d-glucose balance in the enterocyte of rat jejunum “in vitro”

An everted sac of male albino rat jejunum (Wistar strain) incubated in vitro is used. Netd-glucose and Na⁺ transport together withd-glucose concentration in the emerging fluid [5], or in the serosal fluid, and in the enterocytes are determined. Celld-glucose concentration does not change significantly in a range between 20–200 moles or between 50–500 moles of netd-glucose transepithelial transport, depending on the experimental conditions.As far as cellulard-glucose and Na⁺ concentration is concerned, the enterocyte behaves as an homeostatic system.The mechanism involved ind-glucose extrusion is extensively discussed. Two hypotheses seem to be possible. First, the mechanism is an active metabolically dependent one, just as it is for sodium transport. Second, the metabolic activity favoursd-glucose facilitated permeability through the basolateral membrane in such a way as to maintain a constant relationship betweend-glucose and Na-extrusion, notwithstanding the fact thatd-glucose concentration gradient across the basolateral membrane lowers by increasing Na and glucose extrusion rate.     

45Ca2+ uptake by dispersed pancreatic islet cells: Effects ofd-glucose and the calcium probe,chlorotetracycline

Uptake of⁴⁵Ca²⁺ was studied in dispersed pancreatic islet cells from non-inbredob/ob-mice. Like whole islets the dispersed cells responded to 20 mMd-glucose with a markedly increased⁴⁵Ca²⁺-labeling of both the lanthanum-nondisplaceable and the lanthanum-displaceable calcium pools. The pronounced effect ofd-glucose could not be reproduced with 3-O-methyl-d-glucose,l-glucose,d-mannose,l-leucine, ord-leucine; however,⁴⁵Ca²⁺ uptake was greater in the presence ofl-leucine as compared withd-leucine.⁴⁵Ca²⁺ uptake by dispersed cells or whole islets was stimulated severalfold by 100 M or more chlorotetracycline. At the concentration of only 10 M, chlorotetracycline had no effect on whole islets and partially inhibited⁴⁵Ca²⁺ uptake by the dispersed cells. The ability ofd-glucose to stimulate⁴⁵Ca²⁺ uptake by islets or dispersed cells remained in the presence of 10 M chlorotetracyline. Islet cell suspensions apparently represent a valid model for studying how Ca²⁺ interacts with the cells. However, when using chlorotetracycline as fluorescent Ca²⁺ probe, attention must be paid to its potential ionophoric activity. At only 10 M, the drug seems to monitor a peripheral pool of Ca²⁺, some of which may reside in normal transport channels.     

A novel stopped-flow method for measuring the affinity of actin for myosin head fragments using μg quantities of protein

Summary The dissociation constant for actin binding to myosin and its subfragments (S1 & HMM) is 1 m at physiological ionic strength. Many of the methods used to measure such affinities are unreliable for a K_d below 0.1 m. We show here that the use of phalloidin to stablise F-actin and fluorescently labelled proteins allows the affinity of actin for myosin S1 to be measured in a simple transient kinetic assay. The method can be used for K_d's as low as 10 nm and we demonstrate that the K_d's can be estimated using only g quantities of material. Furthermore we suggest how this method may be adapted for ng quantities of protein. This will allow the affinity of actin for myosin fragments to be estimated for proteins which are difficult to obtain in large quantities i.e. from biopsy material or from proteins expressed in baculovirus.     

Effect of depolarizing and hyperpolarizing agents on the membrane potential difference of primary cultures of rabbit aorta vascular smooth muscle cells

Vascular smooth muscle cells of rabbit aorta were enzymatically dispersed, kept in primary culture, and studied between days 1 and 7 in a bath rinsed with Ringer-like solution at 37°C. The electrical membrane potential difference (PD) was measured with microelectrodes. The mean value of PD was –50±0.4 mV (n=53). Cromakalim (BRL 34915), 1 mol/l and 10 mol/l, hyperpolarized the membrane potential by 9±1 mV (n=11) and 15±1 mV (n=53) respectively. Glibenclamide (10 mol/l) abolished the hyperpolarizing effect of cromakalim (n=6). Simultaneous addition of cromakalim and glibenclamide (both 10 mol/l, n=11) and glibenclamide itself (10 mol/l, n=7) had no effect on PD. In patch-clamp experiments in outside-out-oriented Ca²⁺-sensitive K⁺ channels, cromakalim increased the open probability (P _o) only slightly and only with a cytosolic Ca²⁺ activity of 1 mol/l. In all other series cromakalim had no effect on the P _o of these channels. Forskolin (10 mol/l) hyperpolarized PD by 6±1 mV (n=13). The nucleotides UTP, ATP and ITP (10 mol/l) depolarized PD by 12±1 mV (n=7), 8±1 mV (n=65) and 5±1 mV (n=6) respectively. GTP, [,-methylene]ATP and adenosine had no significant effect. Mn²⁺ (1 mmol/l, n=18), Ni²⁺ (1 mmol/l, n=13), Co²⁺ (1 mmol/l, n=11), Zn²⁺ (1 mmol/l, n=6) and the Ca²⁺-channel blockers verapamil and nifedipine (both 0.1 mmol/l, n=6) did not attenuate the depolarization induced by 10 mol/l ATP. Fetal calf serum (100 ml/l, n=7) depolarized PD by 11±2 mV. This effect was not abolished by nifedipine or by replacing NaCl by choline chloride. The data indicate that PD of vascular smooth muscle cells is depolarized by P₂ agonists and hyperpolarized by the K⁺-channel opener cromakalim. The effect of cromakalim is antagonized by glibenclamide. The effect of cromakalim is probably not mediated by the K⁺ channel identified in excised patches.Supported by DFG Gr 480/10     

On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation

The Ca²⁺ channel subunits _1C-a and _1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba²⁺ current (I _Ba) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 M cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 M PKA and 1 M okadaic acid. The activity of the _1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 M H 89 and was not increased by superfusion with 5 M forskolin plus 20 M isobutylmethylxanthine (IBMX). The _1C-a·₂·₂/ complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 M H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the _1C-a·₂·₂/ channel with 10 M PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca²⁺ current (I _Ca) of cardiac myocytes increased threefold during internal dialysis with 5 M PKA or 25 M microcystin and during external superfusion with 0.1 M isoproterenol or 5 M forskolin plus 50 M IBMX. These results indicate that the L-type Ca²⁺ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.     

Binding of α-actinin to F-actin or to tropomyosin F-actin is a function of both α-actinin concentration and gel structure

Summary We have studied by electron microscopy as well as by measurements of low shear viscosity, rigidity and binding, the effect of-actinin on the gel formed at 37° C with F-actin and with tropomyosin-decorated F-actin. Contrary to previous reports in the literature,-actinin at nanomolar concentrations is an efficient actin gelling protein, even at 37° C, provided that the concentration of actin (or of tropomyosin-decorated F-actin) is low (1.2–2.4 m). The binding of-actinin to F-actin, as a function of actin concentration, is anomalous. The amount of bound-actinin increases when actin concentration increases from 0 to 1.2 m but does not change significantly when actin concentration is further increased up to 48 m. A similar result is obtained with tropomyosin-decorated F-actin.These observations can be explained by an hypothesis that binding is a function of the-actinin — F-actin association constant as well as of the rigidity of the gel. When the concentration of actin increases, the rigidity of the gel also increases and more work is required to bring two actin filaments to the reaction distance with-actinin and, consequently, a larger-actinin concentration is required to attain the same ratio of bound-actinin to actin monomers in the filaments.     

In vitro effects of the antiallergy compound,CI-949, on interleukin-1 and 2 release,and on mitogen and alloantigen responsiveness

CI-949 (5-methoxy-3-(1-methylethoxy)-1-phenyl-N-1H-tetrazol-5-yl-1H-indole-2-carboxamide,l-arginine salt), an antiallergy compound was found to be a weak inhibitor of IL-1 release from LPS-stimulated murine peritoneal exudate cells and human peripheral blood leukocytes, with IC₅₀s of 186.2 and 267.9 M, respectively. CI-949 was also a poor inhibitor of release of IL-2 from Con A-stimulated rat spelenocytes (37% inhibition at 100 M). CI-949 did produce concentration-related inhibition of the response of human lymphocytes to PHA and Con A (IC₅₀s=44.7 and 21.5 M, respectively) as well as in the mixed lymphocyte reaction (MLR) (IC₅₀=16.8 M). The clinical significance of these latter findings is unknown at present.     

Nitric oxide mediates the anti-adrenergic effect of adenosine on calcium current in isolated rabbit atrioventricular nodal cells

The aim of this study was to determine if adenosine exerts an anti-adrenergic effect on rabbit isolated atrioventricular (AV) nodal cells and, if so, the dependence of this effect on nitric oxide (NO) production. Inward Ca current,I _Ca, was measured in AV nodal cells, enzymatically isolated from rabbit hearts. Isoprenaline (0.1 M) increasedI _Ca from 676 ± 59 to 1102 ± 86 pA (n = 25). This isoprenaline-induced increase inI _Ca, (178 ± 15 % of control) was abolished in the presence of 10 M adenosine (I _Ca 100 ± 2 % of control,n = 9, P < 0.05). This effect of adenosine was completely blocked by the A₁ receptor antagonist CPDPX (8-cyclopentyl 1, 3-dipropylxanthine, 0.1 M). In cells pre-treated with the NO synthase inhibitor,l-nitro-arginine methyl ester (l-NAME, 1 mM) the isoprenaline-induced increase inI _Ca(208 ± 39 % of control,n = 7) was not reduced by the addition of 10 M adenosine (195 ± 32% of control). Co-incubation of cells inl-NAME withl--arginine (1 mM, the endogenous substrate of NO synthase) restored the adenosine-induced attenuation ofI _Ca. In these cells, isoprenaline increasedI _Ca (157 ± 7% of control,n = 6), and, following addition of adenosine (10 M)I _Ca was reduced to 107 ± 8% (P < 0.05). The NO-releasing agent SIN-1 (3-morpholino-sydnonimine, 100 M) inhibitedI _Ca augmented by isoprenaline (n = 5). It is concluded that adenosine exerts an anti-adrenergic effect on the AV node via A, receptors to attenuate a catecholamine-stimulated increase inI _Ca and that this action involves the intracellular production of NO.     

Concentration-dependent effects of tetracaine on excitation-contraction coupling in frog skeletal muscle fibres

Summary The effects of low (10–100 m) concentrations of tetracaine on intramembrane charge movement and on the rate of calcium release (R_rel) from the sarcoplasmic reticulum (SR) were studied in cut skeletal muscle fibres of the frog using the voltage clamp technique. The fibres were mounted in a single or double vaseline gap chamber to study the events near the contraction threshold or in a wide membrane potential range. Although the hump component of charge movement (Q) was suppressed to some extent, the voltage dependence and the parameters of the Boltzmann distribution were not modified significantly at tetracaine concentrations below 50 m. At 50 and 100 m of tetracaine the midpoint voltage of the Boltzmann distribution was shifted to higher membrane potentials and the steepness was decreased. The total available charge remained the same at all concentrations tested. Using fura-2 to measure calcium transients at 100 m tetracaine the threshold for calcium release was found to be significantly shifted to more positive membrane potentials. Tetracaine reversibly suppressed both the early inactivating peak and the steady-level of R_rel but the concentration dependence of these effects was markedly different. The inactivating component of calcium release was decreased with a Hill coefficient of approximately 1 and half effective concentration of 11.8 m while the steady-level was decreased with a Hill coefficient of greater than 2 and a half effective concentration of 47.0 m. These results favour two sites of action where tetracaine would suppress the calcium release from the SR.     

Sorbitol metabolism in inner medullary collecting duct cells of diabetic rats

Intracellular accumulation of sorbitol, generated fromd-glucose via the aldose reductase pathway, is thought to play an important role in diabetic complications such as lens cataracts and neuropathy. In order to elucidate the effect of diabetes on the renal inner medulla, another sorbitol-rich tissue, male Wistar rats were treated with a single dose of streptozotocin (60 mg/kg body weight, i.p.). Six wecks later total inner medullary tissue (IM) or isolated inner medullary collecting duct (IMCD) cells were prepared. In diabetic IM tissue, sorbitol content was 1.8-fold higher than in control IM tissue (134±17 vs. 74±22 mol/g tissue protein). Sorbitol production in both normal and diabetic IMCD cells was strongly dependent on extracellulard-glucose concentration. In normal cells, for example, sorbitol production was 90±9 mol sorbitol/g protein x h at 45 mMd-glucose compared to 13±1 mol/g protein x h at 5 mM. At identicald-glucose concentrations sorbitol synthesis in diabetic IMCD cells was, however, always significantly higher than in control cells (122% of control at 15 mM and 126% of control at 45 mM). In addition, aldose reductase activity in diabetic IM was found to be augmented. The maximal velocity was 4.2 times higher (97±22 U/g protein vs. 23±7 U/g protein) while theK _m of the enzyme remained unchanged. Membrane permeability for sorbitol or the response to changes in extracellular osmolarity was not significantly different in diabetic IMCD cells and normal cells with correspondingly high intracellular sorbitol concentrations. Similarly the kinetic parameters ofd-glucose uptake were not altered by streptozotocin treatment. These results suggest that increased medullary sorbitol content in diabetic rats is a result of increased sorbitol synthesis due to a higher extracellulard-glucose concentration and augmented aldose reductase activity in face of an unaltered sorbitol permeability of the plasma membrane.     

Differential regulation of the activation of human eosinophils,macrophages, and neutrophils: Effect of the allergic mediator release inhibitor CI-949

The allergic mediator release inhibitor CI-949 [5-methoxy-3-(1-methylethoxy)-1-phenyl-N-1H-tetrazol-5-yl-1H-indole-2-carboxamide,l-arginine salt] was evaluated for its effect on the activation of human eosinophils, macrophages, and neutrophils by the phagocytic stimulus serum-opsonized zymosan (SOZ). CI-949 inhibited the SOZ-stimulated respiratory burst of eosinophils, measured as the generation of superoxide anion, with an IC₅₀ of 22.8 M. At concentrations up to 100M, CI-949 had no inhibitory effect against superoxide anion generation by human macrophages, while inhibiting the response of human neutrophils by only 24.8 percent. CI-949 exhibited a different profile of inhibitory activity against lysosomal enzyme release by these cells. At 100 M, CI-949 had no inhibitory effect against release of eosinophil peroxidase while inhibiting release of the macrophage lysosomal enzyme N-acetyl--d-glucosaminidase by only 11.7 percent. In contrast, CI-949 inhibited the release of the neutrophil primary granule enzyme myeloperoxidase with an IC₅₀ of 21.4M, while inhibiting release of lysozyme from secondary granules with an IC₅₀ of 99.3M. These results demonstrate that oxygen radical generation and lysosomal enzyme release by human eosinophils, macrophages, and neutrophils are differentially regulated by CI-949. These results suggest that these inflammatory cells may have distinct stimulus-response coupling mechanisms.     

Der basale Noradrenalinspiegel im peripheren ven?sen Blut des Menschen

Zusammenfassung 1. Mit einer in den wesentlichen Punkten auf das Verfahren von Häggendal zurückführenden Technik wurden die Katecholamine, insbesondere der Noradrenalinspiegel, im venösen Armvenenblut bei kreislaufgesunden Probanden zwischen 20 und 68 Jahren unter Ruhebedingungen untersucht.2. Die Noradrenalin-Konzentration betrug im Mittel aus 95 Einzeluntersuchungen 0,282 g/l Plasma (Häggendal 0,300 g), die Adrenalin-Konzentration war 0,056 g/l (Häggendal 0). Während die Kreislaufgrößen (Mitteldruck und Herzfrequenz)nach 20 min Ruhe und 10 min nach dem Einsetzen der Kanüle schon praktisch basale Werte erreichten, ist dies beim venösen Noradrenalin-Spiegel erst ca. 20 min nach dem letzten vorausgegangenen Stress der Fall.3. Die Aufgliederung des Materials nach dem Alter der Probanden zeigt eine ansteigende Tendenz der Noradrenalinkonzentration mit zunehmenden Jahren.4. Die Aufgliederung von insgesamt 238 Proben aus 95 Versuchen nach dem Mitteldruck und der Herzfrequenz zeigt jeweils eine signifikante positive Korrelation sowohl zwischen Druck als auch Frequenz und NA-Gehalt des venösen Blutes.5. Der mittlere NA-Spiegel von 16 weiblichen Probanden lag mit 0,250 g etwas unter dem Gesamtdurchschnitt, sowohl nach der Alters- als auch nach der Druck-Aufgliederung; keine Signifikanz.

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Summary 1. Catecholamines, especially noradrenaline, are determined in the cubital vein blood of normal individuals aged between 20 and 68. A slightly modified Häggendal-procedure is used.2. The average concentration of noradrenaline is 0.282 g/l plasma (Häggendal 0.3 g), the concentration of adrenaline 0.056 g/l (Häggendal 0). Blood pressure and heart rate are back to normal 10 min after the insertion of the cannula, the noradrenaline level, however, only 20 min after the last antecedent stress.3. A classification of the results according to age shows increasing noradrenaline levels with increasing age.4. A classification according to average blood pressure and heart rate shows significantly positive correlations between pressure, heart rate, and noradrenaline content of the venous blood.5. The average noradrenaline concentration in 16 females is 0.25 g/l plasma as compared to 0.28 g total average. The difference is not regarded as significant.

     

Transformation of ψ − Saccharomyces cerevisiae to ψ + with DNA co-purified with 3 μm circles

Summary DNA enriched for supercoiled plasmids prepared from the 3 m plasmid-enriched, [ ⁺], [2 m°] strain 6-1G-P188 and from the [2 m⁺] [⁺] strain LL20 can be used to transform a ^– recipient strain to ⁺. Fractionation of the former preparation by electrophoresis showed that the 3 Mm plasmid band contained the transforming activity.     

Modulation of bradykinin responses in airway smooth muscle by epithelial enzymes

We have studied the effect of epithelium removal on responses of guinea pig trachea to bradykinin (BK). BK (1 nM–10 M) gave a concentration-dependent relaxation when epithelium was present (E+: EC₅₀=10±3 nM). Epithelium removal resulted in a biphasic response to BK with relaxation at low concentrations (E–: EC₅₀=3.0±1.0 nM) and a recontraction to baseline at higher concentrations (EC₅₀=2.0±1 M). Phosphoramidon (10 M), an inhibitor of neutral endopeptidase (NEP), which cleaves BK into inactive peptides, potentiated relaxation (EC₅₀=1.0±0.9 nM and 0.1±0.1 nM in E+ and E respectively) and contraction in trachea with intact epithelium (EC₅₀=0.08±0.03 M). Inhibition of cyclooxygenase by indomethacin (5 M), inhibited relaxation to BK in E+ tracheal segments, resulting in a slight contraction (EC₅₀=1.0 M), whereas a potent contractile response was observed in E–segments (EC₅₀ 1.6 M, maximal contraction >1 g). In the presence of both indomethacin and phosphoramidon BK caused contraction, even in the presence of epithelium (EC₅₀=0.2±0.11 M), and the response in the absence of epithelium was similar to the response observed in trachea with intact epithelium (EC₅₀=0.25±0.1 M). The contractile effect of BK on airway smooth muscle may be inhibited by a protective role of epithelium, due to release of relaxant prostanoids and by degradation by epithelial NEP. In asthma, bronchoconstrictor responses to BK may be partly explained by loss of airway epithelium.     

Role of nitric oxide in modulating permeability of hamster cheek pouch in response to adenosine 5′-diphosphate and bradykinin

The goal of this study was to determine the role of the synthesis and release of nitric oxide in modulating alterations in microvascular permeability of the hamster cheek pouch in response to adenosine 5-diphosphate and bradykinin. We used intravital fluorescent microscopy to examine the permeability of the hamster cheek pouch to agonists before and following application of enzymatic inhibitors of nitric oxide, N^G-monomethyl-l-arginine (l-NMMA; 0.01, 0.1, and 1.0 M) and N^w-nitro-l-arginine methyl ester (l-NAME; 0.01, 0.1, and 1.0 M). Increases in permeability of the hamster cheek pouch were quantitated by the formation of microvascular leaky sites. ADP and bradykinin produced an increase in the number of venular leaky sites, and superfusion ofl-NMMA andl-NAME significantly decreased ADPand bradykinin-induced increases in microvascular permeability. To determine the specificity of nitric oxide blockade on microvascular permeability, we examined changes in permeability in response to adenosine, and examined the effects ofd-NMMA on microvascular permeability. Adenosine-induced increases in permeability were not altered by treatment withl-NMMA, andd-NMMA did not inhibit ADP-induced increases in microvascular permeability. Thus, these findings suggest that production of nitric oxide, in response to application of ADP and bradykinin, has a role in modulating macromolecular permeability of the hamster cheek pouch in vivo.     

Tension development and calcium sensitivity in skinned muscle fibres of the frog

Calcium activated isometric tension development was measured in single skinned muscle fibres of the ileofibularis muscle of the frog. The experiments were carried out at 5°C, pH=6.9, 1 mM free Mg²⁺ and an ionic strength of 160 mM. A Hill curve was fitted to the isometrically developed tension at different Ca²⁺ concentrations by means of a non-linear least mean square approximation. At a sarcomere length of 2.15 m, the Ca²⁺ concentration for half maximum tension (K) was 1.6 M. This Ca²⁺ concentration decreased with increasing sarcomere length; at 2.7 m, K was 1.1 M and at 3.1 m, K was 0.9 M. Therefore, Ca sensitivity is increased at larger sarcomere lengths. Consequently, the optimal sarcomere length for tension development shifted to larger values when the Ca²⁺ concentration was lowered. Osmotic compression of the fibre at 2.15 m by means of 5% Dextran also caused an increase in Ca sensitivity (K was 1.0 M). At 2.7 m, addition of 5% Dextran hardly affected the Ca sensitivity. The possible role of the interfilament spacing in the explanation of these results discussed.     

Mechanical properties of mammalian single smooth muscle cells I. A low cost large range microforce transducer

Summary A transducer has been developed for measuring the minute forces generated during isometric contractions (1.0–10.0N) of single smooth muscle cells from the pig urinary bladder and the human uterus. In addition to its high sensitivity, resolution and stability (100 mVN^–1, <0.1N and <2.0N h^–1), the transducer features a very wide range (100–140N) with good linearity, enabling measurement of contractions as well as passive force-length characteristics within one uninterrupted measurement session. Since the transducer features an independent and interchangeable force to displacement conversion system, different force ranges can be realized by inserting force conversion systems with different compliances.     

Effect of inhibitors of glycosylation and carbohydrate processing on invasion of malignant mouse MO4 cells in organ culturef

Inhibitors of glycosylation and carbohydrate processing were used to investigate the role of carbohydrates exposed at the cell surface in invasion. Malignant mouse MO₄ cells were confronted with embryonic chick heart in organ culture, an assay shown to be relevant for a number of aspects of invasionin vivo. Tunicamycin (1·0g/ml), 2-deoxy-d-glucose (100mm),-OH-norvaline (1·0mm), and Monensin (0·1g/ml) reversibly inhibited the invasion of MO₄ cells. At these concentrations the drugs also inhibited the growth of MO₄ cells. 1-Deoxynojirimycin (10mm), swainsonine (0·4g/ml), and Marcellomycin (0·1 g/ml) permitted invasion. Marcellomycin also reversibly inhibited the growth of MO₄ cells. These results show that drugs known to interfere with the glycosylation or processing of carbohydrate chains of glycoproteins in different ways have different effects on the invasion of MO₄ cellsin vitro.Part of this work has been presented at the conference on Treatment of Metastasis: Problems and Prospects, held at the Strand Palace Hotel, London, 15–17 October 1984.     

Permeability to Ca2+ of the acetylcholine receptor channel of denervated mouse muscles in the presence of Na+ and of some other cations

Calcium uptake caused by exposure to azelainylcholine and the additional membrane slope conductance caused by the same agonist were compared in partially depolarized mouse soleus muscles denervated for 3–5 days. Ca uptake was estimated from the amount of⁴⁵Ca retained after a 2 min exposure to the tracer (1 min in the presence of azelainylcholine) and a subsequent 17 min period of tracer washout. The amount taken up in the presence of Na⁺ was 0.152 m mole/kg fresh muscle. The uptake was by about 60% higher when Na⁺ was replaced by N-methyl-d-glucamine. For 10 other monovalent cations Ca uptake was less than with Na. Ca uptake was not related to the molecular weight, size or structure of the cation. The slope conductance in the presence of 10 M azelainylcholine was 4 S and it was 21% of that value when Na⁺ was replaced by N-methyl-d-glucamine, i.e. conductance was decreased when Ca uptake was increased. This discrepancy points to a major difference in the way cations such as Ca²⁺ and K⁺ pass the receptor channel.     

Roles of inositol trisphosphate and protein kinase C in the spontaneous outward current modulated by calcium release in rabbit portal vein

We examined the effects of heparin, guanosine nucleotides, protein kinase C (PKC) modulators, such as phorbol 12,13-dibutylate (PDBu) and H-7 on Ca²⁺-dependent K⁺ currents in smooth muscle cells of the rabbit portal vein using the whole-cell patch-clamp technique, to explore the effects of PKC on the oscillatory outward current (I _oo). Neomycin (30 M), an inhibitor of phospholipase C, and intracellular applications of heparin (10 g/ml) and guanosine 5-O-(2-thiodiphosphate) (GDP[S]; 1 mM) partly but consistently inhibited the generation of I _oo, whereas a higher concentration of heparin (100 g/ml) transiently enhanced then suppressed the generation of I _oo. Inhibition of I _oo generation by heparin was more powerful at the holding potential of + 20 mV than at –20 mV. Inositol 1,4,5-trisphosphate (InsP ₃; 30 M) continuously generated I _oo at holding potentials more positive than –60 mV. Noradrenaline (10 M) and caffeine (3–20 mM) transiently augmented, then reduced the generation of I _oo. Heparin (10 g/ml) completely inhibited responses induced by InsP ₃ and noradrenaline, but not those induced by caffeine. Intracellular application of guanosine 5-triphosphate (GTP; 200 M) or low concentrations of guanosine 5-O-(3-thiotriphosphate) (GTP[S]; 3 M) continuously augmented the generation of I _oo. High concentrations of GTP[S] (10 M) transiently augmented, then inhibited I _oo. Neither GTP[S] nor noradrenaline induced the transient augmentation or the subsequent inhibition of I _oo when applied in the presence of GDP[S] (1 mM), neomycin (30 M) or heparin (10 g/ml). PDBu (0.1 M) reduced the generation of I _oo but failed to produce an outward current following application of caffeine (3–5 mM). This action of PDBu was inhibited by pretreatment with H-7 (20 M). In the presence of H-7, GTP[S] continuously enhanced the generation of I _oo. The suppression of the generation of I _oo during application of noradrenaline (10 M) was reduced by pretreatment with H-7. Thus both InsP₃ and protein kinase C contribute to the generation of I _oo in smooth muscle cells of the rabbit portal vein and heparin is not a specific InsP ₃ antagonist on the InsP ₃-induced Ca²⁺-release channel (PIRC). InsP ₃ opens PIRC and protein kinase C may deplete the stored Ca²⁺ by either inhibiting the reuptake of Ca²⁺ or by enhancement of the releasing actions of InsP ₃.