Dopamine and adenosine 3',5'-monophosphate responses of single mammalian sympathetic neurons.

Acetylcholine (ACh), dopamine, and dibutyryl-adenosine 3',5'-monophosphate (dbcAMP) were applied iontophoretically to the rabbit superior cervical ganglion cells from triple-barreled micropipettes, and the response was recorded by intracellular techniques. All ganglion cells tested responded to the depolarizing action of ACh, whereas less than half of the cells that responded to ACh were hyperpolarized by dopamine. This effect was blocked by low concentrations of haloperidol. None of the cells examined responded to dbcAMP applied by iontophoresis. Hence, the present result is not consistent with the concept that a cyclic AMP mechanism underlies the hyperpolarizing effect of dopamine.

Unique alignment and texture of biological apatite crystallites in typical calcified tissues analyzed by microbeam X-ray diffractometer system.

Preferential orientation of biological apatite (Ap) crystallites in typical calcified tissues of rabbit ulna, rabbit skull, and monkey dentulous mandible was investigated using a microbeam X-ray diffractometer, with a beam spot of 100 microm in diameter, to clarify relationship between the Ap orientation and mechanical function. Preferential alignment of the c-axis of the biological Ap was evaluated by the relative intensity between (002) and (310) diffraction peaks. Preferential alignment of biological Ap in each calcified tissue varied depending on the shape and stress condition in vivo; that is, the c-axes of biological Ap in the rabbit ulna and the rabbit skull bone were preferentially observed as a one-dimensional orientation along the longitudinal axis and a two-dimensional orientation along the surface, respectively. Precise analysis of the preferential alignment along the skull surface showed an elliptical distribution of the c-axis of biological Ap elongating along the suture inside the skull surface of both lamina exterior and interior. The c-axis of biological Ap in a monkey dentulous mandible basically aligned along the mesiodistal direction in the flat bone, but this alignment changed along the normal direction to the flat bone surface parallel to the biting direction near the tooth, due to the force of mastication. It was concluded that the microscale measurement of biological Ap texture is one of the useful new methods for evaluating mechanical function and stress distribution in vivo in calcified tissues.

Effects of anion exchange resin as phosphate binder on serum phosphate and iPTH levels in normal rats.

In order to investigate the characteristics of anion exchange resins that may safely and effectively bind dietary phosphate in digestive tract, phosphate binding experiments were carried out in vitro and in vivo with normal rats by comparing anion exchange resins, PAA-B (which has the same chemical structure as Sevelamer HCl) and Dowe 1x8, with CaCO3. In in vitro phosphate binding experiments, PAA-B bound 32.3% less phosphate than CaCO3 at pH 7. In the rat dietary phosphorus excretion experiments, PAA-B, Dowex 1x8, and CaCO3 increased fecal phosphorus excretion by 62.7, 32.3, and 84.0%, respectively. Famotidine significantly reduced the phosphate binding of CaCO3. When phosphate solution was orally administered, PAA-B depressed serum phosphorus augmentations immediately after administration and thereafter effectively depressed serum iPTH. This suggests that anion exchange resins with most primary and secondary amino type anion exchange groups, have bright prospects in the treatment of hyperphosphatemia.

Characterizations of critical processes in liquid-liquid phase separation of the elastomeric protein-water system: microscopic observations and light scattering measurements.

Biological self-assembly process of tropoelastin in an extracellular space, viewed as a key step of the elastogenesis, can be mimicked by the temperature-dependent coacervation of the elastin-related polypeptide-water system. Early and late stages of the phase separation behavior of the bovine neck ligamental alpha-elastin-water system were examined respectively by the laser light scattering photometry and phase contrast microscopy. Changes in the hydrodynamic size of molecular assemblies and visible microcoacervate droplet size were traced as a function of the concentration of alpha-elastin and temperature. Near the critical point, alpha-elastin concentration of 0.11 mg/mL and temperature of 21.5 degrees C, the phase separation was initiated after fast increase of the hydrodynamic size of primary aggregates as scattering particles and followed by the appearance of larger microcoacervate droplets with a broad size distribution. Whereas in the off-critical region, slow decrease of the hydrodynamic size of primary particles induced phase separation with smaller droplets of a narrow size distribution. Observation of the phase separation processes in the alpha-elastin-water system with metal chlorides and hydrophobic synthetic model polypeptide-water system indicated that the fast and slow molecular assembly processes were based on the fundamental hydrophobic interactions and involvements of electrostatic interactions between charged amino acid residues, respectively.

Effects of metal cations on coacervation of alpha-elastin.

The quasi-elastic light scattering studies were carried out to investigate effects of metal cations such as Ca2+ and Na+ on the early stage of coacervation process of alpha-elastin, a chemical fragmentation product originated from the biological elastomeric protein elastin, in aqueous solutions. In particular, our attention was focused on changes of two types of dynamical behaviors found in the earlier work, which are a remarkable increase and a monotonous decrease in the hydrodynamic radius R of molecules with temperature for critical and off-critical concentrations of alpha-elastin, respectively. For the critical alpha-elastin concentration, an addition of Ca2+ was found to exert little effects on the steep temperature profile of R observed in the absence of Ca2+. On the other hand, an addition of a slight amount of Na+ resulted in a monotonous decrease in R, but its further addition restored a remarkable increase in R similar to the critical behaviors in the salt-free system. In the case of off-critical sample, the addition of either Ca2+ or Na+ above a certain concentration induced a change in R from a monotonous decrease to a remarkable increase. For both critical and off-critical concentrations of alpha-elastin, Ca2+ and Na+ brought about an elevation and a lowering of the temperature at which the sample started to be turbid, respectively.

Postsynaptic facilitatory effects of theophylline on amphibian neuromyal transmission.

This study concerns the effects of theophylline on nerve-muscle transmission of the frog; it was of particular interest to evaluate the facilitatory actions of theophylline at the postsynaptic sites. At concentrations of up to 5 mM, theophylline exerted negligible effects on the end-plate resting potential or on the passive membrane characteristics. The major effects of theophylline (0.5--5.0 mM) were exerted on the end-plate potentials (EPPs), miniature EPPs, acetylcholine (ACh) potentials, and on the end-plate current. The amplitude of these parameters was markedly increased; furthermore, the half-decay time of the EPP and, particularly, of the end-plate current were markedly affected. On the other hand, the time course of the ACh potentials was not significantly affected by theophylline. Spontaneous and evoked release of ACh were not affected by theophylline (0.5--5 mM). Altogether, these results indicate that, in amphibia, the neuromyal facilitation induced by theophylline is mainly due to its postsynaptic actions. Furthermore, some of these data as well as results of others indicate that these effects of theophylline are not due to its anticholinesterase properties. It is suggested that theophylline may act directly on the cholinergic receptor or ionic conductance modulator and that it may stabilize the ACh-receptor complex.

A kinetic analysis of the facilitatory action of adrenaline.

The 22Na+-efflux from skeletal muscle cells of frog (Rana nigromaculata) was measured in Ringer solutions containing different concentrations of K+ (0.1 to 30 mM). The effects of adrenaline (30 microM) and ouabain (10 microM) on the 22Na+-efflux were investigated for the purpose to clarify the mechanism of the facilitatory effect of adrenaline on Na+ - K+ pump. The rate coefficient for the ouabain-sensitive 22 Na+-efflux increases with increasing extracellular K+ concentrations and adrenaline potently facilitates these rate coefficients. On the basis of Michaelis-Menten type kinetics assumed for the reaction between pump site and extracellular K+, it is concluded that adrenaline decreases the dissociation constant (Km), and increases the maximum Na+-efflux.

Beta-adrenergic modulation of the Na+-K+ pump in frog skeletal muscles.

Adrenaline markedly increased the ouabain-sensitive 22Na+-efflux by stimulating the Na+-K+ pump in frog skeletal muscle. The facilitatory effects of adrenaline had the following properties. The effects of adrenaline on the ouabain-sensitive Na+-efflux were observed at concentrations greater than 0.1 microM and the magnitude increased with concentration up to 10 microM. At a concentration of 30 microM, adrenaline markedly augmented the ouabain-sensitive Na+-efflux, but other biogenic amines were less effective (noradrenaline and dopamine) or ineffective (histamine and serotonin). The increase of Na+-efflux induced by 1 microM adrenaline was blocked by 3 microM propranolol, but not by 3 microM phenoxybenzamine. The properties of the facilitatory action of adrenaline on the ouabain-sensitive Na+-efflux suggest that beta-adrenoceptors have an important role in modulating the Na+-K+ pump activity in the skeletal muscle membrane. The protein complex localized in excitable membranes, namely the Na+-K+ ATPase-beta-adrenoceptor complex, may be the functional unit which operates the membrane machinery driving the Na+-K+ pump.

Alpha-elastin coacervate as a protein liquid membrane: effect of pH on transmembrane potential responses.

A protein liquid membrane composed of coacervated alpha-elastin, a chemical fragmentation product of the biological elastic fiber protein, functioned as an amphoteric liquid ion-exchange membrane. Ionic permselectivities of the alpha-elastin coacervate membrane to a series of metal chlorides were investigated for the concentration-cell systems by the ordinary electrochemical measurements. Effects of pH on the transmembrane potential responses for NaCl, CaCl2, and MgCl2 systems were examined. Only in the Ca(2+)-containing system did potential responses stay at constant levels against the pH changes, whereas in the other systems, increasing pH caused potential changes, indicating an improvement of cationic permselectivity across the alpha-elastin coacervate membrane. It was suggested that the characteristic Ca2+ transport mechanisms across the alpha-elastin coacervate membrane are related in some way to the polypeptide backbone interactions specific and selective to Ca2+ ions.

Characteristic interaction of Ca2+ ions with elastin coacervate: ion transport study across coacervate layers of alpha-elastin and elastin model polypeptide, (Val-Pro-Gly-Val-Gly)n.

Ion transport characteristics across a macrocoacervate layer membrane composed of aqueous elastin model polypeptides with a specific repeating pentapeptide sequence, H-(Val-Pro-Gly-Val-Gly)n-Val-OMe (n > or = 40), were investigated. Transmembrane potential responses for NaCl. MgCl2, and CaCl2 concentration-cell systems were measured and examined systematically by comparing with those across a coacervate membrane composed of bovine neck ligamental alpha-elastin. In the case of the NaCl and MgCl2 systems, potential responses across these protein liquid membranes were different noticeably from each other depending upon the molecular structure with and without charged peptide side chains, whereas in the CaCl2 systems the transmembrane potential responses across the noncharged polypentapeptide coacervate membrane were comparable with those across the alpha-elastin coacervate membrane carrying both the positively and negatively charged amino acid residues as an amphoteric ion-exchange membrane. These results indicated that mechanisms of major Ca2+ ion transport are based on the specific and selective interactions with electrically neutral sites of elastin, such as the polypentapeptide backbone chain.

pH-induced coacervation in complexes of bovine serum albumin and cationic polyelectrolytes.

Turbidity and light scattering measurements, along with phase contrast microscopy, were used to follow the processes leading to coacervation when aqueous solutions of bovine serum albumin (BSA) and poly-(dimethyldiallylammonium chloride) (PDADMAC) were brought from pH = 4 to 10. The state of macromolecular assembly of complexes formed between BSA and PDADMAC prior to and during the pH-induced coacervation could be characterized by specific pH values at which recognizable transitions took place. In addition to the two characteristic pH values (pHcrit and pH phi) previously identified through turbidimetry, other transitions were explicitly established. On the basis of the pH-induced evolution of scattering intensity measurements, we concluded that the formation of soluble primary protein-polymer complexes is initiated at pHcrit and proceeds until "pH'crit". A subsequent increase in scattering intensity at "pHpre" may arise from the assembly of quasi-neutralized primary complexes as their net positive charge decreases with increase in pH. Subsequently, a maximum in scattering intensity at pH phi is observed coincident with the appearance of turbidity and also corresponding to the first microscopic observation of coacervate droplets. The temperature independence of pHcrit and pH phi suggests that hydrophobic contributions are negligible for the initial BSA-PDADMAC interactions and the subsequent coacervation process. The pH dependence of scattering intensity profiles allowed the identification of two other transitions beyond pH phi. Spherical microcoacervate droplets first observed around pH phi subsequently displayed morphological changes at "pHmorph", followed by the transformation to solid or flocculant substances at pHprecip.