Direct measurement of probenecid and its glucuronide conjugate by means of high pressure liquid chromatography in plasma and urine of humans

Probenecid with its phase-I metabolites, and phase-II glucuronide conjugate can be analysed by a gradient high pressure liquid chromatographic method. Probenecid glucuronide in plasma with pH 7.4 is not stable and declines to 10% of the original value within 6 h (t_1/21 h). Probenecid glucuronide is stable in urine with pH 5.0, moderately unstable at pH 6.0 (t_1/210 h), and unstable at pH 8.0 (t_1/20.5 h). Probenecid glucuronide is stable in water and 0.01 mol/l phosphoric acid in the autosampler of the high pressure liquid chromatograph. The decrease in concentration in water is 5.5% during 9 h and 0% in diluted acid. Probenecid glucuronide and the phase-I metabolites were not detectable in plasma. The main compound in fresh urine is the phase-II conjugate probenecid glucuronide (62% of a 500 mg dose); the phase-I metabolites are present and only a trace of probenecid is present. The percentage of the dose of the phase-I metabolites varies between 5 and 10, while hardly any probenecid is excreted unchanged (0.33%).     

5-Hydroxytryptamine-induced contractions of the human isolated saphenous vein: involvement of 5-HT2 and 5-HT1D-like receptors,and a comparison with grafted veins

Summary The receptors mediating the contractile effect of 5-hydroxytryptamine (5-HT) on the human isolated saphenous vein, obtained from 42 patients undergoing coronary bypass surgery, have been further characterized using a number of 5-HT-related drugs. The rank order of agonist potency was 5-carboxamidotryptamine (5-CT) 5-HT > methysergide sumatriptan -methyl-5-HT 5-methoxy-3-(1,2,3,6-tetrahydropyridin-4-yl)-1-Hindolesuccinate (RU 24969) 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI) > 2-methyl-5-HT > 8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT). Flesinoxan was inactive as an agonist. Ketanserin (1 mol/l) hardly affected sumatriptan-induced contractions but it caused a rightward shift of the upper part of the concentration-response curve of 5-HT and 5-CT. The same concentration of ketanserin caused a parallel rightward shift of the concentration-response curves of -methyl-5-HT and DOI with pK_B values of 7. 1 and 7.1, respectively. The responses to sumatriptan were antagonized by methiothepin (0.1 mol/l), metergoline (0.1 and 1 mol/l), rauwolscine (1 mol/l) and cyanopindolol (1 mol/l); the calculated pK_B values were 7.3, 6.9, 7.3, 6.7 and 6.5, respectively. Contractions to 5-HT were antagonized by methysergide (1 mol/l), methiothepin (0.1 mol/l; pKB = 7.1), ICS 205-930 (1 mol/l; pK_B = 5.9) and flesinoxan (30 mol/l; pK_B = 5.3). Remarkably, the contractions elicited by 2-methyl-5-HT were not attenuated by ICS 205-930, but were antagonized by methiothepin (0.1 mol/l) and, more markedly, by ketanserin (1 mol/l).There was a high correlation between the functional pD₂ values of 5-HT₁-like receptor agonists (5-CT, 5-HT, methysergide, sumatriptan, RU 24969 and 8-OH-DPAT) and their reported binding affinities for the 5-HT_1D receptor in human or calf brain membranes. Such a correlation for the antagonism of sumatriptan-induced responses was less marked than for the agonists, but of the 5-HT₁-like receptor subtypes it was the highest for the 5-HT_1D receptor identified in human or calf brain membranes.In 3 patients, undergoing heart transplantation, saphenous vein which had previously functioned as a graft for 6–11 years, was dissected out from the heart. Though the contractions to potassium were significantly smaller in the grafted veins, the pD₂ and E_max values (calculated as percentage of potassium-induced contractions) for 5-HT and sumatriptan were similar to those found in the veins obtained directly from the lower leg.It is concluded that contractions in the human isolated saphenous vein induced by 5-HT are mediated by 5-HT₂ receptors as well as by a 5-HT₁-like receptor resembling the 5-HT_1D subtype found in brain membranes. It is also to be noted that 2-methyl-5-HT, considered selective for the 5-HT₃ receptor, contracts the saphenous vein mainly via 5-HT₂ receptors.This study was supported by the Netherlands Heart Foundation, grant 89.252 Send offprint requests to W. A. Bax at the above address     

Chronic β1-adrenoceptor blockade sensitises the H1 and H2 receptor systems in human atrium: role of cyclic nucleotides

We have reported that chronic treatment of patients with ₁-adrenoceptor blockers sensitises isolated atrial preparations to adrenaline, noradrenaline and 5-HT. We have now examined the effect of chronic treatment with -adrenoceptor blockers on responses to histamine of human right atrial appendages. We compared the effects of histamine on contractile force, cyclic AMP and cyclic GMP levels as well as cyclic AMP-dependent protein kinase (PKA) activity and explored the arrhythmogenic effects of histamine in preparations obtained from patients chronically treated or not treated with -adrenoceptor blockers.Histamine increased contractile force in paced preparations; the effects were blocked by the H₂ receptor antagonist famotidine (0.1–30 mol/1). The maximum inotropic response to histamine was doubled and the inotropic potency of histamine 0.4 log units greater in atria from -adrenoceptor blocker-treated compared to non -adrenoceptor blocker-treated patients. Histamine elicited frequency-dependent arrhythmias that were blocked by famotidine (30 mol/1) but not by mepyramine (1 mol/1). The incidence of arrhythmias was higher in atria from -adrenoceptor blocker-treated compared to untreated patients. Histamine increased both cyclic AMP and cyclic GMP levels, as well as PKA activity, significantly more in atria from -adrenoceptor blocker-treated compared to those from untreated patients. Mepyramine 1 mol/l prevented the histamine-evoked increase in cyclic GMP levels, reduced the inotropic hyperresponsiveness and abolished the hyperresponsiveness in cyclic AMP levels and PKA activity observed in patients chronically treated with blockers. Sodium nitroprusside 10 mol/l caused smaller increases of cyclic GMP levels than histamine and restored the contracile force depressed by mepyramine to its original level in atria from -adrenoceptor blocker-treated patients.The evidence is consistent with sensitisation of both the histamine H₁ and histamine H₂ receptor systems by chronic ₁-adrenoceptor blockade. H₁ receptor-mediated increases in cyclic GMP, enhanced through an as yet unknown mechanism by chronic ₁-adrenoceptor blockade, may inhibit phosphodiesterase 3 activity, thereby causing enhanced histamine-evoked increases in cyclic AMP levels and PKA activity, and accounting partially for the increased inotropic responses to histamine through H₂ receptors.     

Poly(Alkylcyanoacrylate) Nanocapsules: Physicochemical Characterization and Mechanism of Formation

Nanocapsules of poly(isobutylcyanoacrylate) and poly(isohexylcyanoacrylate) were prepared by addition of the monomer to an organic phase and subsequent mixing of the organic phase to an aqueous phase containing poloxamer 188, 238 or 407. Gel permeation chromatography indicated that in contrast to literature reports, polymerization occurred in the organic phase and nanocapsules were obtained by interfacial precipitation of the polymer without any significant change of the molecular weight. Addition of SO₂ to the organic phase before the introduction of the monomer allowed preparation of nanocapsules with a lower molecular weight. Nanospheres were prepared in a similar way albeit using an organic phase that was completely miscible within the aqueous phase so that solid spheres were obtained. Density gradient centrifugation revealed that nanocapsules had a density intermediate between nanospheres and an emulsion prepared in the same way without addition of monomer to the organic phase. Further, the process used to prepare nanocapsules had a high yield since no oil droplets or nanospheres were obtained by this process. Zeta potential of the nanocapsules and spheres was found to be related to the molecular weight of the polymer: values as high as –42 mV were obtained for low molecular weight nanocapsules (MW 1000) compared to –l0mV for the emulsion and the high molecular weight nanocapsules (MW 100 000). Surface charge of the nanocapsules and molecular weight of their polymeric wall conditioned the adsorption capacity of poloxamers. Moreover, the highest adsorption was measured with the most hydrophobic poloxamer. These observations agree with previous work conducted on hydrophobic surfaces.     

Effect of terfenadine and ranitidine on histamine and suxamethonium wheals

Summary The effects of the H-1 receptor blocker terfenadine and the H-2 receptor blocker ranitidine have been experimentally tested in wheals induced with histamine and suxamethonium chloride.Ranitidine alone in the standard and a four-fold higher dose did not notably reduce the size of the wheal and flare as compared to placebo. As expected, both parameters were markedly reduced by the H-1 antihistamine terfenadine in full and half-dosage. A combination of the two drugs both in standard and modified dosage resulted in the greatest reduction in the wheal and flare.Histamine and suxamethonium-induced wheals reacted in a similar manner to the antihistamines.     

Role of Brain Tissue Localized Purine Metabolizing Enzymes in the Central Nervous System Delivery of Anti-HIV Agents 2′-β-Fluoro-2,3′-Dideoxyinosine and 2′-β-Fluoro-2′,3′-Dideoxyadenosine in Rats

Purpose. This study examines the central nervous system (CNS) delivery of 2--fluoro-2,3-dideoxyadenosine (F-ddA) and 2--fluoro-2,3-dideoxyinosine (F-ddl), acid stable analogues of dideoxyadenosine (ddA) and dideoxyinosine (ddI) having reduced susceptibility to purine salvage pathway enzymes important in the metabolism of ddA and ddI, adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP), respectively. Their CNS delivery compared to that for ddI provides insight into the role of brain tissue ADA and PNP in these processes. Methods. Brain and cerebrospinal fluid (CSF) concentration-time profiles were obtained for F-ddI during and after intravenous infusions of F-ddl, and for both F-ddA and F-ddI after F-ddA infusions in normal rats or rats pre-treated with the ADA inhibitor 2-deoxycoformycin (DCF). Rate constants for CNS entry, efflux and metabolism were estimated by computer fits using plasma concentration-time profiles as the driving force functions. Results. The CNS delivery of F-ddI did not differ significantly from that for ddI. F-ddA, which is more lipophilic than F-ddI, provided higher brain ( 8×) and CSF ( 11×) concentrations of total dideoxynucleoside (F-ddA and F-ddI) compared to F-ddI. Deamination by brain tissue ADA to form F-ddI reduced CNS levels of intact F-ddA but provided higher brain parenchyma (5×) and CSF/plasma (3×) ratios of F-ddI relative to F-ddI controls. Thus, F-ddA functions in part as a CNS-activated prodrug of F-ddI. DCF pre-treatment inhibited brain tissue ADA, abolishing the prodrug effect, and enhancing F-ddA concentrations in both brain parenchyma (5×) and CSF (6×). Conclusions. PNP metabolism does not appear to play a role in the low CNS delivery of ddI. On the other hand, deamination of F-ddA by brain tissue ADA is an important process, such that F-ddA functions in part as a CNS-activated prodrug of F-ddI. Enhanced CNS uptake of intact F-ddA can be achieved with ADA inhibition.     

The Influence of Sodium Glycocholate and Other Additives on the in vivo Transfection of Plasmid DNA in the Lungs

Purpose. A plasmid containing the luciferase marker cDNA was constructed to test non viral gene delivery formulations in vivo. Methods. A scale up procedure was devised to produce up to gram quantities of plasmid. Sufficient quantities were generated to process and test the DNA with various additives and to generate a spray-dried powder formulation of the plasmid. Male Sprague-Dawley rats (250 g) were intratracheally instilled with 200–250 µl of solution containing 200 µg plasmid ± lipid [DC Chol:DOPE 1:1 molar (2mg/kg)] growth factors [KGF (10 mg/kg), EGF (5 mg/kg)], permeation enhancers [sodium glycocholate (0.01 to 10% w/v)), sodium deoxycholate (1% w/v), beta-cyclodextrin (1% w/v)], surfactant [Tween 80 (1% w/v)], a mucolytic [N-acetylcysteine (10% w/v)] and positively charged synthetic polymers [PVAVAM 6 and 14%]. Animals were sacrificed 24 hr post-dose and the lungs were assayed for luciferase using a chemiluminescent assay. Results. The relative ability of the materials to promote luciferase production in the lungs was permeation enhancer >> DNA alone lipid, mucolytic, surfactant, growth factor > polymer. Protein production in the lungs ranged from 10 times below the DNA control (16 pg) using the polymers (1.5 pg) to 125 times greater than the control using the permeation enhancer (2050 pg). The transfection capabilities of the majority of additives was low. The enhancing effects of sodium glycocholate were dose-dependent and perhaps associated with the critical micelle concentration. Although the bile salt was the most successful of the tested compounds, it resulted in significant mortality when used at concentrations greater than 1 % w/v. Conclusions. The results suggest that transfection can be significantly enhanced by additives such as NaGC but some toxicity may be unavoidable.     

Nitric oxide influences the release of histamine and glutamate in the rat hypothalamus

To investigate the influence of nitric oxide (NO) on the release of histamine and glutamate, the anterior hypothalamus of anaesthetized rats was superfused through a push-pull cannula either with artificial cerebrospinal fluid (CSF) or with various drugs dissolved in CSF.Hypothalamic superfusion with the NO-donating compounds linsidomine (200 mol/l) or diethylamine-NO (DEANO, 100 mol/l) led to a pronounced and sustained decrease in the histamine release rate, whereas the release rate of glutamate was enhanced. Superfusion with the inhibitor of NO synthase L-N^G-nitro-L-arginine methyl ester (L-NAME, 200 mol/l) increased the histamine release rate. The inhibitory effect of 200 mol/l linsidomine was abolished by atropine (10 mol/l). Superfusion with the glutamate receptor agonists glutamate (100 mol/l) or N-methyl-D-aspartate (NMDA, 50 mol/l) enhanced the histamine release rate. In the presence of linsidomine, the releasing effect of NMDA was not changed.These findings demonstrate that the release of histamine in the hypothalamus is diminished by endogenous NO. This effect of NO on histamine release seems to be due to enhanced release of acetylcholine from vicinal cholinergic neurons via stimulation of muscarinic acetylcholine receptors located presynaptically on histaminergic neurons. The NO-induced glutamate release seems to exert a subordinate stimulatory effect on histamine release. Finally, the inhibition of histamine release by NO is not due to blockade of NMDA receptors.This work was supported by the Jubiläumsfonds der österreichischer Nationalbank     

Liposomes Containing Interferon-Gamma as Adjuvant in Tumor Cell Vaccines

Purpose. Liposomal systems may be useful as a cytokine supplementin tumor cell vaccines by providing a cytokine reservoir at the antigenpresentation site. Here, we examined the effect of liposomeincorporation of mIFN on its potency as adjuvant in an established tumor cellvaccination protocol in the murine B16 melanoma model. Adjuvanticityof the mIFN-liposomes was compared to that achieved bymIFN-gene transfection of the B16 tumor cells. Furthermore, we studiedwhether liposomal incorporation of mIFN indeed increases theresidence time of the cytokine at the vaccination site. Methods. C57B1/6 mice were immunized with i) irradiated IFN-genetransfected B16 melanoma cells or ii) irradiated wild type B16 cellssupplemented with (liposomal) mIFN, followed by a challenge withviable B16 cells. The residence time of the (liposomal) cytokine at thesubcutaneous (s.c.) vaccination site was monitored using radiolabeledmIFN and liposomes. Results. Immunization with irradiated tumor cells admixed withliposomal mIFN generated comparable protection against B16 challenge asimmunization with mIFN-gene modified tumor cells. Irradiated tumorcells admixed with soluble mIFN did not generate any protectiveresponses. Radiolabeling studies indicated that free mIFN rapidlycleared from the s.c. injection site. Association of [¹²⁵I]-mIFNwith liposomes increased the local residence time substantially: liposomalassociation of mIFN resulted in a prolonged local residence time ofthe cytokine as reflected by a 4-fold increase of the area under thecurve. The amount of released cytokine in the optimal dose rangecorresponds to the amount released by the gene-transfected cells. Moderate but significant CTL-activity against B16 cells was found for miceimmunized with irradiated cells supplemented with mIFN-liposomescompared to untreated control animals. Conclusions. Prolonged presence of mIFN at the site of antigenpresentation is crucial for the generation of systemic immune responsesin the B16 melanoma model. These studies show that liposomalencapsulation of cytokines is an attractive strategy for paracrine cytokinedelivery in tumor vaccine development.     

Histamine H3A receptor-mediated inhibition of noradrenaline release in the mouse brain cortex

Summary Mouse brain cortex slices preincubated with ³H-noradrenaline were superfused with physiological salt solution containing desipramine plus a drug with ₂-adrenoceptor antagonist properties, and the effects of histamine receptor ligands on the electrically (0.3 Hz) evoked tritium overflow were studied. The evoked overflow (from slices superfused with phentolamine) was inhibited by histamine (pIC₃₅ 6.53), the H₃ receptor agonist R-(–)--methylhistamine (7.47) and its S-(+)-enantiomer (5.82) but not influenced by the H₁ receptor agonist 2-(2-thiazolyl)-ethylamine 3.2 mol/l and the H₂ receptor agonist dimaprit 10 mol/l. The inhibitory effect of histamine was not affected by the H₁ receptor antagonist dimetindene 1 mol/l and the H₂ receptor antagonist ranitidine 10 ol/l. The concentration-response curve of histamine (determined in the presence of rauwolscine) was shifted to the right by the H₃ receptor antagonists thioperamide (apparent pA₂ 8.67), impromidine (7.30) and burimamide (6.82) as well as by dimaprit (6.16). The pA₂ values of the four drugs were compared with their affinities for H_3A and H_3B binding sites in rat brain membranes (West et al. 1990 Mol Pharmacol 38:610); a significant correlation was obtained for the H_3A, but not for the H_3B sites. The results suggest that noradrenaline release in the mouse brain cortex is inhibited by histamine via H_3A receptors and that dimaprit is an H₃ receptor antagonist of moderate potency. Send offprint requests to E. Schlicker at the above address     

N-methyl-d-aspartate (NMDA)-stimulated noradrenaline (NA) release in rat brain cortex is modulated by presynaptic H3-receptors

In superfused rat brain cortex slices and synaptosomes preincubated with [³H]noradrenaline the effect of agonists or antagonists at presynaptic H₃ receptors on NMDA-evoked [³H]noradrenaline release was investigated. In experiments on slices, histamine and the preferential H₃ receptor agonist R-(–)--methylhistamine inhibited NMDA-evoked tritium overflow (IC₂₀ values 0.27 mol/l or 0.032 mol/l, respectively); S-(+)--methylhistamine (up to 10 mol/l) as well as the selective H₁ receptor agonist (2-(2-thiazolyl)ethylamine) and the selective H₂ receptor agonist dimaprit (each up to 10 mol/l) were ineffective. The H₃ receptor antagonist thioperamide abolished the inhibitory effect of histamine whereas the preferential H₁ receptor antagonist dimetindene and the preferential H₂ receptor antagonist ranitidine were ineffective. In experiments on synaptosomes, histamine and R-(–)--methylhistamine inhibited NMDA-evoked tritium overflow, whereas 2-(2-thiazolyl)ethylamine or dimaprit had no effect. The inhibitory effect of histamine was abolished by thioperamide. When tritium overflow was stimulated by NMDA in the presence of -conotoxin GVIA (which by itself decreased the response to NMDA by about 55%), R-(–)--methylhistamine did not inhibit NMDA-evoked overflow. It is concluded that NMDA-evoked noradrenaline release in the cerebral cortex can be modulated by inhibitory H₃ receptors. NMDA receptors and H₃ receptors are both located presynaptically and may interact at the same noradrenergic varicosity. An unimpaired function of the N-type voltage-sensitive calcium channel probably is a prerequisite for the inhibition of NMDA-evoked noradrenaline release by H₃ receptor stimulation. Correspondence to: M. Göthert at the above address     

A dispersion model of hepatic elimination: 2. Steady-state considerations-influence of hepatic blood flow,binding within blood,and hepatocellular enzyme activity

The dispersion model of hepatic elimination is based on the distribution of residence times of blood elements within the liver. The model has two asymptotic solutions corresponding to the wellstirred model (complete mixing of blood elements) and the parallel-tube model (no variation in residence times of blood elements). The steady-state form of the dispersion model relevant to pharmacokinetic analysis is developed and explored with respect to changes in blood flow, in binding within blood, and in hepatocellular enzyme activity. Literature data are used to evaluate discrepancies among the predictions of the dispersion, well-stirred, and tube models. It is concluded that the dispersion model is consistent-with the data. The limitations of steady-state perfusion experiments to estimate the residence time distribution of blood elements within the liver are considered.     

Regulation of gastric acid secretion by histamine H3 receptors in the dog: an investigation into the site of action

The involvement of histamine H₃ receptors in the regulation of gastric acid secretion was investigated in the conscious dog with gastric fistula, by the use of the selective agonist (R)-methylhistamine and the selective antagonist thioperamide. (R)-methylhistamine (0.3–1.2 mol/kg/h) induced a dose-related inhibition of the acid secretion induced by pentagastrin and by bombesin, maximum inhibition not exceeding 60–65%. The inhibitory effect of the H₃ agonist (0.6 mol/kg/h) was inhibitited by thioperamide (0.1 mol/kg/h), suggesting that the effect was entirely mediated by H₃ receptors. Thioperamide was also able to enhance the acid response to submaximal doses of pentagastrin and bombesin. The acid secretion induced by histamine was not modified by (R)a-methylhistamine (0.3–1.2 mol/kg/h) but it was significantly enhanced by thioperamide (0.1 mol/kg/h). Neither (R)-methylhistamine nor thioperamide significantly modified the increase in plasma gastrin levels induced by bombesin. In conclusion these data demonstrate that histamine H₃ receptors may represent an effective mechanism for the negative control of stimulated gastric acid secretion in the dog; however, since the inhibition was mainly evident against stimuli which involve the release of histamine, a location of H₃ receptors in paracrine cells of the gastric mucosa rather than in gastrin producing cells or parietal cells seems more likely. Correspondence to: G. Bertaccini at the above address     

The cardiovascular effects of intraventricularly administered histamine in the anaesthetised rat

Summary In urethane-anaesthetised rats intraventricular (i.c.v.) injections of histamine (0.1–10.0 g) elicited dose-related rises in both the resting blood pressure and heart rate. These cardiovascular effects of histamine were antagonised in a dose-dependent manner by i.c.v. pretreatments with the histamine H₁-receptor antagonists mepyramine (10, 50 and 100 g) and diphenylpyraline (100 and 200 g). Pretreatment with the histamine H₂-receptor antagonist metiamide (100 and 200 g i.c.v.) failed to modify either of the responses. A dose-related antagonism of the hypertensive response to histamine i.c.v. was elicited by phentolamine (100 and 200 g i.c.v.) but the positive chronotropic effect was not modified by this pretreatment. The cardiovascular responses to histamine i.c.v. were abolished by mecamylamine (5.0 mg/kg i.v.) and greatly reduced by 6-hydroxydopamine (3×250 g i.c.v.), but only the tachycardia was significantly modified by atropine (100 g i.c.v.) and propranolol (1 mg/kg i.v.). Propranolol (100 g i.c.v.), bilateral vagotomy, or acute bilateral adrenal demedullation failed to modify the cardiovascular responses to histamine i.c.v. The results suggest that histamine is able to modify the resting blood pressure and heart rate by independent central modes of action, which involve central adrenergic and cholinergic mechanisms.Preliminary findings of this study were presented at the Autumn meeting of the British Pharmacological Society (Finch and Hicks, 1975).     

Pharmacokinetics,anticonvulsant efficacy,and adverse effects of trans-2-en-valproate after acute and chronic administration in amygdala-kindled rats

Summary Effects of adenosine and nucleotides on the release of previously stored [3H]-noradrenaline were studied in rabbit brain cortex slices. The slices were stimulated twice, in most experiments by 6 electrical field pulses delivered at 100 Hz.Adenosine and the nucleotides AMP, ADP, ATP, AMPS, ADPS, ATPyS, ,-imido-ATP and ,-methyl-ene-ATP all reduced the evoked overflow of tritiated compounds. For purines for which concentration-response curves were determined, the order of potency was adenosine > ATP ATPyS ,-imido-ATP ADP > ,-methylene-ATP. AMP 30 Etmol/l and AMPS 30 mol/l were approximately equieffective with 30 mol/l of adenosine and ATPS, and ADPS, 30 mol/l was approximately equieffective with 30 mol/l of ADP. ,-Methylene-ADP, 2-methylthio-ATP, UTP and GTPS did not change the evoked overflow of tritium. ,-Methylene-ATP caused an increase; however, the increase was small and became significant only after 59 min of exposure to ,-methylene-ATP or when the slices were stimulated by 30 pulses, 10 H₂. Neither adenosine deaminase (100 U/l) nor the blocker of 5-nucleotidase, ,-methylene-ADP (10 mol/l), attenuated the inhibition caused by ATP, ATPyS and ,-methylene-ATP, despite the fact that adenosine deaminase abolished the effect of adenosine. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX, 10 nmol/l) shifted the concentration-response curves of adenosine, ATPyS, ,-imido-ATP and ,-methylene-ATP to the right by very similar degrees. 8(p-Sulphophenyl)-theophylline (30 and 300 mol/l) also markedly antagonized the inhibition produced by ATPS. ,-Methylene-ATP (10 and 30 mol/l) and suramin (100 gmol/l) did not modify the effects of adenosine, ATPS and ,-methylene-ATP.It is concluded that nucleotides themselves can inhibit the release of noradrenaline in the rabbit brain cortex. The nucleotides and adenosine seem to act at the same site, i.e., the A₁ subtype of the P₁-purinoceptor. The results support the notion that metabolically stable, phosphate chain-modified nucleotides such as ATPS, ,-imido-ATP and ,-methylene-ATP can be potent P₁ agonists. No evidence was found for presynaptic P_2X-, P_2Y- or P₃-purinoceptors. Send offprint requests to I. von Kugelgen at the above address     

Comparison of the densities of 5-HT4 receptors, β1- and β2-adrenoceptors in human atrium: functional implications

We measured in human atrium the density of 5-HT₄ receptors, labelled with [¹²⁵I]-SB 207710 (1-butyl-4-piperidinyl) methyl 8-amino-7-iodo-1, (4-benzodioxan-5-carboxylate), and compared it with the density of ₁- and ₂-adrenoceptors, labelled with (–)-[¹²⁵I]-cyanopindolol. [¹²⁵I]-SB 207710 (5–1200 pmol/l) labelled a small population of saturable binding sites (B _max 4 fmol/mg protein) with a pK_D of 9.7 and with 5-HT₄ receptor characteristics, as assessed with competing ligands. The density of atrial binding sites with 5-HT₄ receptor characteristics was 10 and 5 times lower, respectively, than the density of ₁- and ₂-adrenoceptors. We suggest that the small 5-HT₄ receptor population may in part explain why the positive inotropic effects of 5-HT are smaller than those of catecholamines mediated through ₁- and ₂-adrenoceptors.     

Changing doctor prescribing behaviour

The aim of this overview was to identify interventions that change doctor prescribing behaviour and to derive conclusions for practice and further research. Relevant studies (indicating prescribing as a behaviour change) were located from a database of studies maintained by the Cochrane Collaboration on Effective Professional Practice. This register is kept up to date by searching the following databases for reports of relevant research: DHSSDATA; EMBASE; MEDLINE; SIGLE; Resource Database in Continuing Medical Education (19751994), along with bibliographies of related topics, hand searching of key journals and personal contact with content area experts. Randomised controlled trials and nonequivalent group designs with pre and postintervention measures were included. Outcome measures were those used by the study authors. For each study we determined whether these were positive, negative or inconclusive. Positive studies (+) were those that demonstrated a statistically significant change in the majority of outcomes measured at level of p 0.05 between the intervention and control groups. Negative studies () showed a significant change in the opposite direction and inconclusive studies () showed no significant change compared to control or no overall positive findings. We identified 79eligible studies which described 96 separate interventions to change prescribing behaviour. Of these interventions, 49 (51%, 41%61%) showed a positive significant change compared to the control group but interpretation of specific interventions is limited due to wide and overlapping confidence intervals.     

Evaluation of P2-purinoceptor antagonists at two relaxation-mediating P2-purinoceptors in guinea-pig taenia coli

The guinea-pig taenia coli possesses two relaxation-mediating receptors for nucleotides: a prototypic P_2Y-purinoceptor, which is activated by adenosine 5-O-(2-thiodiphosphate) (ADPßS), and a separate receptor for ,-methylene ATP (,-MeATP). Effects of several as yet incompletely characterized P₂-purinoceptor antagonists at these receptors were examined.The concentration-relaxation curve of ADPßS was shifted to the right by reactive blue 2, suramin, 8-(3,5-dinitro-phenylenecarbonylimino)-1,3,5-naphthalenetrisulphonic acid (XAMR0721; at 1000 M only), pyridoxalphosphate-6-azophenyl-2,5-disulphonic acid (iso-PPADS), pyridoxal 5-phosphate, trypan blue and Evans blue (at 320 M only). Schild plots for the antagonism of reactive blue 2, suramin, iso-PPADS and pyridoxal 5-phosphate against ADPßS had slopes <1. The concentration-relaxation curve of ,-MeATP was shifted to the right by reactive blue 2, suramin, XAMR0721, iso-PPADS, pyridoxal 5-phosphate and trypan blue but not by Evans blue (320 M). Schild plots for the antagonism of suramin, XAMR0721 and iso-PPADS against ,-MeATP had slopes >1. Only XAMR0721 differed clearly in potency against the two nucleotides: it was considerably more potent against ,-MeATP than against ADPßS. 2-Methylthio ATP (MeSATP; 1 M) and ATP (100 M) were degraded by pieces of taenia coli. All antagonists except trypan blue attenuated the degradation of either or one of the two nucleotides.The selective effect of XAMR0721 against ,-MeATP confirms the existence of two relaxation-mediating P₂-purinoceptors in guinea-pig taenia coli. Comparison of the apparent affinities of the antagonists for the two taenia coli receptors with affinities for the P_2X-purinoceptor of the rat vas deferens shows that reactive blue 2, suramin, iso-PPADS, pyridoxal 5-phosphate and trypan blue have little selectivity for any of the three receptors. XAMR0721, which has been shown to possess relatively high affinity for the P_2Y-purinoceptor in turkey erythrocytes, was very weak at the P_2Y-receptor of the taenia, thus supporting the existence of pharmacologic P_2Y-receptor subtypes. Evans blue, with little effect in the taenia coli but a marked effect in the rat vas deferens, is the most selective P_2X-(versus P_2Y-) purinoceptor antagonist presently known, although its effect on the degradation of nucleotides must be kept in mind.     

Resistance of adrenergic neurotransmission in the toad heart to adrenoceptor blockade

Summary Stimulation of sympathetic nerves to the toad heart produced increases in both the rate and force of cardiac beat. Although these responses were abolished by treatment with bretylium (10^–6 mol ·l^–1) or 6-hydroxydopamine (100 mg·kg^–1), and surgical sympathetic denervation, they were not abolished by treatment with propranolol (10^–6 mol·l^–1) and phentolamine (3×10^–6 mol·l^–1), either alone or in combination. The responses remaining after adrenoceptor blockade could not be ascribed to the effects of neurally released dopamine, ATP, adenosine, histamine or a variety of neuropeptides, although the participation of an as yet unidentified co-transmitter cannot be ruled out. Quantitative analysis of the interactions between propranolol and adrenaline on cardiac adrenoceptors, after blockade of -receptors and amine uptake mechanisms, revealed that these interactions do not comply with the conditions for simple competitivity. Therefore, in addition to its action on -receptors, adrenaline seems to be producing excitation of the toad heart by acting on adrenoceptors which cannot be classified as either -or -receptors. These results, together with the existence of close neuromuscular gaps (<50nm) in the toad heart, are consistent with the hypothesis that sympathetic excitation of the toad heart is mediated by both extra-junctionl -adrenoceptors, and junctional adrenoceptors which are neither -nor -receptors.     

Dissociation of phosphoinositide hydrolysis and positive inotropic effect of histamine mediated by H1-receptors in guinea-pig left atria

Summary The present study was undertaken to determine whether the phosphoinositide hydrolysis is responsible for the positive inotropic effect of histamine in guinea-pig left atria. Histamine induced hydrolysis of phosphoinositides and a positive inotropic effect in a concentration-dependent manner. These effects were antagonized by chlorpheniramine (0.1 mol/l) but not by cimetidine (10 mol/l). At a concentration of 1 mol/l histamine produced a dual-component positive inotropic response composed of an initial increasing phase and a second and late developing, greater positive inotropic phase. Histamine (10 mol/l) caused a gradual increase in the formation of [³H]inositol trisphosphate (IP3) and a significant increase in the [³H]IP₃ level was detected 10 min after the stimulation. Thus, the increase in IP₃ did not precede the increase in force of contraction. The phospholipase C inhibitors 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (100 mol/l) and neomycin (100 mol/l) significantly reduced the histamine-induced [³H]inositol monophosphate accumulation. However, pretreatment with the phospholipase C inhibitors did not affect the positive inotropic effect of histamine, either in its extent or in its pattern. The phorbol esters 12-O-tetradecanoylphorbol-13-acetate (TPA) (100 nmol/l) and phorbol-12,13-dibutyrate (PDBu) (100 nmol/l) also significantly inhibited the phosphoinositide hydrolysis induced by histamine. The inhibitory effect of the phorbol esters on the phosphoinositide response was completely abolished in the presence of 10 mol/l 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), a protein kinase C inhibitor. TPA significantly attenuated the positive inotropic effect of histamine without changing the dual-component pattern, whereas PDBu merged two distinct components of the histamine inotropic response into one and potentiated the early part of the positive inotropic effect. However, neither of the changes which the phorbol esters produced in the positive inotropic response to histamine was blocked by H-7. In addition, H-7 itself failed to modify the positive inotropic effect of histamine. These results indicate that histamine induces hydrolysis of phosphoinositides in guinea-pig left atria that is mediated by H₁-receptors, but this biochemical event does not appear to contribute to the H₁-receptor-mediated positive inotropic action. Send offprint requests to Y. Hattori at the above address