Sensitivity to dihydropyridines, ω-conotoxin and noradrenaline reveals multiple high-voltage-activated Ca2+ channels in rat insulinoma and human pancreatic β-cells

High-voltage-activated (HVA) Ba²⁺ currents of rat insulinoma (RINm5F) and human pancreatic -cells were tested for their sensitivity to dihydropyridines (DHPs), -conotoxin (-CgTx) and noradrenaline. In RINm5F cells, block of HVA currents by nimodipine, nitrendipine and nifedipine was voltage- and dose-dependent (apparent K _D<37 nM) and largely incomplete even at saturating doses of DHPs (mean 53%, at 10 M and 0 mV). Analysis of slow tail currents in Bay K 8644-treated cells indicated the existence of Bay K 8644-insensitive channels that turned on at slightly more positive voltages and deactivated more quickly than Bay K 8644-modified channels. DHP Ca²⁺ agonists and antagonists in human -cells had similar features to RINm5F cells except that DHP block was more pronounced (76%, at 10 M and 0 mV) and Bay K 8644 action was more effective, suggesting a higher density of L-type Ca²⁺ channels in these cells. In RINm5F cells, but not in human -cells, DHP-resistant currents were sensitive to -CgTx. The toxin depressed 10–20% of the DHP-resistant currents sparing a residual current (25–35%) with similar voltage-dependent characteristics and Ca²⁺/Ba²⁺ permeability. Noradrenaline (10 M) exhibited different actions on the various HVA current components: (1) it prolonged the activation kinetics of -CgTx-sensitive currents, (2) it depressed by about 20% the size of DHP-sensitive currents, and (3) it had little or no effects on the residual DHP- and -CgTx-resistant current although intracellularly applied guanosine 5-O-(3-thiotriphosphate) (GTP--S) prolonged its activation time course. The first action was clearly voltage-dependent and most evident in RINm5F cells that displayed neuronal-like processes. The second was observed more frequently, was voltage-independent and fully blocked by saturating doses of nifedipine (10 M). Both actions were prevented by intracellular perfusion with guanosine 5-O-(2-thiodiphosphate) (GDP--S). Our data suggest that beside a majority of L-type channels, RINm5F and human pancreatic -cells may express a variable fraction of DHP-insensitive channels that may be involved in the control of insulin secretion during -cell activity.     

Ca-dependent K channels in smooth muscle cells permeabilized by β-escin recorded using the cell-attached patch-clamp technique

Using the cell-attached patch-clamp technique, the activity of single, Ca-dependent K channels was recorded in single smooth muscle cells permeabilized by -escin. The conductance and the relationship between the open probability of the channels and pCa recorded in permeabilized cells were very similar to those obtained in excised inside-out patches. At pCa 7, application of 30 M acetylcholine (ACh) or 0.1 M substance P (SP) together with 1 mM guanosine 5-trisphosphate to permeabilized cells elicited transient bursts of channel openings similar to those which occur in intact cells. Transient activation was also observed when 2–30 M inositol trisphosphate (IP₃) was applied to permeabilized cells. This single channel activity was inhibited by pretreatment with low-molecular-weight heparin at 50–100 g/ml. Channel activity at pCa 7.0 was greatly enhanced by 200 M cyclic adenosine monophosphate. These results provide direct evidence that single Ca-dependent K channel activity is regulated by the transmitters ACh and SP, as well as a second messenger, IP₃, via the release of intracellular Ca from intracellular sites which are blocked by heparin. This novel approach is valuable in elucidating second messenger mechanisms involved in the regulation of single channel activity by transmitters and autacoids, since permeabilization by -escin preserves the entire system of receptor-operated signal transduction and allows intracellular application of second messengers at fixed concentrations.     

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.     

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.     

Effects of ryanodine on skinned skeletal muscle fibers of the rabbit

The mechanism(s) of ryanodine-induced contracture of skeletal muscle were studied in skinned fibers from soleus (SL) and adductor magnus (AM) (slow- and fast-twitch skeletal muscles) of rabbits. Pieces of SL or AM were homogenized (sarcolemma disrupted). Single fibers were dissected from the homogenate and mounted on photodiode force transducers. At concentrations 1–50 M, ryanodine slightly but significantly increased the submaximal Ca²⁺-activated tension development of the contractile proteins in skinned fibers of AM but not of SL. Ryanodine in uptake phase or release phase increased caffeine-induced tension transients in the SR of both muscle types; however, no dose-response relation was found. Ryanodine 1 M decreased, however, the second control tension transients in a dose-dependent manner. The depression was nearly irreversible and activity-dependent. The concentrations of ryanodine that inhibited the second control tension transients by 50% were 10 M and 5 M for SL and AM, respectively, following ryanodine administration in the release phase, and 100 M and 30 M, respectively, for these preparations after the drug was present in the uptake phase. The quantity of calcium released from the SR by Triton X-100 and caffeine in the second control tension transient was unchanged by ryanodine at all concentrations tested when compared with that of the absence of ryanodine. The present findings suggest that the ability of ryanodine to increase immediate calcium release from the SR, and in AM but not SL, to increase the sensitivity of the contractile proteins to Ca²⁺ underlies the contracture caused by this agent in intact skeletal muscles. The delayed decreased Ca²⁺ efflux by caffeine, as evidenced by depression of tension transient with no change in the calcium content may be responsible for the decreased twitch tension caused by this agent.     

Pyramidal tract of the cat: axon size and morphology

Summary The purpose of this work was to determine the number and morphology of pyramidal tract (PT) axons in the cat, using electron microscopy, modern methods of fixation, and computer-assisted morphometric analysis. Sections taken at the level of the medullary pyramids in three animals were fixed and magnified up to 10,000 x to produce photomicrographs. Morphological data were entered into computer files for analysis by tracing axon perimeters on micrographs mounted on a digitizer tablet. The number of axons per PT averaged 415,000, of which 88% were myelinated and 12% were unmyelinated. 90% of the myelinated axons fell in the diameter range 0.5–4.5 m. Axons larger than 9 m diameter accounted for 1% of the total; the largest were 20–23 m. Myelinated axon mean diameter was 1.98 m; because of the skewed distribution, with many small axons and a few very large axons, median diameter was 1.60 m. Size distribution was relatively uniform throughout the PT cross section, with all sizes represented in all regions. However, the more medial regions had a higher proportion of small fibers than the more lateral regions: mean medial diameter was 1.85 m while mean lateral diameter was 2.09 m. Myelin sheath thickness averaged 7.9% of fiber diameter for axons up to 11 m, but was constant at 0.9 m for larger fibers. Myelinated fibers were distorted from the circular shape in cross section, with a mean circularity index (or form factor) of 0.85, which implies that the fibers could swell about 15% without rupture of the cell membrane. Unmyelinated fibers averaged 0.18 m diameter (range 0.05–0.6 m); the largest unmyelinated axons were larger than the smallest myelinated axons. It is concluded that previous work greatly underestimated the number of axons in the cat pyramidal tract.     

Effect of lysolecithin on blood vessel reactivity and of some ethers and esters of glycerophosphocholine and phosphocholine,respectively, on aggregation of rat platelets

Infusion of lysolecithin (LPC; e.g. 88 g/ml for 0.5–1.0 min) did not significantly impair the vasopressor action of norepinephrine (NE), prostaglandin F₂ (PGF₂) and extract of posterior pituitary (EPP) in the isolated perfused hind legs of rats. In other words, vascular smooth muscle behaves differently from the smooth muscle of the guinea-pig small intestine, since, in the latter, contractions evoked by acetylcholine, prostaglandins etc., are inhibited by LPC. Triton X 100 which, by comparison, was used as a detergent effective on the guinea-pig small intestine, depressed the vasopressor effect of NE, PGF₂ and EPP.LPC, at low concentrations (40 mol/l), potentiated (15% max.) ADP-induced platelet aggregation (PA) in rat PRP but, at high concentrations, inhibited PA (IC₅₀=390 mol/l). 2-Hexadecylglycerophosphocholine and its short-chain 1-alkyl ethers, which are structurally related to platelet-activating factor, as well as some long-chain alkanol phosphocholine esters, were somewhat more active than LPC. Dipalmitoyllecithin (4–700 mol/l) was without any effect.     

Modulation of lymphocyte activating factor activity (interleukin 1 like activity) and acute phase proteins in pertussis-induced air pouch inflammation by muramyl dipeptide

We have studied the effect of the macrophage activator, muramyl dipeptide (MDP) on immune inflammation induced in the rat six day subcutaneous air pouch. Treated animals received either 100 g or 200 g MDP at the time of challenge and twenty four hours before exudate harvest. Using the thymocyte co-mitogenic assay for lymphocyte activating factor (LAF), 100 g MDP enhanced LAFactivity whereas 200 g caused inhibition. Increased dilution of 200 g exudate in this assay removed this inhibition. Similarly, at the lower dose, MDP caused enhanced production of the acute phase protein alpha 1 glycoprotein, whereas the higher dose had no effect. The present study suggests that macrophage activity can be manipulatedin vivo to produce LAF and naturally occurring inhibitors of LAF. These studies indicate that the stimulation of LAF inhibitors by MDP may be a potential theerapeutic action.     

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.     

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.     

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.     

Differential regulation of human eosinophil,macrophage, and neutrophil functions by the allergic mediator release inhibitor CI-959

The cell activation inhibitor CI-959 (5-methoxy-3-(1-methyl-ethoxy)-N-1H-tetrazol-5-ylbenzo[b]thiophene-2-carboxamide, monosodium salt) was evaluated for its effect on the activation of human eosinophils, macrophages, and neutrophils by the phagocytic stimulus serum-opsonized zymosan (SOZ). CI-959 inhibited the respiratory burst of eosinophils and neutrophils, measured as the generation of superoxide anion, with IC₅₀s of 9.6 and 14.5 M, respectively. In contrast, 100 M CI-959 inhibited superoxide anion generation by human macrophages by only 22.7%. The compound exhibited a different inhibition profile for lysosomal enzyme release from these cells. At 100 M, CI-959 inhibited the release of eosinophil peroxidase and macrophageN-acetyl--d-glucosaminidase by only 19.5 and 25.6%, respectively. In contrast, CI-959 inhibited the release of the neutrophil primary granule enzyme myeloperoxidase with an IC₅₀ of 7.5 M, while inhibiting release of lysozyme from secondary granules by only 11.4% at 100 M. These results demonstrate that oxygen radical generation and lysosomal enzyme release by human leukocyte populations are differentially regulated by CI-959.     

Effects of vector cutting on its recombination with the chromosomal immunoglobulin gene in hybridoma cells

We have analyzed the effects of linearizing vector DNA on the frequency and pathway of its recombination with the homologous chromosomal gene. The pSV2neo vector bearing a 4.3-kb fragment encoding the mouse immunoglobulin heavy chain constant (C) region was cut either at sites within the C segment or outside C and then transferred to hybridoma cells bearing a mutant gene. The frequency of recombinant cells producing normal was then measured. For most cut sites, whether in regions of homology or of nonhomology, linearization of the transferred DNA enhanced the recombination frequency between the vector and chromosomal genes. When the vector was either uncut or cut at SacI in the region of homology, G418-resistant m⁺ recombinants were found to have integrated the vector by a single reciprocal homologous crossover; the enzyme site (SacI) used for cutting was present in the recombinants. By contrast, when the vector had been linearized at Pvul or SfiI in the region of nonhomology, vector integration involved nonhomologous crossovers, either between transferred DNA molecules or between transferred and chromosomal DNA, and the vector cut sites were absent in these recombinants. Some recombinants were found to have an unaltered as well as recombinant gene, suggesting that the nonhomologous recombination process might have involved sister chromatids.     

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.     

A diffractometer using a lateral effect photodiode for the rapid determination of sarcomere length changes in cross-striated muscle

A simple method is devised to record rapid sarcomere length changes of muscle fibres using a lateral effect diode. In the standard position the diffractometer records length changes between 1.65 and 3.8 m, the output being linear 1 V/m with a frequency response of –3 dB at 1.2 kHz. The absolute error is<0.05 m between 1.65 and 2.80 m and <0.1 m between 2.81 and 3.30 m. The resolution of length changes is<0.005 m over the whole range. By varying the detector position the length range can be extended to either side, and spatial resolution can be improved at the expense of length range.     

The peritrophic membrane ofIxodes ricinus

A peritrophic membrane was found in all three stages ofIxodes ricinus at no later than 18 h after their placement on rabbits. It was found to remain intact until at least 11, 30 and 10 days after repletion in larvae, nymphs and females, respectively. In blood-feedingI. ricinus, the peritrophic membrane is an uneven single layer with a thickness of about 0.03–0.48 m in larvae, 0.03–0.79 m in nymphs and 0.04–0.93 m in females. It covers the whole surface of the midgut epithelium at a distance of about 0.2–0.8 m. After repletion, the peritrophic membrane becomes thicker and thicker and more and more winding and simultaneously becomes multi-layered mainly in its arched parts. The distance between the peritrophic membrane and the midgut epithelium increases considerably and in the arched parts can reach as much as about 13 and 16 m in metamorphosing larvae and nymphs, respectively and 25 m in ovipositing females.Dedicated to Prof. J. Eckert (Zürich) on the occasion of his 60th birthday     

Fibre types inLimulus telson muscles: morphology and histochemistry

Summary Using a variety of techniques, we have demonstrated the presence of at least two fibre types inLimulus median telson levator muscle. By light and electron microscopy, large (21 56 m² mean cross-sectional area) fibres have A-bands of 4.1 m, one-half I bands of 2.15 m and Z lines 0.5 m in width. Few mitochondria are found in these fibres, which comprise 54% of those present in a given microscope field and which occupy 82% of the total cross-sectional area. Small fibres (484 m² mean cross-sectional area) have A bands of 6.3 m, one-half I bands of 3.1 m and Z lines between 0.5 and 1.0 m in width and are rich in mitochondria. Although small fibres comprise nearly one-half (46%) of the fibres in a field, they occupy only 18% of the total cross-sectional area.Histochemical staining for alkaline-stable myofibrillar ATPase activity and mitochondrial reduced -nicotinamide adenine nucleotide (-NADH) tetrazolium reductase activity confirms the presence of two fibre types. The large fibres react positively for the myofibrillar ATPase activity and negatively for the mitochondrial enzyme activity. The reverse is seen with the small fibres. Some fibres of intermediate size, having intermediate staining characteristics, were also observed. Native gel electrophoresis of both myofibrillar and purified myosin preparations supports the observed differences in myofibrillar ATPase activity in that two myosin isozymes are resolved on pyrophosphate gels. Although the thick filaments isolated from unstimulated small fibres are longer (>6.0 m) than those isolated from unstimulated large fibres (4.26 m), all have a similar appearance with respect to the arrangement of myosin heads on their surfaces, and similar diameters. The implications of the observed heterogeneity of fibre types is discussed with reference to previously reported phenomena inLimulus telson muscle, including changes in length of thick filaments on fibre stimulation and the shape of the length-tension curve obtained from fibre bundles.     

High frequency recombination and the expression of genes cloned on chimeric yeast plasmids: Identification of a fragment of 2-μm circle essential for transformation

Summary We have carried out experiments aimed at explaining the observed variations in transformation frequencies when Saccharomyces cerevisiae or Saccharomyces carlbergensis are transformed with chimeric plasmids that contain one of 4 possible EcoRI fragments of the yeast 2-m circle. These plasmids fall into 2 classes when used to transform 2 different yeast his3 auxotrophs, one (strain LL20) harbours indigenous 2-m circle, and the other (strain YF233) is devoid of this plasmid. Hybrid plasmids containing either the 2.4 mega-dalton (mD) R-form EcoRI fragment (pYF88) or the l.4 mD L-form EcoRI fragment (pYF177) of 2-m circle transform either of the two hosts at a high frequency (50,000 colonies per Mg in LL20 and 10,000 colonies per g in YF233). Hybrid plasmids containing the 1.5 mD R-form EcoRI fragment (pYF87) or the 2.5 mD L-form EcoRI fragment (pYF178) of the 2-m circle transform LL20 at a reduced frequency (6,000–16,000 colonies per g) and YF233 at extremely low frequencies (1–5 colonies per g). All plasmids retrieved from strain YF233 that had been transformed with pYF88 or pYF177 were identical to the original transforming plasmid. Of the plasmids retrieved from strain LL20 that had been transformed with pYF87 and pYF178, approximately half had acquired an extra copy of the 2-m circle. Of the plasmids retrieved from strain LL20 that had been transformed with pYF88 and pYF177, an average of only approximately 13% had acquired an extra copy of 2-m circle. Taken together, these observations indicate that the transformation of yeast by a plasmid lacking the ability to replicate (pYF87 and pYF1780) occurs by the recombinational acquisition of 1 copy of the host 2-m circle, which serves to supply the incoming plasmid with missing essential sequences. A comparison of 2-m circle DNA fragments carried by pYF88 and pYF177 indicates that the region of 2-m circle required for high frequency transformation is a 1.2 mD segment that is common to the 2.4 mD R-form and 1.4 ml) L-form EcoRI fragments. This region extends from the EcoRI cut site adjacent to the PstI site, through to the end of the inverted repeat. However, the inverted repeat sequence alone is not sufficient to bestow high frequency transformation of yeast.     

Morphological studies on equine arteritis virus

Summary Electron microscopy of purified preparations of equine arteritis virus (EAV) revealed enveloped, spherical particles with an average diameter of 55 m. The envelope was found to carry tiny projections on the surface, 3 to 5 m in length. No detailed structure of an internal component could be seen.In sections of EAV infected BHK cells 24 hours after inoculation, viral particles were shown to bud from the cytoplasmic matrix into cisternae of the endoplasmic reticulum, the process starting in the vicinity of the Golgi apparatus. The mature particles had an average diameter of 50 m, with an inner core measuring 25m.This study was supported by grant no. B70-16X-744-05-6068-19195 from the Swedish Medical Research Council.     

A simple method for measurement of inulin in nanoliter samples using glass capillaries

Summary A simple method using glass capillaries instead of microcuvettes for measurement of inulin in nanoliter samples is given. Inulin was determined with anthron reagent (5 or 10 nl samples +3 l anthron reagent). Glass capillary tubes (o.d.=1 mm, i.d.=0.68 mm, length=150 mm) in which the chemical reaction took place during incubation at 56°C were directly introduced into the optical system of a Zeiss spectrophotometer PMQ II with sphere attachment and objective.Extinction was measured vertically to the axis of the capillary. The changes of extinction of 20 different capillaries with the blank at different positions was only 1.13×10^–3. The exactness of measurement in the concentration range of 100 200 400 750 1500 3000 mg-% inulin was for 5nl/3 l: 19.8 11.0 6.7 4.7 3.0 2.2%. 10nl/3 l: 13.0 8.4 5.1 3.9%.This method of measurement may also be applicable for other colorimetric reactions with nanoliter samples.This work was supported by Fonds zur Förderung der wissenschaftlichen Forschung.