Stimulation of the adenosine A3 receptor reverses vascular hyporeactivity after hemorrhagic shock in rats.

AIM: To investigate whether adenosine A(3) receptors (A(3)AR) stimulation restore vascular reactivity after hemorrhagic shock through a ryanodine receptor (RyR)-mediated and large conductance calcium-activated potassium (BK(Ca)) channel-dependent pathway. METHODS: Rat hemorrhagic shock model (40 mmHg) and vascular smooth muscle cell (VSMC) hypoxic model were used. The expression of A(3)AR was determined by Western blot and RT-PCR. The effect of A(3)AR stimulation on RyR-mediated Ca(2+) release in VSMCs was analyzed by the Fura-3/AM loading Ca(2+) imaging. The modulation of vascular reactivity to norepinephrine (NE) by A(3)AR stimulation was monitored by an isolated organ tension instrument. RESULTS: Decrease of A(3)AR expression is consistent with the loss of vasoreactivity to NE in hemorrhagic shock rats. The stimulation of A(3)AR with a selective agonist, IB-MECA, could partly but significantly restore the vasoreactivity in the rats, and this restorative effect could be counteracted by MRS1523, a selective A(3)AR antagonist. In hypoxic VSMCs, RyR activation by caffeine significantly evoked the rise of [Ca(2+)] compared with the control cells, a phenomenon closely associated with the development of vascular hyporeactivity in hemorrhagic shock rats. The stimulation of A(3)AR with IB-MECA significantly blocked this over activation of RyR-mediated Ca(2+) release. RyR activation by caffeine and BK(Ca) channel activation by NS1619 attenuated the restoration of vasoreactivity to NE resulting from A(3)AR stimulation by IB-MECA after hemorrhagic shock; this attenuation effect could be antagonized by a selective BK(Ca) channel blocker. CONCLUSION: These findings suggest that A(3)AR is involved in the modulation of vasoreactivity after hemorrhagic shock and that stimulation of A(3)AR can restore the decreased vasoreactivity to NE through a RyR-mediated, BK(Ca) channel-dependent signal pathway.

[The role of interferon-gamma in prevention of hydrogen peroxide-induced oxidative injury to vascular endothelium].

OBJECTIVE: To observe the protective effects of interferon-gamma (IFN-gamma) on hydrogen peroxide (H(2)O(2))-induced oxidative injury to vascular endothelial cell (VEC), and to explore the role of 26S proteasome protection against IFN-gamma-induced endothelial oxidative injury. METHODS: A H(2)O(2)-induced VEC oxidative injury to cell and isolated organ models were reproduced in the current study. The VEC oxidative damage in cellular level was evaluated by the contents of lactate dehydrogenase (LDH) and malondialdehyde (MDA) in culture medium, and the degree of oxidative injury of VEC in isolated organ level was evaluated by acetylcholine (Ach)-induced endothelium- dependent vascular relaxation. RESULTS: H(2)O(2) triggered the oxidative injury in cultured VEC in a dose- and time-dependent manner, characterized by an increased contents of LDH and MDA in culture medium (P<0.05 or P<0.01). The Ach-induced endothelium- dependent vascular relaxation was decreased, and as shown by a decrease in 10(-5) mol/L Ach induced maximum reaction of relaxation of vasculature from (95.82+/-9.25)% to (12.61+/-2.96)% (P<0.01). The damage to VEC induced by H(2)O(2) was diminished significantly after incubation with IFN-gamma (20 microg/L) for 48 hours, characterized by a decreased production of LDH and MDA (both P<0.05) and restoration of endothelium- dependent vasodilation, and Ach-induced maximum reaction of vascular relaxation was increased to (72.68+/-18.82)% (P<0.01). 26S proteasome inhibitor lactacystin could partly antagonize the protective effects of IFN-gamma on H(2)O(2)-induced oxidative injury. CONCLUSION: IFN-gamma possesses protective effects on H(2)O(2)-induced oxidative injury to vascular endothelium, and its mechanism is at least partly related with 26S proteasome.

HSD is a better resuscitation fluid for hemorrhagic shock with pulmonary edema at high altitude.

To investigate the fluid tolerance of hemorrhagic shock with pulmonary edema (HSPE) at high altitude in unacclimated rats and the beneficial effect of 7.5% hypertonic saline/6% dextran (HSD). One hundred seventy-six Sprague-Dawley rats, transported to LaSa, Tibet, 3,760 m above the sea level, were anesthetized with sodium pentobarbital (30 mg/kg, i.p.) within 1 week. Hemorrhagic shock with pulmonary edema was induced by bloodletting (50 mmHg for 1 h) plus intravenous injection of oleic acid (50 microL/kg). Seventy-seven rats were equally divided into 11 groups (n = 7/group) including sham-operated control group; hemorrhagic shock control group; HSPE control group; HSPE plus 0.5-, 1.0-, 1.5-, 2.0-, or 3.0-fold volumes of lactated Ringer's solution (LR) groups; and HSPE plus 4, 6, and 8 mL/kg of HSD groups. Hemodynamic parameters including mean arterial blood pressure, left intraventricular systolic pressure, and the maximal change rate of intraventricular pressure rise or decline (+/-dp/dtmax) were observed at baseline and at 15, 30, 60, and 120 min after infusion; blood gases were measured at 30 and 120 min after infusion, and the water content of lung and brain was determined at 120 min after infusion. Additional 99 rats were used to observe the effect of these treatments on the survival time of HSPE rats; 0.5 volume of LR infusion slightly increased the mean arterial blood pressure, left intraventricular systolic pressure, and +/-dp/dtmax and prolonged the survival time of HSPE animals as compared with the HSPE group (P < 0.05 - 0.01); it did not increase the water content of lung and brain and had no marked influences on blood gases. One volume of LR infusion had somewhat improved the hemodynamic parameters for HSPE animals, but had no apparent effect on the survival time and the water content of lung and brain. Lactate Ringer's solution infusion, 1.5, 2, and 3 volumes, significantly deteriorated the hemodynamic parameters, increased the water content of lung, and decreased the survival time of HSPE animals. Hypertonic saline/6% dextran (4 - 8 mL/kg) significantly increased the hemodynamic parameters, improved the blood gases, decreased the water content of lung and brain, and prolonged the survival time of HSPE rats. Among the three dosages of HSD, 6 mL/kg of HSD had the best effect. The tolerance of fluid infusion for hemorrhagic shock with pulmonary edema at high altitude is significantly decreased. More than one volume of LR infusion would aggravate the pulmonary edema and exacerbate the resuscitation effect, but only one volume of LR cannot reach the effective volume resuscitation. Small volume of HSD could better resuscitate hemorrhagic shock with pulmonary edema at high altitude.

Subclass opioid receptors associated with the cardiovascular depression after traumatic shock and the antishock effects of its specific receptor antagonists.

The present available opioid receptor antagonists such as naloxone and naltrexone are not highly receptor selective. They may antagonize mu opioid receptors to affect the pain threshold of the patients with traumatic shock while they exert antishock effects. Therefore, they are not suitable for traumatic shock. It is very important to elucidate the subclass of opioid receptors that are closely associated with cardiovascular depression of traumatic shock and then choose their specific receptor antagonists to treat it. Traumatic shock was used in pentobarbital-anesthetized Wistar rats by right femur fracture plus hemorrhage (fixed hemorrhage at a rate of 20 mL/kg in experiment 1 or hemorrhage to 40 mmHg mean arterial blood pressure for 60 min in experiments 2 and 3), and the changes of myocardial and brain opioid receptors after traumatic shock, the antagonizing effects of mu, delta, and kappa opioid receptor antagonists on the cardiovascular depression of traumatic shock and the antishock effects of delta and kappa opioid receptor antagonists ICI174,864 and Nor-binaltorphimine (Nor-BNI) were observed. The results indicate that after traumatic shock, the number of myocardial and brain delta and kappa opioid receptors were significantly increased that were significantly associated with the decreased cardiovascular functions. mu Opioid receptors in the heart and brain did not change significantly. Intracerebral ventricular administration of ICI174,864 and Nor-BNI significantly antagonized the decreased cardiovascular function after traumatic shock and increased the survival rate of traumatic shock rats, but mu opioid receptor antagonist beta-funaltrexamine did not. Meanwhile, intravenous administration of delta and kappa opioid receptor antagonists ICI174,864 and Nor-BNI also significantly increased the mean arterial blood pressure, improved the hemodynamic parameters, and prolonged the survival rate of traumatic shock rats. These findings suggest that opioid receptors are involved in the cardiovascular depression of traumatic shock, and the subclass receptors are mainly delta and kappa opioid receptors. delta and kappa opioid receptor antagonists have good beneficial effects on traumatic shock.

Effect of nitric oxide-induced tyrosine phosphorylation of calcium-activated potassium channel alpha subunit on vascular hyporesponsiveness in rats.

OBJECTIVE: To study the effect of nitric oxide-induced tyrosine phosphorylation of large-conductance calcium-activated potassium (BK(Ca)) channel alpha subunit on vascular hyporesponsiveness in rats. METHODS: A total of 46 Wistar rats of either sex, weighing 250 g +/- 20 g, were used in this study. Models of vascular hyporesponsiveness induced by hemorrhagic shock (30 mm Hg for 2 hours) in vivo and by L-arginine in vitro were established respectively. The vascular responsiveness of isolated superior mesenteric arteries to norepinephrine was observed. Tyrosine phosphorylation of BK(Ca) alpha subunit was evaluated with methods of immunoprecipitation and Western blotting. RESULTS: In the smooth muscle cells of the superior mesenteric arteries, the expression of BK(Ca) alpha subunit tyrosine phosphorylation increased following hemorrhagic shock, and L-arginine could induce BK(Ca )channel alpha subunit tyrosine phosphorylation in a time- and dose-dependent manner. L-NAME (Nomega-nitro-L-arginine-methyl-ester), a nitric oxide synthetase inhibitor, could partly restore the decreased vasoresponsiveness of the superior mesenteric arteries after hemorrhagic shock in rats. Down-regulating the protein tyrosine phosphorylation with genistein, a widely-used special protein tyrosine kinase inhibitor, could partly improve the decreased vasoresponsiveness of the superior mesenteric arteries induced by L-arginine in vitro, while up-regulating the protein tyrosine phosphorylation with Na(3)VO(4), a protein tyrosine phosphatase inhibitor, could further decrease the nitric oxide-induced vascular hyporesponsiveness, which could be partly ameliorated by 0.1 mmol/L tetrabutylammonium chloride (TEA), a selective BK(Ca )inhibitor at this concentration. CONCLUSIONS: Nitric oxide can induce the tyrosine phosphorylation of BK(Ca) alpha subunit, which influences the vascular hyporesponsiveness in hemorrhagic shock rats or induced by L-arginine in vitro.

Opioid receptor antagonists increase [Ca2+]i in rat arterial smooth muscle cells in hemorrhagic shock.

AIM: To examine the effects of opioid receptor antagonists and norepinephrine on intracellular free Ca2+ concentration ([Ca2+]i) in mesenteric arterial (MA) smooth muscle cells (SMC) isolated from normal and hemorrhagic shocked rats in the vascular hyporesponse stage. METHODS: The rat model of hemorrhagic shock was made by withdrawing blood to decrease the artery mean blood pressure to 3.73-4.26 kPa and keeping at the level for 3 h. [Ca2+]i of vascular smooth muscle cells (VSMC) were detected by the laser scan confocal microscopy. RESULTS: In the hyporesponse VMSC of rats in hemorrhagic shock, selective delta-, kappa-, and mu-opioid receptor antagonists (naltrindole, nor-binaltorphimine, and beta-funaltrexamine, 100 nmol/L) as well as norepinephrine 5 micromol/L significantly increased [Ca2+]i by 47 %+/-13 %, 37 %+/-14 %, 33 %+/-10 %, and 54 %+/-17 %, respectively, although their effects were lower than those in the normal rat cells (the increased values were 148 %+/-54 %, 130 %+/-44 %, 63 %+/-17 % and 110 %+/-38 %, respectively); and the norepinephrine-induced increase in [Ca2+]i was further augmented by three delta-, kappa-, and mu-opioid receptor antagonists (50 nmol/L, respectively) application (from 52 %+/-16 % to 99 %+/-29 %, 146 %+/-54 % and 137 %+/-47 %, respectively). CONCLUSION: The disorder of [Ca2+]i regulation induced by hemorrhagic shock was mediated by opioid receptor and alpha-adrenoceptor, which may be partly responsible for the vascular hyporesponse, and the opioid receptor antagonists improved the response of resistance arteries to vascular stimulants in decompensatory stage of hemorrhagic shock.

The effect of different volumes of fluid resuscitation on traumatic-hemorrhagic shock at high altitude in the unacclimated rat.

The effects of different volumes of fluid resuscitation on traumatic hemorrhagic shock in unacclimated rats to high altitude were investigated. Seventy-eight Wistar rats were transported to LaSa, Tibet, 3760 meters above sea level, and traumatic hemorrhagic shock was induced by right-femur fracture plus bleeding to 45 mmHg of mean arterial pressure (MAP) for 1 h under the anesthesia of sodium pentobarbital (40 mg/kg, i.p.). Experiments were conducted in two series. In series I, 36 rats were equally divided into six groups: sham-operated, untreated (traumatic shock without fluid infusion), and with lactated Ringer's resuscitation (LR) of 1.0, 1.5, 2.0 or 3.0 times the shed blood (1, 1.5, 2, 3 vol LR group). MAP, left intraventricular systolic pressure, the maximal change rate of intraventricular pressure rise or decline (+/-dp/dtmax), the maximal physiological velocity of contractile element shortening, and the area of left intraventricular pressure-dp/dt vector loop (Lo) were observed at 30, 60, 90, and 120 min and the blood gases were determined at 30 and 120 min after resuscitation. Meanwhile the survival time was observed after the observation period. In series II, 42 rats were used to observe the effects of different volumes of fluid resuscitation on water content of lung and brain and hematocrit. One and 1.5 vol LR resuscitation significantly lifted MAP, left intraventricular systolic pressure, +dp/dtmax, and Lo, partially improved the blood gases and significantly prolonged the survival time. Although 2 and 3 vol of LR resuscitation caused apparent hemodilution and lung edema, they only partially improved hemodynamic parameters. Meanwhile 2 and 3 vol of LR resuscitation decreased the survival time. These results suggest that 1 and 1.5 vol of LR resuscitation can effectively resuscitate traumatic hemorrhagic shock at high altitude. More than two volumes of LR resuscitation would deteriorate the resuscitation outcome.

[Beneficial effect of thyrotropin-releasing hormone in combination with HSD on hemorrhagic shock with pulmonary edema at high altitude in the rat].

OBJECTIVE: To study the effects of thyrotropin-releasing hormone (TRH) in combination with hypertonic saline/dextran (7.5% NaCl + 6% Dextran 40, HSD ) on hemorrhagic shock with pulmonary edema in the rats which were recently brought to high altitude. METHODS: Forty-nine SD rats, transported to Lasa, Tibet, which was 3,760 meters above the sea level, were anesthetized one week later with sodium pentobarbital (30 mg/kg, intraperitoneal). Hemorrhagic shock with pulmonary edema was induced by hemorrhage (50 mm Hg maintained for 1 hour,1 mm Hg=0.133 kPa) plus intravenous injection of oleic acid (50 microl/kg). They were equally divided into seven groups (n=7): normal control, hemorrhagic shock, hemorrhagic shock with pulmonary edema (HSPE), HSPE plus TRH (5 mg/kg), HSPE plus HSD (4 ml/kg), and HEPE plus TRH and HSD in combination. Hemodynamic parameters including mean arterial blood pressure(MAP), left intraventricular systolic pressure (LVSP) and the maximal change rate of intraventricular pressure rise or decline (+/- dp/dt max) were observed at 15, 30, 60 and 120 minutes, blood gases were analyzed at 30 and 120 minutes, and the water content of lung and brain was determined at 120 minutes after drug administration. RESULTS: TRH or HSD used alone or in combination significantly increased MAP, LVSP and +/- dp/dt max (P<0.05 or P<0.01 ), ameliorated acid-base imbalance, and decreased the water content of lung and brain. The effect of the two in combination was superior to either drug used alone. CONCLUSION: TRH in combination with HSD can be used in the treatment of hemorrhagic shock with pulmonary edema at high altitude.

[Effect of different volumes of fluid resuscitation on hemorrhagic shock with pulmonary edema at high altitude in the unacclimated rat].

OBJECTIVE: To study the effects of different volumes of fluid resuscitation on hemorrhagic shock with pulmonary edema at high altitude in the unacclimated rat. METHODS: One hundred and twenty-six SD rats transported to Lasa, Tibet, 3 760 meters above the sea level, were anesthetized one week later with sodium pentobarbital (30 mg/kg, intraperitoneal). Hemorrhagic shock with pulmonary edema model was induced by hemorrhage (50 mm Hg for 1 hour, 1 mmHg=0.133 kPa) plus intravenous injection of oleic acid (50 microl/kg). Experiments were then conducted in two parts. Sixty-three rats in part I were equally divided into nine groups (n=7): normal control, hemorrhagic shock control, hemorrhagic shock with pulmonary edema (HSPE) without fluid infusion, HSPE plus infusing lactated Ringer's solution (LR) with 0.5-, 1-, 1.5-, 2- or 3- fold volume shed blood, and 1 volume of LR plus mannitol (10 ml/kg). Hemodynamic parameters including mean arterial blood pressure (MAP), left intraventricular systolic pressure (LVSP) and the maximal change rate of intraventricular pressure rise or decline (+/- dp/dt max) were observed at 15, 30, 60 and 120 minutes after infusion, blood gases were measured at 30 and 120 minutes after infusion and the water content of lung and brain was determined at 120 minutes after infusion. In part II, additional 63 rats were used to observe the effect of different volumes of fluid resuscitation on survival time of HSPE rats. RESULTS: 0.5 volume of LR infusion significantly improved MAP, LVSP and +/- dp/dt max, prolonged the survival time of HSPE animals (all P<0.01), while it did not increase the water content of lung and brain and had no marked influence on blood gases. One volume of LR infusion slightly improved hemodynamic parameters, prolonged the survival time and increased the water content of lung. More than 1 volume of LR infusion including 1.5-, 2- and 3- fold volume LR deteriorated the hemodynamic parameters and decreased the survival time of shocked animal, meanwhile they apparently increased the water content of lung. One volume of LR plus mannitol (10 ml/kg) infusion did not improve the hemodynamic parameters and blood gases; also it did not decrease the water content of lung. CONCLUSION: The tolerance to fluid infusion for the unacclimated animal subjected to hemorrhagic shock with pulmonary edema at high altitude is significantly decreased. 0.5-1 volume of LR infusion appears to be beneficial effect on resuscitation at high altitude, while over 1 volume of LR infusion would aggravate pulmonary edema and exacerbate fluid resuscitation effect.

Opioid receptor antagonists modulate Ca2+-activated K+ channels in mesenteric arterial smooth muscle cells of rats in hemorrhagic shock.

Previous study has indicated a significant enhancement of activity of large-conductance Ca2+-activated K+ channel (BKCa) in mesenteric arterial vascular smooth muscle cells isolated from rats in vascular hyporesponsive stage of hemorrhagic shock. In the present study, the effect of opioid receptor antagonism on BKCa activity in the vascular smooth muscle cells of rats in the hyporesponse stage of hemorrhagic shock was investigated by using inside-out configuration of the patch-clamp technique. The results showed that naloxone (10 microM) down-regulated the activity of BKCa by reducing open probability (Po) and open frequency of the channels. The reduction of Po resulted from a decrease of mean open time and an increase of the slow closed time constant. Naltrindole and nor-binaltorphimine (100 nM) had the similar effects to that of naloxone, but no significant effect of beta-funaltrexamine (100 nM) on the activity of the channels could be found. These results suggest that delta- and kappa-opioid receptors, but not mu-receptors, may be involved in the regulation of BKCa in vascular hyporesponse stage, and that inhibition of BKCa may be one of the mechanisms of the opioid receptor antagonists improving the response of resistance arteries to vasoactive stimulants during the decompensatory stage of hemorrhagic shock.

Protective effects of apocynin on "two-hit" injury induced by hemorrhagic shock and lipopolysaccharide.

AIM: To evaluate the protective effects of apocynin on "two-hit" injury in rats. METHODS: "Two-hit" injury model of rat was induced by hemorrhagic shock (40 mmHg for 45 min) followed by iv administration of lipopolysaccharide (LPS, 150 microg/kg). Rats were randomized into seven groups: Sham, LPS, hemorrhage, hemorrhage/LPS, and hemorrhage/LPS+apocynin (2.5, 5.0, and 10.0 mg/kg). Apocynin was dissolved in the resuscitation fluid (normal saline, NS) and administered iv for 2 h. After LPS or NS administration, the survival rates at 8 h, 16 h, 24 h, and 48 h were monitored. The content of malondialdehyde (MDA) was measured in lung at 3 h and 6 h after iv LPS and in serum before hemorrhage, after hemorrhage, and at 0, 0.5, 1, 2, 4, and 6 h after iv LPS. Myeloperoxidase (MPO) activity in lung and liver was examined at 3 h and 6 h after iv LPS/NS. RESULTS: After "two-hit" injury, the survival rates of rats at 8 h, 16 h, 24 h, and 48 h were 64.3 %, 35.7 %, 28.6 %, and 14.3 % respectively, there were significant differences as compared to sham group (P<0.05 or P<0.01, respectively), the MDA level in lung and serum were significantly enhanced (P<0.01) as compared to sham group, and MPO activity in lung and liver after "two-hit" injury was also significantly increased (P<0.01). Apocynin treatment enhanced the mean arterial pressure (MAP) of hemorrhagic shock rats dose-dependently (P<0.05), increased the survival rate of "two-hit" injury rats, decreased the serum and lung MDA content, and downregulated MPO activity in lung and liver. CONCLUSION: Apocynin could preventively ameliorate "two-hit" injury in rats induced by hemorrhagic shock and LPS insult.

Modulation of Ca2+ channels by opioid receptor antagonists in mesenteric arterial smooth muscle cells of rats in hemorrhagic shock.

The effects of hemorrhagic shock on Ba currents ( ) via Ca channels and the regulation of the channels in the vascular hyporesponse stage of hemorrhagic shock by opioid receptor antagonists were examined by using the whole-cell recording of patch-clamp technique in mesenteric arterial smooth muscle cells of rats. The results showed that hemorrhagic shock induced an inhibition of Ca channels in the cells; 10 micro M of naloxone and 100 n of naltrindole, nor-binaltorphimine, and beta-funaltrexamine increased the in the cells of rats in shock. After inhibition of protein kinase C by using 1-(5-isoquindinesulfonyl)-2-methylpiperazine via electrodes, the enhancement of by the antagonists was not observed. These results suggested that the inhibition of Ca channel induced by hemorrhagic shock was mediated by delta-, kappa-, and mu -opioid receptors in the cells and may be partly responsible for vascular hyporesponse. The enhancement of was mediated by activation of protein kinase C and may be responsible for the antagonist-caused improvement in the response of resistance arteries to vasoactive stimulants at the decompensatory stage of hemorrhagic shock.