Calcium dependence of phenylephrine-, endothelin-, and potassium chloride-stimulated atrial natriuretic factor secretion from long term primary neonatal rat atrial cardiocytes

Since calcium is involved in both excitation-secretion and excitation-contraction coupling, it was of interest to evaluate its involvement in atrial natriuretic factor (ANF) release from atrial cardiocytes. In medium containing physiological levels of calcium (1.4 mM), the secretion of ANF from primary atrial cells was stimulated from 3- to 6-fold by a variety of agents including KCl, phenylephrine, and endothelium (ET). However, in medium containing 2 nM calcium, KCl was incapable of increasing ANF secretion above basal levels, while the stimulatory effects of phenylephrine and ET were only partially diminished. Nifedipine or verapamil could mimic the effects of the 2 nM calcium medium on KCl-, phenylephrine-, and ET-stimulated ANF secretion. Kinetic studies indicated that during the initial 5 min of ET-stimulated secretion the cells exhibited little requirement for extracellular calcium; however, the requirement was more apparent during the sustained secretion observed between 10 min and 2 h of secretagogue exposure. Additionally, the stimulation of ANF secretion by ET increased to a maximum of about 15-fold over basal by 10-min after ET application; subsequent to this time there was an apparent functional desensitization wherein the rate of secretion decreased by approximately 3-4-fold and remained at this level for the duration of secretagogue exposure up to 2 h. All forms of stimulated secretion could be inhibited through ionomycin-mediated depletion of intracellular calcium pools. Taken together, these results indicate that atrial cardiocytes require both extracellular and intracellular calcium to support maximal rates of stimulated ANF secretion, and that intracellular calcium pools may be used during the early phase of secretion, while the extracellular source of calcium may be important for the sustained phase of secretion.     

Miniature endplate currents of muscle fibers from rat diaphragm during galantamine-induced acetylcholinesterase inhibition

Miniature endplate currents (MEPC) were recorded in muscle fibers of rat diaphragm using voltage clamp technique during acetylcholinesterase (AChE) inhibition induced by various concentrations of galantamine. Their amplitude and time course began to increase at a galantamine concentration of 3.16·10^–8 g/ml. Increased concentrations of galantamine produced a greater effect. Maximum amplitude and time course were reached at a concentration of 10^–6 g/ml. The input resistance of muscle fibers increased under the effects of galantamine. In all cases MEPC fell exponentially. At a concentration of 10^–5 g/ml galantamine produced a curarelike effect; amplitude and time course of decay increased to a lesser extent than at a concentration of 10^–6 and the decay in MEPC became biphasic. Following washout of galantamine (10^–5 g/ml) the time course of MEPC first rose, then fell, returning to the initial level in 3 h, and decay again became exponential. Changes in MEPC parameters under the effects of different concentrations of galantamine and washout were closely correlated. A positive correlation was found between the time course of decay and MEPC amplitude both in the presence and absence of AChE inhibition. It is postulated that the functional importance of synaptic AChE in repressing the postsynaptic action of acetylcholine is limited and that parameters of postsynaptic response may therefore be used to evaluate its action.A. A. Ukhtomskii Institute of Physiology, A. A. Zhdanov State University, Leningrad. Translated from Neirofiziologiya, Vol. 17, No. 5, pp. 607–614, September–October, 1985.     

Automated monitoring of cell concentration and viability using an image analysis system

In order to automate measurements of cell concentration and viability in a suspended animal cell culture, we have developed anin situ microscopic image analysis system with an effective cell recognition algorithm. With a small amount of sample, this system can measure the cell density rapidly and aseptically. In addition, it can measure a cell size histogram including cell debris small particle distribution. These small particles have been found to be related to the viability of the mouse-mouse hybridoma STK1 cell line. By using cell debris small particle density as an indicator of cell viability, the developed system provides non-destructive viability monitoring without trypan blue staining.     

Purification and properties of thiamine-binding proteins from sesame seed

Three thiamine-binding proteins of 17-19 kDa (STBP-I, II, and III) were purified from sesame seed (Sesamum indicum L.). Each of the proteins was composed of two subunits of equal molecular mass and each subunit consisted of a large polypeptide and a small polypeptide linked by a disulfide bond(s). They were rich in glutamic acid (or glutamine) and arginine. Their binding activities were optimal at neutral pH. They bound specifically free thiamine but not thiamine phosphates. STBP-I had higher affinity for thiamine than STBP-II or STBP-III. STBP-II and STBP-III bound one molecule of thiamine per molecule, and STBP-I bound 0.5 molecule. The amino acid composition and structure of the STPBs were similar to those of 2S storage proteins.     

A sulfotransferase model: Covalent participation of a macrocyclic oxime in the hydrolysis of 2,4-dinitrophenyl sulfate

The liberation of 2,4-dinitrophenolate ion from 2,4-dinitrophenyl sulfate (DNPS) in aqueous organic solvent with 0.1 N sodium hydroxide was accelerated upon addition of an equimolar amount of Oxime-I (10-hydroxy-11-hydroxyimino[20]-paracyclophane) to the sulfate ester. Oxime-I was found to undergo covalent participation at the oxime group to afford oxime O-sulfonate. The rate acceleration with Oxime-I was larger than that with β-CD (cycloheptaamylose). The catalytic efficiency of Oxime-I has been ascribed primarily to the tighter inclusion of the substrate ester into the more hydrophobic Oxime-I cavity provided by the effective apolar paracyclophane skeleton, as well as to the greater nucleophilicity of the oxime group than of the hydroxyl group in β-CD. Consequently, Oxime-I may be considered as a conventional model for arylsulfatases and sulfotransferases, providing the effective binding process for the substrate.     

Studies of ANF processing and secretion using a primary cardiocyte culture model.

In contrast to most other endocrine peptides ANF is stored in the heart as part of a larger prohormone, often called pro-ANF, yet is found in the circulation as a 28 amino acid peptide, called ANF. It has been shown that the conversion of the 126 amino acid pro-ANF to ANF occurs in the heart. This paper summarizes studies from our laboratory that have used a primary neonatal rat heart cell culture system to investigate the location and mechanism of this relatively unusual processing event. We have found that in culture the maintenance of the cells in a glucocorticoid-containing serum-free medium is required to observe processing as occurs in vivo. The cells contain the prohormone while ANF accumulates in the medium. Various experiments with protease inhibitors, pulse-chase biosynthetic labeling, incubation of cells with ANF-related peptides, and enrichment of cultures for myocytes have resulted in our conclusion that the processing of pro-ANF takes place most likely within the cardiac myocyte just prior to, but in concert with secretion. We have expanded on the use of this processing-competent atrial myocyte culture system to investigate mechanisms of stimulated ANF secretion. It has been shown that the activation of several phospholipase C-coupled receptors (e.g., alpha 1-adrenergic and endothelin receptors) produces a robust release of ANF, but only in cultures that have been maintained under appropriate conditions. Further, it is apparent that the phenylephrine- or endothelin-mediated release of ANF depends in part on influx of extracellular calcium (Ca2+o), while the remaining component of stimulated release may depend on mobilization of intracellular calcium. It also appears that these agonists produce an initial phase of stimulated release, occurring within the first 5 min of agonist exposure, independent of Ca2+o, and a sustained phase that persists as long as the agonists remain on the cells, and depends on the presence of Ca2+o and thus calcium influx. Taken together our studies indicate that the hormonal environment may be an important factor directing the development of differentiated endocrine functions by atrial myocytes and may be involved in the regulation of ANF expression, biosynthesis, and secretion.     

Real-Time Monitoring of Slow-Wave Sleep by Electroencephalogram Variance

Based on statistical variance as an index of electroencephalogram (EEG) parameters, we monitored slow-wave sleep in both humans and rats in real time and on-line with a widely used personal computer. This EEG variance method may be a useful tool to carry out biological rhythm research, including sleep studies.     

Observations on the effect of visual and olfactory ablation on the swimming behavior of migrating adult chum salmon,Oncorhynchus keta

The behavior of chum slamon,Oncorhynchus keta, was studied using ultrasonic telemetry in the waters off the Okhotsk coast of Hokkaido from 1979 to 1981. Thirty-six adult fish were outfitted with a 50 KHz ultrasonic transmitter provided with either a depth sensor, depth/illumination sensors, or depth/ temperature sensors. Twenty-one of the experimental fish were used as controls and left intact. Of the remaining fish, six had their sight destroyed, seven had their olfactory nerves severed or their nares filled with wax, one had both senses destroyed, and one had a sham operation, but had only a shallow transverse cut made in the skin over the olfactory nerves. Control fish and the fish with the sham operation initially swam in a horizontal zigzag pattern while fish with an obliterated sense swam in a similar pattern but to a reduced extent. The horizontal and vertical speeds of the intact fish were faster than those of the fish with a destroyed sense. Amplitude of vertical movements of the intact fish stretched from surface to bottom, while fish with destroyed vision tended towards midwater. Fish without an olfactory sense tended to be at the surface or near the bottom. Regular vertical movement would be effective in refreshing olfactory epithelia which might become acclimated when exposed to one odor. The vertical movements can be found in the horizontal zigzag movements in coastal and near shore migrations, as well as in the stream phase of homing. The zigzag movements, both horizontal and vertical, allow the fish to sense the water masses and locate the correct tributary.     

Different seasonal feeding activity of diverse herbivores in two temperate forests

The aim of the present study was to understand the effects of abiotic conditions on seasonal feeding activity of diverse herbivores on the same oak tree species in two different forests. We tracked changes in herbivore feeding activities on an oak tree species (Quercus serrata) in two localities: a low elevation small hillock forest patch (Muan, MN) and a middle elevation mountain forest patch (Mt. Jirisan, JR). A total of five sites were selected in each of two forest localities. Data for leaf expansion, leaf chemical qualities, leaf damage ratio, and numbers of lepidopteran caterpillars were collected during spring (May) and summer (July to August), 2012. Leaf expansion rate was higher at the low hillock forest than the mid‐mountain forest from spring to summer. Nitrogen and carbon content decreased seasonally at both localities. Lepidopteran larval diversity was high in the mid‐mountain forest, and two‐way ANOVA showed that species richness of lepidopteran larvae was significantly affected by the interaction between season and locality. Leaf damage by all herbivores was higher in the low hillock forest than the mid‐mountain forest in spring, but was higher in the mid‐mountain forest in summer. Relative proportion of general herbivores increased from spring to summer in the mid‐mountain forest, but not in the low hillock forest. Canonical Correspondence Analysis (CCA) ordination showed that altitude‐ and season‐related variables were significant species and environment interaction factors. Our data indicate that locality and temperature disproportionally affected the feeding activities of diverse herbivores in two different temperate forests.