription-polymerase chain reaction in cells from atria and ventricles. In atrial cardiomyocytes, both AT1A and AT1B receptor genes were expressed, whereas in ventricular cardiomyocytes, only the AT1A receptor gene was expressed. These data demonstrate that neonatal cardiomyocytes possess Ang II receptors of the AT1 receptor subtype that are linked to [Ca2+]i signaling pathways. The different Ang II-induced [Ca2+]i responses between atrial and ventricular cells may be related to differences in the distribution of AT1 receptor subtype subvariants.

Central and peripheral noradrenergic responses to 14 days of spaceflight (SLS-2) or hindlimb suspension in rats.

INTRODUCTION: The purposes of this work were to assess the influence of microgravity on the central and peripheral noradrenergic activity to reevaluate SLS-1 mission findings and to compare it with that of simulated microgravity in rats. METHODS: The norepinephrine (NE) contents of the brainstem cell groups (A1, A2, A5, and A6) and organs (heart and kidneys) involved in blood pressure regulation were determined in rats after a 14-d spaceflight (SLS-2 with animals sacrificed 6 h after landing) and after a 14-d hindlimb suspension followed with 6 h of recovery. RESULTS: After SLS-2 spaceflight, NE contents were not significantly different between flight and ground-based rats either in A1 (5.2 +/- 0.5 vs. 5.7 +/- 0.4 pmol/structure), rostral A2 (12.1 +/- 0.5 vs. 11.1 +/- 0.9 pmol/structure), caudal A2 (3.2 +/- 0.6 vs. 4.3 +/- 0.5 pmol/structure) and A5 (4.4 +/- 0.4 vs. 4.3 +/- 0.5 pmol/structure) nuclei or in cardiac atria (98.6 +/- 7.5 vs. 83.4 +/- 8.9 pmol.mg-1 protein), ventricles (38.3 +/- 2.2 vs. 44.1 +/- 2.8 pmol.mg-1 protein) and kidneys (13.4 +/- 0.8 vs. 17.7 +/- 1.5 pmol.mg-1 protein). NE content was unchanged in A6 nucleus after SLS-2 comparing with control rats (respectively 4.1 +/- 0.3 vs. 4.5 +/- 0.5 pmol/structure), while it was depleted after SLS-1 mission (2.9 +/- 0.3 vs. 8.8 +/- 0.7 pmol/structure, p < 0.001) probably in relation with the stressful conditions on return to Earth. Similarly, no alterations between suspended and control rats were noted in central and peripheral NE contents after 14 d of suspension and after 6 h of recovery, whereas NE turnover studies evidenced large changes in the activities on structures on suspension and on recovery. CONCLUSION: These results suggest that only NE turnover determination will provide information about the role of the sympathetic system in the cardiovascular deconditioning. This raises the problem of the necessity to experiment inflight (injections, sacrifice) in order to avoid the recovery effects of the few hours following the landing.

Modulation of Ca2+ transients in neonatal and adult rat cardiomyocytes by angiotensin II and endothelin-1.

Vasoactive peptides may exert inotropic and chronotropic effects in cardiac muscle by modulating intracellular calcium. This study assesses effects of angiotensin II (ANG II) and endothelin-1 (ET-1) on intracellular free calcium concentration ([Ca2+]i) in cultured cardiomyocytes from neonatal and adult rats. [Ca2+]i was measured microphotometrically and by digital imaging using fura 2 methodology. Receptor subtypes through which these agonists induce responses were determined pharmacologically and by radioligand binding studies. ANG II and ET-1 increased neonatal atrial and ventricular cell [Ca2+]i transients in a dose-dependent manner. ANG II (10(-11) to 10(-7) M) failed to elicit [Ca2+]i responses in adult cardiomyocytes, whereas ET-1 increased [Ca2+]i in a dose-dependent manner. The ETA receptor antagonist BQ-123 significantly reduced (P < 0.05) ET-1 induced responses, and the ETB receptor agonist IRL-1620 (10(-7) to 10(-5) M) significantly increased (P < 0.05) [Ca2+]i in neonatal and adult cardiomyocytes. ET-1 binding studies demonstrated 85% displacement by BQ-123 and approximately 15% by the ETB receptor agonist sarafotoxin S6c, suggesting a predominance of ETA receptors. Competition binding studies for ANG II failed to demonstrate significant binding on adult ventricular myocytes, indicating the absence or presence of very few ANG II receptors. These data demonstrate that ANG II and ET-1 have stimulatory [Ca2+]i effects on neonatal cardiomyocytes, whereas in adult cardiomyocytes, ANG II-induced effects are insignificant, and only ET-1-induced responses, which are mediated predominantly via ETA receptors, are preserved. Cardiomyocyte responses to vasoactive peptides may thus vary with cardiac development.

Endothelin-1 and angiotensin II receptors in cells from rat hypertrophied heart. Receptor regulation and intracellular Ca2+ modulation.

This study investigates the cellular localization and regulation of endothelin-1 (ET-1) and angiotensin II (Ang II) receptors and the effects of ET-1 and Ang II on [Ca2+]i in cardiac hypertrophy due to volume overload in the rat. Radioligand binding assays and [Ca2+]i measurements by fura 2 methodology were performed on isolated ventricular cardiomyocytes and fibroblasts from the heart of rats with a 4-week aortocaval shunt. In the hypertrophied myocardium, ET-1 and Ang II concentrations were unchanged in ventricles. Ventricular ET-1 receptors had a cell-specific distribution: > 90% of ET receptors in cardiomyocytes are of the ETA subtype, whereas fibroblasts had a nearly equal proportion of the ETA and ETB subtypes. ET-1 receptor densities, affinities, and ET-1-induced [Ca2+]i were not significantly different from control in both ventricular cell types from hypertrophied myocardium. Ang II specific binding was very low on isolated ventricular cardiomyocytes, suggesting few receptors in control conditions. However, [Ca2+]i responses induced by Ang II at concentrations > 10(-8) mol/L were detectable and were significantly higher in hypertrophied cardiomyocytes. Ang II receptor density (exclusively AT1) on fibroblasts was significantly reduced (42,970 +/- 3330 versus 73,870 +/- 7940 sites per cell for control cells, P < .01), but AT1 receptor affinity was unchanged after volume overload. The maximum increase in [Ca2+]i evoked by 10(-6) to 10(-4) mol/L Ang II was significantly lower in fibroblasts from overloaded hearts. In conclusion, ET-1 receptor proportion is cell specific, with cardiomyocytes possessing predominantly the ETA subtype and fibroblasts possessing both ETA and ETB receptors. Plasma and cardiac ET-1 concentrations and ET-1 receptor regulation on both ventricular cell types are not altered in cardiac volume overload, suggesting that cardiac ET-1 may not play a significant role in this model. Cardiac hypertrophy induced a significant downregulation of AT1 receptors on fibroblasts, whereas total binding and [Ca2+]i sensitivity to Ang II were significantly enhanced in hypertrophied cardiomyocytes. This suggests that cardiac Ang II may be involved in the pathophysiology of the cardiac hypertrophy of volume overload.

Basal and angiotensin II-induced cytosolic free calcium in adult rat cardiomyocytes and fibroblasts after volume overload.

This study investigates basal and angiotensin II (Ang II)-induced [Ca2+]i concentrations in cells from hearts of rats that have undergone cardiac hypertrophy due to volume overload. [Ca2+]i measurements assessed by digital imaging using fura 2 methodology were performed on isolated ventricular cardiomyocytes and fibroblasts from adult rat hearts with a 4-week aortocaval shunt. Long-term aortocaval shunt induced a significant increase in atrial (72%) and ventricular (41%) weights and a large elevation in plasma atrial natriuretic peptide-(1-98) concentration (160%). For adult cardiomyocytes [Ca2+]i measurements are reported as diastolic (average of the lowest points) and systolic intracellular Ca2+ values (average of the maximum points corresponding to the diastolic points) over a 30-second time interval. Basal diastolic [Ca2+]i (99 +/- 4.1 nmol/L for experimental cells versus 90 +/- 4.8 for control cells) was not altered, whereas basal systolic [Ca2+]i was significantly greater in ventricular cardiomyocytes from overload hearts (155 +/- 2.3 versus 129 +/- 4.4 nmol/L for control cells, P < .05). Ang II increased intracellular Ca2+ spike frequency in a concentration-dependent manner in cardiomyocytes from control and overload myocardium. Basal and Ang II-induced intracellular Ca2+ spike frequencies were not modified in cardiomyocytes from hypertrophied hearts. Basal [Ca2+]i in ventricular fibroblasts from overload myocardium was significantly increased (128 +/- 5.1 nmol/L for fibroblasts from hypertrophied hearts versus 104 +/- 3.5 for control cells, P < .05). Ang II-induced [Ca2+]i was lower in fibroblasts from overload myocardium (P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Choroidal responses in microgravity. (SLS-1, SLS-2 and hindlimb-suspension experiments).

Fluid and electrolyte shifts occurring during human spaceflight have been reported and investigated at the level of blood, cardiovascular and renal responses. Very few data were available concerning the cerebral fluid and electrolyte adaptation to microgravity, even in animal models. It is the reason why we developed several studies focused on the effects of spaceflight (SLS-1 and SLS-2 programs, carried on NASA STS 40 and 56 missions, which were 9- and 14-day flights, respectively), on structural and functional features of choroid plexuses, organs which secrete 70-90% of cerebrospinal fluid (CSF) and which are involved in brain homeostasis. Rats flown aboard space shuttles were sacrificed either in space (SLS-2 experiment, on flight day 13) or 4-8 hours after landing (SLS-1 and SLS-2 experiments). Quantitative autoradiography performed by microdensitometry and image analysis, showed that lateral and third ventricle choroid plexuses from rats flown for SLS-1 experiment demonstrated an increased number (about x 2) of binding sites to natriuretic peptides (which are known to be involved in mechanisms regulating CSF production). Using electron microscopy and immunocytochemistry, we studied the cellular response of choroid plexuses, which produce cerebrospinal fluid (CSF) in brain lateral, third and fourth ventricles. We demonstrated that spaceflight (SLS-2 experiment, inflight samples) induces changes in the choroidal cell structure (apical microvilli, kinocilia organization, vesicle accumulation) and protein distribution or expression (carbonic anhydrase II, water channels,...). These observations suggested a loss of choroidal cell polarity and a decrease in CSF secretion. Hindlimb-suspended rats displayed similar choroidal changes. All together, these results support the hypothesis of a modified CSF production in rats during long-term (9, 13 or 14 days) adaptations to microgravity.

Modulation of Ca2+ transients in neonatal and adult rat cardiomyocytes by endothelin-1.

Endothelin-1 (ET-1) may exert inotropic and chronotropic effects in cardiac muscle by modulating intracellular Ca2+. This study assesses effects of ET-1 on intracellular free Ca2+ concentration ([Ca2+]i) in neonatal and adult rat cardiomyocytes. [Ca2+]i was measured by Fura 2 methodology. ET receptor subtypes were determined by binding studies. ET-1 increased neonatal and adult [Ca2+]i and spike frequency in a dose-dependent manner. It decreased [Ca2+]i amplitude in neonatal cardiomyocytes but had no effect on [Ca2+]i amplitude in adult cells. The ET(A) receptor antagonist BQ-123 reduced ET-1-induced responses and the ETB receptor agonist IRL-1620 increased [Ca2+]i. ET-1 binding studies demonstrated 85% displacement by BQ-123 and 15% by the ETB receptor agonist sarafotoxin S6c, suggesting a predominance of ET(A) receptors. These data demonstrate that in neonatal and adult cardiomyocytes ET-1 has stimulatory effects on [Ca2+]i which are mediated predominantly via ET(A) receptors. Therefore, ET-1 may influence cardiac development and function.

Cardiac and plasma atrial natriuretic peptide after 9-day hindlimb suspension in rats.

To determine atrial natriuretic peptide (ANP) adaptation to simulated weightlessness, immunoreactive plasma (ir-NH2- and ir-COOH-terminals) and atrial (ir-COOH-terminal) ANP levels, atrial mRNA expression, immunoreactive cardiocyte ANP levels (ir-NH2- and ir-COOH-terminals), and ultrastructural observations of granules in atrial cardiocytes were assessed in male Wistar rats after a 9-day hindlimb suspension. Plasma ir-NH2- and ir-COOH-terminal ANP concentrations decreased by 17 (P < 0.05) and 37% (P < 0.05), respectively, in suspended rats. A concomitant ir-COOH-terminal ANP content reduction was also observed in left (31%; P < 0.01) and right atria (25%; P < 0.05). Atrial ANP mRNA expression was severely depleted in the right atrium and less so in the left atrium after 9 days of hindlimb suspension. Immunocytochemistry observations demonstrated lowered NH2- and COOH-terminal ANP immunoreactivities in left and right atria from suspended rats. A reduced number of storage granules (dense granules) in both atria was also noted on ultrastructural analysis. It was concluded that ANP biosynthesis, storage, and release were decreased after a 9-day hindlimb suspension.

Comparison of the effects of spaceflight and hindlimb-suspension on rat pituitary vasopressin and brainstem norepinephrine content.

To compare actual spaceflight to ground-based simulation (hindlimb-suspension), we measured the norepinephrine (NE) content in A1, A2, A5 and A6 (locus coeruleus) and the vasopressin content in the neurohypophysial system. The experimental period was of 9 days' duration. The NE content in the locus coeruleus decreased significantly in rats flown for 9 days (67%, p < 0.001), but showed no significant changes after hindlimb-suspension. These results demonstrated that suspended rats adapted better to weightlessness-simulation than flown rats to actual microgravity. In rats flown aboard SLS-1, the vasopressin content was significantly increased in the posterior pituitary (71%, p < 0.01), and was decreased in the hypothalamus (49%, p < 0.05). In 9-day suspended rats pituitary vasopressin levels were unchanged, while in the hypothalamus a significant decrease was noted (21%, p < 0.05). It was concluded that spaceflight changes in pituitary vasopressin levels and in the locus coeruleus NE content were consistent with a stress reaction, occurring during and/or after landing. These results confirmed that hindlimb-suspension model constitutes a valid and less stressful [correction of lesstressful] ground-based simulation of microgravity in rats.

Norepinephrine content in discrete brain areas and neurohypophysial vasopressin in rats after a 9-d spaceflight (SLS-1).

The norepinephrine (NE) content in discrete brain areas and the vasopressin content in the neurohypophysial system were assessed in rats after a 9-d spaceflight and after a recovery period (9 d). The NE content in the locus coeruleus decreased significantly in spaceflight rats (2.9 +/- 0.3 vs. 8.9 +/- 0.7 pmol.structure-1 for control rats, p < 0.001), but showed no difference between control and flight animals after a 9-d recovery. These findings were probably due to an acute stress undergone during landing. The NE content was unchanged in the A2 and A5 cell groups. In rats flown aboard SLS-1, the vasopressin content was increased in the posterior pituitary (1.47 +/- 0.1 vs. 0.86 +/- 0.1 micrograms.structure-1, for control rats, p < 0.01), and was significantly decreased in the hypothalamus (8.95 +/- 2.0 vs. 17.6 +/- 2.2 ng.structure-1, for control rats, p < 0.05). We conclude that the NE depletion in the locus coeruleus and the alteration in vasopressin release were consistent with an acute stress, likely occurring during and/or after landing. These changes tend to mask the actual neuroendocrine modifications caused by microgravity.

Central and peripheral norepinephrine turnover after hindlimb suspension in the rat.

After a 9-d hindlimb suspension, the turnover rate of norepinephrine (NE) in rats was determined in A1, A2 (rostral and caudal), A5 and A6 cell groups, as well as in peripheral target organs (heart and kidneys). The NE turnover rate decreased after hindlimb suspension respectively in caudal A2 (67.5%, p < 0.001), rostral A2 (63%, p < 0.001) and in A5 cell groups (62.5%, p < 0.001), but remained unchanged in A1 and A6 regions. The peripheral sympathetic outflow response was selectively modified: in suspended rats, the NE turnover was mainly decreased in atria (79%, p < 0.001) and in ventricles (44%, p < 0.001); there were no biochemical changes in kidneys. It was concluded that a 9-d hindlimb suspension: 1) impaired the noradrenergic neuron activity of A2 and A5 cell groups, which are involved in the central cardiovascular regulation, and particularly in the baroreceptor reflex mechanism; and 2) mainly altered the cardiac NE turnover and induced a selective response of peripheral target organs.