The role of the paracellular pathway in the net transport of calcium across the colonic muscosa

Summary Concentration dependent calcium fluxes across the colon descendens of the rat were measured in a modified Ussing chamber. Mucosa (m) to serosa (s) calcium flux showed a saturable component, whereas s to m calcium flux was linearly related to the calcium concentration. At low calcium concentrations net absorption and at concentration above 2.5 mmol/l net secretion of calcium was observed. The results obtained from the unidirectional calcium fluxes when clamping the transepithelial electrical potential agree well with those of the concentration dependence of the calcium fluxes: (1) Only m to s flux has a voltage independent component. (2) Calcium s to m movement is totally voltage dependent. (3) Diffusional s to m calcium flux is greater than the diffusional fraction of the m to s calcium flow.Dexamethasone, known to stimulate water absorption in the colon descendens by an activation of sodium transport, had no effect on the cellular mediated m to s calcium transport but significantly increased paracellular s to m flux parallel to that of the extracellular marker mannitol. This increase in paracellular s to m calcium and mannitol flux was completely abolished by amiloride, which is known to suppress the dexamethasone-induced stimulation in sodium and water absorption.The results demonstrate that the increased paracellular s to m calcium and mannitol flow is oppositely directed to the dexamethasone-induced net fluid movement as it could be expected on the basis of Ussing's anomalous solvent drag effect.Supported by a grant of the sonderforschungsbereich 38, Membrane Research, Universität des Saarlandes     

Cellular and paracellular calcium transport in the rat ileum and the influence of 1α,25-dihydroxyvitamin D3 and dexamethasone

Summary Concentration dependence of unidirectional calcium fluxes across the rat ileum freed from the serosa and the muscularis externa were measured in a modified Ussingchamber. Mucosa (m) to serosa (s) calcium flux showed a saturable component, whereas s to m calcium flux was linearly related to the calcium concentration between 0.125 mmol/1 and 5 mmol/1. At all calcium concentrations used net secretion of calcium was observed. The s to m flux of the simultaneously measured paracellular marker mannitol at all calcium concentrations was remarkably higher than the m to s flux, resulting in net mannitol secretion. The results obtained from the calcium fluxes when clamping the transepithelial electrical potential agree well with those of the concentration dependence of the calcium fluxes: 1. Only m to s flux has a voltage independent, transcellular component. 2. Calcium s to m flux is totally voltage dependent, i.e. diffusive. 3. Diffusional s to m calcium flux is about 80% greater than the diffusional fraction of the m to s flux. Omitting glucose from the bathing solution effected a decrease of the transepithelial electrical potential and of the short circuit current by 91% and 85% respectively; net calcium secretion was almost abolished and net mannitol secretion remarkably reduced. Addition of glucose, which stimulates water absorption in the ileum as a metabolic substrate, activated m to s but significantly more pronounced s to m calcium flux parallel to that of mannitol. Dexamethasone, known to stimulate sodium and water absorption in the ileum by activation of the Na,K-ATPase, effected an increase of the transepithelial electrical potential difference and of the short circuit current by about 100% but had no influence on tissue resistance; m to s and more pronounced s to m calcium flux was stimulated after the induction by dexamethasone and net calcium secretion was increased by 70%. After pretreatment with 1,25-dihydroxyvitamin D3 tissue resistance was decreased by about 42%. The vitamin had no effect on net calcium or mannitol secretion but significantly increased bidirectional calcium and mannitol fluxes. Flux measurements in clamped preparations revealed: 1. 1,25-dihydroxyvitamin D₃ and dexamethasone has no effect on the cellular-mediated m to s calcium transport; 2. diffusive calcium flux in m to s and in the opposite direction, from s to m, is increased by the vitamin and by the hormone. In conclusion the net ileal calcium and mannitol secretion is the consequence of an asymmetry of the paracellular flux with a prevalence of the s to m flux over that in m to s direction. It is hypothesized that this prevalence is caused by an anomalous solvent drag effect. Paracellular calcium flux in both directions is increased by 1,25-dihydroxyvitamin D3 and dexamethasone by different mechanisms, as indicated by the changes in the electrical parameters of the tissue. Send offprint requests to U. Karbach at the above address     

The excretion of thallium (I)-ions into the gastrointestinal tract in situ of rats

1. The excretion of Tl⁺-ions from blood into the lumen was investigated on the stomach and an segments of jejunum, ileum, colon ascendens, and colon descendens in situ of anesthetized rats using the pendular perfusion technique. The kidneys of the rats were tied-off. Tl⁺-ions were administered i.v. at the beginning of the experiments as ²⁰⁴Tl-(Tl₂SO₄). The amount of ²⁰⁴Tl-labeled Tl⁺-ions in the perfusion fluid of the gastrointestinal sections was calculated from the ²⁰⁴Tl concentration and the fluid volume.
2. Excretory activity for Tl⁺-ions is highest in the the jejunum; it is followed by the colon ascendens. The ileum and the colon descendens show nearly equal excretory activity which is just half of that measured in the jejunum. The amount of Tl⁺-ions excreted into the stomach is negligible.
3. At the end of the experiment in the perfusion fluid of all segments of the intestine the concentration of ²⁰⁴Tl is statistically significently higher than that in plasma.
4. In jejunal and colonic (descendens) segments the amount of Tl⁺-ions excreted increases with increasing i.v. doses tested from 2×10^?8 up to 2×10^?6 moles/kg body weight.

     

Intestinal 5-Fluorouracil Absorption: Use of Ussing Chambers to Assess Transport and Metabolism

We have employed an in vitro system to study transport and metabolism of organic molecules by gastrointestinal tissues. Such a system would aid in the evaluation of the potential for oral delivery of organic molecules. Transport and metabolism of 5-fluorouracil (5-FU) were studied using rabbit intestinal preparations. Unidirectional fluxes and metabolism were measured in vitro in Ussing chambers under short-circuit conditions. Results from these studies reveal that in ileum, proximal, and distal colon, steady-state fluxes of 5-FU (10 µM added to both bathing solutions) are established after 30 min and remain constant for at least 110 min. Transport of 5-FU under sink conditions with 10 µM 5-FU present in the mucosal or serosal bathing solution alone demonstrated similar rates of transport as under nonsink conditions. The concentration dependence of 5-FU fluxes indicates that the mucosal (m)-to-serosal (s) flux is composed of both a saturable and a linear component over the range of 1–100 µM in the ileum, whereas the s-to-m flux in the ileum and both fluxes in the colon are linear functions of concentration. Over the concentration range employed and the time course of these studies, 5-FU had no effect on the electrical properties of the ileum or colon. In the ileum, the m-to-s but not the s-to-m flux of 5-FU was reduced by (1) serosal ouabain (0.1 mM); (2) reduction of the bathing solution Na concentration; and (3) addition of uracil, thy mine, thymidine, uridine, 2-deoxyuridine, or uridine-5-monophosphate. These results indicate that 5-FU absorption in the ileum occurs by a Na-dependent mechanism that is inhibited by uracil and structurally related compounds. In distal colon, no evidence for an active transport mechanism was obtained. High-performance liquid chromatography (HPLC) analysis reveals that both ileum and distal colon metabolize 5-FU to more polar compounds. Metabolism in ileum is quantitatively greater than in distal colon. Metabolites are found predominantly on the side to which transport has occurred, suggesting that metabolism occurs concomitantly with transport. Since the intestinal cells metabolize 5-FU to more polar compounds and active absorption is inhibited in a competitive manner by related compounds, these results may provide an explanation for the variable oral activity reported for 5-FU.     

Determination of Transport Rates for Arginine and Acetaminophen in Rabbit Intestinal Tissues in Vitro

The in vitro Ussing technique was employed to examine transport rates for acetaminophen and arginine across rabbit intestinal tissues. Mannitol and transepithelial conductance were used to monitor the integrity of rabbit intestinal tissues and the basal and stimulated short-circuit current were measured to assess functional viability. Transepithelial transport of acetaminophen, arginine, and mannitol was determined in rabbit jejunum, ileum, and distal colon. Transepithelial transport of arginine in the ileum and jejunum was composed of both passive (nonsaturable) (P _m = 0.06) and saturable components (K _T = 0.6-0.7 mM; J _max = 0.3-0.4 µmol/hr · cm²). The saturable component of arginine fluxes was abolished by pretreatment of the tissue with serosal ouabain (0.1 mM). In the distal colon, both unidirectional arginine fluxes were nonsaturable. In the segments examined, both unidirectional fluxes of acetaminophen were nonsaturable over the concentration range from 0.1 to 30 mM. These results provide values for maximal permeabilities attained by molecules traversing both the cellular and the paracellular pathways and, by comparison to their in vivo bioavailabilities, provide selection criteria for evaluating drug candidates for oral activity.     

Thallium(I) secretion across the isolated mucosa of rat descending colon

Unidirectional Tl⁺-fluxes across the isolated mucosa of rat descending colon were measured under short circuit (SCC) or voltage clamp conditions (VCC). Under SCC a serosal-to-mucosal net flux (J_net= –0.22 nmol × cm^–2 × h^–1) was observed that agrees with the voltage-independent component measured under VCC. At 7 °C the secretory net flux was abolished. In controls both unidirectional fluxes were unsaturable between 0.1 and 500 mol/l thallium (I). After furosemide or withdrawal of Cl^– or Na⁺ J_net was abolished. Ouabain decreased serosal-to-mucosal Tl⁺ flux and an energy-dependent absorptive net flux of Tl⁺ was observed, characterized by a rather low K_m (10,2 mol/l), V_max (3.8 nmol×cm^–2 × h^–1) and apparent activation energies (E= 10.7 kcal × mol^–1) typically for enzyme catalaysed reactions or narrow channel interactions. The data suggest that in the mucosa of rat descending colon Tl⁺ ions share, at least in part, the same transport systems at K⁺.Dedicated to Professor Dr. W. Rummel, Homburg/Saar (FRG) on the occasion of his 65th birthday (October 21, 1986)     

The effect of naringenin on the intestinal and renal transport of organic solutes

Summary Naringenin (4,5,7-trihydroxy-flavanone) inhibits the accumulation of glycine, -methyl-glucoside, p-amino-hippurate and N¹-methyl-nicotinamide in dog renal cortex slices. It also inhibits oxygen consumption by this tissue. Since the sensitivity of amino-acid uptake to the drug is less than the sensitivity of oxygen consumption, the inhibition of this transport might be secondary to an effect on intermediary metabolism. The inhibition of PAH uptake occurs at a lower concentration of the drug, and so naringenin may affect this process at the membrane level.Naringenin inhibits the transport of sugars and amino-acids by dog, guinea-pig and rat small intestine. Both steady-state accumulation and initial rates of entry are affected. Amino-acid uptake is depressed both in the presence or absence of sodium ions. The inhibition is reversible provided short contact times are employed. According to kinetic analysis, naringenin appeared to be a fully non-competitive inhibitor of phenylalanine influx. Examination of the unidirectional transmural fluxes of phenylalanine across guinea-pig intestine revealed that only the mucosal-serosal flux was affected, and then only if the flavanone was added to the solution bathing the mucosal face of the tissue. Naringenin does not inhibit mucosal Na⁺-K⁺-ATPase, but it does alter the intracellular ion concentrations.Although some of the results can be explained in terms of an effect of naringenin on metabolism, others can not. It is argued that naringenin has a direct action on cell membranes.Some of these results have been presented to the Physiological Society and a short report has appeared in their proceedings (Robinson, 1979)     

Mechanistic Studies on Effervescent-Induced Permeability Enhancement

Purpose. To determine the mechanism(s) by which effervescence induces penetration enhancement of a broad range of compounds ranging in size, structure, and other physiocochemical properties across rat and rabbit small intestinal epithelium. Methods. Effervescent induced penetration enhancement was investigated in vitro by utilization of a modified Ussing chamber diffusion cell apparatus and in vivo by single-pass intestinal perfusion. Results. Carbon dioxide (CO₂) bubbling directly onto rabbit ileum epithelium induced an increase in drug permeability. Mechanistic studies indicated that effects due to CO₂ bubble evolution, such as increased drug dissolution rates, mucus thinning/stripping, and pH buffer effects did not contribute to increases in drug flux. Cellular enzyme (5-ND and LDH) and total protein release assays did not indicate cell membrane perturbation and/or damage. CO₂ bubbling induced a reduction in transepithelial electrical resistance (TEER) indicating epithelial disruption due to a structural change of the paracellular pathway. This was further substantiated by a MW dependence on paracellular marker flux. In addition, tissue recovery was relatively rapid, 20 min. Conclusions. CO₂ bubbling directly onto the intestinal epithelium induced enhanced drug permeability due to an alteration of the paracellular pathway. This, in addition to fluid flow and membrane hydrophobicity concepts, may account for observed increases in drug flux.     

Rate Control in Transdermal β-Estradiol Reservoir Membrane Systems: The Role of Membrane and Adhesive Layer

Purpose. The aim of our study was to clarify the kinetic performance of a membrane controlled reservoir system (MCRS) for -estradiol (E₂) under in vitroconditions by determination of the role of membrane and adhesive layer on E₂flux control. Methods. E₂and ethanol fluxes across EVA membrane or membrane coated with adhesive from saturated solutions in defined ethanol/PBS mixtures were measured in the symmetric and asymmetric configuration. Physicochemical parameters of the EVA membrane were determined. Results. The E₂flux across the 9% EVA membrane steadily increased with increasing ethanol concentrations in both configurations, due to enhanced uptake of E₂by the polymer and increasing membrane diffusivity. Permeation across the EVA membrane coated with an adhesive layer in the symmetric and asymmetric configuration increased up to maximum values of 0.80 ± 0.14 (g × cm^–2× h^–1and 0.37 ± 0.02 g × cm^–2× h^–1, respectively, at 62.5% (v/v) ethanol. The fluxes then decreased with further increase in the volume fraction of ethanol due to a dramatically reduced permeability of the adhesive layer. For the asymmetric case, a linear dependence of E₂on ethanol fluxes was observed. Conclusions. The E₂flux from MCRS is strictly dependent on reservoir ethanol concentrations, whereas the adhesive layer represents the rate controlling barrier at high ethanol levels (>70% v/v).     

Effects of ethacrynic acid on electrolyte and fluid transport by the guinea pig gallbladder

Summary The effect of ethacrynic acid on fluid and electrolyte transport by the guinea pig gallbladder was investigated in vitro. 10^–4M ethacrynic acid, applied to the serosal side, inhibited fluid and sodium chloride absorption. The reduction in salt absorption was accounted for by a 3 Eq/cm²h decrease in the unidirectional fluxes of Na and Cl from mucosa to serosa with no change in the fluxes from serosa to mucosa. Ethacrynic acid (10^–4 M) had no effect on HCO₃–Cl exchange, PGE₁-induced fluid secretion and inulin permeability. At 10^–3 M, ethacrynic acid markedly increased both the serosa to mucosa fluxes of Na and Cl, and the inulin permeability. Examination by light and electron microscopy of gallbladder tissue treated with 10^–3 M ethacrynic acid revealed large intracellular vacuoles and occasionally ruptured apical cell membranes. Only slight morphological changes were seen by 10^–4 M ethacrynic acid with no changes in the controls and ouabain treated gallbladders. The effects of ethacrynic acid are remarkably different from those of furosemide which has been previously shown to inhibit only the HCO₃ secretion leaving fluid and NaCl absorption unchanged.This work was supported by a grant from the Deutsche Forschungsgemeinschaft given to the Sonderforschungsbereich 160. Eigenschaften biologischer Membranen, Projekt C2     

Pharmacological characterizations of recombinant human 5-HT1D1Dα and 5-HT1Dβ receptor subtypes coupled to adenylate cyclase inhibition in clonal cell lines: apparent differences in drug intrinsic efficacies between human 5-HT1D subtypes

Recombinant human 5-HT_1D and 5-HT_1D receptor subtypes were stably expressed in NIH-3T3 fibroblasts (1D cell line) and Y-1 adrenocortical tumor cells (1D cell line), respectively, for pharmacological evaluations of serotonergic compounds to inhibit forskolin-stimulated CAMP accumulation (FSCA). [³H]LSD saturation studies indicated that 5-HT_1D receptor expression levels were slightly higher in the 1D cell line (B _max = 1334 ± 134 fmol/mg protein) than in the (1D) cell line (B _max = 900 ± 900 fmol/mg protein). 5-HT inhibited FSCA with similar potencies (EC₅₀ 2 nM) in both assay systems. The rank order of agonist potencies in both clonal cell lines matched their pharmacological profiles previously determined in binding studies: dihydroergotamine >- 5-carboxamidotryptamine (5-CT) > LSD >- 5-HT > sumatriptan > 1-naphthylpiperazine (1-NP) > yohimbine > 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH DPAT) > 1-(2,5-dimethoxy4-iodophenyl)-2-aminopropane (DOI), with K_i/EC₅₀ ratios greater than unity. Methiothepin acted as a silent antagonist at both human 5-HT_1D and 5-HT_1D receptors with apparent dissociation constants (K_b values) of 12 ± 1 nM and 3 ± 1 nM, respectively. Whereas GR 127,935, metergoline, DOI, and quipazine acted as full agonists in the 1D cell line, these compounds behaved as partial agonists in the 1D cell line.To determine whether high levels of receptor reserve might mask partial agonist activity in the two second messenger assay systems, studies were performed using the irreversible receptor alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). The relationships between receptor occupancy and inhibition of FSCA were determined for 5-HT, sumatriptan, and 1-NP in both clonal cell lines after partial receptor inactivation using Furchgott analysis. Hyperbolic relationships between receptor occupancy and second messenger response were determined for 5-HT in both transfected cell lines. Steep hyperbolic relationships were also found for sumatriptan and 1-NP in the 1D cell line whereas nearly linear relationships were observed for these two compounds in the 1D cell line. Moreover, K_A/EC₅₀ ratios of these compounds were significantly larger in the (1D)(10–32) as compared to the 1D (0.9–2.5) cell line. These data are consistent with the hypothesis that the two heterologous expression systems contain a differential amount of receptor reserve. Despite the presence of an apparently larger receptor reserve in the 1D cell line, GR 127,935, metergoline, DOI, and quipazine behaved as partial agonists.Although the potencies (EC₅₀ values) of compounds matched their respective affinity constants (K_i values) for the closely-related 5-HT_1D subtypes, differences in intrinsic activities were observed for a few compounds between the two 5-HT_1D receptor expression systems. Since receptor reserve is dependent on the properties of both the assay system and drug, the observed variations in intrinsic activity, although influenced by the variable amounts of receptor reserve in the two transfected cell lines, reflect primarily system-independent differences in the intrinsic efficacy of the tested compounds at the two human 5-HT_1D receptors. Higher intrinsic efficacies of compounds at the human 5-HT_1D receptor relative to the human 5-HT_1D subtype may be responsible for the higher intrinsic activities observed in the (1D) cell line, even though receptor reserve is apparently lower in this system.Abbreviations CRC Concentration-response curve - FSCA forskolin-stimulated cAMP accumulation - KA pseudo-dissociation constants - 5-CT 5-carboxamidotryptamine - 5-HT 5-hydroxytryptamine - 5MeOT 5-methoxytryptamine - PAPP 1-[2-(4-aminophenyl) ethyl] -4-(3-trifluoromethylphenyl)-piperazine - 1-NP 1-(1-naphthyl) piperazine - 8-OH-DPAT 8-hydroxy-2-(di-n-propylamino) tetralin - DOI 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane - MK-462 (N,N-dimethyl-2-[5-(1, 2, 4-triazol-l-yl methyl)-1H-indole3-yl]ethylamine - GR 127,935 (2-methyl-4-(5-methyl-[1, 2, 4]oxadiazol-3-yl)-biphenyl-4-carboxylic acid [4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-amide) - GR 46611 5-[4-methoxybenzyl ethylene]-1H-indole3-yl]ethyl amine) - L-694,247 (2-[5-[3-(4-methylsulfonylamino)benzyl-1, 2, 4-oxadiazol-5-yl]-1H-indole3-yl]ethylamine)     

Effect of Medium-Chain Glycerides on Physiological Properties of Rabbit Intestinal Epithelium in Vitro

Medium chain glycerides (MCGs) have been reported to enhance intestinal absorption of hydrophilic drugs. However, the mechanisms involved in absorption enhancement are not well understood. The effects of MCGs (CapMul MCM) on physiological properties of rabbit ileum and distal colon, including active ion transport, transepithelial resistance (R_t) and passive permeability, have been investigated in vitro. CapMul MCM inhibited active ion transport (measured as a decrease in short-circuit current, I_sc) in both intestinal segments in a concentration-dependent manner. The inhibition of I_sc was rapidly reversible (within 100 min) upon removal of CapMul MCM. The data indicate that CapMul MCM preferentially affected ion transport by villus cells in the ileum and surface cells in the distal colon. Ion transport in crypt cells in both segments was not significantly altered. R_t of the ileum was not significantly affected by 5% CapMul MCM, while mannitol transport was 6 fold enhanced. Treatment of distal colon with 1% CapMul MCM reduced R_t by 95%, while mannitol transport was 100 fold enhanced. In a parallel experiment, mucosal(m)-to-serosal(s) transport of cephalexin, a -lactam antibiotic, in the ileum was about 40% reduced in the presence of 5% CapMul MCM, whereas transport in the s-to-m direction was 2.5 fold enhanced. Treatment of the distal colon with 1% CapMul MCM resulted in 25 fold enhancement of cephalexin transport in either direction. These results suggest that absorption enhancement by MCGs results from an increased permeability of the intestine confined to the villus or surface epithelium.     

Simultaneous study of the pharmacokinetics of intravenous and oral nicardipine using a stable isotope

Summary The systemic elimination of nicardipine has been studied by an initial oral administration of nicardipine followed 1.25 h later by intravenous injection of the deuterium-labelled molecule (D₃ nicardipine).To check that intravenous kinetics was not modified by the oral administration, an i.v. injection of unlabelled nicardipine (D₀ nicardipine) was also given. The study was carried out in six healthy male volunteers, aged between 24 and 27 years, according to a Latin square cross-over design.Similar values were found for each kinetic parameter after i.v. administration regardless of whether it was administered alone by that route or with an oral dose. The plasma level-time curves of nicardipine were described by a three open compartment model. The total plasma clearance was about 800 ml/min, the volume of distribution was of the order of 1 l/kg and the half-life of -elimination ranged from 4 to 5 h. The elimination rate constant was independent of the route of administration.     

Antagonists that differentiate between α2A- and α2D-adrenoceptors

Four antagonists were examined for their ability to differentiate _2A from the orthologous _2Dadrenoceptors. The antagonists were (2S,12bS) 1, 3-di-methylspiro(1, 3, 4, 5, 6, 6, 7,12b-octahydro-2H-benzo[b]furo[2,3-a]quinolizine)-2,4-pyrimidin-2-one (MK 912), 2-[2-(methoxy-1, 4-benzodioxanyl)imidazoline (RX 821002), efaroxan and benoxathian. The ₂-autoreceptors in rabbit brain cortex were chosen as _2A- and the a₂-autoreceptors in guinea-pig brain cortex as _2D-adrenoceptors. Slices of the brain cortex were preincubated with ³H-noradrenaline and then superfused and stimulated electrically by brief pulse trains (4 pulses, 100 Hz) that led to little, if any, ₂-autoinhibition. 5-Bromo-6-(2-imidazolin-2-ylamino)quinoxaline (UK 14,304) was used as an ₂-adrenoceptor agonist.UK 14, 304 decreased the stimulation-evoked overflow of tritium. The antagonists shifted the concentration-inhibition curve of UK 14, 304 to the right in an apparently competitive manner. Dissociation constants of the antagonists were calculated from the shifts. MK 912, RX 821002 and efaroxan had markedly higher affinity for (guinea-pig) _2D-adrenoceptors (pK _d values 10.0, 9.7 and 9.1, respectively) than for (rabbit) _2A-adrenoceptors (pK _d 8.9, 8.2 and 7.6, respectively). Benoxathian had higher affinity for _2A- (pK _d 7.4) than for _2D-adrenoceptors (pK _d 6.9). Ratios calculated from the K _d values of the four compounds differentiated between _2A and _2D up to 100 fold. It is concluded that MK 912, RX 821002, efaroxan and benoxathian are antagonists with high power to differentiate _2A- from _2D-adrenoceptors.     

Interaction between accumbens D1 and D2 receptors regulating rat locomotor activity

The effect of intra-accumbens injections of various dopaminergic agonists and antagonists on the rat locomotor activity has been evaluated in automated open fields. Locomotor stimulation has been observed after local administration ofd-amphetamine (10 g), apomorphine (10 g), as well as of solution containing the D₁ agonist SKF 38 393 and D₂ receptor agonist LY 171 555 (quinpirole) in doses (10 and 4 g, respectively) which were inactive when both drugs were administered separately. On the other hand separate injections of metoclopramide (0.1 g) and SCH 23 390 (0.5 g) (D₂ and D₁ receptor antagonists) very potently inhibited animals' locomotor activity. The data indicate that concomitant stimulation of both accumbens D₁- and D₂-receptor related mechanisms is a necessary condition to increase rat motility. Moreover, it seems that accumbens D₁ receptors may be differently involved in the control of facilitatory versus inhibitory motor processes.     

Movement of thallium (I) ions in vitro

1. Movement of thallium (I) ions across the mucosal epithelium of descending rat colon, stripped of the muscularis externa in vitro was determined under voltage clamp conditions.
2. The unidirectional fluxes of thallium (I) ions from mucosal to serosal side (M → S) and in the opposite direction (S → M) were measured at 0 mV. A net flux of thallium (I) ions was observed from serosal to mucosal side (0.216 nmoles · cm^?2 · h^?1) in other words, the mucosal epithelium is apparently asymmetric for the movement of thallium (I) ions.
3. The investigations were carried out on the range of ?30 mV up to +30 mV from mucosal to serosal side. Thallium (I) ions move from mucosal to serosal side (M → S) by diffusion only (P_i = 0.119 cm · h^?1), whereas into the opposite direction (S → M) thallium (I) ions in addition to a voltage-dependent component are transported against an electrical gradient.

     

Phospholipase A2 and mediation of the activation of short-circuit current in the rat colonic mucosa

Summary Melittin (0.5–2 g ml^–1) increased the short-circuit current (I_sc) in mucosa-submucosa and mucosa preparations of the rat colon descendens in a dose-dependent manner. In the preparation with the submucosal plexus, quinacrine and indomethacin completely blocked the effect of melittin, indicating activation of phospholipase A₂ and production of prostaglandins induced by the drug. The melittin response was also partially sensitive to the lipoxygenase inhibitor, nordihydroguaiaretic acid. Complete inhibition by tetrodotoxin and atropine gives evidence for the involvement of cholinergic neurons in the mediation of the response induced by melittin. In contrast, in the preparation without the submucosal plexus the effect of melittin was only partially inhibited by quinacrine, indomethacin, or by neuronal blockers, suggesting direct interactions of melittin with the epithelium in addition.The effect of melittin resembles to the action of bradykinin, which is neuronally mediated and quinacrine-sensitive in the mucosa-submucosa preparation, and quinacrine-resistant and not neuronally mediated in the mucosa preparation. In the mucosa-submucosa preparation, the melittin response is even partially sensitive to the bradykinin receptor antagonist [D-Phe⁷]-bradykinin. The results provide evidence for the presence of a quinacrine-sensitive phospholipase A₂ in the preparation with and that without the submucosa. Send offprint requests to: M. Diener at the above address     

Epithelium selectively controls hypersensitization of the response of smooth muscle to leukotriene D4 by endogenous prostanoid(s) in guinea-pig trachea

Summary The effect of epithelium removal on the sensitivity of smooth muscle to carbachol and leukotriene D₄ (LTD₄) was investigated in guinea-pig isolated tracheal preparations untreated and treated with a cyclooxygenase inhibitor, flurbiprofen (1 ol/1). The pD₂ value of carbachol was not changed by epithelium removal or by flurbiprofen-treatment, alone or in combination. On the other hand, the pD₂ of LTD₄ was higher in the tracheal strips without epithelium than in the intact preparations. In preparations devoid of epithelium, the pD₂ value of LTD₄ was decreased by treatment with flurbiprofen, suggesting that the sensitivity of the smooth muscle (namely, epithelium-free preparations) to LTD₄ is enhanced by intramurally produced excitatory prostaglandin(s) [PG(s)]. However, in intact preparations with epithelium, no such sensitized response to LTD₄ was observed. After flurbiprofen-treatment, in the continued presence of prostaglandin F₂ (PGF₂, 200 nmol/l), the sensitivity to LTD₄ was partially recovered in the epithelium-free preparations, but not in the intact ones. These results suggest that epithelium diminishes the sensitizing effect of intramural excitatory PG(s) on responsiveness of tracheal smooth muscle to LTD4, possible via a non-prostanoid substance(s).Send offprint requests to I. Takayanagi     

Effect of SR 142801 on nitric oxide-dependent and independent responses to tachykinin NK3 receptor agonists in isolated guinea-pig colon

We have determined the ability of the novel nonpeptide tachykinin (TK) NK₃ receptor antagonist, SR 142801, [(S) -(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl) piperidin-3-yl) propyl)-4-phenylpiperidin-4-yl)-N-methylacetamide] in inhibiting the nitric oxide (NO)-independent prejunctional inhibition of cholinergic twitches and the NO-dependent relaxation produced by the NK₃ receptor selective agonist, senktide, in the circular muscle of the guinea-pig proximal colon. Under moderate load (10 mN) and isometric recording of mechanical activity, single pulse electrical field stimulation (EFS) produced atropine- and tetrodotoxin-sensitive twitch contractions of mucosa-free circular muscle strips from the guinea-pig proximal colon. In the presence of NK₁ and NK₂ receptor antagonists (SR 140333 0.01 M and GR 94800 0.1 M, respectively) the NK₃ receptor selective agonist, senktide (EC₅₀ 33 pM) and the NK₃ receptor preferring natural TK, neurokinin B (NKB, EC₅₀ 13 pM) produced a concentration-dependent slowly developing inhibition of cholinergic twitches. Senktide (1 nM) did not affect the contractile response to acetylcholine (1 gM) indicating that depression of evoked twitches occurs prejunctionally. The inhibitory effect of senktide was 'unaffected when evoked in the presence of the cyclooxygenase inhibitor (S)-ketoprofen (10 M), guanethidine (10 M), naloxone (0.3 M), the GABA_B receptor antagonist 2-hydroxysaclofen (10 M) or the combined application of the adenosine A₁ and A₂ receptor antagonists, 8-cyclopentyl-1,3-dipropylxanthine (10 M) and 3,7-dimethyl-l-propargylxanthine (30 M) respectively.In the presence of NK₁ and NK₂ receptor antagonists, the NO-synthase inhibitor l-nitroarginine (L-NOARG 30-100 M) did not affect twitch inhibition induced by senktide (EC₅₀ 33 pM). The response to NKB (EC₅₀ 95 pM) was slightly reduced by L-NOARG, yet the bulk of the inhibitory effect of both agonists on cholinergic twitches was substantially independent of NO generation. SR 142801 (0.1–0.3 M) produced a moderate rightward shift of the concentration-response curve to senktide without depression of the Emax to the agonist, yielding an apparent pK_B value of 7.65.Under low resting tone (3 mN) and isotonic recording of mechanical activity, mucosa-free circular muscle strips from the guinea-pig proximal colon gained a high intrinsic tone suitable for testing the response to relaxant agents. In the presence of atropine (1 M), guanethidine (3 M), SR 140333 (0.01 [M) and GR 94800 (0.1 LM), senktide (EC₅₀ 50 pM) produced a concentration-dependent relaxation of the strips, which was blocked by L-NOARG. SR 142801 (0.01–0.1 M) produced a large rightward shift of the L-NOARG-sensitive concentration-response curve to senktide yielding an apparent pKB value of 8.62. Under isometric recording condition, SR 142801 (0.1 M) did not affect twitch inhibition produced by 3 nM clonidine. Under isotonic recording condition, SR 142801 did not affect the L-NOARG-sensitive relaxation produced by EFS.The present results indicate that NK₃ receptor stimulation produces a NO-dependent relaxation of the guinea-pig colon and a substantially NO-independent prejunctional inhibition of cholinergic twitches. The variable affinities of SR 142801 in antagonizing various senktide-induced neuromodulatory effects in the guinea-pig intestine suggest a possible intraspecies heterogeneity of NK₃ receptors in the enteric nervous system.     

Nicorandil shortens action potential duration and antagonises the reduction of V max by lidocaine but not by disopyramide in guinea-pig papillary muscles

Summary Conventional microelectrode techniques were used to examine whether or not nicorandil, which shortens action potential duration (APD), modifies the lidocaine- or disopyramide-induced time-dependent reduction of V _max in guinea-pig papillary muscles. First, effects of 0.1 and 1 mmol/l nicorandil were examined on the frequency dependence of V _max and on the recovery process of V _max. Second, the frequency-dependent reduction of V _max by 20 mol/l antiarrhythmic drugs was examined in the presence and absence of 1 mmol/l nicorandil at stimulation frequencies of 1/120 Hz–5 Hz. Third, the recovery process of V _max in the presence of 20 mol/l antiarrhythmic drugs was examined, with and without 1 mmol/l nicorandil, by applying test stimuli at various diastolic intervals after conditioning stimuli. 1 mmol/l nicorandil greatly shortened APD₉₀ to 30–40% of control without changing the frequency dependence of V _max, the recovery process of V _max, and the resting potential. The lidocaine-induced, frequency-dependent reduction of V _max was significantly antagonised by 1 mmol/l nicorandil, but the disopyramide-induced reduction was not. The recovery process of V _max slowed in the presence of lidocaine was antagonised by 1 mmol/l nicorandil as follows: the time to get the full recovery of V _max was shortened by nicorandil with a significant decrease in the zero time-intercept (from 0.54 to 0.38) but with an insignificant change in the recovery time constant (from 130 ms to 121 ms). In contrast, the recovery process of V _max slowed in the presence of disopyramide (a zero time intercept of 0.13 and a recovery time constant of 50 s) was not significantly antagonised by 1 mmol/l nicorandil. In conclusion, nicorandil having an action potential-shortening action antagonises the lidocaine-induced, time-dependent reductions of V _max, but not the disopyramide-induced reductions. These results suggest that: (1) lidocaine and disopyramide preferentially bind to inactivated and activated sodium channels, respectively, because lidocaine's effects are dependent on and disopyramide's effects are independent of APD (during which sodium channels are in the inactivated state); and (2) nicorandil is a useful drug for estimating whether a sodium channel-blocking action of class I antiarrhythmic drugs is due to an inactivated channel block or an activated channel block. These time-dependent reductions of V _max by both lidocaine and disopyramide were well simulated by the guarded receptor hypothesis. Send offprint requests to M. Kojima