Generation of nonadrenergic inhibitory junction potentials in the smooth muscle of the guinea-pig gastrointestinal tract after replacing potassium ions with cesium ions

Nonadrenergic inhibitory junction potentials (IJPs), evoked by intramural nerve stimulation, were studied in the smooth muscle of the guinea-pig stomach, cecum, and colon, using a modified sucrose-gap technique. After incubating smooth muscle preparations for 4–9 h in potassium-free Krebs solution, IJPs were abolished, but reappeared when cesium ions (6 mM) were added to the Krebs solution. Under these conditions, in the majority of cases the amplitude of the IJP was half as small, and the latency and duration were significantly longer, than in normal conditions; also ATP, but not adenosine, caused hyperpolarization of the smooth muscle membrane. The amplitude of the IJP depended on the extracellular concentration of cesium. In all types of preparation, in cesium-containing Krebs solution, apamin usually abolished the IJP and responses to ATP. These results are consonant with the purinergic hypothesis of inhibitory neuromuscular transmission. The generation of the IJP in these potassium-free conditions depends on cesium ions, which pass through the small-conductance apamin-sensitive, calcium-dependent potassium channels.A. A. Bogomoletz Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 22, No. 5, pp. 634–641, September–October, 1990.     

EMG muscle scanning: Comparison to attached surface electrodes

Electromyographic (EMG) muscle scanning measures brief samples of integrated muscle action potentials from individual muscles using a hand-held scanner with post-style electrodes. This scanning technique is widely used by biofeedback practitioners to quickly assess muscle activity in the diagnosis of musculoskeletal disorders. In an effort to compare muscle scanning with the established technique using attached surface electrodes, ten healthy subjects (25–35 years old) were scanned using 2-second sampling at five bilateral muscle sites while simultaneously monitoring the same sites with surface electrodes. This was repeated using 10-second scanning samplings. Pearson's product-moment correlations between scanning for 2 seconds and prolonged surface recording at all sites were 0.54–0.89. Scanning for 10 seconds improved the correlations to 0.68–0.91. EMG scanning for 2 seconds compares favorably with attached surface electrode recording. Comparisons are further improved by 10-second scans.     

Hydrochlorothiazide action on the apical Cl−, Ca2+ and K+ conductances in rabbit gallbladder epithelium. Presence of an apamin-sensitive,Ca2+-activated K+ conductance

In the rabbit gallbladder epithelium, hydrochlorothiazide (HCTZ) was shown to inhibit the transepithelial NaCl transport and the apical Na⁺-Cl^– symport, to depolarize the apical membrane potential and to enhance the cell-to-lumen Cl^– backflux (radiochemically measured), this increase being SITS-sensitive. To better investigate the causes of the depolarization and the Cl^– backflux increase, cells were punctured with conventional microelectrodes on the luminal side (incubation in bicarbonate-free saline at 27°C) and the apical membrane potential (V _m) was studied either with prolonged single impalements or with a set of short multiple impalements. The maximal depolarization was of 3–4 mV and was reached with 2.5 × 10^–4 m HCTZ. It was significantly enhanced by reducing luminal Cl^– concentration to 30 mm; it was abolished by SCN^–, furosemide, SITS; it was insensitive to DPC. SITS converted the depolarization into a hyperpolarization of about 4 mV; this latter was apamin, nifedipine and verapamil sensitive. It was concluded that HCTZ concomitantly opens apical Cl^– and (probably) Ca²⁺ conductances and, indirectly, a Ca²⁺-sensitive, apamin inhibitable K⁺ conductance: since the intracellular Cl^– activity is maintained above the value predicted at the electrochemical equilibrium, the opening of the apical Cl^– conductance depolarizes V _mand enhances Cl^– backflux. In the presence of apamin or verapamil, to avoid the hyperpolarizing effects due to HCTZ, the depolarization elicited by this drug was fully developed (7–10 mV) and proved to be Ca²⁺ insensitive. On this basis and measuring the transepithelial resistance and the apical/basolateral resistance ratio, the Cl^– conductance opened by HCTZ has been estimated and the Cl^– backflux increase calculated: it proved to be in the order of that observed radiochemically. The importance of this Cl^– leak to the lumen in the overall inhibition of the transepithelial NaCl transport by HCTZ has been evaluated.This research was supported by Ministero dell'Università e della Ricerca Scientifica e Tecnologica, Rome, Italy. We are very grateful to prof. G. Meyer and dr. G. Bottà for helpful discussion and criticism.     

Regulation of Energy Transduction and Electron Transfer in Cytochrome c Oxidase by Adenine Nucleotides

Cytochrome c oxidase from bovine heart contains seven high-affinity binding sites for ATP or ADP and three additional only for ADP. One binding site for ATP or ADP, located at the matrix-oriented domain of the heart-type subunit VIaH, increases the H⁺/e^– stoichiometry of the enzyme from heart or skeletal muscle from 0.5 to 1.0 when bound ATP is exchanged by ADP. Two further binding sites for ATP or ADP, located at the cytosolic and the matrix domain of subunit IV, increases the K _M for Cytochrome c and inhibit the respiratory activity at high ATP/ADP ratios, respectively. We propose that thermogenesis in mammals is related to subunit VIaL of cytochrome c oxidase with a H⁺/e^– stoichiometry of 0.5 compared to 1.0 in the enzyme from bacteria or ectotherm animals. This hypothesis is supported by the lack of subunit VIa isoforms in cytochrome c oxidase from fish.     

Extra-organ sources of parasympathetic innervation of the small intestine in the treitz ligament region

Neuronal sites in the dorsal motor nucleus of the vagus nerves sending out axons to the duodeno-jejunal junction and the upper jejunum were located during acute experiments on cats using retrograde horseradish peroxidase transport techniques. A large proportion of these cells are found in the ventrolateral region of the nucleus at distances of 1.0–2.7 mm from the obex. Morphological aspects of nodose ganglion neurons responsible for afferent intestinal innervation were also investigated, showing the largest numbers of such cells to be located in the medial and caudal portions of the ganglion.I. P. Pavlov Institute of Physiology, Russian Academy of Sciences, St. Petersburg. Translated from Neirofiziologiya, Vol. 24, No. 4, pp. 423–430, July–August, 1992.     

Enrichment of Triadic and Terminal Cisternae Vesicles from Rabbit Skeletal Muscle

An enriched triad and terminal cisternae preparation was achieved from skeletal muscle through alterations of the differential centrifugation and muscle homogenization protocols. Both yield and specific activity (pmoles of radioligand binding per mg protein) were optimized for ³H-PN200-l10 (transverse tubule marker) and ³H-ryanodine (terminal cisternae marker) binding sites. By pelleting crude microsomes between 2,000 an 12,000 × g without any rehomogenizations, we improved both the yield and specific activity of transverse tubule and terminal cisternae markers in crude microsomes by approximately 4-fold to 1000–3000 pmoles binding sites (starting material: approximately 400 grams wet weight fast twitch skeletal muscle), with 10–15 pmoles/mg. Rehomogenization of the 1,000 × g pellet, which is typically discarded, allowed recovery of an additional 5000 pmoles PN200-110 binding sites and an additional 8000 pmoles ryanodine binding sites. Crude microsomes from the rehomogenized 1,000 × g pellets typically displayed specific activities of 20–25 pmoles binding/mg for both ³H-PN200-110 and ³H-ryanodine. Separation of crude microsomes on a sucrose gradient increased specific activity up to a maximum of 50 pmoles/mg in a specific fraction, a five- to ten-fold increase over standard triadic or terminal cisternae preparations. The mean specific activity for enriched triads was 30–40 pmoles/mg for both PN200-110 and ryanodine in pooled fractions, while pooled fractions of enriched terminal cisternae displayed low ³H-PN200-110 binding (3–5 pmoles/mg) and high ³H-ryanodine-specific activity (30–40 pmoles/mg).     

Transmitter release at the active zone of motor nerve endings

Transmitter release sites were located in the motor nerve ending of the frog cutaneous-pectoris muscle using three extracellular electrodes. Transmitter release sites were found to be grouped in a direction cutting across the nerve ending and reflecting transmitter release and active release zones (AZ). Measurements from these groups showed that most transmitter release takes place at the center of the AZ, declining towards the periphery and to either side of this zone. All AZ were found to take place in spontaneous release with a low extracellular concentration of calcium ions present, compared with only a proportion in evoked release. Advantages of the triple as opposed to the dual micro-electrode technique are analyzed. It was found that transmitter release in spatially isolated AZ at the nerve ending leads to a polymodal distribution pattern of the amplitude of uniquantal signals during extracellular recording. The part played by AZ in transmitter release is discussed.S. V. Kurashov Medical Institute, Ministry of Health of the RSFSR, Moscow. V. I. Ul'yanov-Lenin State University, Kazan'. Translated from Neirofiziologiya, Vol. 22, No. 3, pp. 318–327, May–June, 1990.     

Buteo Nesting Ecology: Evaluating Nesting of Swainson’s Hawks in the Northern Great Plains

Swainson’s hawks (Buteo swainsoni) are long-distance migratory raptors that nest primarily in isolated trees located in areas of high grassland density. In recent years, anthropogenic conversion of grassland habitat has raised concerns about the status of the breeding population in the northern Great Plains. In 2013, we initiated a study to investigate the influence of extrinsic factors influencing Swainson’s hawk nesting ecology in north-central South Dakota and south-central North Dakota. Using ground and aerial surveys, we located and monitored nesting Swainson’s hawk pairs: 73 in 2013 and 120 in 2014. We documented 98 successful breeding attempts that fledged 163 chicks; 1.52 and 1.72 fledglings per successful nest in 2013 and 2014, respectively. We used Program MARK to evaluate the influence of land cover on nest survival. The top model, S _{Dist2Farm+%Hay}, indicated that nest survival (fledging at least one chick) decreased as nests were located farther from farm sites and as the percent of hay cover increased within 1200-m of the nest site (34.4%; 95% CI = 27.6%–42.3%). We used logistic regression analysis to evaluate the influence of landscape variables on nest-site selection; Swainson’s hawks selected for nest sites located closer to roads. We suggest that tree belts associated with farm sites, whether occupied or not, provide critical breeding sites for Swainson’s hawks. Additionally, poor breeding success may be related to the late migratory behavior of this species which requires them to occupy marginal habitat due to other raptors occupying the most suitable habitat prior to Swainson’s hawks arriving to the breeding grounds.     

Computer assisted quantitative densitometric analysis of 125I-apamin binding sites in the central nervous system

The binding sites for 125I-monoiododerivative of apamin in the central nervous system of rat, guinea-pig, chicken and frog were analysed and compared by computer assisted quantitative densitometric autoradiography on X-ray film. The highest level of binding sites in the rat and guinea-pig brain was found in the limbic-olfactory system and in the substantia gelatinosa of the spinal cord. In the chicken brain apamin binds preferentially to the tectum opticum and nuclei isthmi. In the frog brain no specific apamin binding sites were found. The role of presented topography for apamin binding sites is discussed in relation to neurotoxic properties of apamin.     

Rat muscle acylphosphatase: Purification, amino sequence, and immunological characterization

Acylphosphatase was purified from rat skeletal muscle essentially by gel filtration and high-performance ion-exchange chromatography. The complete amino acid sequence was reconstructed by using the sequence data obtained from tryptic, peptic, andS. aureus V8 protease peptides. The protein consists of 96 amino acid residues and is acetylated at the NH₂-terminus. The immunological cross-reactivity of acylphosphatase from rat and horse skeletal muscle was examined by ELISA. The reaction with rabbit antiserum revealed the presence of at least five antigenic sites on rat enzyme, two of which are common to horse muscle enzyme. Anti-rat antibodies also recognize the peptide that corresponds to the initial part of the molecule, which varies greatly from equine enzyme. Two completely new antigenic sites are herein described: the first can be considered the main antigenic site and is located within positions 21–36, the second is in the COOH-terminal part of the molecule. A mixture of immunoreactive peptides gives strong antibody-antigen reaction inhibition (94%).     

Calcium-activated potassium channels in liver cells

Activation of certain membrane receptors increases the concentration of Ca²⁺ in the cytosol of hepatocytes. Since in most species these cells possess a P_K(Ca) mechanism, the outcome is a rise in P_K. This can be blocked by quinine, apamin and certain neuromuscular blocking agents. The binding of labelled apamin to hepatocytes has been studied under physiological conditions, and the relationship between the binding sites and K⁺ channels is discussed. The physiological role of the P_K(Ca) mechanism in hepatocytes is unclear, though it is largely responsible for ‘adrenaline hyperkalaemia’.     

Architecture of the media of the arterial vessels in the dog brain: A scanning electron-microscopic study

Summary The architecture of the media of arterial vessels in dog brain was investigated using scanning electron microscopy. The arrangement and shape of the circularly-oriented smooth muscle cells varied with vessel diameter: The arteries (>100 m in diameter) had 4–10 layers of spindle-shaped smooth muscle cells; the muscular arterioles (30–100 m), 2–3 layers of spindle-shaped smooth muscle cells; the terminal arterioles (10–30 m), a compact layer of spindle-shaped smooth muscle cells with more dominant nodular or rod-like processes and thin lateral processes; and the precapillary arterioles (5–15 m), a less compact layer of branched smooth muscle cells.Longitudinally-oriented muscles were observed in the medio-adventitial border. The distribution and arrangement of these muscles varied with vessel size: in the large arteries (> 300 m in diameter), at the branching sites only; in the small arteries (100–300 m), at both the branching and non-branching sites; in the muscular arterioles, at both the branching and non-branching sites in a reticular arrangement with some muscle cells having an asteroid appearance; in the terminal aterioles, only asteroid-like muscle cells were found at the branching and non-branching sites.     

Carbon dioxide efflux and pCO2 in soils of threeQuercus ilex montane forests

Soil CO₂ efflux and pCO₂ in the soil atmosphere were measured during one year at three montane sites of Mediterranean sclerophyllous forests in NE Spain. Two sites were located in the upper and lower slopes of a small catchment in the Prades mountains (mean precipitation 550 mm year^–1), and a third site was located on a lower slope in the Montseny mountains (mean precipitation 900 mm year^–1). The three sites were similar in bedrock and vegetation, but differed in soil characteristics and water availability. Seasonal variation of CO₂ efflux and soil pCO₂ were affected by soil temperature and, to a lesser extent, by soil moisture. Annual mean soil CO₂ efflux (considered as soil respiration) was similar at Montseny and at the comparably located site at Prades (83 ± 18 S.E. vs. 75 ± 9 mg CO₂ m^–2 hour^–1 , respectively), and was highest at the Prades upper slope site (122 ± 22 mg C0₂ m^–2 hour^–1 ). Despite those relatively similar CO₂ effluxes, mean soil pCO₂ was much higher at both Prades sites than at Montseny. Soil pCO₂ always increased with depth at Prades while maxima pCO₂ at Montseny were often at 20–30 cm depth. A model based on gas diffusion theory was able to explain why soil pCO₂ was much higher at Prades than at Montseny, and to reproduce the shape of the vertical profile of pCO₂ at the Prades soils. Nevertheless, the model failed to simulate the soil pCO₂ maximum found at 20–30 cm depth at the Montseny site. Model simulations using a time-variable CO₂ production rate suggested that pCO₂ maxima at intermediate depth could be the result of a transient situation instead of an equilibrium one.     

Regulation of postsynaptic alpha-1 adrenoceptors in smooth muscle of the cat nictitating membrane

Findings from radioligand research into smooth muscle in the cat nictitating membrane revealed the presence of specific [³H]prazosin binding sites corresponding to alpha-1 adrenoceptors. After smooth muscle sympathectomy, numbers of alpha-1 adrenoceptors rose without any significant change in their affinity. Incubating previously sympathectomized smooth muscle with noradrenaline led to a decline in the number of alpha-1 adrenoceptors — again without alteration in binding affinity. It was deduced that numbers of alpha-1 adrenoceptors are controlled by the sympathetic nervous system.S. V. Kurashov Medical Institute, Kazan'. Translated from Neirofiziologiya, Vol. 21, No. 6, pp. 735–741, November–December, 1989.     

Expression and motor effects of secretin in small and large intestine of the rat

Immunocytochemistry and in situ hybridization revealed abundant secretin expressing cells on duodenal villi with a gradual decrease throughout the small intestines of the rat. They were absent in pancreas, stomach and colon. Secretin caused relaxation of rat intestinal longitudinal muscle in vitro. Studies on colon revealed that the secretin-evoked response was unaffected by apamin, tetrodotoxin, L-NAME, VIP or PACAP pretreatment; secretin itself caused desensitization. Addition of VIP or PACAP when the secretin-evoked relaxation was maximal evoked a further relaxation suggesting the presence of distinct receptors. Secretin causes relaxation via activation of secretin receptors located on the smooth muscle and not via any of the related VIP/PACAP receptors.     

The Ca2+-dependent slow K+ conductance in cultured rat muscle cells: characterization with apamin.

The interaction of apamin, a bee venom neurotoxin, with rat skeletal muscle cell membranes has been followed using both an electrophysiological and a biochemical approach. Voltage-clamp analyses have shown that apamin, at low concentrations, specifically blocks the Ca2+-dependent slow K+ conductance in rat myotubes and myosacs . A specific binding site for apamin in rat muscle cell membranes has been characterized with the use of a highly radiolabelled apamin derivative [( 125I]apamin). The dissociation constant for the apamin-receptor complex is 36-60 pM and the maximal binding capacity is 3.5 fmol/mg of protein. [125I]Apamin binding to rat muscle membranes is displaced by quinine and quinidine with K0.5 values of 110 microM and 200 microM, respectively.     

Synaptic activation of thoracic interneurons by reticulospinal fibers of the lateral funiculus

Synaptic responses of different functional groups of interneurons in segments T10 and T11 to stimulation of the ipsilateral and contralateral medullary reticular formation were investigated in anesthetized cats with only the ipsilateral lateral funiculus remaining intact. Activation of reticulospinal fibers of the lateral funiculus with conduction velocities of 30–100 m/sec was shown to induce short-latency and, in particular, monosynptic EPSPs in all types of cells tested: in interneurons excited by group Ia muscle afferents, in cells activated only by high-threshold cutaneous and muscle afferents (afferents of the flexor reflex), in cells activated mainly by descending systems, and, to a lesser degree, in neurons connected with low-threshold cutaneous afferents. These cell populations are located mainly in the central and lateral parts of Rexed's lamina VII. Most neurons in laminae I–V of the dorsal horn, except six cells located in the superficial layers of the dorsal horn, received no reticulofugal influences. The functional organization of connections of the lateral reticulospinal tract with spinal neurons is discussed and compared with the analogous organization of the medial reticulospinal tract, and also of the "lateral" (cortico- and rubrospinal) descending systems.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 10, No. 2, pp. 150–161, March–April, 1978.     

Spatial analysis of limb bud myogenesis: A proximodistal gradient of muscle colony-forming cells in chick embryo leg buds

The regional distribution of myogenic cells in developing chick leg buds has been investigated using an in vitro clonal assay. Leg buds were embedded in gelatin and sectioned at intervals of 100–300 μm utilizing a vibratome, and cells dissected from prospective myogenic areas were analyzed for their ability to form colonies containing multinucleated myotubes. The results show that muscle colony-forming (MCF) cells from stage 23 ( to 4-day incubation) are exclusively of the early morphological type, and are found in the proximal two-thirds of the bud. Late-type MCF cells are first obtained from the proximal sections of stage 24–25 (4- to day) buds; in succeeding stages (26–29), late MCF cells supercede the early MCF cell type in the proximal regions, and extend into progressively more distal sections in a graded fashion. Results from sequential sections suggest that early and late MCF cells are located within the same muscle groups. The proportion of late MCF cells continues to increase throughout this period, until by stage 31 (7 days) only the most distal myogenic regions (the toe muscle regions) have an appreciable proportion of early MCF cells. Clonal plating efficiencies increase throughout the period of analysis, and by stage 31 precisely dissected myogenic regions yield plating efficiencies as high as 36% with greater than 95% of these colonies differentiating as muscle.     

Sequence analysis of a PM2-DNA anti-Z-IgG-binding region

An anti-Z-antibody-binding region between PM2-DNA map units 0.05 and 0.18, containing approx. 25% of the bound PM2 antibody molecules (1,2) has been sequenced. Analysis of this PM2 DNA sequence from map units 0.00 to 0.175 demonstrates that alternating purine/pyrimidine tracts capable of adopting the left-handed conformation are present within this antibody-binding region. Longer (GC)n-rich tracts are clustered together and comprise seven alternating purine/pyrimidine-rich areas (48%–84%) ranging from 19 to 142 nucleotides in length. The DNA located between these alternating purine/pyrimidine-rich areas exhibit a low level (0%–19%) of this sequence arrangement. There is a very strong correlation between the alternating purine/pyrimidine-rich areas and the anti-Z-DNA-IgG-binding sites. Nucleotides 1461–1583 of the PM2-DNA genome encode the bacteriophage capsid protein IV. One of the PM2 left-handed sites is located within this protein-coding sequence; a B-to-Z transition within this site may be involved in protein-IV gene regulation in vivo.     

Periphyton nutrient limitation and other potential growth-controlling factors in Lake Okeechobee,U.S.A.

Periphyton nutrient limitation was assessed in Lake Okeechobee, a large, shallow, eutrophic lake in the southeastern U.S.A. Nutrient assays were performed to determine if the same nutrients that limit phytoplankton also limit periphyton growth in the lake. Nutrient diffusing clay substrates containing agar spiked with nitrogen, phosphorus, or both, along with nutrient-free controls, were incubated at four sites in the lake. Three sites were located in a pelagic–littoral interface (ecotone) and one site was located in the interior littoral region. Incubations lasted for 20–26 days, and were repeated on a quarterly basis between 1996 and 1997, to incorporate seasonal variability into the experimental design. The physical and chemical conditions at each site also were measured. Periphyton biomass (chlorophyll a and ash-free dry mass) was highest at the littoral and northern ecotone sites. At the littoral site, nitrogen limited biomass in four of five incubations, although the largest biomass differences between the treatments and controls (3 g cm^–2 as chl) were probably not ecologically significant. Periphyton biomass at the western and southern ecotone sites was low compared to the other two sites. Increases in water column depth and associated declines in light penetration strongly correlated with periphyton growth and suggested that they may have limited growth most often at all three ecotone sites. Nitrogen also was found to limit periphyton growth approximately 20% of the time at the ecotone sites and phosphorus was found to limit growth once at the west site.