Peripheral hypotensive and central hypertensive effects of cimetidine

Rapid intravenous (i.v.) injections of high doses (16–128 mol/kg) of cimetidine induced a short-lasting (5–15 min) hypotension in anaesthetized rats. Diastolic pressure was reduced more than systolic pressure, suggesting vasodilatation. Heart rate was not affected. Diphenhydramine pretreatment (100 mol/kg i.v.) did not antagonize the hypotensive effect of cimetidine. However, in the presence of diphenhydramine, cimetidine induced bradycardia. Intracerebroventricular administration of cimetidine or metiamide (2 mol/rat) increased the blood pressure and heart rate. It is concluded that the hypotension after i.v. cimetidine is mediated by peripheral mechanisms. Since diphenhydramine pretreatment had no antagonistic effect cimetidine-induced hypotension could not be due to indirect H₁-receptor stimulation caused by histamine liberation.     

Analysis of mutations introduced into the chromosomal immunoglobulin μ gene

We have introduced a pSV2neo-derived vector that contains a 2-base-pair (bp) deletion in its immunoglobulin gene constant region into hybridoma cells bearing a single copy of the wild-type chromosomal immunoglobulin gene. Homologous recombination between the transferred mutant C region and the wild-type chromosomal C region is expected to introduce the 2-bp deletion into the chromosomal gene, generating recombinant cells synthesizing noncytolytic IgM. Analysis of the DNA in independent noncytolytic transformants indicates that in one case the gene has the structure expected for correct homologous recombination. Unexpectedly, the remaining transformants, bear chromosomal gene deletions.     

Intrinsic cholinergic excitation in the rat neostriatum: Nicotinic and muscarinic receptors

Summary An in vitro slice technique was employed to study the receptors involved in intrinsic cholinergic excitation in the rat neostriatum. The locally evoked synaptic potentials were suppressed by antinicotinic agents, mecamylamine (10 M), d-tubocurarine (3 M) or hexamethonium (100 M), but not by the antimuscarinic agent atropine (100 M). If the slices were exposed to an acetylcholinesterase (AChE)-inhibitor (paraoxon 1–20 M, physostigmine 0.1–0.5 M), the synaptic potentials were potentiated. The amplitude of the orthodromic population spike increased, and it was further facilitated when the stimulus frequencies were raised from 1–3 Hz to 10–30 Hz. The frequency facilitation following exposure to an AChE-inhibitor was blocked by atropine (1–100 M). Intracellular recording indicated that a slow depolarizing potential caused the frequency potentiation of the orthodromic discharges. Apparently rat neostriatum is similar to cholinergic systems in sympathetic ganglia and spinal Renshaw cells, in that nicotinic receptors mediate fast excitation and muscarinic receptors mediate slow excitation.     

The significance of respiration for thoracic duct flow in relation to other driving forces of lymph flow

Experiments were performed to study the effect of respiratory intrathoracic pressure changes upon thoracic duct lymph propulsion as compared to other forces driving lymph flow in anaesthetized and artificially ventilated dogs. The effect of an open bilateral pneumothorax upon thoracic duct flow and protein composition was determined at rest, with passive limb movement and during saline infusion. The effect of hyperventilation was also tested.Thoracic duct flow was 30 l/min/kg, 45 l/min/kg and 60 l/min/kg at rest, with passive limb movement and saline infusion, respectively. These flows were decreased by opening the pneumothorax by 11 l/min/kg, 12 l/min/kg and 8 l/min/kg, respectively, and returned to the control level after the thorax was closed. The lymph protein concentration and lymph albumin to globulin ratio were not changed significantly. During hyperventilation, lymph flow was increased and showed a retarded decrease after hyperventilation had ceased. Lymph protein composition was not changed significantly by hyperventilation.The data confirm that lymph is propelled in anaesthetized dogs by respiratory intrathoracic pressure changes. The significance of this respiratory pump decreases, when lymph flow is increased by activation of the tissue pump or vis a tergo. Consequently, the respiratory pump may be assumed to play a secondary role in lymph propulsion in the conscious state when the other forces driving lymph flow are more predominant.Presented in part at the 48th meeting of the Deutsche Physiologische Gesellschaft [18]Supported by the Deutsche Forschungsgemeinschaft     

Efficacy of the two-microelectrode voltage clamp technique in crayfish muscle

Crayfish muscle fibres of different dimensions were voltage clamped and white noise current was injected into the fibres at various distances from the voltage clamp current electrode. The clamp current was measured and power spectral densities were calculated. This method revealed the efficacy of the voltage clamp in these fibres. In large fibres (l=1.8–2.0 mm; =100–180m) a space clamp was achieved only for a band width f=40Hz. At a distance of 100m from the clamp electrodes f was 250–500Hz. In fibres of medium size (l=1.0–1.3mm; =60–120m) f was about 80Hz and about 800 Hz at a distance of 100m. In experiments with very small muscle fibres (l=400–600m; =30–50m) f was more than 500Hz. The improvement of the space clamp for the smaller muscle fibres resulted mainly from the reduced total membrane capacity,c _m, of these fibres. The limitations of the space clamp could be derived from the impedance properties of the fibres. The band width of the space clamp correlated with the band width for which the square of the absolute impedance, |Z _p|², of the muscle fibre could be described by a simple RC-model. This correlation was demonstrated in a model circuit.Power density spectra of membrane current fluctuations were measured also. To optimize the resolution of these measurements the contribution of instrumental noise was minimized. The effects of instrumental noise are discussed.This investigation was supported by the Deutsche Forschungs-gemeinschaft     

Benoxaprofen inhibits the adhesion of human monocytes to cultured vascular endothelium

Pretreatment of human monocytes with benoxaprofen for at least 2 h produced a dose-dependent abrogation of their adhesion to monolayers of cultured porcine endothelium with 0.05 g/ml and 50.0 g/ml of the drug inducing a mean 33% and 83% inhibition of adhesion respectively. When the endothelium was treated with the drug there was no modification of monocyte adhesion. In contrast, pretreatment of endothelium with 5.0 and 50.0 g/ml benoxaprofen for at least 6 h, resulted in a mean 35% and 31% inhibition of polymorphonuclear cell (PMN) adhesion in 6/11 experiments. This inhibitory effect was not seen when drug-treated PMNs were added to endothelium. An impairment of monocyte chemotactic migration was only apparent with high concentrations of the drug (50 g/ml). These results suggest that an important anti-inflammatory property of benoxaprofen is the inhibition of monocyte adhesion to vascular endothelium.     

Criteria for testing the susceptibility ofStreptococcus pneumoniae to cefotaxime and its desacetyl metabolite using 1 μg or 30 μg cefotaxime disks

In vitro susceptibility tests were performed with 350 selected strains ofStreptococcus pneumoniae to evaluate disk diffusion tests with 30 g and 1 g cefotaxime disks. Zones were compared to MICs of cefotaxime with and without its desacetyl metabolite. Cefotaxime was two to eight times more active than desacetyl cefotaxime, but the two compounds were additive when combined in vitro. For 30 g disks, zone size breakpoints were 27 mm, 28–30 mm and 31 mm for resistant, intermediate and susceptible, respectively. For 1 g disks, those zone size criteria were reduced to 13 mm, 14–16 mm and 17 mm. The 30 g disk that is currently available for testing other species can be used for testing pneumococci; however, the 1 g disk has some important advantages.     

Scanning electron microscopy of merogonous stages ofEimeria falciformis var.pragensis inMus musculus

Asexual stages ofEimeria falciformis var.pragensis in Swiss-Webster mice were studied by scanning electron microscopy. Sporozoites were present in the cecum and colon 2 h post-inoculation (PI) and measured 11.3×2.1 m (9–13.9×2–2.2 m). Sporozoites penetrated epithelial cells with an extended anterior end and were constricted at the site of entry. Asexual generations were found in the cecum and colon epithelial cells. In meronts found at days 3–6 PI, merozoites matured synchronously, were oriented in the same direction, and were arranged in a helical pattern. Such meronts measured 11.3×6.4 m (8–13.7×5–7.4 m) and contained 8–12 meroxoites, which measured 11.9×1.5 m (7.4–15.7×1.3–1.8 m). Meronts which were present at day 7 PI measured 9.5×8.2 m (9–10.5×7–9.5 m) and contained 20–50 small merozoites which budded asynchronously from a central residuum. At days 3–7 PI, parasitized epithelial cells had shorter and fewer or no microvilli. The lumenal plasmalemma of the host cell was often disrupted or absent in cells containing mature meronts and escaping merozoites. At day 6 PI, phagocytic cells appeared on the epithelial surface, some of which were in contact with merozoites. Small foci of exposed basal lamina were present at day 7 PI in areas where cells had sloughed from the epithelium.     

Length-dependent effects of osmotic compression on skinned rabbit psoas muscle fibers

The goal of this study was to characterize the interrelationship between sarcomere length and interfilament spacing in the control of Ca²⁺ sensitivity in skinned rabbit psoas muscle fibers. Measurements were made at sarcomere lengths 2.0, 2.7 and 3.4 m. At 2.7 m the fiber width was reduced by 17% relative to that at 2.0 m and the pCa₅₀ for force development was increased by 0.3 pCa units. In the presence of 5% Dextran T-500 the fiber width at sarcomere length 2.0 m was also decreased by 17% and the Ca²⁺ sensitivity was increased to the same value as at 2.7 m. In contrast, at sarcomere length 2.7 m the addition of as much as 10% Dextran T-500 had no effect on Ca²⁺ sensitivity. At sarcomere length 3.4 m there was an additional 7% compression and the Ca²⁺ sensitivity was increased slightly (0.1 pCa units) relative to that at 2.7 m. However at 3.4 m the addition of 5% Dextran T-500 caused the Ca²⁺ sensitivity to decrease to the level seen at 2.0 m. Given that the skinning process causes a swelling of the filament lattice it is evident that the relationship between sarcomere length and Ca²⁺ sensitivity observed in skinned fibers may not always be applicable to intact fibers. These data are consistent with measurements of Ca²⁺ in intact fibers which indicate that there might be a decline in Ca²⁺ sensitivity at long sarcomere lengths.     

Effect of epidermal growth factor on non-steroidal anti-inflammatory drug-induced intestinal damage

Epidermal growth factor (EGF, 10 g/kg po, ip, or sc, BID, and 20 g/kg iv) had no protective activity in the indomethacin-induced intestinal lesion model (6 h model). In the ethanol-induced gastric lesion model, EGF (10 g/kg sc) reduced lesions by 52% and reduced gastric acid secretion by 68% (5 g/kg iv). In the 24 h indomethacin-induced intestinal lesion model, pretreatment with EGF (10 g/kg sc, BID; 1 day before and during indomethacin treatment) had no beneficial effects. Therefore, EGF had no protective effects against non-steroidal antiinflammatory drug (NSAID)-induced intestinal lesions at doses that protect against the necrotizing action of ethanol and that inhibit gastric acid secretion in the rat.     

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.     

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.

     

Effects of PAF and PAF antagonists on the shape of venous endothelial cellsin vitro

Three platelet-activating factor (PAF) antagonists were tested for their ability to prevent or reduce PAF-induced shape changes of large vein endothelial cellsin vitro. BN52021 had a significant protective action at concentrations of 1 M and 0.1 M, but at 100 M had a damaging effect of its own. CV3988 (0.1 M and 1 M) and L652, 731 (20 M) did not reduce the responses to PAF, and at higher concentrations (CV3988 10 M and 100 M, L652, 731 100 M) both compounds alone caused significant changes of shape. BN52021 (0.1 M) was also effective against leukotriene (LT) C₄, at 1 M against bradykinin and LTE₄, and at 10 M against LTD₄ and the calcium ionophore A23187. BN52021 (10 M) was ineffective against shape changes induced by histamine, prostaglandin (PG) E₂ and lysophosphatidylcholine (LPC). Neither indomethacin (100 M) nor verapamil (20 M) altered the response to PAF.Using electron spin resonance (ESR) spectrometry it was shown that the damaging effects of LPC and CV3988 may be due partly to their detergent properties. It is suggested that the mechanism by which PAF alters the shape of large vein endothelial cells is primarily receptor mediated.     

Efficient selection of μ m − mutants from μm-expressing myeloma cells by treatment with ricin A-conjugated anti-μ antibody

We have developed an efficient system for obtaining myeloma mutants defective intrans-acting factors required for immunoglobulin (Ig) gene expression. The system consists of a myeloma cell line designed for this purpose and an efficient method for selecting mutants from it. The cell line is X63.653 transfected with the gene, whose tailpiece sequence was replaced with the transmembrane sequence of human EGF receptor to hold on the cell surface and whose CH1 sequence was removed to prevent from being retained in the endoplasmic reticulum. It efficiently and stably expressed chains of IgM on the cell surface ( _m ⁺ ) without light chains. To obtain mutants lacking _m ( _m ^– ) from the _m ⁺ cell line by selectively killing _m ⁺ cells, a method with ricin A-conjugated anti- antibody was more reliable than complement lysis mediated by anti- antibody. Applying the system, we obtained a variety of _m ^– mutants.     

Mechanism of modulation of single sodium channels from skeletal muscle by the β 1-subunit from rat brain

We studied the molecular mechanism of the rat skeletal muscle -subunit (_I) gating kinetics modulation by the brain ₁-subunit by heterologous expression of single sodium channels from _I and ₁ in Xenopus laevis oocytes. Coexpression of ₁ reduced mean open time at –10 mV to 21% when compared to channels expressed by _I alone. Channels formed by _I exerted multiple openings per depolarization, which occurred in bursts, in contrast to the channels formed by the _I/₁ complex that opened in average only once per depolarizing voltage pulse. Macroscopic current decay (mcd), as evidenced by reconstructed open probability vs. time , was greatly accelerated by ₁, closely resembling mcd of sodium currents from native skeletal muscle. Generally was larger for channels expressed from the pure _I subunit.From our single channel data we conclude that ₁ accelerates the inactivation process of the sodium channel complex.     

In vitro activity of the aryl-fluoroquinolones A-56619 and A-56620 and evaluation of disk susceptibility tests

The activity of two new quinolones, A-56619 and A-56620, was compared in vitro to that of norfloxacin and ciprofloxacin against 6,699 bacterial isolates in four separate clinical laboratories. The overall percentage of strains susceptible to designated concentrations were as follows: 99.1% for norfloxacin (MIC4.0 g/ml), 96.1% for ciprofloxacin (MIC1.0 g/ml), 96.8% for A-56620 (MIC 2.0 g/ml) and 96.1% for A-56619 (MIC 4.0 g/ml). For disk diffusion susceptibility tests 10 g A-56619 disks are tentatively recommended with interpretive standards of 18mm for susceptibility and 13mm for resistance; 5 g A-56620 disks may be used with tentative standards of 19mm for susceptibility and 14mm for resistance.     

Expression of surface membrane IgG on pokeweed mitogen-reactive anti-tetanus toxoid antibody-producing cells

Anti-tetanus toxoid antibody-producing cells, differentially expressing surface membrane IgM, were analyzed for the additional expression of surface membrane IgG. ⁺ and ^– cells were rosetted with anti--ox red blood cells and separated by density centrifugation into fractions enriched or depleted or ⁺ cells. These B-cell subsets were assayed for the production of IgM and IgG anti-tetanus toxoid antibody and total IgM and IgG. The results indicated that the majority of anti-tetanus toxoid antibody synthesis in the ^– fraction was by ⁺ cells. In the ⁺ fraction, however, both IgM and IgG anti-tetanus toxoid antibody production was detected in the ^+– and ⁺⁺ fraction. The inclusion of isotype-specific antisera during the first 2 days of culture further established that was expressed on the surface of the majority of the precursors for IgG anti-tetanus antibody productionin vitro. Studies performed to determine the culture requirements of ^– and ⁺ cells revealed that production of IgG anti-tetanus toxoid antibody by both cell subsets was dependent on T cells and pokeweed mitogen. However, some ^– cells could produce IgG in the presence of T cells alone.     

In vitro efficacy and fungicidal activity of voriconazole againstAspergillus andFusarium species

Twenty-nineAspergillus isolates and 25Fusarium isolates underwent in vitro antifungal susceptibility testing by a broth macrodilution procedure adapted from the National Committee for Clinical Laboratory Standards guidelines. The MIC50s of both voriconazole and amphotericin B were 0.5 g/ml and 1 g/ml against species ofAspergillus andFusarium, respectively, while the MIC90s of both agents were 1 and 2 g/ml. Voriconazole was more active in vitro than amphotericin B: the geometric mean MICs of voriconazole and amphotericin B againstAspergillus spp. were 0.36 g/ml and 0.64 g/ml, respectively. Voriconazole also demonstrated fungicidal activity againstAspergillus spp., with 86% (24/29) of isolates exhibiting minimum lethal concentrations of 4 g/ml.     

2 μm plasmid copy number in different yeast strains and repartition of endogenous and 2 μm chimeric plasmids in transformed strains

By using the renaturation kinetics technique we tried to get informations about the maintenance of the 2 m plasmid in yeast cells. For this purpose we determined the 2 m plasmid copy number: in various yeast strains, in a special set of mutants, in cells treated with ethidium bromide and cycloheximide and in different yeast strains obtained by transformation with 2 m chimeric plasmids.According to the strain used the proportion of 2m DNA varied from 1.1% to 3.9%, which corresponds to 24 to 88 2 m molecules per haploid genome. The particular multiresistant mutant, where the frequent loss of oligomycine resistance is correlated with the loss of extractible covalently closed circular DNA, contained 39 2 m copies per haploid genome. In the partial revertant oligomycine sensitive all the 2 m DNA sequences were lost. (Less than 0.1 copy per haploid genome.)Ethidium bromide did not affect the 2 m copy number while cycloheximide induces an increase of 36%.When a strain containing 88 2 m DNA copies per haploid genome is transformed with 2 m chimeric plasmids there is no significative change in the total number of plasmid: 36 copies of endogenous and 44 of chimeric plasmid per haploid genome. When 2 m chimeric plasmids were introduced in our 2 m-less strain despite the stability of the transformants, there is only 8 copies per haploid genome.     

Inhibitory effect of phloretin on histamine release from isolated rat mast cells

The ability of the flavonoid phloretin to inhibit histamine release from rat mast cells varied considerably with the releasing agent investigated. The response to the combination of the ionophore A23187 and the phorbol ester TPA and to suboptimal concentrations of the ionophore (0.5 M) was potently inhibited (IC₅₀ about 5 M), whereas phloretin was less potent against responses to the ionophore (1 M) IC₅₀ of 17 M), to antigen alone and in combination with TPA (IC₅₀ of 30–50 M), to TPA in the absence of calcium (IC₅₀ of 50 M) and to compound 48/80 in the absence and presence of calcium (IC₅₀ of 60–90 M). The inhibition by phloretin at concentrations above 10M was partly counteracted by glucose (5 mM) indicating effects on oxidative metabolism. The flavonoid quercetin was equally potent in inhibiting histamine release induced by antigen, the ionophore at different concentrations and in combination with TPA (IC₅₀ of 20M). Although not conclusive, the results are consistent with an inhibition of protein kinase C by phloretin at concentrations below 10 M. At higher concentrations unspecific actions become apparent and phloretin therefore seems to be of limited utility as a probe for signal-pathways in cell responses.