Pulmonary vasoreactivity to serotonin during hypoxia is modulated by ATP-sensitive potassium channels

Barman, Scott A. Pulmonary vasoreactivity to serotoninduring hypoxia is modulated by ATP-sensitive potassium channels. J. Appl. Physiol. 83(2): 569-574, 1997.The role of ATP-sensitive K⁺-channel modulation in thecanine pulmonary vascular response to serotonin during hypoxia wasdetermined in the isolated blood-perfused dog lung. Pulmonary vascularresistances and compliances were measured by using vascular occlusiontechniques. Under normoxia, serotonin(10⁵ M) significantlyincreased precapillary and postcapillary resistances and pulmonarycapillary pressure and decreased total vascular compliance bydecreasing both microvascular and large-vessel compliances. Duringhypoxia, the effect of serotonin was potentiated on both precapillaryand postcapillary resistance and capillary pressure, as well as onmicrovascular compliance and large-vessel compliance. Under normoxia,the ATP-sensitive K⁺-channelopener cromakalim (10⁵ M)inhibited the serotonergic response on postcapillary resistance andmicrovascular compliance, whereas during hypoxia cromakalim inhibitedthe potentiated effect of serotonin on both precapillary andpostcapillary resistance, capillary pressure, and both microvascular and large-vessel compliances. These results indicate that canine pulmonary vasoreactivity to serotonin is heightened under hypoxic conditions and that ATP-sensitiveK⁺ channels modulate the pressorresponse to serotonin, an effect that is more pronounced duringhypoxia.

     

Leukotriene E4 causes pulmonary vasoconstriction, not inhibited by meclofenamate

Leukotriene E4 (LTE4) appears to be a rather stable product of the lipoxygenase pathway. Its action in the pulmonary circulation is unknown. Therefore we investigated its effect on the circulation of isolated rat lungs perfused with a cell- and plasma-free solution. Synthetic LTE4 in doses from .15 micrograms to 5 micrograms/.25 ml .9% NaCl injected as a bolus in the pulmonary artery during normoxia caused a fast, transient perfusion pressure increase within seconds. This was followed by a slow rise in baseline perfusion pressure (normoxia) over 25 min. In addition, 5 micrograms LTE4 caused edematogenic lung damage. Injection of 1.5 micrograms LTE4 during hypoxic vasoconstriction caused fast, transient pressure rises, similar to normoxic conditions. 6-keto-PGF1 alpha and TXB2 were measured in the lung effluent before and after LTE4 injection. Neither 6-keto-PGF1 alpha nor TXB2 production changed after LTE4 injection. Meclofenamate (.5 micrograms/ml) increased the fast, transient and the slow, sustained pressure rise. We conclude that LTE4 caused direct pulmonary vasoconstriction unrelated to cyclooxygenase products.     

Pulmonary vasoconstriction induced by mitral valve obstruction in sheep

We hypothesizedthat left atrial hypertension results in pulmonary vasoconstriction,which is obscured by the expected passive decrease in pulmonaryvascular resistance. The objectives of this study were todemonstrate and quantify the vasoconstrictive changes that occur in thepulmonary circulation during experimental left atrial hypertension, todetermine the site of vasoconstriction, and to explore its mechanism.Sheep were instrumented for measurement of pulmonary arterial (Ppa),left atrial (Pla), and systemic arterial pressures (Psa) with a Foleyballoon catheter to variably obstruct the mitral valve. Distalpulmonary arterial wedge pressure (Ppaw) was determined by using a 5-FrSwan-Ganz catheter that was advanced until it wedged with the balloondeflated. Cardiac output (CO) was estimated by thermodilution;pulmonary vascular resistances (PVR) were calculated as mean (Ppa  Pla)/CO = total PVR, (Ppa  Ppaw)/CO = upstream PVR, and(Ppaw  Pla)/CO = downstream PVR. We studied 15 awake sheep atbaseline and during increases in Pla of 10 and 20 cmH₂O, with and without inhalationof ~36 parts per million of nitric oxide. Left atrial hypertensionresulted in elevation of Ppa. CO decreased only slightly at both levels of Pla elevation. Nitric oxide inhalation caused a significant decreasein PVR, which was greater as Pla increased. This vasodilator effect wasmost striking in downstream vessels. Experiments with phentolamine,atropine, and ibuprofen failed to reveal the mechanism of the reactivepulmonary vasoconstriction.

     

S-nitrosoglutathione inhibits alpha1-adrenergic receptor-mediated vasoconstriction and ligand binding in pulmonary artery

Endogenous nitric oxide donor compounds (S-nitrosothiols) contribute to low vascular tone by both cGMP-dependent and -independent pathways. We have reported that S-nitrosoglutathione (GSNO) inhibits 5-hydroxytryptamine (5-HT)-mediated pulmonary vasoconstriction via a cGMP-independent mechanism likely involving S-nitrosylation of its G protein-coupled receptor (GPCR) system. Because catecholamines, like 5-HT, constrict lung vessels via a GPCR coupled to G(q), we hypothesized that S-nitrosothiols modify the alpha1-adrenergic GPCR system to inhibit pulmonary vasoconstriction by receptor agonists, e.g., phenylephrine (PE). Rat pulmonary artery rings were pretreated for 30 min with and without an S-nitrosothiol, either GSNO or S-nitrosocysteine (CSNO), and constricted with sequential concentrations of PE (10(-8)-10(-6) M). Effective cGMP-dependence was tested in rings pretreated with soluble guanylate cyclase inhibitors {either 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or LY-83583} or G kinase inhibitor (KT-5823), and a thiol reductant [dithiothreitol (DTT)] was used to test reversibility of S-nitrosylation. Both S-nitrosothiols attenuated the PE dose response. The GSNO effect was not prevented by LY-83583, ODQ, or KT-5823, indicating cGMP independence. GSNO inhibition was reversed by DTT, consistent with S-nitrosylation or other GSNO-mediated cysteine modifications. In CSNO-treated lung protein, the alpha1-adrenergic receptor was shown to undergo S-nitrosylation in vitro using a biotin switch assay. Studies of alpha1-adrenergic receptor subtype expression and receptor density by saturation binding with 125I-HEAT showed that GSNO decreased alpha1-adrenergic receptor density but did not alter affinity for antagonist or agonist. These data demonstrate a novel cGMP-independent mechanism of reversible alpha1-adrenergic receptor inhibition by S-nitrosothiols.     

Pulmonary uptake of PGE2 is inhibited by dipyridamole in rat isolated lungs

The metabolism of prostaglandin E₂ (PGE₂) is decreased by dipyridamole (20 μM) in rat isolated perfused lungs. The inhibition of the metabolism is reversible as the decreased metabolism returned to the control level when pulmonary infusion of dipyridamole was abolished. After pulmonary injection of ¹⁴C-PGE₂ (10 nmol) the radioactivity appeared more rapidly in the effluent when dipyridamole was infused into pulmonary circulation. Dipyridamole in vitro did not change the activity of 15-hydroxyprostaglandin dehydrogenase (15-OH-PGDH) in the 100, 000 × g supernatant fraction of homogenized lungs. Thus, the decreased metabolism seems to be due to the inhibition of the uptake of PGE₂ into the lungs. When the rats were pretreated with dipyridamole in drinking water for one week the activity of 15-OH-PGDH in the 100, 000 × g supernatant fraction of the lungs was not changed significantly.     

Ghrelin inhibited serotonin release from hippocampal slices

Ghrelin (Ghr) is a peptide produced peripherally and centrally. It participates in the modulation of different biological processes. In our laboratory we have shown that (a) Ghr administration, either intracerebroventricular or directly into the hippocampus enhanced memory consolidation in a step down test in rats (b) the effect of Ghr upon memory decreases in animals pretreated with a serotonin (5-HT) reuptake inhibitor, Fluoxetine, suggesting that Ghr effects in the hippocampus could be related to the availability of 5-HT. It has been demonstrated that Ghr inhibits 5-HT release from rat hypothalamic synaptosomes. Taking in mint these evidences, we studied the release of radioactive 5-HT to the superfusion medium from hippocampal slices treated with two doses of Ghr (0.3 and 3 nm/μl). Ghr inhibited significantly the 5-HT release in relation to those superfused with artificial cerebrospinal fluid (ACSF) (H = 9.48, df = 2, p ≤ 0.05). In another set of experiments, Ghr was infused into the CA1 area of hippocampus of the rats immediately after training in the step down test and the 5-HT release from slices was studied 24 h after Ghr injection showing that in this condition also the 5-HT release was inhibited (H = 11.72, df = 1, p ≤ 0.05). In conclusion, results provide additional evidence about the neurobiological bases of Ghr action in hippocampus.     

Leukotriene E4 causes pulmonary vasoconstriction,not inhibited by meclofenamate

Leukotriene E₄ (LTE₄) appears to be a rather stable product of the lipoxygenase pathway. Its action in the pulmonary circulation is unknown. Therefore we investigated its effect on the circulation of isolated rat lungs perfused with a cell- and plasma-free solution. Synthetic LTE₄ in doses from .15 μg to 5μg/ .25 ml .9% NaCl injected as a bolus in the pulmonary artery during normoxia caused a fast, transient perfusion pressure increase within seconds. This was followed by a slow rise in baseline perfusion pressure (normoxia) over 25 min. In addition, 5 μm LTE₄ caused edematogenic lung damage. Injection of 1.5 μg LTE₄ during hypoxic vasoconstriction caused fast, transient pressure rises, similar to normoxic conditions. 6-keto-PGF_1α and TXB₂ were measured in the lung effluent before and after LTE₄ injection. Neither 6-keto-PGF_1α nor TXB₂ production changed after LTE₄ injection. Meclofenamate (.5 μg/ml) increased the fast, transient and the slow, sustained pressure rise. We conclude that LTE₄ caused direct pulmonary vasoconstriction unrelated to cycloxygenase products.     

DNA-dependent protein kinase is inhibited by trifluoperazine

The DNA-dependent protein kinase (DNA-PK) is a serine/threonine nuclear kinase, important for the repair of DNA double strand breaks (DSB). Cells defective in DNA-PK show increased sensitivity to ionising radiation and different DNA-damaging drugs, such as cisplatinum. Increased sensitivity to cisplatinum has previously been noted in the presence of phenothiazines. We tested a panel of phenothiazines and one thioxanthen for any influence upon the activity and expression of DNA-PK in a nonsmall cell lung cancer cell line, U-1810. The activity of DNA-PK was completely inhibited in cell lysate and in purified enzyme by 200 microM TFP. DNA-PKcs and Ku86 cleavage were evident in U-1810 cells after 30 min incubation with 100 microM TFP, along with changes in the cells consistent with apoptosis. Our study suggests that phenothiazines and thioxanthens, acting through DNA-PK, have the potential to enhance the effects of DNA damaging agents.     

Food intake is inhibited by oral oleoylethanolamide

Oleoylethanolamide (OEA) may be an endogenous regulator of food intake, and intraperitoneal injection of this compound decreases food intake in 24 h-starved rats. It is generally believed that this kind of lipid amide is rapidly catabolized in the gastrointestinal tract, thereby preventing its use as an oral antiobesity compound. We now show that oral OEA inhibits food intake dose dependently at 90 min after food presentation to starved rats. Food intake was reduced by 15.5% (P < 0.01) by administration of 10 mg/kg OEA. [(3)H]OEA was used to assess the degree of catabolism in the gastrointestinal tract. The endogenous level of this acylethanolamide was increased 11 times in the intestinal tissue (to 3.91 +/- 0.98 nmol/g tissue, mean +/- SEM) at 90 min after food presentation, based on the finding of 0.48% of the dose as intact OEA. These findings reveal unexpected properties of orally administered OEA, which may have potential as a cheap and safe antiobesity drug.     

Putative serotonin reuptake inhibitor-induced spawning and parturition in freshwater bivalves is inhibited by mammalian 5-HT2 receptor antagonists

Selective serotonin reuptake inhibitors (SSRIs) can mimic the physiological actions of serotonin, and in bivalve molluscs they induce zebra mussel spawning and fingernail clam parturition. We have elucidated further the pharmacology of SSRI-induced spawning and part-urition by blocking these reproductive processes with two mammalian 5-HT(2) receptor antagonists, cyproheptadine and mianserin. These two antagonists were potent inhibitors of both spawning and parturition induced by the SSRIs fluvoxamine, fluoxetine, and zimelidine. In zebra mussels, both cyproheptadine and mianserin significantly blocked spawning induced by fluvoxamine and by zimelidine. In the fingernail clams Sphaerium spp., both cyproheptadine and mianserin blocked fluvoxamine-induced parturition. A possible mechanism of action for SSRI-induced spawning and parturition in bivalves is suggested.     

Human carbonyl reductase 1 is an S-nitrosoglutathione reductase

Human carbonyl reductase 1 (hCBR1) is an NADPH-dependent short chain dehydrogenase/reductase with broad substrate specificity and is thought to be responsible for the in vivo reduction of quinones, prostaglandins, and other carbonyl-containing compounds including xenobiotics. In addition, hCBR1 possesses a glutathione binding site that allows for increased affinity toward GSH-conjugated molecules. It has been suggested that the GSH-binding site is near the active site; however, no structures with GSH or GSH conjugates have been reported. We have solved the x-ray crystal structures of hCBR1 and a substrate mimic in complex with GSH and the catalytically inert GSH conjugate hydroxymethylglutathione (HMGSH). The structures reveal the GSH-binding site and provide insight into the affinity determinants for GSH-conjugated substrates. We further demonstrate that the structural isostere of HMGSH, S-nitrosoglutathione, is an ideal hCBR1 substrate (Km = 30 microm, kcat = 450 min(-1)) with kinetic constants comparable with the best known hCBR1 substrates. Furthermore, we demonstrate that hCBR1 dependent GSNO reduction occurs in A549 lung adenocarcinoma cell lysates and suggest that hCBR1 may be involved in regulation of tissue levels of GSNO.     

Acetylcholinesterase is not inhibited by pyridoxal 5'-phosphate

Acetylcholinesterase activity was assayed in the absence and presence of pyridoxal 5'-phosphate. If substrate hydrolysis was measured by the pH-stat method, its rate was not significantly affected by pyridoxal 5'-phosphate. In the spectrophotometric assay, however, this compound led to an apparent decrease in rate. The discrepancy between the two assays is explained by stray-light artefacts produced by pyridoxal 5'-phosphate at the wavelengths of the spectrophotometric assay.     

Megakaryocyte polyploidy is inhibited by lysyl oxidase propeptide

Megakaryocytes (MKs), the platelet precursors, undergo an endomitotic cell cycle that leads to polyploidy. Lysyl oxidase propeptide (LOX-PP) is generated from lysyl oxidase (LOX) pro-enzyme after proteolytical cleavage. We recently reported that LOX, a known matrix cross-linking enzyme, contributes to MK lineage expansion. In addition, LOX expression levels are ploidy-dependent, with polyploidy MKs having minimal levels. This led us to test the effects of LOX-PP on the number and ploidy of primary MKs. LOX-PP significantly decreases mouse bone marrow MK ploidy coupled with a reduction in MK size. MK number is unchanged upon LOX-PP treatment. Analysis of LOX-PP- or vehicle-treated MKs by western blotting revealed a reduction in ERK1/2 phosphorylation and in the levels of its downstream targets, cyclin D3 and cyclin E, which are known to play a central role in MK endomitosis. Pull-down assays and immunochemistry staining indicated that LOX-PP interacts with α-tubulin and the mictotubules, which can contribute to decreased MK ploidy. Thus, our findings defined a role for LOX-PP in reducing MK ploidy. This suggests that high-level expression of LOX in aberrantly proliferating MKs could play a part in inhibiting their polyploidization via LOX-PP.     

Olfactory bulb axonal outgrowth is inhibited by draxin

Olfactory bulb (OB) projection neurons receive sensory input from olfactory receptor neurons and precisely relay it through their axons to the olfactory cortex. Thus, olfactory bulb axonal tracts play an important role in relaying information to the higher order of olfactory structures in the brain. Several classes of axon guidance molecules influence the pathfinding of the olfactory bulb axons. Draxin, a recently identified novel class of repulsive axon guidance protein, is essential for the formation of forebrain commissures and can mediate repulsion of diverse classes of neurons from chickens and mice. In this study, we have investigated the draxin expression pattern in the mouse telencephalon and its guidance functions for OB axonal projection to the telencephalon. We have found that draxin is expressed in the neocortex and septum at E13 and E17.5 when OB projection neurons form the lateral olfactory tract (LOT) rostrocaudally along the ventrolateral side of the telencephalon. Draxin inhibits axonal outgrowth from olfactory bulb explants in vitro and draxin-binding activity in the LOT axons in vivo is detected. The LOT develops normally in draxin−/− mice despite subtle defasciculation in the tract of these mutants. These results suggest that draxin functions as an inhibitory guidance cue for OB axons and indicate its contribution to the formation of the LOT.