Conjugates of Chitinase with Fluorescein Isothiocyanate or Lissamine Rhodamine as Specific Stains for Chitin In Sztu

The fluorescent chitinase technique is based on the specific affinity of the enzyme for its substrate and applicable when an enzyme can be coupled with a fluorescent dye. Fluorescent chitinase specifically stained chitinous structures in several fungi and an insect, but failed to stain other polysaccharides in bacterial and algal cell walls. Freezing-microtome sections of Drosophila and fungal mycelia 6 μ thick were fixed in acetone for 5 min, then stained and mounted in fluorescent chitinase. Staining of smears of unsectioned fungal material required 5 min in absolute acetone, 5 min in 95% ethanol-1 N aqueous acetic acid (1:1), 10 min in 0.2 M phosphate buffer, PH 5.7, 1 sec in enzyme-dye conjugate, and 10 min in carbonate-bicarbonate buffer (0.2 M, pH 10.7, for chitinase-FITC; pH 7.6, for chitinase-LRBC). Preparations are viewed microscopically with ultraviolet light.     

Galanin-induced relaxation in gastric smooth muscle cells is mediated by cyclic AMP

Galanin has numerous effects on gastrointestinal motility in different species; however, its cellular basis of action in mediating these effects is unclear. Dispersed gastric smooth muscle cells have been shown to possess high-affinity galanin receptors that increase cAMP and cause relaxation. Recent studies show some smooth muscle relaxants such as VIP cause relaxation by both cAMP-dependent and -independent mechanisms. It is unknown if galanin's cellular basis of relaxation is similar or different from that of VIP. To investigate galanin's relaxant effect and compare it to VIP's effect, dispersed smooth muscle cells from guinea pig stomach were prepared by collagenase digestion. The mean length in resting cells was 110 ± 2 μm and, with carbachol treatment, contracted to 89 ± 2 μm. VIP and galanin alone had no effect on cell length, but each caused a dose-dependent inhibition of carbachol-induced contraction and both had an EC₅₀ of 3–7 nM. Galanin (1 μM) and VIP (1 μM) increased cellular cAMP from 118 ± 10 pmol/10⁶ cells in control to 212 ± 14 and 214 ± 12 pmol/10⁶ cells, respectively. The protein kinase A inhibitor, Rp-cAMPS, at 100 μM, completely inhibited the relaxant effect of an EC₅₀ concentration of galanin (3 nM), but only inhibited that by VIP by 80% (p < 0.05). Adding the nitric oxide inhibitor, -NNA ( ), at 100 μM did not alter the length of resting cells or inhibit carbachol-induced contraction. However, -NNA (100 μM) decreased VIP-induced relaxation by 45%, whereas it had no effect on galanin-induced relaxation. To determine the ability of each peptide to activate nitric oxide, the incorporation of [³H]arginine into [³H]citrulline was determined. Galanin (1 μM) did not cause nitric oxide generation whereas VIP (1 μM) increased nitric oxide generation above the control by 97 ± 14% (p < 0.01). These results demonstrated that with galanin, in contrast to VIP, nitric oxide is not involved in its ability to cause gastric smooth muscle cell relaxation. The relaxant action of galanin can be accounted for completely by its ability to activate protein kinase A and therefore resembles recent results with β-adrenergic agents.     

Role of calcium in the bombesin-induced intestinal CCK release in rats

In the isolated vascularly perfused rat duodenojejunum, vascular infusion of bombesin (100 nM) provoked an early, transient (6 min) release of CCK (500% of basal), followed by a sustained response (400% of basal). The calcium chelator EGTA (2 mM) reduced the early peak and abolished the second phase of CCK release. A similar variation was evoked by verapamil (10 μM), whereas diltiazem (100 μM), nifedipine (50 μM), and ω-conotoxin (100 nM) had no significant effect. It is concluded that bombesin-induced CCK release from rat intestine is dependent on the availability of extracellular calcium and on the activation of calcium channels sensitive to blockers of the phenylalkylamine family.     

Effects of peptide YY and its analogues on chloride ion secretion in fed and fasted rat jejunum

The aim of our study was to determine whether a meal modifies the antisecretory response induced by PYY and the structural requirements to elicit antisecretory effects of analogue PYY(22–36) for potential antidiarrhea therapy. The variations in short-circuit current (Isc) due to the modification of ionic transport across the rat intestine were assessed in vitro, using Ussing chambers. In fasted rats, PYY induced a dose- and time-dependent reduction in Isc, with a sensitivity threshold at 5 × 10⁻¹¹ M (ΔIsc −2 ± 0.5 μA/cm²). The reduction was maximal at 10⁻⁷ M (Isc −23 ± 2 μA/cm²), and the concentration producing half-maximal inhibition was 10⁻⁹ M. At 10⁻⁷ M, reduction of Isc by PYY reached 90% of response to 5 × 10⁻⁵ M bumetanide. The PYY effect was partly reversed by 10⁻⁵ M forskolin (Isc +13.43 ± 2.91 μA/h·cm², p < 0.05) or 10⁻³ M dibutyryl adenosine 3′,5′ cyclic monophosphate (Isc +12 ± 1.69 μA/cm², p < 0.05). Naloxone and tetrodotoxin did not alter the effect of PYY. In addition, PYY and its analogue P915 reduced net chloride ion secretion to 2.85 and 2.29 μEq/cm² (p < 0.05), respectively. The antisecretory effect of PYY was accompanied by dose- and time-dependent desensitization when jejunum was prestimulated by a lower dose of peptide. The antisecretory potencies exhibited by PYY analogues required both a C-terminal fragment (22–36) and an aromatic amino acid residue (Trp or Phe) at position 27. At 10⁻⁷ M the biological activity of PYY was lower in fed than fasted rats (p < 0.001). Our results confirm the antisecretory effect of PYY, but show that the fed period is accompanied by desensitization, similar to the transient desensitization observed in the fasted period with cumulative doses. This suggests that PYY may act as a physiological mediator that reduces intestinal secretion.     

Determination of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione in plasma by direct injection into a coupled column liquid chromatographic system

The chemical substance 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) is in clinical use for the treatment of hereditary tyrosinemia type 1. In the present study, the plasma concentration of NTBC was determined by a coupled column liquid chromatographic method. A 20-μl volume of plasma was diluted with phosphate buffer, pH 2, and injected into a small precolumn (BioTrapAcid C₁₈) with a mobile phase containing sulfuric acid. The precolumn was based on the restricted access principle, i.e., retention of NTBC within the lipophilic pores, while polar and large endogenous compounds were eluted with the void volume. NTBC was transferred to the analytical column using a mobile phase with a high content of acetonitrile. The compound was monitored by UV detection at 278 nm. The standard curve was linear between 0.3 and 69 μM, and the between-day precision (RSD) was 3% (n=6 days) at 13.8 μM and 14% (n=6 days) at 0.3 μM NTBC in plasma. The quantitation limit was approximately 0.3 μM using 20 μl of plasma.     

Stimulation by melanocortins of neurite outgrowth from spinal and sensory neurons in vitro

Using ELISAs for B-50/GAP43 and neurofilament (NF), we tested ACTH(1–24), -MSH, ACTH(4–10), and an ACTH(4–9) analogue (ORG2766) for their ability to induce sprouting and neuritogenesis from spinal and sensory neurons. Dissociated fetal rat spinal cord neurons or neonatal rat dorsal root ganglion (DRG) cells were cultured with peptide and assayed after 24, 48, or 96 h. In spinal neurons, -MSH and ACTH(1–24) induced the expression of B-50 dose dependently. After 24 h -MSH had a stimulatory effect (from 10 nM onwards), with a maximum at 100 μM (36% increase). After 96 h the maximal effect of 100 μM -MSH on B-50/GAP43 was lower (19%). ACTH(1–24) (100 μM) stimulated B-50/GAP43 by 19%. Neurofilament levels (96 h) were elevated maximally by 64% at 100 μM -MSH. In DRG neurons a bell-shaped dose-response curve was found for -MSH, the maximal effect being observed after 48 h at 100 nM: 54% for B-50/GAP43 and 22% for NF. In both culture systems neither ACTH(4–10) nor ORG2766 was effective. We conclude that -MSH stimulates the expression of B-50/GAP43 (sprouting) and the formation of NF (neurite elongation) and may therefore be considered a neurotrophic factor.     

Determination of pyronaridine in blood plasma by high-performance liquid chromatography for application in clinical pharmacological studies

A method is described for the determination of pyronaridine in plasma using high-performance liquid chromatography with fluorescence detection. The method involves liquid-liquid extraction with phosphate buffer (pH 6.0, 0.05 M) and diethyl ether-hexane (70:30%, v/v) and chromatographic separation on a C₁₈ column (Nucleosil, 250 × 4.6 mm I.D., 5 μm particle size) with acetonitrile-0.05 M phosphate buffer pH 6.0 (60:40%, v/v) as the mobile phase (1 ml/min) and detection by fluorescence (λ_ex = 267 nm, λ_em = 443 nm). The detector response is linear up to 1000 ng and the overall recoveries pyronaridine and quinine were 90.0 and 60.3%, respectively. The assay procedure was adequately sensitive to measure 10 ng/ml pyronaridine in plasma samples with acceptable precision (< 15% C.V.). The method was found to be suitable for use in clinical pharmacological studies.     

Determination of acyclovir and its metabolite 9-carboxymethoxymethylguanine in serum and urine using solid-phase extraction and high-performance liquid chromatography

A reversed-phase ion-pair high-performance liquid chromatography method for the determination of acyclovir and its metabolite 9-carboxymethoxymethylguanine is described. The samples are purified by reversed-phase solid-phase extraction. The components are separated on a C₁₈ column with a mobile phase containing 18% acetonitrile, 5 mM dodecyl sulphate and 30 mM phosphate buffer, pH 2.1, and measured by fluorescence detection using an excitation wavelength of 285 nm and an emission wavelenght of 380 nm. Detection limits are 0.12 μM (plasma)) and 0.60 μM (urine) for acyclovir, and 0.26 μM (plasma) and 1.3 μM (urine) for metabolite. Correlation coefficients that were better than 0.998 were obtained normally. This analytical method, which enables simultaneous measurement of parent compound and metabolite, has been used in kinetics studies and for therapeutic drug monitoring in different patient groups with variable degrees of renal dysfunction.     

Counterstaining Golgi-Cox Impregnations with Luxol Fast Blue as a Myelin Stain

Immerse pieces of brain tissue 4 wk in solutions A and B, mixed just before use: A. K₂Cr₂O₇, 1 gm; HgCl₂, 1 gm; boiling distilled water, 85 ml. Boil A for 15 min, cool to 2 C and add: B. K₂CrO₄, 0.8 gm; Na₂WO₄, 0.5 gm; distilled water, 20 ml. Rinse in water and immerse 24 hr in LiOH, 0.5 gm; KNO₃, 15 gm; distilled water, 100 ml. Wash 24 hr in several changes of 0.2% acetic acid and then for 2 hr in tap water. Dehydrate and embed in celloidin. Process a 60 μ section through 70 and 95% ethanol, a 3:1 mixture of absolute ethanol and chloroform, and toluene. Immerse it for 5 min in a solution containing methyl benzoate, 25 ml; benzyl alcohol, 100 ml; chloroform, 75 ml. Orient the section on a chemically clean slide and let air-dry 5-10 min. Process through toluene, 3:1 ethanol-chloroform and 95% ethanol. Place the section for 5-60 min at 60 C in a solution made up of: Luxol fast blue G (Matheson, Coleman and Bell), 1 gm; 95% ethanol, 1000 ml; 10% acetic acid, 5 ml. Hydrate to water and immerse in 0.05% Li₂CO₃ for 3-4 min. Differentiate in 70% ethanol and place in water. Immerse for 5-15 min in a mixture of two solutions: A. cresylechtviolet (Otto C. Watzka, Montreal), 2 gm; 1 M acetic acid, 185 ml; B. 1 M sodium acetate, 15 ml; distilled water, 400 ml; absolute ethanol, 200 ml. Dehydrate to 3:1 ethanol-chloroform. Clear in toluene and apply a coverslip. The technique produces fast Golgi-Cox impregnated neurons against a background of counterstained myelinated fibers. Patterns of the myelinated fibers can be used to localize impregnated neurons.     

Quantitation of harringtonine and homoharringtonine in serum by high-performance liquid chromatography

Harringtonine and homoharringtonine are naturally occurring alkaloids with demonstrated antineoplastic activity against certain types of leukemias in cell cultures, experimental animals, and initial clinical trials. Sample preparation consists of addition of the internal standard (one compound used as the internal standard for the other), solvent extraction with methylene chloride, washing with ammonium formate, and evaporation to dryness. The residue is dissolved in the mobile phase (40% methanol—60% 0.1M ammonium formate) and an aliquot is chromatographed on μC₁₈ reversed-phase column (flow-rate 1.5 ml/min). Peaks are detected with a spectrophotofluorimeter by monitoring the emission at 320 nm with excitation wavelength of 280 nm. Limit of detection is 10 ng/ml (20 nM) for both compounds; reproducible quantitation can be made to 30 ng/ml (60 nM).     

Isolation of a renin-like enzyme from the leech Theromyzon tessulatum

This article reports the purification of a renin-like enzyme (an aspartyl protease) from head parts of the leech Theromyzon tessulatum. After four steps of purification including gel permeation and anion exchange chromatographies followed by reversed-phase HPLC, this enzyme was purified to homogeneity. The renin-like enzyme (of 32 kDa) hydrolyses at neutral pH and at 37°C, the Leu¹⁰-Leu¹¹ bond of synthetic porcine angiotensinogen tetradecapeptide yielding the angiotensin I and the Leu¹¹-Val¹²-Tyr¹³-Ser¹⁴ peptide as products, with a specific activity of 1.35 pmol AI/min/mg (K_m 22 μM; K_cat 2.7). The hydrolysis of angiotensinogen is inhibitable at 90% by pepstatin A (IC₅₀ = 4.6 μM), consistent with a renin activity. This is the first biochemical evidence of renin-like enzyme in invertebrates.     

Simultaneous determination of 5-fluorouracil and uracil by high-performance liquid chromatography using four serial columns

A sensitive assay was developed for the quantitation of 5-fluorouracil (5-FU) and uracil using liquid–liquid extraction (LLE) and HPLC with UV detection. Analyses were performed with four μBondapak C₁₈ columns connected in series using 20 mM acetic acid with 1% ACN as mobile phase. The calibration curves were linear across the range of 26–1000 ng ml⁻¹ (0.21–7.8 μM) for 5-FU and 1.0–14.0 μg ml⁻¹ (0.01–110 μM) for uracil. This assay has been implemented to determine the plasma concentrations for pharmacokinetic studies for 5-FU and uracil in conjunction with clinical trials.     

The Calcitonin Gene-Related Peptide Activates Both cAMP and NO Pathways to Induce Relaxation of Circular Smooth Muscle Cells of Guinea-Pig Ileum

Rekik, M., M. Delvaux, J. Frexinos, and L. Bueno. Calcitonin gene-related peptide activates both cAMP and NO pathways to induce relaxation of circular smooth muscle cells of guinea-pig ileum. Peptides 18(10) 1517–1522, 1997.—The direct effects and the intracellular pathways of rCGRP were investigated on smooth muscle cells (SMC) isolated by enzymatic digestion from the circular and longitudinal layers of guinea-pig ileum. In circular SMC, rCGRP inhibited CCK8-induced contraction in a concentration-dependent manner (Cmax = 100 μM and EC₅₀ = 0.7 ± 0.4 nM). Preincubation of SMC with 1 μM Rp-cAMPs, a cAMP antagonist, abolished the relaxing effect of rCGRP; moreover, preincubation of SMC with 100 μM L-NAME, an inhibitor of NOS, inhibited the relaxing effect of rCGRP. hCGRP(8-37), a selective antagonist of rCGRP receptors, inhibited the rCGRP-induced relaxation in a concentration dependent manner whereas the vasoactive intestinal polypeptide (VIP) antagonist had no significant effect. In longitudinal SMC, rCGRP-induced relaxation was abolished by Rp-cAMPs, whereas L-NAME had no effect. In conclusion, rCGRP triggers different intracellular pathways to induce relaxation of circular or longitudinal intestinal SMC; cAMP is involved in cells from both layers while nitric oxide (NO) is involved only in relaxation of circular SMC.     

Histological Localization of Microelectrode Placement in Brain by Ferrocyanide and Silver Staining

After recordings had been taken from a microelectrode used for mapping nerve impulses, a current of 100 μa from the positive pole of a direct current generator was run through the electrode for 5 sec while it was still in place. On terminating the experiment, in which the use of several electrodes was possible, 50-75 ml of a 1:1 mixture of 4% potassium ferrocyanide and 4% acetic acid was injected into each common carotid artery, and the brain left in situ for 0.5 hr. It was then removed and the electrode-bearing part fixed 5-6 hr in a 1:1 mixture of 40% formalin and 95% ethyl alcohol at 55 °C. This specimen was washed in running water 5-10 min, the electrodes removed and frozen sections of 40-80 μ cut and placed in 95% alcohol. Sections were stained 5-10 min at 25-30°C in 10% silver nitrate solution in 75-80% alcohol acidified by 3-4 drops of glacial acetic acid per 50 ml, washed 4-5 sec in each of 2 baths of 95% alcohol, and reduced while being agitated constantly in a 2% solution of pyrogallol and 6-7% formalin in 75-80% alcohol. Washing in 95% alcohol, clearing in clove oil or methyl salicylate followed by xylene and mounting in synthetic resin or balsam completed the process. Sites of electrolysis at the tips of electrodes (under magnification) were blue before silver staining and black after staining. Axons stained brown to black on a yellow background.     

Hemoglobin and iron-evoked oxidative stress in the brain: protection by bile pigments, manganese and S-nitrosoglutathione

In the present in vitro and in vivo study we investigated the pro-oxidant effects of hemoglobin, as well as the antioxidant effects of its metabolites, in the brain. Incubation of rat brain homogenates with hemoglobin (0-10 μM) but not hemin induced lipid peroxidation up to 24 h (EC₅₀ = 1.2 μM). Hemoglobin's effects were similar to ferrous ion (EC₅₀ = 1.7 μM) and were blocked by the chelating agent deferoxamine (IC₅₀ = 0.5 μM) and a nitric oxide-releasing compound S-nitrosoglutathione (IC₅₀ = 40 μM). However, metabolites of hemoglobin — biliverdin and bilirubin — inhibited brain lipid peroxidation induced by cell disruption and hemoglobin (biliverdin IC₅₀ = 12-30 and bilirubin IC₅₀ = 75-170 μM). Biliverdin's antioxidative effects in spontaneous and iron-evoked lipid peroxidation were further augmented by maganese (2 μM) since manganese is an antioxidative transition metal and conjugates with bile pigments. Intrastriatal infusion of hemoglobin (0-24 nmol) produced slight, but significant 20-22% decreases in striatal dopamine levels. Whereas, intrastriatal infusion of ferrous citrate (0-24 nmol) dose-dependently induced a greater 66% depletion of striatal dopamine which was preceded by an acute increase of lipid peroxidation. In conclusion, contrary to the in vitro results hemoglobin is far less neurotoxic than ferrous ions in the brain. It is speculated that hemoglobin may be partially detoxified by heme oxygenase and biliverdin reductase to its antioxidative metabolites in the brain. However, in head trauma and stroke, massive bleeding could significantly produce iron-mediated oxidative stress and neurodegeneration which could be minimized by endogenous antioxidants such as biliverdin, bilirubin, manganese and S-nitrosoglutathione.     

A Simple Stain for Myelin in Frozen Sections: A Modification of Mahon's Method

A simple and rapid method for demonstrating myelinated nerve fibers in frozen sections of the central and peripheral nervous system is described. Material fixed by perfusion with mixed aldehydes gives the best results but the method also works on specimens fixed by immersion in formaldehyde. Frozen sections varying in thickness from 15-50 μm are mounted on slides subbed with chrome alum-gelatin. After hydration (60-140 min), Sections are mordanted (20-40 min) in 2.5% iron alum and rinsed briefly in three changes of distilled H₂O (total 2 min). Staining is for 60-180 min in 40 cc freshly made 10% alcoholic hematoxylin diluted with 165 cc distilled H₂O to which 15 cc saturated Li₂CO₂is added. the sections are washed in distilled H₂O (5-15 min) and dehydrated in graded alcohols without differentiation in mordant, and covered. Myelin stains a dark blue-purple against a light grey background. Fiber tracts, as well as individual myelinated fibers, are clearly demonstrated.     

Improved method for the routine analysis of acetylcholine release in vivo: quantitation in the presence and absence of esterase inhibitor

An improved high-performance liquid chromatographic (HPLC) method using electrochemical detection (ED) is described capable of routinely measuring the low levels of acetylcholine (ACh) typically found in rat brain microdialysis samples. Microdialysis was performed in the striatum of the urethane anesthetized rat using a 4-mm membrane length, high recovery (40% at 1.0 μl/min; ambient conditions), loop-design probe perfused with an artificial cerebrospinal fluid (aCSF) solution containing physiologically normal calcium levels (1.2 mM). The HPLC method utilizes a polymeric stationary phase to resolve choline (Ch) from ACh. These analytes are then converted to hydrogen peroxide (H₂O₂) by a solid-phase reactor (containing immobilized choline oxidase and acetylcholinesterase enzymes). The H₂O₂ is detected amperometrically and quantitated on a platinum (Pt) working electrode (+300 mV; with a unique analytical cell featuring a solid-state palladium reference electrode). Two designs of the Pt working electrode were examined, differing only in the support material used (Kel-F or PEEK). The Kel-F/Pt electrode had a limit of detection (LOD) for both analytes of <30 fmol per 10 μl with a signal-to-noise ratio of 3:1. Striatal microdialysis perfusates were monitored for ACh and Ch over a 0–1000 nM range of neostigmine (NEO) in the CSF perfusion medium. Using the 4-mm probe, basal ACh and Ch levels were detected with a NEO level as low as 10 nM and were found to be 37 ± 3 fmol and 22 ± 1 pmol per 10 μl (mean ± S.E.M., n = 6 replicates) respectively. In similar experiments using 3-mm concentric probes comparable (lower) levels of ACh were found with the 50 and 1000 nM NEO doses (n = 4–21 animals). ACh could not be reliably quantitated when animals were perfused with the 10 nM dose of NEO (n = 4). The PEEK/Pt electrode had an improved LOD of < 20 fmol per 10 μl due to a two- to three-fold decrease in the background noise component. Basal striatal levels of ACh in the absence of NEO approached the LOD and were found to be 15 ± 2 fmol per 10 μl; Ch was 5 ± 1 pmol per 10 μl (n = 2, mean of five basal samples). The analytical system requires very little maintenance; a simple electrochemical electrode cleaning step eliminates the need for routine polishing of the Pt electrode and the mobile phase is stable for up to one week. Both ACh and Ch are resolved in under 7 min making this method highly suitable for analysis of microdialysis samples.     

Determination of transdermal sildenafil in nude mouse skin by reversed-phase high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatographic method was developed for the determination of sildenafil transdermal permeation of nude mouse skin. A reversed-phase column with UV detection at 224 nm was used for chromatographic separation. The mobile phase consisted of 32% acetonitrile with 0.2% phosphoric acid in water at pH 5.3 adjusted with 10 M NaOH with the flow-rate set at 1.0 ml/min. The limit of quantitation achieved was 5 ng/ml, and the calibration curve showed good linearity over the concentration range of 5–500 ng/ml. The relative standard deviations of within- and between-day analyses were all within 15%. Sildenafil was found to be stable between pH 3 and 12 during 24-h incubation with skin. After transdermal administration of 15.8 μg/ml of sildenafil to nude mouse skin, it was detected as early as 15 min. The transport amount of sildenafil could be quantitated and, at pH 8–11, had the highest permeation rate in nude mouse skin.     

Staining Mitochondria With Hematoxylin After Formalin-Sublimate Fixation; A Rapid Method

Mitochondria were stained in liver, kidney, pancreas, adrenal and intestinal mucosa of rat and mouse. Tissues 1 mm thick, were fixed in a mixture of saturated aqueous HgCl₂, 90 ml; formalin (37-38% HCHO), 10 ml, at room temperature (25°C) for 1 hr. Deparaffinized sections 3-4μ thick were treated with Lugol's iodine (U.S.P.) followed by Na₂S₂O₃ (5%), rinsed in water and the ribonucleic acid removed by any of the following procedures: 0.2 M McIlavaine's buffer, pH 7.0, 2 hr, or 0.2 M phosphate buffer, pH 7.0, 2 hr at 37°C; 0.1% aqueous ribonuclease, 2 hr at 37°C; 5% aqueous trichloracetic acid overnight at 37°C; or 1% KOH at room temperature for 1 hr. After washing in water, sections were treated with a saturated solution of ferric ammonium alum at 37°C for 8-12 hr and colored by Regaud's ripened hematoxylin for 18 hr. They were then differentiated in 1% ferric ammonium alum solution while under microscopic observation.     

Comparative effects on blood pressure and heart rate of dynorphin A(1–13) in anterior hypothalamic area, posterior hypothalamic area, nucleus tractus solitarius, and lateral cerebral ventricle in the rat

The objective of this study was to explore the effects of the endogenous opioid peptide dynorphin A(1–13) on the CNS regulation of blood pressure and heart rate. Wistar rats, anesthetized with pentobarbital and halothane, received dynorphin A(1–13) microinjected into the anterior hypothalamus area (AHA), the posterior hypothalamic area (PHA), the nucleus tractus solitarius (NTS), or the lateral cerebral ventricle (ICV). Dynorphin A(1–13), 20 (12 nmol) or 30 μg ICV, produced significant (p < 0.05) reductions in blood pressure and heart rate. Naloxone, 50 μg/kg ICV, completely prevented the blood pressure response and significantly (p < 0.05) blunted the heart rate response to the highest dynorphin concentration, 30 μg ICV (18 nmol). Dynorphin A(1–13), 5 μg, in the NTS significantly (p < 0.05) decreased systolic and diastolic blood pressure and heart rate with the response being evident 10 min and persisting for 30 min after injection. In contrast, the same dose of dynorphin A(1–13) in the AHA produced an immediate, marked, and significant (p < 0.05) decrease in systolic and diastolic blood pressure and heart rate that attained its maximum 1–3 min and returned rapidly towards baseline levels. Dynorphin A(1–13), 5 or 10 μg in the posterior hypothalamic area, was not associated with any change in blood pressure or heart rate. Injection of the diluent at any site was not associated with any changes in blood pressure or heart rate. The maximum change in blood pressure with dynorphin was greater in the AHA than NTS, and the maximum change in heart rate was greater in the NTS than AHA. These data indicate a potential role for dynorphin as a modulator of the CNS regulation of blood pressure and cardiac rate, and this is mediated in part through different areas in the brain that maybe localized to the anterior hypothalamic area and nucleus tractus solitarius but not the posterior hypothalamic area.