Transient modification of baroreceptor response during tilt-induced vasovagal syncope.

AIMS: Normally, arterial baroreceptors attempt to minimize systemic hypotension by initiating reflex vasoconstriction and tachycardia. However, in the setting of vasovagal syncope (VVS), these usual compensatory mechanisms either fail to be triggered or the response is inadequate. We hypothesized that in VVS prone individuals, arterial baroreceptor response (BRR) is normal under most conditions, but that a transient functional BRR disturbance occurs during an evolving vasovagal faint and may in part account for failure of the usual compensatory response. METHODS AND RESULTS: This study assessed BRR in the baseline state and again in association with either VVS induced head-up tilt (HUT) or after a prolonged period of upright posture without VVS. To minimize impact on HUT outcome, BRR was estimated non-pharmacologically by measuring blood pressure and heart rate changes, induced when subjects were returned to the supine position after undergoing diagnostic 70 degrees HUT evaluation. Beat to beat heart rate and arterial blood pressure changes were recorded in 13 patients with syncope and another 16 individuals with negative HUT (control group). Baseline BRR was initially evaluated at the end of a 3 min symptom free HUT (HUT#1), and the measurement was repeated after a 45 min duration HUT in the control group or in conjunction with syncope in VVS prone individuals (HUT#2). Baseline BRR did not differ significantly in controls and VVS prone individuals (controls: 3.37+/-1.56, VVS prone: 6.0+/-2.02 ms/mmHg, p=0.27). Further, at the end of 45 min HUT#2, BRR was unaltered from baseline in control subjects (4.92+/-1.36 ms/mmHg, p=0.48), but was markedly reduced from baseline value in individuals who experienced a faint, -3.30+/-0.81 ms/mmHg (p<0.0003 vs baseline). CONCLUSION: Compared with individuals who do not manifest VVS during HUT, VVS prone individuals appear to demonstrate functional diminution of baroreceptor responsiveness. This altered response may undermine the normal expected compensatory response to evolving systemic hypotension. The basis for this transient disturbance in baroreceptor responsiveness is currently unknown.     

Systemic administration of lipopolysaccharide induces release of nitric oxide and glutamate and c-fos expression in the nucleus tractus solitarii of rats.

There is increasing recognition that communication pathways exist between the immune system and brain, which allows bidirectional regulation of immune and brain responses to infection. The endotoxin lipopolysaccharide (LPS) has been reported to elicit release of cytokines and expression of inducible nitric oxide synthase (iNOS) in peripheral organs. Whereas LPS given systemically causes endotoxic shock, little is known about its central nervous system action, particularly the induction of iNOS. Nitric oxide (NO) and glutamate in the nucleus tractus solitarii (NTS) are important mediators of central cardiovascular regulation. We have previously demonstrated that intravenous injections of LPS increased the NO precursor L-arginine-induced depressor effect in the NTS. The present study investigated further the effects of LPS on the release of NO and glutamate in the NTS and the expression of c-fos, an immediate early response gene product, in neural substrates for central cardiovascular control. In vivo microdialysis coupled with chemiluminescence and electrochemical detection techniques were used to measure extracellular levels of NO and glutamate in the rat NTS. Immunohistochemistry was used for the examination of c-fos protein expression. We found that intravenous infusion of LPS (10 mg/kg) produced a biphasic depressor effect, with an early, sharp hypotension that partially recovered in 15 minutes and a secondary, more prolonged hypotension. In the NTS, a progressive increase of extracellular glutamate and NO levels occurred 3 and 4 hours after LPS was given, respectively. The effects of LPS on the induction of delayed hypotension and NO formation in the NTS were abolished by pretreatment with the iNOS inhibitor aminoguanidine. Finally, c-fos protein expression in the NTS and related structures for cardiovascular regulation was observed after LPS challenge. Taken together, these data suggest that an endotoxin given systemically can elicit delayed increases of glutamate release and iNOS-dependent NO production in the NTS and activate the central neural pathway for modulating cardiovascular function.     

Functional interaction between nucleus tractus solitarius NK1 and 5-HT3 receptors in the inhibition of baroreflex in rats

OBJECTIVE: Previous data showed that in the nucleus tractus solitarius (NTS), 5-HT(3) receptors are critically involved in the inhibition of cardiac baroreceptor reflex response occurring during the defense reaction. Since stimulation of NTS NK(1) receptors has been found to inhibit the baroreflex bradycardia, we examined in this study whether this reflex response is inhibited during the defense reaction via an interaction between NK(1) and 5-HT(3) receptors. METHODS: For this purpose, we analyzed in urethane-anaesthetized rats the effects of intra-NTS GR205171, a selective NK(1) receptor antagonist, on the baroreflex bradycardia inhibition observed either during the defense reaction triggered by electrical stimulation of the dorsal periaqueductal grey matter (dPAG) or after NTS 5-HT(3) receptor activation. RESULTS: Intra-NTS GR205171, reversed, in dose-dependent manner, the inhibitory effect of dPAG stimulation on baroreflex bradycardia. This reversion was of 49% when both sinus carotid and aortic baroreceptors were stimulated by phenylephrine, and of 84% when aortic depressor nerve was stimulated. Similarly, intra-NTS GR205171 reversed partially or almost totally the inhibitory effect of local microinjections of phenylbiguanide, a 5-HT(3) receptor agonist, on baroreflex bradycardia induced either by phenylephrine administration or aortic nerve stimulation, respectively. CONCLUSION: These results strongly suggest that NK(1) receptors contribute downstream to the 5-HT(3) receptor-mediated inhibition of the aortic but not carotid cardiac baroreflex response occurring during the defense reaction, therefore implying that baroreceptor afferent inputs may be differentially modulated depending on their origin. This differentiation may be useful for a better understanding of baroreflex dysfunction in disease-induced conditions.     

Effect of Okadaic Acid, a Protein Phosphatase Inhibitor, on Heat Stress-Induced HSP72 Synthesis and Thermotolerance

Heat stress proteins (HSPs), in particular HSP72, seem to play a major role in cell protection against lethal stresses such as hyperthermia or ischemia. HSP synthesis is negatively regulated by protein phosphatases, which are implicated in dephosphorylation processes. In the present study, we have investigated the effect of okadaic acid (OA, a protein phosphatase inhibitor) on heat stress-induced HSP72 synthesis and thermotolerance in smooth muscle cells (SMC).SMC were heat stressed (42°C for 20 minutes) in the presence of 250 nM OA (HS+OA cells) or its vehicle (HS+V cells). Control (OA or V) cells were not heat stressed. HSP72 mRNA expression was determined 1, 1.5, 3, and 6 hours after heat stress by RT-PCR, and HSP72 synthesis was determined 6, 12, 24, 48, and 72 hours after heat stress by Western blotting. SMC survival of lethal hyperthermia (47°C for 90 minutes) was assessed 6, 24, and 48 hours after heat stress by a tetrazolium assay.The maximal expression of HSP72 mRNA was markedly prolonged in HS+OA cells (until 6 hours after heat stress) compared to HS+V cells (1 hour after heat stress). The kinetics of HSP72 synthesis and thermotolerance of SMC were not different between HS+OA and HS+V cells. Baseline HSP72 mRNA and protein expression were similar in control V and OA cells.In conclusion, okadaic acid treatment of SMC potentiated HSP72 mRNA expression without affecting heat stress-induced HSP72 synthesis and thermotolerance.     

Impact of hyperthermic preconditioning on postischemic hepatic microcirculatory disturbances in an isolated perfusion model of the rat liver

Sublethal hyperthermia and the following recovery from this heat exposure, referred to as hyperthermic preconditioning, elicits a transient state of tolerance to oxidative insults through an intracellular protective response: stress response. The impact of hyperthermic preconditioning on hepatic microcirculatory disturbance, which is one of the determinants of ischemia/reperfusion-induced injury of the liver, was investigated by using intravital fluorescence microscopy. Thirty minutes of ischemia and a subsequent 120 minutes of reperfusion was induced in an in situ isolated perfusion model of Sprague-Dawley rats. Heat stress was given by whole-body hyperthermia, and a subsequent recovery was allowed for 18 or 48 hours, respectively. Postischemic decrease in sinusoidal perfusion rate and sinusoidal diameter, leukocyte stagnation in sinusoids, and leukocyte adhesion in postsinusoidal venules were significantly attenuated in both hyperthermia-pretreated groups. A recovery of bile production, a reduction of liver enzyme release, and an attenuation of tissue edema and histological damage were also observed. A marked expression of heat shock protein (HSP) 70 and heme oxygenase (HO-1)/HSP32 was correlatively observed in the liver tissue coincident with the induction of these protective effects. Hyperthermic preconditioning provides a continuous long-term and constant inhibitory effect (up to 48 hours after heat exposure) on postischemic injury of the liver through the attenuation of microcirculatory disturbances. These beneficial effects might be associated with a concomitant increase in HSP70 and HO-1/HSP32 expression.     

脑缺血大鼠脑组织热休克蛋白70的表达

目的　研究脑缺血时大鼠脑组织热休克蛋白 70 (HSP70 )的表达及不同脑缺血时间对HSP70表达的影响。方法　选取雄性SD大鼠 4 0只 (体重约 2 0 0g)随机分为 4组 ,每组 10只。其中一组为正常对照组 ,一组为脑缺血 2min组 ,一组为脑缺血 6min组和脑缺血 12min。实验后取大鼠脑组织匀浆并用WesternBlot法测HSP70的表达。结果　脑缺血组脑组织HSP70的表达明显增高 ,其中脑缺血 6min和 12min组与对照组相比有显著性差异 (P <0 0 5) ;脑缺血 12min与6min相比亦有显著性差异 (P <0 0 5)。结论　HSP70在脑缺血时的脑组织存在高表达 ,脑缺血时间越长 ,HSP70的表达越高。     

Elevated circulating levels of heat shock protein 70 are related to systemic inflammatory reaction through monocyte Toll signal in patients with heart failure after acute myocardial infarction

BACKGROUND: Recent studies have shown that heat shock protein (HSP) 70 may serve as a "damage signal" to the immune system and could be the endogenous ligand for Toll-like receptor (TLR) 4 mediating synthesis of inflammatory cytokines. AIMS: To explore the relationship between circulating HSP70 levels and activation of monocyte TLR4 and myocardial damage after AMI. METHODS AND RESULTS: This study examined circulating HSP70 and monocyte TLR4 levels in 52 patients with AMI and 20 controls, and analyzed ex vivo inflammatory cytokine productions using HSP70-stimulated monocytes. Circulating HSP70 levels were higher in AMI patients on day 1 after onset than in controls and remained elevated in AMI patients 14 days after onset. HSP70 levels were positively correlated with monocyte TLR4, plasma interleukin-6 and tumor necrosis factor-alpha levels in AMI patients. HSP70 levels 14 days after onset were higher in AMI patients with heart failure (n=15) than in those without heart failure. In our in vitro study, HSP70-stimulated monocytes resulted in dose-dependent TLR4 expression and release of inflammatory cytokines. TLR4 antibody inhibited inflammatory cytokines release. CONCLUSIONS: Elevated circulating levels of HSP70 may be involved in TLR4 signal-mediated immune response and the progression of heart failure after AMI.     

Acute changes in 3H-PAC and 125I-PYY binding in the nucleus tractus solitarii and hypothalamus after a hypertensive stimulus

Activation of alpha-2-adrenergic and neuropeptide Y (NPY) receptors in the nucleus tractus solitarii (NTS) induces hypotension and bradycardia. On the contrary, activation of angiotensin II (Ang II) receptors leads to hypertension. Acute changes in binding parameters of alpha-2-adrenergic, NPY and Ang II receptors were evaluated in the NTS and paraventricular hypothalamic nucleus (PVN) of rats after a hypertensive stimulus employing quantitative receptor autoradiography. Saturation experiments showed a decrease in the number (Bmax) of alpha-2-adrenergic binding sites in the NTS 6 hours after coarctation-induced hypertension. Furthermore, the affinity of NPY receptors was diminished as seen by the increase in the KD value of 125I-PYY. Tyrosine hydroxylase and NPY immunoreactivities were increased in the NTS and ventral medulla. Binding of 125I-Ang II was not changed in the NTS. Binding of all ligands analyzed was not altered in the PVN. The results suggest an acute down-regulation of alpha-2-adrenergic and NPY receptors involved with hypotension in response to hypertensive stimulus, which might be related to an increased availability of catecholamines and NPY in the NTS.     

Failure of exogenous ATP and creatine phosphate to modify the course of E coli endotoxin shock in the cat.

Exogenous adenosine triphosphate (ATP) (in combination with MgCl2) and also creatine phosphate (CrP), have been administered intravenously to endotoxin-shocked cats. Untreated shocked cats exhibited systemic hypotension during the first hour and again from four hours. Cardiac output fell progressively, and markedly elevated arterial lactate levels were evident within one hour of endotoxin administration. Treatment with ATP (10 mg/kg every 30 minutes) during shock led to rapid hemodynamic deterioration in all cats; most of the cats were dead before completion of dosing (at three hours). Long-lasting systemic hypotension and bradycardia were associated with this ATP administration and marked hypoglycemia developed in the survivors. Neither ATP (62 mg/kg) administered before endotoxin, nor CrP (500 mg/kg; administered either prior to endotoxin, or one hour afterwards) significantly modified the hemodynamic or metabolic changes associated with endotoxin shock in this species. Neither ATP nor CrP increased survival (assessed at five hours). Other workers have demonstrated improved survival from shock with ATP treatment. There may be species differences in the responsiveness to exogenous ATP or, alternatively, a difference in the role of high-energy phosphates in different types of shock.     

ACUTE CHANGES IN 3H-PAC AND 125I-PYY BINDING IN THE NUCLEUS TRACTUS SOLITARII AND HYPOTHALAMUS AFTER A HYPERTENSIVE STIMULUS

Activation of alpha-2-adrenergic and neuropeptide Y (NPY) receptors in the nucleus tractus solitarii (NTS) induces hypotension and bradycardia. On the contrary, activation of angiotensin II (Ang II) receptors leads to hypertension. Acute changes in binding parameters of alpha-2-adrenergic, NPY and Ang II receptors were evaluated in the NTS and paraventricular hypothalamic nucleus (PVN) of rats after a hypertensive stimulus employing quantitative receptor autoradiography. Saturation experiments showed a decrease in the number (B_max) of alpha-2-adrenergic binding sites in the NTS 6 hours after coarctation-induced hypertension. Furthermore, the affinity of NPY receptors was diminished as seen by the increase in the KD value of ¹²⁵ I-PYY. Tyrosine hydroxylase and NPY immunoreactivities were increased in the NTS and ventral medulla. Binding of ¹²⁵ I-Ang II was not changed in the NTS. Binding of all ligands analyzed was not altered in the PVN. The results suggest an acute down-regulation of alpha-2-adrenergic and NPY receptors involved with hypotension in response to hypertensive stimulus, which might be related to an increased availability of catecholamines and NPY in the NTS.     

外源性皮质酮对大鼠主动脉内皮细胞热休克蛋白70表达的影响

目的在细胞水平观察皮质酮对热休克蛋白70（HSP70）表达的影响及量效动态变化。方法采用培养的大鼠主动脉内皮细胞,观察在不同剂量皮质酮（0．1μM,50μM,100μM）干预2h～72h后,内皮细胞热HSP70的表达情况。结果皮质酮干预大鼠主动脉内皮细胞后,HSP70蛋白表达在4h达到高峰,其中干预浓度为0．1μM组的HSP70蛋白含量增加最为明显,到72h时与正常对照组已无显著差异。结论不同干预剂量的皮质酮均可促使大鼠主动脉内皮HSP70表达增加,应激时大量分泌的皮质酮可能是通过HSP70表达增加参与内皮细胞的保护。     

Arginine induced acute pancreatitis alters the actin cytoskeleton and increases heat shock protein expression in rat pancreatic acinar cells

Arginine induced acute pancreatitis was evaluated as a novel and distinct form of experimental pancreatitis with particular attention to the actin cytoskeleton and expression of heat shock or stress proteins. Arginine induced a dose related necrotising pancreatitis in rats, as shown by histological evaluation, and an increase in serum amylase. Severe pancreatitis induced by 4.5 g/kg arginine was accompanied by dramatic changes in the actin cytoskeleton, as visualised with rhodamine phallodin. Intermediate filaments were also disrupted, as visualised by cytokeratin 8/18 immunocytochemistry. Arginine pancreatitis was accompanied by a stress response with a large increase in the small heat shock protein HSP27, as well as HSP70, peaking at 24 hours and localised to acinar cells. There was a lower increase in HSP60 and HSP90 and no effect on GRP78. HSP27 was also shifted to phosphorylated forms during pancreatitis. A lower dose of arginine (3.0 g/kg) induced less pancreatitis but a larger increase in HSP70 and HSP27 expression and phosphorylation of HSP27. Thus HSP expression can be overwhelmed by severe damage. The present work in conjunction with earlier work on caerulein induced pancreatitis indicates that changes in the actin cytoskeleton are an early component in experimental pancreatitis.     

Both thermal and non-thermal stress protect against caerulein induced pancreatitis and prevent trypsinogen activation in the pancreas.

BACKGROUND AND AIM: Recent studies have indicated that prior thermal stress causes upregulation of heat shock protein 70 (HSP70) expression in the pancreas and protects against secretagogue induced pancreatitis. The mechanisms responsible for the protective effect are not known. Similarly, the effects of prior non-thermal stress on HSP70 expression and pancreatitis are not known. The current studies were designed to specifically address these issues. METHODS: In the current studies pancreatitis was induced by administration of a supramaximally stimulating dose of caerulein 12 hours after thermal stress and 24 hours after non-thermal (that is, beta adrenergic stimulation) stress. RESULTS: Both thermal and non-thermal stresses caused pancreatic HSP70 levels to rise and resulted in increased expression of HSP70 in acinar cells. Both forms of stresses protected against caerulein induced pancreatitis and prevented the early intrapancreatic activation of trypsinogen which occurs in this model of pancreatitis. CONCLUSIONS: These results suggest that both thermal and non-thermal stresses protect against pancreatitis by preventing intrapancreatic digestive enzyme activation and that HSP70 may mediate this protective effect.     

Protection against necrosis but not apoptosis by heat-stress proteins in vascular smooth muscle cells: evidence for distinct modes of cell death

We have reported previously that cultured vascular smooth muscle cells (VSMC) isolated from spontaneously hypertensive rats (SHR) show higher proliferation and cell death than normotensive controls. In addition to protecting cells against death, heat stress proteins (HSPs) appear to play a role in cell proliferation. This investigation examines the involvement of HSP72 and HSP27 in altered SHR VSMC proliferation and death. We have performed detailed discriminatory analysis to characterize which type of VSMC death is induced by heat stress (HS) and serum deprivation. Serum deprivation induced apoptosis (caspase-3 cleavage and DNA laddering) and secondary necrosis, the 2 processes being a continuum of each other. In contrast, acute HS (46 degrees C, 30 minutes), which inhibited BN. lx and SHR VSMC proliferation by 2-fold, increased necrosis (by 5-fold and 2-fold, respectively) but not apoptosis. HSP72 and HSP27 expression evoked in VSMC by mild HS (44 degrees C, 15 minutes) 6 hours before acute HS prevented the inhibition of proliferation and induction of necrosis with no effect on serum deprivation-induced or staurosporine-induced apoptosis. This induced expression of HSP72 and HSP27 did not eliminate the higher basal proliferation, apoptosis, and necrosis of SHR VSMC compared with BN.lx VSMC, suggesting that these HSPs are not involved in altered SHR VSMC proliferation and death. Also, although apoptosis and necrosis may be a continuum, in VSMC the 2 processes may be distinguished by HS, in which only necrosis is prevented by prior HSP accumulation. This observation may be of use in designing strategies for cellular protection.     

Mitochondria are selective targets for the protective effects of heat shock against oxidative injury.

Heat shock (HS) proteins (HSPs) induce protection against a number of stresses distinct from HS, including reactive oxygen species. In the human premonocytic line U937, we investigated in whole cells the effects of preexposure to HS and exposure to hydrogen peroxide (H2O2) on mitochondrial membrane potential, mass, and ultrastructure. HS prevented H2O2-induced alterations in mitochondrial membrane potential and cristae formation while increasing expression of HSPs and the protein product of bcl-2. Protection correlated best with the expression of the 70-kDa HSP, hsp70. We propose that mitochondria represent a selective target for HS-mediated protection against oxidative injury.     

Heat shock proteins and autophagy in rats with cerulein-induced acute pancreatitis

Background/Aims

Heat shock proteins (HSPs) protect rats from cerulein-induced acute pancreatitis (AP) by preventing the subcellular redistribution of cathepsin B and the activation of trypsinogen. Autophagy plays a critical role in the secretion of digestive enzymes and triggering of cerulein-induced AP via the colocalization of trypsinogen and lysosomes. Therefore, using a rat cerulein-induced AP model, we investigated whether HSPs prevent AP by regulating autophagy.

Methods

Twelve hours after fed standard laboratory chow and water, the experimental groups (cerulein, water-immersion [WI]-cerulein and heat-shock [HS]-cerulein) and the control groups (control, WI, and HS) received one intraperitoneal injection of cerulein (50 µg/kg) or saline, respectively. All of the rats were sacrificed at 6 hours after injection. The severity of the AP was assessed based on the serum amylase level and the histological and electron microscopy findings. Western blotting was also performed for HSP60/70 and LC3B-II.

Results

WI and HS induced HSP60 and HSP70, respectively. The induced HSP60/70 effectively prevented the development of cerulein-induced AP. Autophagy developed in the rats with cerulein-induced AP and was documented by the expression of LC3-II and electron microscopy findings. The WI-stressed rats and HS-treated rats did not develop cerulein-induced autophagy.

Conclusions

HSPs exert protective effects against cerulein-induced AP in rats by inhibiting autophagy.     

Salusin β Within the Nucleus Tractus Solitarii Suppresses Blood Pressure Via Inhibiting the Activities of Presympathetic Neurons in the Rostral Ventrolateral Medulla in Spontaneously Hypertensive Rats

Salusin β is a newly identified bioactive peptide, which shows peripheral hypotensive, mitogenic and proatherosclerotic effects. The present study was undertaken to investigate the role of salusin β within the nucleus tractus solitarii (NTS) and the underlying mechanism in regulating blood pressure and heart rate (HR) in spontaneously hypertensive rats (SHR). Our results showed that bilateral or unilateral microinjection of salusin β (0.4–40 pmol) into the NTS in SHR decreased mean arterial pressure and HR in a dose-dependent manner. Bilateral microinjection of salusin β (4 pmol) within NTS improved baroreflex sensitivity functions in SHR. Pretreatment with glutamate receptors antagonist kynurenic acid (5 nmol) into the NTS in SHR did not alter the salusin β (4 pmol) induced hypotension and bradycardia. Likewise, bilateral vagotomy also did not alter the salusin β (4 pmol) induced hypotension and bradycardia. However, pretreatment with GABA_A receptors agonist muscimol (100 pmol) within the rostral ventrolateral medulla (RVLM) in SHR almost completely abolished the hypotension and bradycardia evoked by intra-NTS salusin β (4 pmol). Our findings suggested that microinjection of salusin β into the NTS produced hypotension and bradycardia, as well as improved baroreflex sensitivity functions, via inhibiting the activities of presympathetic neurons in the RVLM in SHR.     

去甲肾上腺素预处理对大鼠供心热休克蛋白70、一氧化氮及一氧化氮合酶表达的影响

目的:探讨去甲肾上腺素预处理是否可诱导心肌热休克蛋白70(HSP70)的合成,并研究其对供心一氧化氮(NO)、一氧化氮合酶(NOS)的影响,探讨去甲肾上腺素预处理心肌保护作用机制。方法:Wistar大鼠18只,分为2组:对照组(C,n=9),腹腔注射0.9%氧化钠注射液0.5 mL,24 h后取离体心脏灌注(Histidine-tryptophan-ketoglutarte,HTK)心脏保护液,4℃保存3 h后建立Langendorff离体心脏灌注模型,灌注(Krebs-Henseleit,K-H)液2 h;实验组(E,n=9)腹腔注射重酒石酸去甲肾上腺素(溶于0.9%氯化钠液中)3.1μmol/kg(0.53 mg/kg),腹腔注射24 h后取离体心脏,处理方法同C组。测定心肌HSP70、NO、NOS的含量以及相关生化指标并做统计学处理比较。结果:HSP70含量E组较C组明显增高(P<0.01),NO、NOS的含量E组较C组明显增多(P<0.01),生化指标E组明显优于C组。结论:去甲肾上腺素预处理能诱导供心心肌组织HSP70、NO、NOS高表达,其对供心具有明显的保护效应,并且其促进心肌NO、NOS的表达,这可能是去甲肾上腺素预处理发挥供心保护作用的机制之一。