Anders Rudehill

The endothelin system in septic and endotoxin shock

The view of the endothelium as a passive barrier has gradually changed as a number of endothelium-derived substances have been discovered. Substances like nitric oxide, prostaglandins and endothelins have potent and important properties, involving not only the circulation as such but also the response to stimuli like inflammation and trauma. The endothelin system, discovered in 1988, has not only strong vasoconstrictor properties, but also immunomodulating, endocrinological and neurological effects exerted through at least two types of receptors. Septic shock, a condition with high mortality, is associated with vast cardiovascular changes, organ dysfunction with microcirculatory disturbances and dysoxia. In the experimental setting, endotoxaemia resembles these changes and is, as well as septic shock, accompanied by a pronounced increase in plasma endothelin levels. The pathophysiology in septic and endotoxin shock remains to be fully elucidated, but several studies indicate that endothelial dysfunction is one contributing mechanism. Activation of the endothelin system is associated with several pathological conditions complicating septic shock, such as acute respiratory distress syndrome, cardiac dysfunction, splanchnic hypoperfusion and disseminated intravascular coagulation. Through the development of both selective and nonselective endothelin receptor antagonists, the endothelin system has been the object of a large number of studies during the last decade. This review highlights systematically the findings of previous studies in the area. It provides strong indications that the endothelin system, apart from being a marker of vascular injury, is directly involved in the pathophysiology of septic and endotoxin shock. Interventions with endothelin receptor antagonists during septic and endotoxin shock have so far only been done in animal studies but the results are interesting and promising.

Neuropeptide Y receptor in pig spleen: binding characteristics, reduction of cyclic AMP formation and calcium antagonist inhibition of vasoconstriction.

Specific, high-affinity binding sites for 125I-porcine neuropeptide Y (NPY) were demonstrated in membranes from the pig spleen. The equilibrium dissociation constant (KD) of the receptor 125I-NPY complex was 532 +/- 87 pM and the maximal number of specific binding sites (Bmax) 23 +/- 3 fmol/mg protein. The Scatchard plot for 125I-NPY binding under equilibrium conditions showed a best-fit to a straight line, whereas the dissociation appeared biphasic. 125I-NPY binding was unaffected by adrenoceptor antagonists and was inhibited by the guanosine triphosphate (GTP) analogue guanylylimidodiphosphate, suggesting regulation by a GTP binding protein. A series of NPY analogues showed a good correlation between binding, inhibition of forskolin-induced cyclic adenosine monophosphate (cAMP) formation and vasoconstrictor activity in vivo. A large carboxyl terminal portion of NPY and the carboxyl terminal amide were essential for binding, inhibition of cAMP formation and vasoconstrictor effects. The NPY fragment 13-36, which has been reported to act only on prejunctional NPY receptors, showed only a 10-fold lower potency than NPY-(1-36) both in binding to splenic membranes and vasoconstrictor activity in vivo. Phenylephrine increased phosphatidyl inositol turnover whereas NPY-(1-36) or -(13-36) did not induce formation of inositol phosphates. The calcium antagonists felodipine and nifedipine attenuated the splenic vasoconstrictor response to NPY in vivo but not the NPY-evoked inhibition of cAMP accumulation or the specific binding of 125I-NPY.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of levosimendan, a novel inotropic calcium-sensitizing drug, in experimental septic shock.

Objective Levosimendan is a novel inodilator that improves cardiac contractility by sensitizing troponin C to calcium. This drug has proved to be effective in treating advanced congestive heart failure but has not been evaluated in septic settings. The purpose of the present study was to study the effects of this drug in a porcine model of endotoxemia. Design Prospective experimental study. Subjects Fourteen landrace pigs. Interventions All animals were anesthetized and catheterized for measurement of central and pulmonary hemodynamics. Ultrasonic flow probes were placed around the renal artery and portal vein to measure blood flow. A tonometer was placed in the ileum to measure mucosal pH. Levosimendan was given to six animals as a bolus (200 &mgr;g·kg−1) followed by a continuous infusion (200 &mgr;g·kg−1· hr−1). Thirty minutes after onset of levosimendan treatment, all animals received endotoxin (20 &mgr;g·kg−1·hr−1 for 3 hrs). Measurements and Main Results At baseline, levosimendan induced a systemic vasodilation with a reduction in blood pressure and an increase in heart rate. A tendency to an increase in cardiac index did not reach statistical significance (p = .055).Cardiac index and systemic oxygen delivery were markedly improved in the levosimendan group during endotoxemia. Systemic vascular resistance and blood pressure were reduced in the levosimendan group. The latter parameter, however, was only different from the control group during the initial phase of endotoxin shock but not at the late, most pronounced phase of shock. Levosimendan also efficiently attenuated endotoxin-induced pulmonary hypertension. Portal venous blood flow and gut oxygen delivery were improved, but no concomitant reduction in endotoxin-induced intestinal mucosal acidosis was observed. Renal blood flow was unaffected, as was the endotoxin-induced increase in plasma endothelin-1-like immunoreactivity.These findings support previous reports of calcium desensitization as a potential component in septic myocardial depression. Furthermore, the vasodilatory properties of this drug were well tolerated in the current model of hypodynamic endotoxin shock, and they may have contributed to improved regional blood flow as seen in the gut as well as improved systemic perfusion by means of reduced biventricular afterload. Conclusion Pretreatment with levosimendan in pigs subjected to endotoxin shock improved cardiac output and systemic and gut oxygen delivery. In addition, pulmonary hypertension largely was attenuated without any adverse effects on gas exchange. These results are promising in several aspects, but the role of levosimendan in the treating circulatory failure in sepsis remains to be established.

Calcitonin gene-related peptide but not substance P mimics capsaicin-induced coronary vasodilation in the pig

The vasodilator effects of the human calcitonin gene-related peptides alpha (hCGRP alpha) and beta (hCGRP beta) were studied in vitro and in vivo in relation to the effects of substance P (SP) and capsaicin on coronary vascular tone in the pig. Both hCGRP alpha and -beta induced a concentration-dependent, long-lasting relaxation of precontracted small (diameter 0.5 mm) pig coronary arteries in vitro. SP was slightly more potent but caused a transient relaxation with a smaller maximal response than CGRP. The relaxation induced by hCGRP alpha and -beta as well as SP was resistant to propranolol and atropine. Capsaicin also induced a long-lasting relaxation of potassium and PGF2 alpha-precontracted coronary arteries. After tachyphylaxis to SP had developed the relaxant effects of CGRP and capsaicin were unchanged. Rubbing the vessels to remove the endothelium completely abolished the relaxant effects of SP while the vasodilation induced by hCGRP alpha as well as capsaicin remained unchanged. Injections of hCGRP alpha, SP or capsaicin into the constantly perfused left anterior descending coronary artery of the pig in vivo caused a dose-dependent decrease in perfusion pressure, suggesting coronary vasodilation. In conclusion, the vasodilator effects of SP in vitro differ from the response to CGRP both with regard to their transient nature, the development of tachyphylaxis and endothelium dependence. The capsaicin-induced coronary vasodilation is therefore more likely to depend on release of CGRP rather than tachykinins from sensory nerves since neither endothelium removal nor SP-tachyphylaxis influenced the capsaicin and CGRP responses.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuropeptide Y (NPY) and the pig heart: Release and coronary vasoconstrictor effects

The effects of electrical stimulation of the stellate ganglia on the arterio-venous concentration differences of neuropeptide Y (NPY)-like immunoreactivity (LI) over the pig heart were studied in vivo in relation to changes in heart rate and left ventricular pressure. Furthermore, the effects of NPY on coronary vascular tone were analysed in vivo and in vitro. Stellate ganglion stimulation at a high frequency (10 Hz) caused a clear-cut, long lasting increase in plasma levels of NPY-LI in the coronary sinus compared to the aorta, suggesting release of this peptide from sympathetic terminals within the heart. The stimulation-evoked overflow of NPY-LI from the heart was enhanced about 3-fold by alpha-adrenoceptor blockade using phenoxybenzamine, suggesting that NPY release is under prejunctional inhibitory control by noradrenaline (NA). Combined alpha- and beta-adrenoceptor blockade abolished most of the positive inotropic response of the heart upon stellate ganglion stimulation, while a considerable positive chronotropic effect remained. After guanethidine treatment, stellate ganglion stimulation still produced a small positive inotropic and chronotropic effect on the heart. The stimulation evoked NPY overflow was markedly reduced by guanethidine indicating an origin from sympathetic nerve terminals. Injection of NPY into the constantly perfused left anterior descending artery in vivo caused a long lasting, adrenoceptor antagonist resistant increase in perfusion pressure, suggesting coronary vasoconstriction. NPY contracted coronary arteries in vitro via a nifedipine-sensitive mechanism. NA dilated coronary vessels both in vivo and in vitro via beta-adrenoceptor activation. It is concluded that sympathetic nerve stimulation increases overflow of NPY-LI from the heart suggesting release from cardiac nerves in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitric oxide inhalation attenuates pulmonary hypertension and improves gas exchange in endotoxin shock

Nitric oxide (10 ppm) inhaled by pigs before or during endotoxin shock induced by an infusion of E. coli lipopolysaccharide. Nitric oxide inhalation selectively attenuated pulmonary hypertension during endotoxin infusion without influencing mean arterial blood pressure and cardiac output. Upon cessation of nitric oxide inhalation, pulmonary artery pressure rapidly increased to levels seen in endotoxin-treated controls. The oxygenation and pH of arterial blood were significantly higher in the animals receiving nitric oxide. A marked increase in arterial plasma noradrenaline and neuropeptide Y was seen in endotoxin-treated control pigs while in the nitric oxide-treated pigs this increase was markedly reduced. The increase in arterial plasma endothelin-1 was not influenced by nitric oxide inhalation. Infusion of L-arginine (substrate for nitric oxide synthesis) also attenuated the pulmonary hypertension but was not selective for the pulmonary vasculature. L-Nitro-arginine (a nitric oxide synthesis inhibitor) initiated a rapid but brief elevation of arterial blood pressure and of pulmonary artery pressure as well as a reduction in cardiac output. Nitric oxide inhalation selectively reduces pulmonary hypertension in porcine endotoxin shock and improves arterial oxygenation and pH with a marked attenuation of sympathetic activation.

Occurrence, specific binding sites and functional effects of endothelin in human cardiopulmonary tissue.

Endothelin (ET)-like immunoreactivity (-LI) was detected in the human cardiopulmonary system, with the highest levels being found in the left anterior descending coronary artery, followed by the lung, right atrium, pulmonary artery, bronchus, pulmonary vein and left ventricle. Chromatographic characterization showed that the ET-LI in the lung and left ventricle corresponded to synthetic ET-1. Specific, high-affinity binding sites for ET-1, with an extremely slow dissociation rate, were found in the lung, right atrium and left ventricle. Displacement studies revealed a rank order of potency of ET-1 greater than ET-2 and sarafotoxin 6b greater than ET-3 and big ET-1. Scatchard analysis indicated a single receptor population in the lung (KD 1.53 x 10(-10) M) and left ventricle (KD 3.0 x 10(-11) M). In functional experiments, ET-1 evoked concentration-dependent, long-lasting vasoconstriction of a higher potency than that evoked by ET-2 and ET-3 in epicardial coronary arteries as well as in pulmonary arteries. ET-1 and ET-2 also showed bronchoconstrictor activity at considerably lower concentrations (threshold 10(-11) M) of ET-1 than those needed to cause vasoconstriction (10(-9) M). ET-LI, mainly consisting of ET-1, occurs in human cardiopulmonary tissue. Specific, high-affinity sites with irreversible binding for ET-1 are found in both the heart and lung. ET-1 is more potent than ET-2 or ET-3 in displacing ET-1 binding and in causing vasoconstriction and bronchoconstriction. Thus, in the human heart and lung, ET-1 seems to be the most abundant and biologically active of the endothelin peptides.

Evidence for co‐transmitter role of neuropeptide Y in the pig spleen

1 The possible involvement of neuropeptide Y (NPY) in relation to noradrenaline (NA) and adenosine triphosphate (ATP) mechanisms in the sympathetic nervous control of the vascular tone and capsule contraction in the blood perfused pig spleen was investigated in vivo. 2 Local injections or infusions of NA, NPY and α‐, β‐methylene ATP (mATP) caused vasoconstriction (perfusion pressure increase) and capsule contraction (increased venous blood flow). ATP only evoked vasodilatation. NPY was about 50 fold more potent than NA as a vasoconstrictor, and the NPY response was more long‐lasting. Reserpine treatment did not change the effects of NPY. 3 Electrical stimulation of the splenic nerves in control animals caused a frequency‐dependent, guanethidine‐sensitive output of both NPY‐like immunoreactivity (‐LI) and NA, suggesting co‐release. The output of NPY‐LI relative to NA was enhanced at high frequency stimulation. Furthermore, α‐adrenoceptor blockade by phentolamine enhanced both the output of NPY‐LI and NA while inhibition of the neuronal uptake of NA with desipramine reduced the low frequency stimulation‐evoked overflow of NPY‐LI. Preganglionic denervation did not change the output of NPY‐LI or NA. 4 Reserpine treatment reduced both the splenic content of NA and NPY‐LI. Preganglionic denervation inhibited the reserpine‐induced depletion of the NPY content but not of NA in terminal areas. The stimulation‐evoked NPY overflow was markedly enhanced, especially at low‐frequency stimulation after resperpine, and the plasma levels of NPY‐LI in the venous effluent were then in the nmolar range (i.e. where exogenous NPY induced vasoconstriction). The perfusion‐pressure increase upon stimulation in reserpine‐treated, preganglionically‐denervated animals was highly correlated (r = 0.91) to the NPY overflow. The functional 0.5 Hz responses were reduced after reserpine, while at higher frequencies the functional effects were of similar magnitude to controls but longer‐lasting. 5 Tyramine induced a release of NA but not of NPY‐LI. Furthermore, the increase in perfusion pressure induced by tyramine was absent after reserpine. 6 After tachyphylaxis to the vasoconstrictor effects of mATP, the nerve stimulation‐evoked, functional response as well as the NA and NPY‐LI overflow were unchanged. After reserpine treatment, both the perfusion‐pressure increase and NPY‐LI overflow to nerve stimulation were reduced after mATP tachyphylaxis. 7 In conclusion, release of NPY rather than ATP may explain the long‐lasting, non‐adrenergic, splenic functional responses in reserpinized animals upon sympathetic stimulation. However, NA is most likely the main splenic transmitter when low‐frequency stimulation is used under control conditions.

Comparison of a single indicator and gravimetric technique for estimation of extravascular lung water in endotoxemic pigs

Objectives:To compare the single thermal indicator dilution (STID) technique for measurement of extravascular lung water (EVLWSTID) with gravimetrically determined EVLW (EVLWGRAV) in anesthetized pigs under sham and endotoxemic conditions. Design:Open experimental comparative animal study. Setting:University animal research laboratory. Subjects:Fifteen anesthetized landrace pigs. Interventions:Endotoxin infusion during 5 hrs in five pigs. Six animals were only anesthetized and rested for 5 hrs. In four additional animals, the impact on EVLWSTID measurements by changes in pulmonary perfusion, ventilation, and the combination of the two was studied. The alterations in ventilation and perfusion were induced by caval balloon inflation, inflation of the pulmonary artery catheter balloon, and bronchial plugging respectively. Measurements and Main Results:The STID technique, with default settings of the intrathoracic blood volume (ITBV) to global end-diastolic volume (GEDV) (i.e., the extrapulmonary intravascular volume between the point of injection of the indicator, and the point of detection) relationship (ITBV = 1.25GEDV), systematically overestimated the EVLW index compared with the gravimetrical method (mean bias of 5.4 mL/kg). By adapting the ITBV to GEDV relationship to the current model (ITBV = 1.52GEDV + 49.7), the accuracy of the STID technique improved. Moreover, the measurement of EVLWSTID proved to be reduced by manipulation of pulmonary perfusion and ventilation. However, the STID technique could detect an increase in EVLW during endotoxemia independent of the ITBV/GEDV relationship used. Conclusion:Despite technological improvement, the dilution techniques for the measurement of EVLW might still be influenced by changes in perfusion and ventilation. The STID technique, in addition, might demand adjustment of the ITBV/GEDV relationship to the particular condition and species subjected to measurement. The STID technique may, however, be a useful tool for monitoring changes of EVLW over time, but further studies are warranted to confirm this.

Bosentan-improved cardiopulmonary vascular performance and increased plasma levels of endothelin-1 in porcine endotoxin shock.

1 To evaluate the possible contribution of endothelin‐1 (ET‐1) to the pathophysiology of porcine septic shock, the non‐peptide, mixed ET‐receptor antagonist, bosentan (RO 47–0203) was administered (5 mg kg−1, i.v.) 30 min before infusion of lipopolysaccharide (LPS) (E. coli., serotype 0111:B4) (15 μg kg−1 h−1) and at 3.5 h of endotoxaemia in six anaesthetized and mechanically ventilated pigs. Six other pigs served as controls and received only LPS infusion. Pulmonary and systemic haemodynamics as well as splenic, renal and intestinal blood flows were measured continuously. Release and synthesis of ET‐1 and Big ET‐1 were also measured. 2 Only three of the six pigs in the control group survived 3 h of LPS infusion while in the bosentan‐treated group all six pigs were alive at that time. A biphasic increase in mean pulmonary arterial pressure (MPAP) and pulmonary vascular resistance (PVR) was seen in control pigs. Pretreatment with bosentan did not influence the first peak but markedly attenuated the second, more prolonged increase in MPAP and PVR. The second dose of bosentan completely restored these parameters to pre‐LPS levels. The LPS‐induced changes in mean arterial blood pressure, heart rate and systemic vascular resistance were similar in both groups, while cardiac output (CO) was significantly higher in the bosentan‐treated group. The second bosentan dose increased CO and splenic and intestinal blood flow without further lowering of blood pressure. 3 Bosentan caused an increase of the basal arterial plasma levels of ET‐1‐like immunoreactivity (LI), from 16.8 ± 1.3 pM to 49.6 ± 10.0 pM (n = 6, P < 0.01). However, the rate of the increase of ET‐1 levels during the LPS infusion was not affected by bosentan. Repeated administration of bosentan during LPS infusion caused an additional increase of ET‐1‐LI levels. Neither the basal levels of Big ET‐LI nor the LPS induced 8 fold increase in Big ET‐LI were changed by bosentan. The level of preproET‐1 mRNA in the lung was increased about 3 fold after 4.5 h of LPS treatment. This elevation was not influenced by bosentan. 4 From these studies using bosentan, a non‐peptide, selective and mixed ET‐receptor antagonist, we conclude that during LPS‐induced shock bosentan can abolish the late phase pulmonary hypertension and improve cardiac output as well as increase blood flow to the splenic and intestinal vascular beds without causing a further decrease in mean arterial blood pressure. Further investigations in the clinical setting are needed to evaluate the use of ET‐receptor antagonists, such as bosentan, in treatment of septic shock.