Co-release of neuropeptide Y and noradrenaline from pig spleen in vivo: importance of subcellular storage, nerve impulse frequency and pattern, feedback regulation and resupply by axonal transport.

The importance of subcellular storage, nerve impulse rate and pattern, and feedback regulation, as well as resupply by axonal transport for the release of noradrenaline and neuropeptide Y-like immunoreactivity, was studied in the blood perfused pig spleen in vivo. Vasoconstrictor responses were recorded as perfusion pressure changes. Subcellular fractionation experiments using sucrose density gradients showed a bimodal distribution of noradrenaline (peak concentrations at 0.8 and 1.1 M sucrose) while only one main peak of neuropeptide Y was present (at 1.1 M sucrose). Overflow suggesting release of noradrenaline and neuropeptide Y-like immunoreactivity could be detected after 10 s stimulation at 10 Hz. The ratio for the output of noradrenaline and neuropeptide Y upon continuous nerve stimulation in control animals decreased with frequency. After inhibition of noradrenaline reuptake by desipramine the vasoconstrictor response and noradrenaline output were enhanced while the corresponding overflow of neuropeptide Y was reduced by 50% at 0.5 Hz. Stimulation with the irregular or regular bursting patterns at high frequencies caused larger perfusion pressure increase and relative enhancement of neuropeptide Y output compared to noradrenaline than a continuous stimulation both before and after desipramine treatment. A similar fractional release per nerve impulse was calculated both for [3H]noradrenaline (5.6 +/- 1.0 x 10(-5) and neuropeptide Y (7.3 +/- 0.3 x 10(-5). After reserpine treatment combined with preganglionic denervation the vasoconstrictor responses were more long-lasting, neuropeptide Y release was enhanced while noradrenaline content and release were reduced by 99%. The difference in neuropeptide Y overflow between continuous and bursting types of stimulation was smaller after reserpine treatment. After prolonged intermittent stimulation with regular bursts (20 Hz) for 1 h the splenic content of neuropeptide Y was reduced by 58%, while no change was observed for noradrenaline. The maximal perfusion pressure increase upon prolonged nerve stimulation after reserpine was similar in control and reserpine-treated animals, but after reserpine the vasoconstrictor response and neuropeptide Y release were subjected to fatigue. Ligation experiments of the splenic nerves revealed the splenic neuropeptide Y content was resupplied by axonal transport with a calculated total tissue turnover time of 11 days. In contrast, axonal transport contributed only to a marginal extent for the resupply of noradrenaline.(ABSTRACT TRUNCATED AT 400 WORDS)

Neuropeptide Y- and alpha-adrenergic receptors in pig spleen: localization, binding characteristics, cyclic AMP effects and functional responses in control and denervated animals.

The localization of neuropeptide Y binding sites in the pig spleen, as revealed by [125I]Bolton-Hunter-labelled porcine neuropeptide Y and alpha 1-adrenergic receptor binding sites, as revealed by [125I](2-beta/4-hydroxy-phenyl/-ethylaminomethyl)-tetralone as radioligand, was compared with the distribution of neuropeptide Y and noradrenaline nerves, the latter revealed by tyrosine hydroxylase and dopamine-beta-hydroxylase, using immunohistochemistry. A large degree of codistribution was obtained between [125I]neuropeptide Y and alpha 1-binding sites in the capsule, trabeculae, blood vessels and the red pulp of the spleen. Neuropeptide Y and tyrosine hydroxylase as well as dopamine-beta-hydroxylase-positive nerves were identical in the spleen and had a similar gross distribution pattern as the [125I]neuropeptide Y and alpha 1 binding sites. In functional studies using the isolated blood-perfused spleen from pentobarbital-anaesthetized pigs, neuropeptide Y, noradrenaline and the alpha 1-selective agonist phenylephrine contracted the capsule and induced vasoconstriction in the spleen in vivo. However, the selective alpha 2-adrenoceptor agonists clonidine and azepexole had no effects on blood flow or perfusion pressure, suggesting that postjunctional alpha-receptors were of the alpha 1 type. Neuropeptide Y inhibited the forskolin-evoked, cyclic adenosine monophosphate formation in vitro. The [125I]neuropeptide Y binding, with an equilibrium-dissociation constant of 503 +/- 73 pM and a maximal number of specific binding sites of 23 +/- 3 fmol/mg protein, the neuropeptide Y-induced perfusion-pressure increase in vivo and the inhibition of forskolin-evoked cyclic adenosine monophosphate formation in vitro were dependent on the amidation of the C-terminal portion of the peptide molecule. Furthermore, the effects of neuropeptide Y were not changed by alpha- and beta-adrenoceptor blockade using prazosin and propranolol. Two weeks after postganglionic denervation the neuropeptide Y and the noradrenaline contents of the pig spleen were reduced by 97% and 99%, respectively. These changes were associated with a selective supersensitivity for the noradrenaline-induced perfusion-pressure increase in vivo compared with the effect of neuropeptide Y. However, a similar potentiation of the noradrenaline effect was induced by the monoamine-uptake blocker desipramine in the absence of denervation, and there was no change in the functional response to phenylephrine after denervation.(ABSTRACT TRUNCATED AT 400 WORDS)

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 alpha-, beta-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, alpha-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 reserpine, 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.

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 micrograms 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 bosentantreated 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.

Differentiated effects on splanchnic homeostasis by selective and non-selective endothelin receptor antagonism in porcine endotoxaemia.

1. The non-selective endothelin (ET) receptor antagonist bosentan has been shown to restore systemic and gut oxygen delivery and reverse intestinal mucosal acidosis in porcine endotoxin shock. 2. To further elucidate the specific role of the ETA as opposed to the ETB receptor and their effects in the splanchnic region a non-selective (ET(MIX)ra) A-182086 and selective ETA (ET(A)ra) PD155080 and ETB (ET(B)ra) A-192621 receptor antagonists were administered, separately or simultaneously (ET(A+B)ra) 2 h after onset of endotoxin shock. These four groups were compared to a control group receiving only endotoxin and vehicle. 3. Thirty-nine pigs were anaesthetized and catheterized for measurement of central and regional haemodynamics. A tonometer in the distal ileum was used for measurement of mucosal PCO2. Blood gases and plasma ET-1-LI levels as well as histological samples from the gut were assessed. Intervention was started 2 h after onset of endotoxemia and the experiments were terminated after 5 h. 4. Endotoxin-induced changes in systemic, gut oxygen delivery and portal hepatic vascular resistance and systemic acidosis were effectively counteracted by both ET(A+B)ra an ET(MIX)ra. ET(A)ra administration was not effective while ET(B)ra proved to be fatal as all animals in this group died prior to full time of the experiment. While both ET(A+B)ra and ET(MIX)ra improved gut oxygen delivery only the latter attenuated the profound endotoxin-induced ileal mucosal acidosis. 5. The lethal effect seen from selective ETB receptor antagonism in the current study may be due to increased ETA receptor activity as plasma levels of ET-1 is increased several fold by blocking the ETB receptor and thereby the plasma-ET-1-clearing function. Furthermore, a loss of endothelial ETB receptor vasodilating properties may also have contributed to the lethal course in the ET(B)ra group. 6. The findings in this study suggest that ET is involved in the profound endotoxin-induced disturbances in splanchnic homeostasis in porcine endotoxaemia. Furthermore, antagonism of both ETA and ETB receptors is necessary to effectively counteract these changes.

Cardiopulmonary dysfunction during porcine endotoxin shock is effectively counteracted by the endothelin receptor antagonist bosentan.

In a porcine endotoxin shock model, the mixed nonpeptide endothelin receptor antagonist bosentan was administered 2 h after onset of endotoxemia (n = 8). Cardiopulmonary vascular changes, oxygen-related variables, and plasma levels of endothelin-1-like immunoreactivity were compared with a control group that received only endotoxin (n = 8). Bosentan abolished the progressive increase in mean pulmonary artery pressure and pulmonary vascular resistance seen in controls. Possible mechanisms include blockade of vasoconstrictive endothelin receptors, and a lesser degree of edema and inflammation indicated by less alveolar protein and a lower inflammatory cell count observed in bronchoalveolar lavage. Further, bosentan restored cardiac index to the pre-endotoxin level by an increase in stroke volume index, improved systemic oxygen delivery, and acid base balance. Because mean arterial blood pressure was unaffected, bosentan reduced systemic vascular resistance. Endotoxemia resulted in an increase in tumor necrosis factor-alpha and endothelin-1-like immunoreactivity plasma levels, the latter being further increased by bosentan. In conclusion, in porcine endotoxemia, treatment with the endothelin receptor antagonist bosentan, administered during fulminate shock, abolished pulmonary hypertension and restored cardiac index. These findings suggest that bosentan could be an effective treatment for reversing a deteriorated cardiopulmonary state during septic shock.

Angiotensin II receptor antagonism increases gut oxygen delivery but fails to improve intestinal mucosal acidosis in porcine endotoxin shock.

The renin angiotensin system is highly activated in shock states and has been suggested to be involved in the pathophysiology of the markedly deteriorated splanchnic circulation seen in septic shock. The purpose of the present study was to elucidate the capability of losartan, a nonpeptide angiotensin II type 1 (AT1) receptor antagonist, to attenuate splanchnic blood flow disturbances and counteract intestinal mucosal acidosis in endotoxin shock. A total of 20 pigs were anesthetized and catheterized. Central and regional hemodynamics were monitored. A tonometer in the ileum was used for measurement of mucosal pH. Onset of endotoxin challenge was followed by losartan administration (n = 10) 2 h later. Ten animals receiving endotoxin only served as controls. The experiments were terminated 5 h after onset of endotoxin challenge. Endotoxin infusion induced an hypodynamic shock with a reduction in cardiac index and systemic oxygen delivery. Losartan reduced both systemic vascular resistance and pulmonary capillary wedge pressure while stroke volume was improved. Pulmonary hypertension induced by endotoxin was significantly reduced by losartan without further changes in gas exchange. The profound reduction in gut oxygen delivery in response to endotoxin was counteracted by losartan administration. However, losartan failed to improve the markedly deteriorated intestinal mucosal pH and mucosal-arterial PCO2gap (i.e., difference in intestinal mucosal PCO2 and arterial PCO2). Also the mucosal-portal venous PCO2gap, used as a monitor of the mucosa in relation to the gut as a whole (including the spleen and pancreas), was greatly increased by endotoxemia but unaffected by losartan administration. In summary, although the angiotensin II type 1 receptor antagonist losartan improved gut oxygen delivery and reduced pulmonary hypertension during established endotoxin shock, it had no effect on intestinal mucosal acidosis. These findings suggest contribution of the angiotensin II type 1 receptor to perfusion disturbances, but not to deterioration of intestinal mucosal homeostasis seen during endotoxemia.

Comparison of gravimetric and a double-indicator dilution technique for assessment of extra-vascular lung water in endotoxaemia.

OBJECTIVE: To compare a molecular double-indicator dilution technique with the gravimetrical reference method for measurement of extra-vascular lung water in porcine endotoxin shock. DESIGN: Open comparative experimental study. SETTING: Animal research laboratory. MEASUREMENTS AND RESULTS: In fourteen anaesthetised, mechanically ventilated landrace pigs, central and pulmonary haemodynamics as well as pulmonary gas exchange were measured. Extra-vascular lung water was quantitated gravimetrically as well as with a molecular double indicator dilution technique. Eight of these animals were subjected to endotoxaemia, the rest serving as sham controls. No difference in extra-vascular lung water was observed between the two methods in sham animals. Furthermore, extra-vascular lung water assessed with the molecular double-indicator dilution technique at the initiation of endotoxin infusion did not differ significantly from the corresponding values for sham animals. Endotoxaemia induced a hypodynamic shock with concurrent pulmonary hypertension and a pronounced deterioration in gas exchange. No increase in extra-vascular lung water was detected with the molecular double-indicator dilution technique in response to endotoxin, whereas this parameter was significantly higher when assessed with the gravimetric method. CONCLUSION: The molecular double-indicator dilution technique showed similar results as the gravimetrical method for assessment of extra-vascular lung water in non-endotoxaemic conditions. However, during endotoxin-induced lung injury the molecular double indicator dilution technique failed to detect the significant increase in extra-vascular lung water as measured by the gravimetric method. These data suggest that the molecular double indicator dilution technique may be of limited value during sepsis-induced lung injury.

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)

Pharmacological characterization of neuropeptide Y and noradrenaline mechanisms in sympathetic control of pig spleen.

The mechanisms underlying the functional effects of neuropeptide Y (NPY)-like immunoreactivity (LI) and noradrenaline (NA) and their release evoked by nerve stimulation were studied with the blood-perfused pig spleen in vivo. Infusion of selective agonists and antagonists suggested the presence of alpha 1- and beta 2-adrenoceptors mediating vasoconstriction and vasodilatation, respectively. NPY caused a slight inhibition of stimulation-evoked [3H]NA release and a clearcut non-adrenergic vasoconstriction. Local pretreatment with phentolamine and prazosin as well as with clonidine and UK 14304 reduced the perfusion pressure response to nerve stimulation. Phentolamine, yohimbine and idazoxan enhanced while clonidine and UK 14304 decreased the output of [3H]NA or NA and NPY-LI. The subsequent addition of propranolol to the alpha-adrenoceptor antagonists was followed by reappearance at a considerable portion of the perfusion pressure response while the output of [3H]NA or NA and NPY-LI was slightly reduced. It is concluded that NPY exerts pre- and post-junctional actions in pig spleen that regulate both NA release and vascular tone. alpha 1-Adrenoceptors are mainly involved in vasoconstriction, and prejunctional alpha 2 mechanisms inhibit both NA and NPY release at a low frequency of stimulation. beta 2-Adrenoceptors mediate vasodilatation when NA release is enhanced with a minor effect on mediator secretion.

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)

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.

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.

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)

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.

Effects on haemodynamics by selective endothelin ET(B) receptor and combined endothelin ET(A)/ET(B) receptor antagonism during endotoxin shock.

The endothelin system is highly activated during endotoxin and septic shock. To investigate this matter the selective non-peptide endothelin ET(B) receptor antagonist A-192621 ([2R-(2alpha,3beta, 4alpha)]-4-(1,3-benzodioxol-5-yl)-1-[2-[2, 6-diethylphenyl)amino]-2-oxoethyl]-2-(4-propoxy-phenyl)-3-py rrolidine carboxylic acid) was administered alone and in combination with the selective non-peptide endothelin ET(A) receptor antagonist PD 155080 (sodium 2-benzo[1, 3]dioxol-5-yl-3-benzyl-4-(4-methoxy-phenyl)-4-oxobut-2-enoat e) during established porcine endotoxin shock. Cardiopulmonary vascular function, metabolic parameters and plasma endothelin-1-like immunoreactivity levels were compared to a control group only receiving endotoxin. Administration of A-192621 alone resulted in cardiovascular collapse and death whereas combining A-192621 with PD 155080 abolished endotoxin induced pulmonary hypertension, enhanced cardiac performance and improved systemic oxygen delivery and acid-base balance. The beneficial effects of mixed endothelin ET(A)/ET(B) receptor antagonisms on the pulmonary and cardiovascular systems may result from blockage of constrictive endothelin receptors in and pulmonary circulation, reduced afterload and a direct inotropic effect. Possible mechanisms for the devastating effects by selective endothelin ET(B) receptor antagonism include increased endothelin ET(A) receptor-mediated vasoconstriction due to lack of endothelin ET(B) receptormediated vasodilation and decreased endothelin clearance from endothelin ET(B) receptor blockade. In conclusion, selective endothelin ET(B) receptor antagonism is deleterious whereas combined endothelin ET(A) and ET(B) receptor antagonism has favourable effects on haemodynamics, suggesting participation of the endothelin system in cardiopulmonary dysfunction during endotoxin shock.

Frequency- and reserpine-dependent chemical coding of sympathetic transmission: differential release of noradrenaline and neuropeptide Y from pig spleen.

The importance of impulse pattern and stimulation frequency for the release of noradrenaline (NA) and the coexisting peptide neuropeptide Y (NPY) in relation to vasoconstriction (perfusion-pressure increase) was studied in the blood-perfused pig spleen in vivo. Splenic nerve stimulation with intermittent bursts at high frequency (20 Hz) caused a several-fold larger release of NPY-like immunoreactivity (-LI) in relation to NA than a continuous stimulation at a low frequency (2 Hz), giving the same total number of impulses. alpha-Adrenoceptor blockade by phentolamine enhanced markedly both NA and NPY release, especially at low stimulation frequency, suggesting prejunctional adrenergic inhibition of release. Addition of propranolol unmasked a large remaining perfusion-pressure response to nerve stimulation. Reserpine treatment reduced the NA content of the spleen as well as the stimulation-evoked NA release by greater than 90%. However, the perfusion-pressure increase in response to nerve stimulation was well maintained. A marked increase in the stimulation-evoked release of NPY-LI occurred after reserpine. Adrenoceptor blockade after reserpine treatment reduced only slightly the perfusion-pressure response in parallel with a decline in NPY output. NPY caused an adrenoceptor-resistant perfusion-pressure increase at plasma concentrations that were in the same range as the maximal increase during nerve stimulations. In conclusion, the present data suggest a frequency-dependent, chemical coding of sympathetic transmission with preferential release of the classical transmitter NA at low, continuous frequencies and release of NPY, mainly at high frequencies. Reserpine treatment enhances markedly NPY release, which may explain why the functional response is largely intact in spite of adrenoceptor blockade and marked NA depletion.

Effects of neuropeptide Y, calcitonin gene-related peptide, substance P, and capsaicin on cerebral arteries in man and animals.

The smooth-muscle tone of pial, middle, and anterior cerebral arteries from humans, cats, and pigs, respectively, was studied in vitro with respect to the effects of capsaicin and various peptides which are present in local perivascular nerves. Neuropeptide Y (NPY) caused concentration-dependent, potent contractions of the cerebral vessels both in the presence and in the absence of endothelium. In contrast to the response to noradrenaline (NA) and K+, the NPY effect was not altered by changes in the extracellular Ca++ concentration. The relaxant action of the calcium antagonist nifedipine on NPY-evoked contraction of cerebral arteries was not inhibited by a Ca++-deficient medium or by a high-Ca++ medium. Calcitonin gene-related peptide (CGRP), substance P (SP), and capsaicin caused relaxation of precontracted cerebral arteries with an intact endothelium. Calcitonin gene-related peptide was the most potent dilatory agent, and removal of the endothelium did not change the CGRP response. In contrast, the ability of SP to cause relaxation was abolished after removal of the endothelium. Capsaicin, which activates sensory nerves, induced long-lasting relaxation in both the presence and absence of endothelium. In conclusion, in contrast to earlier reported data, the contractile effect of NPY seems to be largely independent of extracellular Ca++, while NA- and K+-induced contractions are dependent on extracellular Ca++. The present results suggest that the relaxant effect of nifedipine on cerebral blood vessels may involve actions other than inhibition of Ca++ influx. The relaxant effect of capsaicin is likely to be induced by release of CGRP rather than SP. The potent effects of these peptides on human pial arteries suggest that neuropeptides may be involved in the control of cerebral blood flow in man.

Intensive care in the neurosurgical unit--a review of 2,492 patients.

The experience with 2,492 patients treated during a 9-year period in a neurosurgical intensive care unit (NICU) integrated into the ordinary neurosurgical wards is reported. The basic admission criterion was the need for ventilator treatment. During the period studied, the stay in the NICU decreased from a mean of 17 to 10 days. With respect to diagnosis, the largest group was patients with brain injuries (36%), followed by subarachnoid hemorrhage (24%), other intracranial bleeding (17%), and tumors (15%). Treatment consisted mainly of controlled ventilation, the duration of which ranged between 1 and 93 days with a mean of 5 days. Invasive monitoring was done relatively seldom, primarily due to a lack of equipment and staff. The incidence of nonneurosurgical complications was low, and the clinical outcome satisfactory. Nevertheless, it is concluded that for the best care of the severely ill neurosurgical patient, a special, well-equipped NICU with an adequate number of highly qualified staff members closely attached to the neurosurgical clinic is crucial.

Elevated plasma levels of neuropeptide Y upon electrical stimulation of the sympathetic chain in humans.

Neuropeptide Y (NPY) a potent vasoconstrictor peptide, is co-stored with the classic neurotransmitter noradrenaline (NA) in certain peripheral sympathetic neurons and has been suggested to be a co-transmitter in vascular control. Surgical interruption of the sympathetic nerve supply of the upper extremity was performed to treat palmar hyperhidrosis in five patients. Intraoperative electrical stimulation (10 Hz; 0.5 ms; 0.5-3.0 mA) of the right and left sympathetic chains for 1 min was employed to determine the correct level for surgical excision. Blood pressure and heart rate were recorded continuously. Blood was sampled from the antecubital vein on the side subjected to stimulation for the determination of NPY-like immunoreactivity (LI) and plasma catecholamines. Blood samples were collected before and during stimulation, as well as 30 s and 2, 5, and 10 min after stimulation. A maximal blood pressure response was observed during the stimulation, but the magnitude varied markedly and was independent of the side on which the sympathetic chain was stimulated. During 3 of the 10 stimulations, the mean arterial blood pressure (MABP) increased 5-10 mm Hg, while in the other 7 an increase of 30-75 mm Hg was seen. No significant changes in heart rate or plasma adrenaline were found. The peak increase in NPY-LI plasma levels was noted 2 or 5 min after stimulation, while the corresponding peak for NA occurred during or 30 s after the stimulation. The maximal changes in plasma NPY-LI and NA were significantly correlated with the changes in blood pressure (NPY-LI, r = 0.80, p <0.01; NA, r = 0.84, p <0.01) as well as with each other (r = 0.95; p <0.001). It is concluded that, after electrical stimulation of the sympathetic chain in humans, plasma levels of both NPY-LI and NA rise, indicating release of these substances from sympathetic neurons. It is therefore possible that the vasoconstrictor peptide NPY is involved, together with NA, in the blood pressure response elicited by the stimulation.