Pontamine Sky Blue Research Articles

Increase in Blood Pressure by Local Injection of Ketamine into the Amygdala in Rats.

To confirm that an increase in blood pressure induced by ketamine is mediated through the central nervous system, we examined the effect of ketamine, applied directly to the amygdala, on blood pressure. Six male Sprague-Dawley rats were used in the study. Under head-restrained and unanesthetized condition, 0.2 µL (5 mg/mL) of ketamine was injected in and around the amygdala at a flow rate of 0.2 µL/min through a glass pipette, and the blood pressure was recorded while monitoring the state of the animals by electroencephalogram and electromyogram. After ketamine injection, the injection site was marked by Pontamine Sky Blue infusion. Blood pressure was increased by ketamine injection into the basolateral and central nuclei of the amygdala, endopiriform nucleus and piriform cortex. In a total of 11 responses, an increase in blood pressure started with a mean latency of 193.5 ± 43.0 s, reached its peak 180.2 ± 23.3 s after the response onset, then gradually returned to the baseline with mean duration of 706.7 ± 113.5 s. The mean fluctuation was 17.1 ± 2.5 mmHg. We revealed that blood pressure fluctuations induced by ketamine are associated with the amygdala. Elucidation of the mechanism of ketamine-induced blood pressure increase will lead to understanding of the mechanism of side effects of ketamine, and will contribute to its appropriate use.

Electrophysiological responses of the ventrolateral periaqueductal gray matter neurons towards peripheral bladder stimulation

IntroductionMany of the currently available therapies for urinary incontinence target the peripheral autonomic system, despite many etiologies residing in the central nervous system. Following previous experiments that determined the ventrolateral column of the periaqueductal gray matter (vlPAG), to be the main afferent station of bladder sensory signals, we aimed for electrophysiological characterization of vlPAG neurons using single unit recording. Methods15 rats were anesthetized and underwent implantation with electrodes at the dome and the neck of the bladder, to electrically stimulate the detrusor. After craniotomy, a glass micropipette was inserted in vlPAG to record neuronal action potentials. The detrusor was stimulated by a series of 20 Hz pulses, for a total duration of 50 s at an intensity of 2 mA, for each vlPAG neuron selected. Single unit recordings were performed on a total of 26 neurons. Confirmation of electrode position was made by iontophoretic ejection of Pontamine sky blue. ResultsThe firing rate of vlPAG neurons decreased significantly during the stimulation period. Peristimulus time histogram (PSTH) analysis showed 24 out of 26 neurons to be unresponsive to stimulation. All recorded vlPAG neurons showed irregular firing patterns. ConclusionsThe change in firing rate may point to an overall inhibitory influence of bladder stimulation on vlPAG neurons. These data suggest an inhibitory relay station at the vlPAG, before sensory bladder signals would affect pontine micturition center. The lack of the inhibitory effect on PSTH may be due to a longer interval between neuronal response and the stimulation.

Determination of Amikacin in Different Pharmaceutical Formulations Using a Resonance Rayleigh Scattering Method with Pontamine Sky Blue

Amikacin (AMK) is a semisynthetic aminoglycoside antibiotic derived from kanamycin A that lacks a strong ultraviolet absorbing chromophore or fluorophore. Greatly enhanced resonance Rayleigh scattering (RRS) signals of AMK can be observed with a conventional fluorescence spectrophotometer when AMK interacts with pontamine sky blue (PSB) and forms an ion-association complex in weakly acidic buffer medium. Based on these characteristics, a sensitive assay for detecting AMK was developed. The maximum RRS peak was observed at 362 nm. The enhancement of the RRS signal was directly proportional to the AMK concentration in the range of 0–1.7 µg/ml, and its detection limit (3σ) was 3.0 ng/ml. The method showed a wide linear range and high sensitivity, and almost no interference could be observed from coexistent substances. In addition, this assay was performed in different pharmaceutical formulations of AMK with satisfactory results. Therefore, the proposed method is promising as an effective means for the determination of AMK. Keywords: Amikacin, resonance Rayleigh scattering, pontamine sky blue, quantitative determination.

Probing Presynaptic Regulation of Extracellular Dopamine with Iontophoresis

Iontophoresis allows for localized drug ejections directly into brain regions of interest driven by the application of current. Our lab has previously adapted a method to quantitatively monitor iontophoretic ejections. Here those principles have been applied in vivo to modulate electrically evoked release of dopamine in anesthetized rats. A neutral, electroactive marker molecule that is ejected purely by electroosmotic flow (EOF) was used to monitor indirectly the ejection of electroinactive dopaminergic drugs (raclopride, quinpirole, and nomifensine). Electrode placements were marked with an iontophoretically ejected dye, pontamine sky blue. We show that EOF marker molecules, acetaminophen (AP) and 2-(4-nitrophenoxy) ethanol, have no effect on electrically evoked dopamine release in the striatum or the sensitivity of electrode. Additionally, we establish that a short, 30 second ejection of raclopride, quinpirole, or nomifensine with iontophoresis is sufficient to affect autoreceptor regulation and the re-uptake of dopamine. These effects vary in lifetime, indicating that this technique can be used to study receptor kinetics.

PANCREATIC PREGANGLIONIC NEURONS: DIFFERENTIAL RESPONSES TO CCK1 AND 5‐HT3 RECEPTOR STIMULATION

The dorsal motor nucleus of the vagus (DMV) is the main source of vagal input to the pancreas. In vitro studies have shown that the DMV contains a mixed population of pancreatic preganglionic neurons (PPNs) but little is known about their responses to pancreatic secretagogues such as cholecystokinin (CCK) and serotonin (5‐HT) in vivo. The aims of this study were (i) to identify DMV PPNs in vivo and (ii) to characterize their responses to CCK and 5‐HT3 receptor activation. Male Sprague‐Dawley rats anesthetised with isoflurane (1.7–2.0%) were used in all experiments. PPNs were recorded extracellularly and their locations were marked using pontamine sky blue. Only PPNs that had an axon in the pancreatic vagus (collision test positive) were studied. Forty‐four PPNs were identified within the rostral, medial and caudal DMV and thirty eight were tested for responses to CCK‐8 and phenylbiguanide (PBG; 5‐HT3 receptor agonist). Their axonal conduction velocities were in the C‐fibre range (< 1 m/s). CCK and PBG (0.1–10 μg/kg, i.v.) produced three types of response: (i) PPNs in the intermediate DMV were inhibited by CCK (n = 18) and PBG (n = 10), (ii) PPNs in the caudal DMV were activated by CCK (n = 5) and PBG (n = 2) and (iii) rostral DMV neurons were not affected significantly by CCK (n = 9) or PBG (n = 7). Pancreatic secretagogues have complex actions on PPNs that may be related to their effects on pancreatic exocrine and endocrine secretion.

Development of a non-derivatization high-performance liquid chromatography method with resonance Rayleigh scattering detection for the detection of sisomicin in rat serum

A resonance Rayleigh scattering (RRS) detection approach was developed to detect sisomicin (Siso) in rat serum following chromatographic separation. The detection principle is based on the enhancement of RRS intensity of ion-association complex formed from aminoglycosides and pontamine sky blue (PSB) used as molecular recognition probe. The high-performance liquid chromatography (HPLC) coupled with RRS detection scheme was implemented post-column by mixing a PSB solution with the column eluent prior to detection. The RRS signal was detected by fluorescence detector at λ ex = λ em = 365 nm. Separation and detection conditions were optimized. Siso and etimicin (Eti) chosen as the internal standard (IS) were separated on a C 18 reversed phase column with the mobile phase consisting of a ternary mixture of 20 mM sodium acetate aqueous solution–methanol (92:8, v/v) containing 0.22% TFA (v/v). The limit of detection (S/N = 3) for Siso was 18 ng. A calibration curve ranged from 25 ng to 700 ng shown to be linear. The presented method was applied for the determination of Siso in rat serum and used for the pharmacokinetics study of Siso in rat.

Localization of pancreatic preganglionic neurons within the rat dorsal vagal nucleus

The dorsal vagal nucleus (DVN) is the main source of the vagal innervation of the pancreas. In vitro studies have demonstrated that the DVN consists of a heterogeneous neuronal population with distinct morphological and electrophysiological characteristics. The aims of this study were (i) to identify DVN pancreatic preganglionic neurons (PPNs) in vivo and (ii) to characterize their responses to cholecystokinin (CCK), a major regulator of pancreatic secretion. Male Sprague Dawley rats anaesthetised with isoflurane (1.5%/100% O2) were used in all experiments. Extracellular single unit recording techniques were used to record the activity of PPNs within the DVN and their locations were marked using Pontamine Sky Blue. PPNs were identified by antidromic activation in response to stimulation of the pancreatic vagus nerve (0.5 Hz, 0.5 ms pulse width, 500μA). They had axonal conduction velocities in the C‐fibre range (< 1 m/s). Twenty‐five PPNs were identified within the rostral, medial and caudal DVN and twenty were tested for responsiveness to CCK. CCK (0.01–10 μg/kg, i.v.) produced three types of responses: (i) PPNs in the medial DVN were completely inhibited by CCK (0.7 ± 0.02 m/s, n = 16), (ii) PPNs in the caudal DVN were slightly activated by CCK (0.6 ± 0.08 m/s, n = 2) and (iii) CCK had no effect on the PPNs in the rostral DVN (0.6 ± 0.01 m/s, n = 2). CCK has complex actions on PPNs that may be related to its effect on exocrine and endocrine secretion. Further investigation of PPNs may allow identification of sub‐groups of DVN neurons that control the pancreas.

NO-cGMP pathway at ventrolateral medullary cardiac inhibitory sites enhances the baroreceptor reflex bradycardia in the rat

The neuronal isoform of the enzyme nitric oxide synthase (nNOS) has been identified in the caudal ventrolateral medulla of the rat close to the location of cardiac vagal motoneurones. Therefore in this study we tested identified ventral medulla cardioinhibitory sites for the involvement of nitric oxide (NO) in the baroreceptor-heart rate reflex pathway. In rats anaesthetised with a mixture of urethane (650 mg kg − 1 ) and chloralose (50 mg kg − 1 ) i.v., blood pressure and heart rate were monitored continuously and using stereotaxic coordinates the ventrolateral caudal brainstem within and around the nucleus ambiguus was systematically explored for sites producing a bradycardia of > 50 bpm, without a change in blood pressure, using D,L homocysteic acid (DLH, 0.2 M) microinjections (50 nl) from a glass micropipette. Identified sites were marked with pontamine sky blue. Microinjection of the NO donor sodium nitroprusside (SNP, 1 mM, 50 nl) at a cardioinhibitory site also produced a significant bradycardia (68 ± 14 bpm) while the NOS inhibitor N G -nitro- l-arginine ( l-NNA) (3 mM, 50 nl) caused a small significant increase in heart rate (5 ± 1 bpm). Baroreceptor reflex gain measured by the response in heart rate to a change in blood pressure induced by phenylephrine i.v. was significantly increased (610 ± 171%, p < 0.05) during the steady state of the response to SNP, whereas it was significantly reduced (73 ± 5%, p < 0.01) by l-NNA injection at a medullary cardioinhibitory site. An inhibitor of soluble guanylyl cyclase, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ, 1 mM, 50 nl) also significantly reduced the baroreceptor reflex gain (63 ± 8%, p < 0.05). The results suggest that a NOS-cGMP signalling system in the baroreceptor reflex pathway distal to the NTS and closer to cardiac vagal motoneurones in the caudal ventral medulla contributes to enhancement of cardiac vagal tone.

A novel technique to perform direct intraganglionar injections in rats

The present work describes a simple method for direct drug administration into the dorsal root ganglion (DRG) in anesthetized rats. This technique does not involve surgery, is easy to learn and allows behavioral testing within minutes after the injection. Based on landmarks that target the L5 DRG, an orifice was created with a guide needle through which a specially designed needle was inserted for solution injection. Its introduction into the ganglia was ensured by the triggering of an ipsilateral hindpaw reflex. The precision of the technique was checked by injections of the biological dye Pontamine Sky Blue (PSB) or C 14-labeled arginine. There was no leakage of the dye to the surrounding tissues after a single 4 μl or three successive 2.5 μl injections (at 30-min intervals). Moreover, identical effects were observed with prostaglandin E 2 (PGE 2), morphine or glibenclamide injected intraplantarly or in the DRG, thus confirming the precision of the method and suggesting that the ganglion cells and peripheral nociceptors may display similar receptor population.

Microiontophoresis electrode location by neurohistological marking: Comparison of four native dyes applied from current balancing electrode channels

Introduction: The re-establishment of electrode position after microiontophoresis with multi-barrelled electrodes has always been a complicated and mostly inaccurate procedure. The present report describes a new method for ‘on line’ neurohistological marking of each recording position during experimental sessions in which in vivo microiontophoresis was combined with extracellular single unit recording. Methods: We tested the effectiveness of four native dyes: pontamine sky blue (PSB), methylene blue (MB), cresyl violet (CV) and toluidine blue (TB). They were ejected from the continuous balancing channels of multi-barrelled microelectrodes during iontophoresis in place of the usual sodium chloride solution. Results: The present results are based on observations obtained from 66 neuronal recordings and 42 labelled sites in the rat neocortex. Results indicate that none of the tested native dyes altered the spontaneous firing rate of the neurons, nor did they have any influence on the applied bioactive compounds. Recording positions were detected as small, labelled spots in brain slices (2–12 μm in diameter) at the end of the recording tracks. Labelling was successful with MB and PSB, but not with CV or TB. In addition, MB provided good labelling with a low net ejection current. Discussion: The present results suggest that using native dyes, especially MB, instead of sodium chloride in current balancing channels during in vivo microiontophoresis provides fast and accurate confirmation of electrode placement. This method saves the experimenter the time consuming further application of secondary labelling procedures after such experiments. Moreover, the MB-labelling we describe here is focal, enabling a more accurate verification of the site of recording and/or application of various neuroactive compounds. In addition, MB labelled accurately and reliably and with very low ejection currents, presumably due to its smaller molecular weight and higher relative mobility.

Excitatory neurotoxic properties of pontamine sky blue make it a useful tool for examining the functions of focal brain parts.

Pontamine sky blue (PSB) is used in brain studies to mark the position of microelectrode and micropipette tips. However, few studies have been made on the effects of PSB on neurons; therefore we examined these effects. When puffed on isolated sensory ganglion cells of rats, PSB increased membrane conductance, depolarized membrane potential, and reduced the amplitude of action potentials. When dripped on frog sympathetic ganglion, much like hexamethonium, PSB decreased the amplitude of compound action potentials of the postganglionic strand. A bath application of PSB to sartorius muscle fibers that had been treated with tetrodotoxin depolarized the membrane potential and increased the frequency and amplitude of miniature end-plate potentials. All these effects were reversible. When injected into the rat's pontine part corresponding to the location of the canine pontine defecation reflex center, PSB produced repetitive colorectal contractions and irreversibly abolished them in response to anal-canal stimulation. The excitatory and blocking effects of PSB and its staining ability make it a useful tool for examining the functions of focal brain parts.

Resonance Rayleigh scattering spectra for studying the interaction of aminoglycoside antibiotics with pontamine sky blue and their analytical applications

In a weakly acid medium, some aminoglycoside antibiotics, such as kanamycin (KANA), gentamicin (GEN), tobramycin (TOB), and neomycin (NEO), or acid bisazo dye pontamine sky blue (PSB) can only produce very weak resonance Rayleigh scattering (RRS) signals. However, when the two agents react with each other to form the ion association complexes, the RRS intensity can be enhanced greatly and a new RRS spectrum and a significant enhancement of the RRS intensity in the wavelength range 350-600 nm can be observed. The maximum scattering peak is at 580 nm. There is a linear relationship between the RRS intensity and the antibiotic concentration in the range 0.01-6.0 microg mL(-1) at 580 nm. This RRS method has therefore been developed for the determination of trace levels of aminoglycoside antibiotics. The detection limits (3 sigma) of the four antibiotics, whose order of sensitivity is KANA>NEO>TOB>GEN, are 5.8-6.9 ng mL(-1). This method has a good selectivity and has been successfully applied to the quick determination of antibiotics not only for injections and ear drops, but clinic serum samples as well. In addition, quantum chemistry-based analysis of the reaction mechanism, the factors influencing the RRS spectra, and the reasons for the enhancement of RRS are discussed.

Anti-inflammatory potency of FR167653, a p38 mitogen-activated protein kinase inhibitor, in mouse models of acute inflammation

The anti-inflammatory effect of FR167653 (1-[7-(4-fluorophenyl)-1,2,3,4-tetrahydro-8-(4-pyridyl)pyrazolo[5,1- c][1,2,4]triazin-2-yl]-2-phenylethanedione sulfate monohydrate), a p38 mitogen-activated protein (MAP) kinase inhibitor, was examined in two mouse models of acute inflammation. Carrageenan-induced paw edema was inhibited by pretreatment with FR167653, anti-tumor necrosis factor (TNF)-α antibody, and NS-398 ( N-(2-cyclohexyloxy-4-nitrophenyl) methanesulfonamide), a selective cyclooxygenase-2 inhibitor. Carrageenan increased TNF-α and prostaglandin E 2 levels in the paw, both of which were suppressed by FR167653. Subcutaneous injection of lipopolysaccharide at the back of mouse caused local increase in vascular permeability determined by leakage of Pontamine sky blue. FR167653 dose-dependently inhibited the lipopolysaccharide-induced plasma leakage. FR167653 also inhibited lipopolysaccharide-induced increases in serum TNF-α level, and skin TNF-α and prostaglandin E 2 levels at the injection site. On the other hand, FR167653 did not reduce arachidonic acid-induced plasma leakage which is not mediated by cyclooxygenase-2. FR167653 exhibits anti-inflammatory effects against both carrageenan-induced paw edema and lipopolysaccharide-induced plasma leakage through inhibiting the synthesis of inflammatory mediators that are regulated by p38 MAP kinase.

Inhibitory effects of cyclic AMP elevating agents on lipopolysaccharide (LPS)-induced microvascular permeability change in mouse skin.

Anti-inflammatory effects of cyclic AMP elevating agents were examined in a mouse model of lipopolysaccharide (LPS)-induced microvascular permeability change. Vascular permeability on the back skin was measured by the local accumulation of Pontamine sky blue (PSB) after subcutaneous injection of LPS (400 microg site-1) from Salmonella typhimurium. Dye leakage in the skin was significantly increased 2 h after injection of LPS. This LPS-induced dye leakage was suppressed by phosphodiesterase inhibitors, including pentoxifylline (160 mg kg-1), milrinone (5 - 10 mg kg-1), rolipram (0.5 - 10 mg kg-1) and zaprinast (5 - 10 mg kg-1). The dye leakage was also inhibited by beta-adrenoceptor agonists, including isoproterenol (0.5 - 5 mg kg-1) and salbutamol (0.05 - 5 mg kg-1), an adenylate cyclase activator, forskolin (5 mg kg-1), and a cell permeable cyclic AMP analogue, 8-bromo-cyclic AMP (8-Br-cAMP, 10 mg kg-1). LPS caused a transient increase in serum TNF-alpha level peaking at 1 h after the injection. This increase in serum TNF-alpha was completely blocked by a pretreatment with pentoxifylline (160 mg kg-1), milrinone (5 mg kg-1), rolipram (1 mg kg-1), zaprinast (10 mg kg-1), salbutamol (0.5 mg kg-1), forskolin (1 mg kg-1) and 8-Br-cAMP (10 mg kg-1). LPS caused an increase in serum IL-1alpha level peaking at 3 h after injection. This increase in serum IL-1alpha was not significantly suppressed by the cyclic AMP elevating agents. Our study suggests that cyclic AMP elevating agents attenuate LPS-induced microvascular permeability change by suppressing TNF-alpha up regulation.

Role of nitric oxide, prostaglandins and tyrosine kinase in vascular endothelial growth factor-induced increase in vascular permeability in mouse skin.

We investigated role of nitric oxide (NO), prostaglandins (PG) and tyrosine kinase in vascular endothelial growth factor (VEGF)-induced increase in vascular permeability in mouse skin. Subcutaneous injection of VEGF (0.5-2.0 ng/site) induced dose- and time-dependent increase in vascular permeability at the injection site determined by a leakage of Pontamine sky blue. VEGF (1 ng/site)-induced dye leakage was partially inhibited by N(G)-nitro-L-arginine methyl ester (an inhibitor for both constitutive and inducible NO synthase) (5 and 10 mg/kg, i.v.) and by aminoguanidine (a selective inducible NO synthase inhibitor) (5-20 mg/kg, i.v.), but not by an inactive enantiomer, N(G)-nitro-D-arginine methyl ester (10 mg/kg, i.v.). Pretreatment with an intraperitoneal injection of indomethacin (a nonselective cyclooxygenase inhibitor) (5 mg/kg) or N-(2-cyclohexyloxy-4-nitrophenyl) methanesulphonamide (a cyclooxygenase-2 selective inhibitor) (1-100 microg/kg) almost completely inhibited the effect of VEGF (1 ng/site). Coadministration of PGE2 (3 and 30 nmol/site) with VEGF did not restore the inhibitory effect of indomethacin on VEGF (1 ng/site)-induced increase in vascular permeability. Lavendustin A (a selective tyrosine kinase inhibitor) (10 and 50 microg/kg, s.c.) dose-relatedly inhibited the VEGF (1 ng/site)-induced increase in dye leakage, whereas its negative control, lavendustin B (10 microg/kg, s.c.) had no effect. Another tyrosine kinase inhibitor, genistein (2.5 mg/kg, s.c.) also inhibited the response. Cycloheximide (a protein biosynthesis inhibitor) (35 mg/kg, s.c.) suppressed the response of VEGF (1 ng/site). Histologically, no cellular infiltration was observed in the area of VEGF injection. These results suggest that increased vascular permeability induced by VEGF is mediated by local production of NO and arachidonic acid metabolites other than PGE2, which are most probably produced by inducible NO synthase and cyclooxygenase-2, respectively. Protein tyrosine kinase-mediated phosphorylation and synthesis of any new proteins are likely to be required in this effect of VEGF in mouse skin.

Role of nitric oxide and prostaglandins in lipopolysaccharide-induced increase in vascular permeability in mouse skin

To examine the possible role of increased vascular permeability in the circulatory shock induced by endotoxin (lipopolysaccharide), we examined whether lipopolysaccharide elicits plasma extravasation in the skin of ddY strain mice. We also studied whether nitric oxide (NO) and prostaglandins may mediate the lipopolysaccharide-induced increase in vascular permeability. Subcutaneous injection of lipopolysaccharide (100–400 μg/site) induced a dose-related and delayed increase in vascular permeability at the injection site as determined by the leakage of pontamine sky blue. Concurrent administration of aminoguanidine (a putative inducible NO synthase inhibitor) (10 mg/kg, i.v.) inhibited the lipopolysaccharide (400 μ/site)-induced dye leakage by 71%. N G-Nitro- l-arginine methyl ester (an inhibitor for both constitutive and inducible NO synthase) (10 and 20 mg/kg, i.v.) inhibited the lipopolysaccharide-induced dye leakage by 36% and 54%, respectively, whereas the inactive enantiomer, N G-nitro- d-arginine methyl ester (10 mg/kg, i.v.), had no effect. Pretreatment with an intraperitoneal injection of dexamethasone (500 μg/kg) or indomethacin (a cyclooxygenase-1 and −2 inhibitor) (5 mg/kg) almost completely inhibited the response induced by lipopolysaccharide, by 96% and 84%, respectively. [ N-(2-Cyclohexyloxy-4-nitrophenyl)methanesulphonamide (a cyclooxygenase-2-specific inhibitor) (0.01–1 mg/kg, i.p.) also induced a dose-related inhibition of dye leakage elicited by lipopolysaccharide: 38% and 80% suppression at the doses of 0.1 and 1 mg/kg, respectively. Cycloheximide (a protein biosynthesis inhibitor) (35 mg/kg, s.c.) suppressed the effect of lipopolysaccharide by 74%. These results suggest that the increase in vascular permeability induced by lipopolysaccharide is mediated by both NO and prostaglandins and that synthesis of inducible NO synthase and cyclooxygenase-2 may be involved in this effect of lipopolysaccharide.

起炎物質のマウス皮膚血管透過性こう進作用における内因性一酸化窒素の役割

We examined the changes in vascular permeability induced by inflammatory mediators in mouse skin by the Pontamine sky blue (PSB) leakage technique. Five min after i.v. administration of PSB, mediators or saline were injected (0.1 ml/site, s.c.) into the back. Sixty min later, the dye accumulated in the skin was determined colorimetrically. Plateletactivating factor (PAF, 45-180 pmol/site), 5-hydroxy-tryptamine (5-HT, 120-960 pmol/site), substance P (100-1, 000 pmol/site) and histamine (550-5, 500 pmol/site) produced dose-related increases in vascular permeability. The dose response curve induced by bradykinin (20-80 nmol/site) was bellshaped. Pretreatment with indomethacin significantly reduced the increase in vascular permeability elicited by the 5 substances tested. Coadministration of prostaglandin (PG) E2 partially reversed the inhibitory effect of indomethacin on the PAF- or histamine induced-increase in vascular permeability. Increases in vascular permeability induced by 5-HT, substance P and bradykinin were inhibited by NG-nitro-l-arginine methyl ester (L-NAME) and methylene blue, but not by D-NAME ; however, PAF-and histamine-induced increases in vascular permeability were not affected by L-NAME.These results suggest that eicosanoids may play a role in the effect of all 5 substances tested, and especially the increases in plasma extravasation induced by PAF and histamine are mediated by the production of PGE2, and that NO may play a role, at least partly, in the case of 5-HT, substance P and bradykinin.

Mutual interactions among cholinergic, noradrenergic and serotonergic neurons studied by ionophoresis of these transmitters in rat brainstem nuclei

In urethane-anesthetized rats, single neuronal activity was recorded in or around the central gray of the caudal mesencephalon to rostral pons with multibarrel microelectrodes for ionophoretic application of acetylcholine, noradrenaline and serotonin. Neurons were classified by spike shape into broad-spike and brief-spike neurons. In the laterodorsal tegmental nucleus, locus coeruleus or dorsal raphe, broad-spike neurons, marked by Pontamine Sky Blue and discriminated in sections processed for histochemistry of reduced nicotinamide adenine dinucleotide phosphate diaphorase or Nissl staining, were presumed to be cholinergic, noradrenergic or serotonergic, respectively. The majority of these neurons were inhibited through autoreceptors, except some laterodorsal tegmental neurons which might not be furnished by autoreceptors. Noradrenaline and serotonin inhibited more than two-thirds of the laterodorsal tegmental neurons tested, while a few neurons were excited by noradrenaline. Though effects of noradrenaline on dorsal raphe neurons and those of serotonin on locus coeruleus neurons were not clear in many neurons tested, neurons affected in these examinations (30%) were all inhibited clearly and no excitatory effect was observed. Acetylcholine exerted inhibition on about one-half of dorsal raphe neurons, while effects of acetylcholine on locus coeruleus neurons were the only case in the present study in which excitation was the major effect, though more than a half of locus coeruleus neurons were not sensitive to this drug. Thus, in this study some new data on the pharmacological properties of the cholinergic laterodorsal tegmental neurons were obtained. In addition, mutual interactions between brainstem cholinergic, noradrenergic and serotonergic neurons were assayed by comparing the pharmacological properties of these neurons tested with a uniform procedure. The interactions between these diffuse projection neurons may be involved in neural mechanisms controling vigilance, wakefulness and/or sleep.

Airway inflammatory effect of hydrogen peroxide in guinea pigs

Reactive oxygens are now considered to be important substances in promoting inflammatory process. Recently, airway inflammation has attracted attention closely linked to bronchial asthma. The present study was undertaken to examine whether hydrogen peroxide, one of the reactive oxygens, could produce airway inflammation. Airway inflammation was assessed by airway vascular permeability in terms of pontamine sky blue (PSB) exudation. Airway resistance was measured with a modified Konzett-Rössler method and was expressed as a change in ventilation overflow. Inhalation of hydrogen peroxide (0.01-1.0 M) markedly caused a PSB exudation in a concentration-dependent manner in all of the trachea, main bronchus, and lungs. The hydrogen peroxide-induced PSB exudation effect was attenuated was attenuated by pretreatment with catalase, although heat-inactivated catalase had no inhibitory effect. Deferoxamine, which inhibits conversion of hydrogen peroxide into hydroxyl radical, decreased the PSB exudation induced by hydrogen peroxide. On the other hand, inhalation of hydrogen peroxide (1.0 M) caused a significant and biphasic increase in ventilation overflow. This airway constriction was suppressed by pretreatment with inhaled catalase, but not by inhaled deferoxamine. These results indicate that hydrogen peroxide causes an intense airway inflammation; this inflammatory effect may be mediated not only by hydrogen peroxide itself but also by hydroxyl radical. Hydrogen peroxide and hydroxyl radical may thus play an important role in bronchial asthma and bronchitis through inducing airway inflammation.

Airway inflammation induced by xanthine/xanthine oxidase in guinea pigs.

Airway inflammation is suggested to play an important role in bronchial asthma. However, there is poor documentation about the effects of reactive oxygens on airway tissues in aspect of airway inflammation. Presently, we investigated whether aerosolized xanthine (X)/xanthine oxidase (XOD) induces airway inflammation in anesthetized guinea pigs. Inhalation of X for 5 min followed by inhalation of XOD for 5 min was performed with an ultrasonic nebulizer in anesthetized animals. Airway inflammation was assessed by airway vascular permeability using Pontamine sky blue. Inhalation of X/XOD produced a marked Pontamine sky blue exudation in the trachea, main bronchus and lungs. The X/XOD-induced increase in Pontamine sky-blue exudation was attenuated by pretreatment with inhaled catalase, but not by superoxide dismutase. Additionally, in the bronchus and lungs, the increase in Pontamine sky-blue exudation was significantly suppressed by deferoxamine. The above results indicate that hydrogen peroxide and hydroxyl radical converted from superoxide anion cause an intense airway inflammation.