Formulation of Vaccine Adjuvant Muramyldipeptides (MDP). 2. The Thermal Reactivity and pH of Maximum Stability of MDP Compounds in Aqueous Solution

The degradation of muramyldipeptides (MDPs) in aqueous solution obeys the rate law k _obs = k _H+_H+ + k _o + k _HO- a _HO- and the Arrhenius equation. For example, the rate constants for degradation of N-acetylmuramyl-L-threonyl-D-isoglutamine, 3, at 25°C are k _H+ = 2.3 × 10^–6 M ^–1 sec^–1, k _o = 8.2× 10^–10 sec^–1, and k _HO– = 0.19 M ^–l sec^–1. The degradation rates are dependent on the side-chain substituents; it is predicted that sterically hindered MDP compounds will show an extended shelf life in aqueous solution. Product studies in the weakly acid pH region (where the pH of maximum stability occurs) show that MDP compounds degrade largely by hydrolysis of the dipeptide side chain. These data show that MDP 3 exhibits a shelf life (t ₉₀) of greater than 2 years in aqueous solutions of pH 4–4.5, the pH of maximum stability.     

Two phases of the prostaglandin F2α-induced contraction in guinea-pig taenia coli involve different Ca2+ channels

Summary Properties of the contraction produced by PGF₂ in the guinea-pig taenia coli were compared to those produced by ACh. Prostaglandin (PG) F₂ (3 × 10^–7 M) and acetylcholine (ACh, 10^–5 M) induced an initial transient contraction (phasic contraction) and a subsequent late contraction (tonic contraction). Both phasic and tonic contractions produced by PGF₂ or ACh were abolished in Ca²⁺-free Krebs solution containing 0.5 mM EGTA. The tonic contractions caused by PGF₂ and ACh were markedly suppressed by -[3-[[2-(3,4-dimethoxy-phenyl)-ethyl]methylamino]-propyl]-3,4,5-trimethoxy--(1-methylethyl) benzeneacetonitrile hydrochloride (D600, > 10^–7 M) as well as nifedipine (5 × 10^–9 M), a Ca²⁺-antagonist. However, the phasic contraction produced by PGF₂, but not by ACh, was greatly inhibited by Mn²⁺ (> 10^–4 M). Furthermore, the phasic contraction caused by PGF₂ was abolished in 18 mM K⁺ Krebs solution with D600 (2 × 10^–7 M), whereas that induced by ACh and the tonic contractions produced by PGF₂ as well as by ACh were unaffected in this high K⁺ solution without D600. Membrane potentials of the tissue in normal (K⁺, 5.9 mM) and 18 mM K⁺ Krebs solution containing D600 were about –55 mV and –43 mV, respectively. In a fluorescence study which used Fura-2 an intracellular free Ca²⁺ indicator in the presence of D600, PGF₂ and ACh increased fluorescence intensity in the tissue, which coupled with the magnitude of contractions. Both the enhanced fluorescence intensity and tension development evoked by PGF₂, but not by ACh, were markedly decreased in 18 mM K⁺ Krebs solution. It is, thus, suggested that the phasic and tonic contractions evoked by PGF₂, unlike those by ACh, may be mediated via certain different Ca²⁺ channels from extracellular Ca²⁺-source.Send offprint requests to S. Usune at the above address     

Coenzyme Q10 attenuates cyanide-activation of the ATP-sensitive K+ channel current in single cardiac myocytes of the guinea-pig

Summary The effect of coenzyme Q₁₀ (CoQ₁₀) on the cyanide (CN^–)-induced ATP-sensitive K⁺ channel current (K_ATP) was examined in single atrial myocytes, using the patch clamp technique. Superfusion of the cells with a CN^–/low glucose bathing solution induced an outward current in the whole-cell clamp condition. Glibenclamide (1 M) abolished this current, indicating that the current was carried through the K_ATP channel. After steady-state activation by CN^–, pinacidil (a K_ATP channel opener, 300 M) failed to further increase the current. In cell-attached patches, CN^–, when applied to the bath, induced bursting openings of an 80 pS channel (the K_ATP channel). In cells preincubated for 30 min in a solution containing CoQ₁₀ (100 g/ml), CN^–-activation of the K_ATP channel was markedly attenuated both at the whole cell and at the single channel level. At the steady-state effect of CN^– in CoQ₁₀-treated cells, pinacidil (300 M) activated the current to the maximum level achieved by CN^– in the control cells. These results suggest that CoQ₁₀ reduces in the CN^–-induced KATP current not by affecting the channel itself but by preventing depletion of intracellular ATP caused by CN^–. Send offprint requests to Y. Kurachi at Mayo Foundation     

Properties of agonist binding at theβ-adrenoceptor of the rat reticulocyte

Summary To study the fundamental differences between agonist and antagonist interaction with the -adrenoceptor of the rat reticulocyte the radiolabeled agonist³H hydroxybenzylisoprenaline (³H HBI) and the radiolabeled antagonist³H dihydroalprenolol (³H DHA) were used.Equilibrium binding experiments with³H HBI revealed all characteristics expected to a -adrenoceptor site, i. e. high affinity binding (K _{D high}=7.4±0.9×10^–9 M), saturability (B _{max high}=230±24 fmoles/mg protein), and stereoselectivity. The rank order of potency for competing agonists was isoprenaline > adrenaline > noradrenaline > dopamine.³H HBI high affinity binding sites amounted to about 25% of -adrenoceptor sites detectable with³H DHA.In competition experiments with³H HBI and (-)isoprenaline[(-)Ipn]aK _{D high}-value for (-)Ipn of 3.1±0.6×10^–8M was obtained corresponding to theK _{D high}-value of (-)Ipn obtained from competition experiments using³H DHA. For (-)propranololK _D-values of 0.9±0.5×10^–8 M and 1.0 ±1.0×10^–8 M were measured using³H HBI and³H DHA respectively.Agonist affinity derived from competition experiments with (-)Ipn versus³H DHA was not affected by temperature changes.Guanylyl-imidodiphosphate [Gpp(NH)p] decreased concentration dependently the number of high affinity binding sites of³H HBI not affecting the respectiveK _D-value. Similar effects were observed after omission of Mg²⁺ from the binding assay or inclusion of Na⁺ in the Mg²⁺-free incubation mixture.The association reaction of³H HBI at the -adrenoceptor revealed two different velocities. The slower phase of the association reaction which represents high affinity binding (80% of equilibrium binding) is not observed in the presence of Gpp(NH)p.A biphasic dissociation of³H HBI binding was induced by 10^–4 M (±)propranolol: 25% dissociated with at _1/2 of 1.3 min whereas the high affinity binding was reversed with at _1/2 of 150 min. This slowly reversible binding of³H HBI however was rapidly reversed by Gpp(NH)p (t _1/2<1 min).It is concluded that the agonist ligand³H HBI permits a direct qualitative and quantitative characterization of the agonist induced high affinity state of the -adrenoceptor. In particular, the kinetic studies strongly support a two step binding model for the agonist--adrenoceptor interaction.This work was supported by a grant from the Deutsche ForschungsgemeinschaftParts of this work were presented at the Spring Meetings of the German Pharmacological Society (Wiemer et al. 1978, 1981 b)Herrn Professor Dr. med. Hans Herken, Pharmakologisches Institut der Freien Universität Berlin, zum 70. Geburtstag gewidmet.     

Expression of the extraneuronal monoamine transporter (uptake2) in human glioma cells

Tritiated methylphenylpyridinium ([³H]MPP⁺), a substrate of the neuronal and extraneuronal noradrenaline transporter (uptake₁ and uptake₂, respectively) and of the organic cation transporter (OCT1), was used to characterize the amine transport system of the established human glioma cell line SK-MG-1.Uptake of [³H]MPP⁺ (25 nM) into SK-MG-1 cells increased linearly with time for up to 15 min. Selective uptake₁ inhibitors (e.g. (+)oxaprotiline) or omission of Na⁺ or Cl^– ions did not affect [³H]MPP⁺ uptake, whereas uptake₂ inhibitors such as O-methyl-isoprenaline (OMI) or corticosterone as well as depolarizing concentrations of K⁺ or Ba²⁺ strongly reduced [³H]MPP⁺ uptake. Initial rates of OMI(100 M)-sensitive [³H]MPP⁺ uptake were saturable, with a K_m of about 17 M and a maximal rate of about 50 pmol/ (min × mg protein). IC₅₀ (or K_i) values for inhibition of [³H]MPP⁺ uptake by substrates and inhibitors of uptake₂ or OCTI were highly significantly correlated with published IC₅₀ values for inhibition of uptake₂ but not with corresponding values for inhibition of OCT1.The results presented here clearly demonstrate that human glioma cells express an uptake₂ transporter. Thus, glial cells in the human central nervous system endowed with this transporter are likely to contribute to the inactivation of neuronally released noradrenaline.     

Cocaine inhibits cromakalim-activated K<Superscript>+</Superscript> currents in follicle-enclosed<Emphasis Type="Italic"> Xenopus</Emphasis> oocytes

The effect of cocaine on K⁺ currents activated by the K_ATP channel opener cromakalim was investigated in follicular cells of Xenopus oocytes. The results indicate that cocaine in the concentration range of 3–500 M reversibly inhibits cromakalim-induced K⁺ currents. The IC₅₀ value for cocaine was 96 M. Inhibition of the cromakalim-activated K⁺ current by cocaine was noncompetitive and voltage independent. Pretreatment with the Ca²⁺ chelator BAPTA did not modify the cocaine-induced inhibition of cromakalim-induced K⁺ currents, suggesting that Ca²⁺-activated second messenger pathways are not involved in the actions of cocaine. Outward K⁺ currents activated by the application of 8-Br-cAMP or forskolin were also inhibited by cocaine. The EC₅₀ and slope values for the activation of K⁺ currents by cromakalim were 184±19 M and 1.14 in the absence of cocaine as compared to 191±23 M and 1.03 in the presence of cocaine (300 M). Cocaine also blocked K⁺ currents mediated through C-terminally deleted form of Kir6.2 (KirC26) in the absence of sulfonylurea receptor with an IC₅₀ value of 87 M, suggesting that cocaine interacts directly with the channel forming Kir6.2 subunit. Radioligand binding studies indicated that cocaine (100 M) did not affect the binding characteristics of the K_ATP ligand, [³H]glibenclamide. These results demonstrate that cromakalim-activated K⁺ currents in follicular cells of Xenopus oocytes are modulated by cocaine.     

Role of ATP-sensitive K+ channels in antinociception induced by R-PIA,an adenosine A1 receptor agonist

The influence of several K⁺ channel-acting drugs on antinociception induced by the adenosine A₁ receptor agonist (–)-N⁶-(2-phenylisopropyl)-adenosine (R-PIA) was evaluated with a tail flick test in mice. The subcutaneous administration of R-PIA (0.5–8 mg/kg) induced a dose-dependent antinociceptive effect. The ATP-sensitive K⁺ (K_ATP) channel blocker gliquidone (2–8 g/mouse, i.c.v.) produced a dose-dependent displacement to the right of the R-PIA dose-response line, whereas the K_ATP channel opener cromakalim (32 g/mouse, i.c.v.) shifted it to the left. Several K_ATP channel blockers dose-dependently antagonized the antinociceptive effect of R-PIA, the order of potency being gliquidone > glipizide > glibenclamide (i.e., the same order of potency shown by these drugs in blocking K_ATP channels in neurons). In contrast, the K⁺ channel blockers 4-aminopyridine and tetraethylammonium did not antagonize the effect of R-PIA. These data suggest that antinociception produced by adenosine A₁ receptor agonists is mediated by the opening of ATP-sensitive K⁺ channels. The present results, together with those of previous studies, further support a role for K⁺ channel opening in the antinociceptive effect of agonists of receptors coupled to G_i/G_o proteins. Correspondence to: José M. Baeyens at the above address     

5-HT3 receptor ligands lack modulatory influence on acetycholine release in rat entorhinal cortex

Summary The objective of this study was to explore the role of 5-HT₃ receptors in modulating potassium (K⁺)-evoked release of [³H]-acetylcholine ([³H]-ACh) from superfused slices of rat entorhinal cortex previously loaded with [³H]-choline. Rat entorhinal cortices were cross-chopped into 300 m slices, superfused with oxygenated Krebs buffer containing 2.5 mmol/1 Ca²⁺ and stimulated with two consecutive exposures of 20 mmol/l K⁺ for 4 min (S₁ and S₂, respectively). Compounds were added 20 min before S₂ stimulation and remained in the superfusion buffer for the duration of the experiment. The S₂/S₁ ratio was then calculated.Stimulated release of [³H]-ACh was dependent on extracellular Ca²⁺ and K⁺ concentration. In Sprague Dawley rats, 2-methyl-5-HT (10^-9–10^-6 mol/l), in the presence of 1 mol/l ritanserin or 1 gmmol/l ondansetron, had no influence on K⁺-evoked release of [³H]-ACh. In slices prepared from Hooded Lister rats, 2 mol/l 5-HT but not 2-Me-5-HT significantly (P<0.05) inhibited K⁺-evoked [³H]-ACh release only 17% in the presence of 1 mol/l ritanserin. However, 2 mol/l 2-Me-5-HT plus 1 nmol/l ondansetron had no effect. High performance liquid chromatography coupled to electrochemical detection (HPLC-ECD) was used to monitor endogenous release of ACh in the above conditions to confirm data from the radiolabelled experiments. No significant inhibition or increase in K⁺-evoked ACh release was observed with either 5-HT₃ receptor agonists or antagonists. 2-Me-5-HT (10^–9 – 10^–5 mol/l) or 1-(m-chlorophenyl)-biguanide (10^–9 – 10^–5 mol/l), when added simultaneously at the S₂ stimulation, in the presence of 1 l/l methysergide, also showed no effect on [³H]ACh release.In entorhinal cortex slices from aged Wistar rats, neither 1-(m-chlorophenyl)-biguanide (2 or 10 ol/l) nor 2-Me-5-HT (2 mol/l) in combination with ritanserin (1 mol/l) or ondansetron (1 nmol/l) elicited any effect on K⁺-evoked [³H]-ACh release. However, release of [³H]-ACh was inhibited by carbachol (10 mol/l) and adenosine (10 mol/l). DuP 996 (3,3-bis(4- pyridinyl-methyl)-1-phenylindolin-2-one) (10^–7 – 10^–5 mol/l), a known releaser of ACh, markedly augmented K⁺-evoked [³H]-ACh release.These studies have failed to confirm the postulated role of 5-HT₃ receptors in modulating cortical ACh release in rat entorhinal cortex slices and suggest that a critical reexamination of the interaction of 5-HT₃ receptor and cortical cholinergic function needs to be addressed.Abbreviations 5-HT serotonin - ACh acetylcholine - HPLC-ECD high performance liquid chromatography - electrical chemical detection - EGTA ethylene glycol bis(-aminoethyl ether)-N,N-tetraacetic acid - 2-ME-5-HT 2-methyl-5-hydroxytryptamine - DuP 996 (3,3-bis(4pyrindinylmethyl)-1-phenylindolin-2-one) A preliminary report of this work was presented at the 1992 Federation of American Societies for Experimental Biology, April 6–9, Anaheim, California, USA (The FASEB J 6A1559) Correspondence to R. M. Johnson at the above address     

Demonstration of a Na+: Mg2+ exchange in human red cells by its sensitivity to tricyclic antidepressant drugs

Summary Iminodibenzyl-, iminostilbene-, dibenzocycloheptadiene-, dibenzooxepine- and dibenzothiepine-derivatives of tricyclic antidepressant drugs were able to inhibit Na⁺-stimulated Mg²⁺ efflux in human erythrocytes at concentrations of 10^–5–10^–3 mol/l. Tricyclic antidepressant drugs belonging to other chemical groups, non-tricyclic antidepressant drugs and phenothiazines were less potent inhibitors (IC₅₀ of 10^–4 mol/l or higher).Imipramine and dothiepine, the most potent compounds, inhibited the Mg" carrier with IC₅₀ of 2.5 and 4 × 10^–5 mol/1 respectively. These IC₅₀ are of similar order of magnitude to those of some classical transport inhibitors (such as furosemide for the [Na⁺K⁺,Cl^–]-cotransport system). In addition, these concentrations of imipramine and dothiepine were free of: i) side effects on other erythrocyte Na and K⁺ transport pathways (with the exception of a slight inhibition of Ca²⁺-sensitive K⁺-channels and [Na⁺,K⁺,Cl^–]- and [K⁺,Cl^–]-cotransport systems) and ii) toxic effects on the membrane leak for divalent or monovalent cations. Therefore, we selected imipramine as an useful tool for investigating fluxes catalyzed by the Na⁺-stimulated Mg²⁺ carrier.Imipramine was tested on the initial rate of ouabain and bumetanide-resistant net Na⁺ influx in Na⁺-depleted, Mg²⁺-loaded erythrocytes. The compound was able to inhibit a Na⁺ influx of about 300–500 mol (l · cells × h)^–1 with an IC₅₀ of about 3 x 10^–5 mol/1. This imipramine-sensitive Na⁺ influx was coupled with an imipramine-sensitive Mg²⁺ efflux in a stoichiometry of 3.03±0.34 (mean±SEM of 7 experiments).Abbreviations MOPS 4-morpholinopropanesulfonic acid - PCMBS p-chloromercuribenzenesulfonate - EGTA ethylene glycol bis-(beta-aminoethyl ether)N,NNN-tetraacetic acid - Tris tris(hydroxymethyl)aminomethane Send offprint requests to R. Garay at the above address     

Evidence against a regulation of Na+/K+-ATPase by Gi proteins. Failure to detect an influence of G proteins on Na+/Ca2+-exchange in cardiac sarcolemmal membranes

In the myocardium the inhibitory guanine nucleotide-binding regulatory proteins (G_i proteins) mediate negative chronotropic and negative inotropic effects by activation of K⁺ channels and inhibition of adenylyl cyclase. The concept of a uniform inhibitory action of G_i proteins on myocardial cellular activity has been questioned by the recent observations of adenosine-induced activation of the Na⁺/Ca²⁺ exchange and a carbachol-induced inhibition of the Na⁺/K⁺-ATPase activity in cardiac sarcolemmal membranes. The aim of the present study, therefore, was to reinvestigate the putative regulation of Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase activity in purified canine sarcolemmal membranes. These membranes were enriched in adenosine A₁ (Maximum number of receptors, B _max 0.033 pmol/mg) and muscarinic M₂ (B _max 2.9 pmol/mg) receptors and contained G_i2 and G_i3, two G_i protein isoforms, and Go, another pertussis toxin-sensitive G protein, as detected with specific antibodies. The adenosine A₁-selective agonist, (–)-N ⁶-(2-phenylisopropyl)-adenosine, and the muscarinic agonist, carbachol, both inhibited isoprenaline-stimulated adenylyl cyclase activity by 25% and 35% respectively, and the stable GTP analogue 5-guanylylimidodiphosphate inhibited forskolin-stimulated adenylyl cyclase activity by 35% in these membranes. The characteristics of Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase activity as well as those of the ouabain-sensitive, K⁺-activated 4-nitrophenylphosphatase, an ATP-independent, partial reaction of the Na⁺/K⁺-ATPase, were in agreement with published data with regard to specific activity, time course of activity and substrate dependency. However, none of these activities were influenced by adenosine, (–)-N ⁶-(2-phenylisopropyl)-adenosine, carbachol, or stable GTP analogs, suggesting that Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase are not regulated by Gi proteins in canine cardiac sarcolemmal membranes.     

The Relationship of pK a and Acute Skin Irritation in Man

The relationship between pK _a and skin irritation in man is studied for a homologous series of benzoic acid derivatives, which permeate through human skin at comparable rates (15–88 µg/cm²/hr). Skin irritation and pK _a are correlated for pK _a 4. Laser Doppler velocimetric assessment of skin blood flow, color meter readings, erythema, edema, and the primary irritation index are all linearly correlated and related to pK _a, erythema at 24 hr appears to be the most sensitive parameter to variation in pK _a when pK _a 4.     

Block of wild-type and inactivation-deficient human ether-a-go-go-related gene K+ channels by halofantrine

Halofantrine is an antimalarial drug developed as a treatment of P. falciparum resistant to chloroquine. However, halofantrine can also induce long QT syndrome (LQTS) and torsades de pointes, a potentially life-threatening ventricular arrhythmia. Drug-induced LQTS is usually caused by block of the human ether-a-go-go-related gene (HERG) channels that conduct the rapid delayed rectifier K⁺ current, I_Kr, in the heart. Here we show that halofantrine preferentially blocks open and inactivated HERG channels heterologously expressed in Xenopus laevis oocytes. The half-maximal inhibitory concentration (IC₅₀) for block of wild-type (WT) HERG was 1.0 M. As we reported previously for other HERG channel blockers, the potency of halofantrine was reduced by mutation to Ala of aromatic residues (Y652, F656) located in the S6 domain, or a Val (V625) located in the pore helix. Halofantrine at a concentration 10 M did not affect the transient outward potassium channel, Kv4.3, the slow delayed rectifier potassium channel, KvLQT1+minK and inward rectifier potassium channel, Kir2.1. An inactivation deficient mutant (G628C/S631C HERG) was only slightly less sensitive (IC₅₀=2.0 M). The rate of block onset by halofantrine at 0 mV was used to estimate the apparent association (k_on) and dissociation (k_off) rate constants for drug binding. For WT and G628C/S631C HERG, k_on was similar (0.0114 and 0.0163 M^–1/s^–1 respectively). In contrast, k_off was significantly faster for G628C/S631C (0.357 s^–1) than WT (0.155 s^–1), and explains the observed decrease in drug potency for the inactivation-deficient mutant channel. We conclude that halofantrine requires channels to open before it can gain access to its binding site located in the central cavity of the HERG channel.     

Cicletanine sulfate: inhibition of anion transport systems and natriuretic activity

Summary In contrast with cicletanine, its urinary sulfoconjugate metabolite (cicletanine sulfate) was active on membrane ion transport in human red blood cells. Cicletanine sulfate was a more potent inhibitor of the Na⁺ dependent [Cl^–/HC0₃ ^–] exchanger (IC₅₀ = 9±3 × 10^–5 mol/l; mean±SD of 4 experiments) than cicletanine (IC₅₀ = 10^–3 mol/l). This inhibitory potency was intermediate between that of xipamide (IC₅₀ = 2 × 10^–5 mol/l) and that of furosemide (IC₅₀ = 2 × 10^–4 mol/l). Moreover, cicletanine sulfate exhibited modest inhibitory potency against the [Na⁺,K⁺,Cl^–]-cotransport system (IC₅₀ = 1±0.3 × 10^–3 mol/I; mean±SD of 4 experiments) and poor inhibitory activity against the [K⁺,CI^–]-cotransport system. Cicletanine sulfate was unable to modify the activity of Cl^–-independent membrane carriers(Na⁺ : H⁺exchanger,Ca ²⁺pump, Na⁺ : Li⁺ countertransport system and Na⁺,K⁺ pump). Following renal intraarterial administration in rats, cicletanine sulfate and not cicletanine, exhibited salidiuretic activity. In conclusion, the urinary sulfo-conjugate of cicletanine is an active anion transport inhibitor and natriuretic metabolite. In fact, this metabolite may be responsible for the salidiuretic action of cicletanine. Send offprint requests to R. Garay at the above address     

Stability of Prostacyclin Analogues: An Unusual Lack of Reactivity in Acid-Catalyzed Alkene Hydration

Prostacyclin analogue 5 undergoes specific acid-catalyzed hydration (k _H+ = 1.9 × 10^–7 M ^–l sec^–1 at 25°C) and a pH-independent oxidation reaction (k ₀ = 1.2 × 10^–10 sec^–1 at 25°C) above pH5. The hydration reaction for 5 is much slower than for other structurally similar exocyclic alkenes, even though the rate-determining step is proton transfer. This slowness of reaction and an analysis of the pH–rate profile show that 5 does not exhibit significant intramolecular general acid catalysis, as does prostacyclin.     

Binding of [3H]-P1075, an opener of ATP-sensitive K+ channels, to rat glomerular preparations

ATP-sensitive K⁺ channels (K_ATP channels) in the kidney have been found in the tubular system and in the afferent arteriole. In this study we have examined the binding of [³H]-P1075 ([³H]-N-cyano-N-(1,1-dimethylpropyl)-N-3-pyridylguanidine), a selective opener of K_ATP channels, in rat glomerular preparations.Equilibrium (saturation, competition) and kinetic experiments indicated that [³H]-P1075 binds to a single class of sites with a dissociation constant of about 3 nM and a maximum binding capacity of 10 fmol mg^–1 glomerular protein. The association rate constant of the complex was 6,5×10⁷ M^–1 min^–1; dissociation occurred with a half-time of 6.2 min. Specific [³H]-P1075 binding was strongly reduced when the metabolic state of the glomerular preparation was impaired during the preparation procedure or the binding assay or when the preparation was subjected to mild collagenase treatment. In different metabolically competent preparations, the amount of specific [³H]-P1075 binding correlated well with the number of vascular endings adherent to the glomeruli; no specific binding was found in mesangial cells in culture. Specific [³H]-P1075 binding was inhibited by representatives of the different classes of K_ATP channel openers and by sulphonylureatype blockers with inhibition constants similar to those obtained in rat aortic rings.It is concluded that rat glomerular preparations possess specific binding sites for K_ATP channel openers with vascular characteristics. The sensitivity of binding to mild collagenase treatment suggests that these sites are located on a membrane protein; in addition, the data suggest that these sites are localized on smooth muscle and/or renin secreting cells of the afferent vascular endings attached to some of the glomeruli. Their estimated density (1,500 m^–2) is much higher than that of K_ATP channels in smooth muscle.     

Simplified methods for the evaluation of the parameters of the time course of plasma concentration in the one-compartment body model with first-order invasion and first-order drug elimination including methods for ascertaining when such rate constants are equal

The many limitations in determining the pharmacokinetic parameters of firstorder invasion of, and elimination from, the onecompartment body model by the method of residuals or by feathering Ct data can be minimized by applying the simplified methods outlined herein. Comparisons of the apparent volumes of distribution, V, calculated on the premises that the Bateman Function represents k_a>k_e or its converse, k_e>k_a,i.e., flip-flop, can permit a proper choice of the correct version. Estimation of k_e can be obtained by regression of (A₀/V)/C(oncentration) on AUC₁/ Cwhere A₀/Vis estimable from knowledge of C_max and t_max since .The ratio of the magnitude of the rate constant of invasion to that of elimination, m=k_a/k_e,is related to k_et_max by the expression k_et_max=ln m/ (m–1)for all possible values of m.A table for the determination of m from values of k_et_max is given. When bioavailability, =A₀/Dose,is known or complete, k_e and Vcan be determined from the respective ordinate and abscissa of the intersection of and Cl(clearance)/k_e,both plotted against arbitrary k_e values. The two functions may not intersect at low values of mdue to errored C-t values but the k_e value when the two curves are closest (k_min)may approximate k_e.The intersections of and k_eAUCT (AUC_trap)plotted against variable k_e values (Method A) provide estimates of k_e from their abscissa values and A/Vfrom their ordinate values when is unknown. Method B appears to give more reliable estimates of k_e at the k_min of the difference plotted against k_e.Since k_min of this plot is 1/t_max when m=1,the identity of the mas unity underlying the C-t data is indicated when either k_mint_max is approximately unity or k_min ispractically synonymous with 1/t_max.This was clearly shown when 12 constructed m=1,C-t cases with 10% random error were evaluated by Method B. Better estimates were effected by all procedures when the raw C-t data were smoothed.We regretfully announce that Dr. Edward Garrett passed away on October 25, 1993, after an extended illness.     

Guanine nucleotide and cation regulation of radioligand binding to R i adenosine receptors of rat fat cells

Summary The modulation of radioligand binding at R _i adenosine receptors of rat fat cells by guanine nucleotides and cations was investigated. Guanine nucleotides (in the order of potency: GTP=GDP>Gpp(NH)p>5-GMP) decreased the binding of the R _i receptor agonist (–)N⁶-phenylisopropyl[³H]adenosine ([³H]PIA), but did not affect binding of the antagonist 1,3-diethyl-8[³H]phenylxanthine ([³H]DPX). Saturation of [³H]PIA binding revealed that GTP (100 mol/l) converts the high affinity form of the R _i receptor into a low affinity form. This effect was confirmed in kinetic experiments. GTP decreased the potency of agonists in competing for [³H]DPX binding, as shown by a 50-fold shift of the K _i-value for (–)PIA, whereas antagonist-induced inhibition of binding remained unchanged. The divalent cations Mg²⁺ and Ca²⁺ produced a slight increase in [³H]PIA binding but did not affect [³H]DPX binding. Mn²⁺ markedly decreased both agonist and antagonist binding at R _i adenosine receptors. Divalent cations reversed the guanine nucleotide-induced decrease of affinity of the R _i receptor. Na⁺ did not significantly affect agonist or antagonist binding but abolished the stimulatory effect of Mg²⁺ on agonist binding in the presence of GTP. Our data indicate that guanine nucleotides convert the R _i adenosine receptor of rat fat cells from a high to a low agonist affinity state and that the modulation of radioligand binding by mono-and divalent cations differs from that of R _i receptors of other tissues.     

Functional role of sodium pump in human placental arteries

Summary Ouabain (10^–7 to 10^–4 M) elicited concentration-dependent contractile responses in human placental arteries. The contractions were reduced by 10^–4 M amiloride and Ca²⁺-free medium, but not affected by 10^–6 M nifedipine or 10^–6 M Bay-K-8644, which markedly reduced or potentiated 75 mM K⁺-induced contractions, respectively. After contracting the vessels with 10^–6 M prostaglandin F₂ in a K⁺-free medium, the subsequent addition of 7.5 mM K⁺ induced a marked relaxation, which was blocked by 10^–6 M ouabain. This glycoside (10^–8 to 10^–4 M) also produced a concentration-dependent reduction of ⁸⁶Rb⁺ uptake. Scatchard analysis of the [³H]-ouabain binding to membrane fractions from human placental arteries suggests a single class of binding sites with a KD of 88.3 nM and a B_max of 345 fmol/mg. 5-Hydroxytryptamine (5-HT; 10^–9 to 10^–5 M) caused concentration-dependent contractions. Single concentrations produced transient responses composed of an initial contraction, followed by a slow fall in tension. Ouabain (10^–8 to 10^–6 M), K⁺-free medium or the reduction of bath temperature (28°C) did not modify contractions but inhibited the relaxant phase of the response. 5-HT (10^–8 to 10^–6 M) increased both total and ouabain-insensitive ⁸⁶Rb⁺ uptake, but the difference between them was not modified. These data indicate that: (1) human placental arteries possess an important sodium pump activity, inhibition or stimulation of which markedly alters vascular tone, (2) ouabain-evoked contractions are produced by Ca²⁺ entry mainly through Na⁺-Ca²⁺ exchange, secondary to intracellular Na⁺ accumulation, (3) the relaxant component of 5-HT response is dependent on the activity of the sodium pump, (4) the activation of Na⁺,K⁺-ATPase activity by this amine is not apparently due to direct effect, and (5) the inhibition of the sodium pump can cause long lasting increases of placental vascular resistance in the presence of physiological concentrations of 5-HT. Send offprint requests to J. Marin at the above address     

Parenteral Peptide Formulations: Chemical and Physical Properties of Native Luteinizing Hormone-Releasing Hormone (LHRH) and Hydrophobic Analogues in Aqueous Solution

The degradation of native LHRH in aqueous buffers of pH 1–10 obeyed the rate equation, k _obs = k _{H +} a _{H +} + k _o + k _HO-(a _HO-) ^x , where x at 60–100°C was 0.64 and temperature independent. Extrapolation to 25°C using the Arrhenius equation and secondary rate constants showed that native LHRH is reasonably stable at pH 5.4, giving a shelf life (t ₉₀) of approximately 5 years. Regarding physical properties, hydrophobic LHRH analogues nafarelin and detirelix were found to be surface active as demonstrated by a decrease in apparent surface tension with increased peptide concentration. The CMC for detirelix at pH 7.4 was determined to be 5.3 × 10^–4 M (0.88 mg/ml), and that for nafarelin, >2 mg/ml. At higher concentrations (4–8 mg/ml), nafarelin and detirelix formed nematic liquid crystals of undulose extinction (birefringence, <0.001). The thermo-dynamic stability of these peptide liquid crystals was probed by determining their melting points (T _cm) in the presence of propylene glycol, a solvent which proved to be efficacious at suppressing gelation and at destabilizing liquid crystals as measured by a reduction in T _cm.     

Characteristics of the [3H]-yohimbine binding on rat brain α2

Summary The labelling of rat cerebral cortex ₂-adrenoceptors with [³H]-yohimbine ([³H]-YOH) was investigated. At 25° C, binding equilibrium was reached in about 10 min and dissociation occurred with a half time of about 1 min. Saturation experiments gave an equilibrium K _D value of 10.13±1.95 nM and a maximum number of sites of 254±22 fmol/mg protein. The [³H]-YOH binding sites exhibited ₂-adrenergic receptor specificity; the order of potency for the antagonists was rauwolscine > yohimbine prazosin > corynanthine. For the agonists, the order was: oxymetazoline > clonidine > (–)-adrenaline > (–)-noradrenaline (–)-phenylephrine. Agonists exhibited shallow curves in inhibiting [³H]-YOH binding, with pseudo-Hill coefficients (n_H) of less than 1.0. These curves were shifted to lower overall affinity and steepened in the presence of 100 M GTP. Antagonist competition curves were also shallow but GTP had no significant effect.Divalent cations at millimolar concentrations decreased the [³H]-YOH binding: IC₅₀ values were about 6.0, 6.8 and 0.3 mM for Ca²⁺, Mg²⁺ and Mn²⁺ respectively.The maximal number of [³H]-YOH binding sites in the cortex was close to that labelled by the agonist [³H]-paraaminoclonidine ([³H]-PAC). The regional distribution of these sites in the brain, examined at a single concentration of [³H]-YOH and [³H]-PAC, showed a similar pattern except in the striatum. Taken together, the results indicate that like [³H]-PAC, [³H]-YOH labels ₂-adrenoceptors in rat brain cortex. They also show that [³H]-YOH is a useful tool for the study of the high and low affinity sites.