Stimulus-Induced Accumulation of Inositol Tetrakis-, Pentakis-, and Hexakisphosphates in N1E-115 Neuroblastoma Cells

When [3H]inositol-prelabelled N1E-115 cells were stimulated with carbamylcholine (CCh) (100 microM), high K+ (60 mM), and prostaglandin E1 (PGE1) (10 microM), a transient increase in [3H]inositol pentakisphosphate (InsP5) accumulation was observed. The accumulation reached its maximum level at 15 s and had declined to the basal level at 2 min. CCh, high K+, and PGE1 also caused accumulations of [3H]inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], [3H]inositol 1,3,4,6-tetrakisphosphate [Ins(1,3,4,6)P4], and [3H]inositol hexakisphosphate (InsP6). Muscarine and CCh induced accumulations of [3H]Ins(1,4,5)P3, [3H]-Ins(1,3,4,6)P4, [3H]InsP5, and [3H]InsP6 with a similar potency and exerted these maximal effects at 100 microM, whereas nicotine failed to do so at 1 mM. With a slower time course, CCh, high K+, and PGE1 caused accumulations of [3H]-inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] and [3H]inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. In an N1E-115 cell homogenate, [3H]Ins(1,4,5)P3, [3H]Ins(1,3,4,5)P4, and [3H]Ins(1,3,4)P3 were converted to [3H]InsP5 through [3H]-Ins(1,3,4,6)P4. The above results indicate that Ins(1,3,4,6)P4, InsP5, and InsP6 are rapidly formed by several kinds of stimulants in N1E-115 cells.     

Rat heart organ culture for the study of trophic substances involved in the maintenance of normal sensitivity to norepinephrine: possible involvement of cAMP and search for other factors

In organ culture conditions, in the absence of in vivo factors, the newborn rat right atria acquire a high sensitivity to agonists similar to that seen before sympathetic innervation and after denervation. In the present study, we examined the effects of various extracts and substances on the development of supersensitivity to norepinephrine (NE) to obtain information on the in vivo factors that regulate myocardial sensitivity. Addition of rat serum, right atrial extract, superior cervical ganglionic extract, vas deferens extract, carbachol, insulin, cortisone, thyroxin, and neuropeptide Y in the culture medium did not prevent the development of supersensitivity. Addition of NE completely inhibited the development of supersensitivity. This effect of NE was blocked by sotalol but not by phentolamine. Addition of calcitonin gene related peptide, forskolin, and 8-bromo-cAMP partially inhibited the development of supersensitivity. These results are consistent with the view that NE released from sympathetic nerve terminals in the newborn atria maintains myocardial sensitivity at normal level by acting on beta-adrenergic receptors, and that the effect may be partially mediated by a rise in intracellular cAMP concentration.     

Synthesis and insulin-mimetic activities of metal complexes with 3-hydroxypyridine-2-carboxylic acid

Metal complexes of 3-hydroxypyridine-2-carboxylic acid (H(2)hpic), [Co(Hhpic)(2)(H(2)O)(2)] (1), [Fe(Hhpic)(2)(H(2)O)(2)] (2), [Zn(Hhpic)(2)(H(2)O)(2)] (3), [Mn(Hhpic)(2)(H(2)O)(2)] (4), and [Cu(Hhpic)(2)] (5) have been synthesized and characterized by mass spectrometry, elemental analysis, magnetic susceptibility, infrared, electronic absorption and electron paramagnetic resonance (EPR) spectroscopies. The solid-state structure of 1 has been established by X-ray crystallography. The EPR spectra of 4 and 5 displayed six and four-line hyperfine splitting patterns, respectively, due to coupling of the unpaired electron with the (55)Mn (I=5/2) nucleus and the (63)Cu (I=3/2) nucleus. In the EPR spectrum of 5, an additional five-line super-hyperfine splitting pattern was observed at 77 K, caused by additional interaction of the unpaired electron with ligand nitrogen atoms (I=1), indicating that the structure of 5 was retained in dimethyl sulfoxide solution. The insulin-mimetic activity of these complexes was evaluated by means of in vitro measurements of the inhibition of free fatty acid (FFA) release from epinephrine-treated, isolated rat adipocytes. Complex 5 was found to exhibit the most potent insulin-mimetic activity among the complexes examined in this study.     

Change in content of sugars and free amino acids in potato tubers under short-term storage at low temperature and the effect on acrylamide level after frying

Changes in the sugar and amino acid contents of potato tubers during short-term storage and the effect on the acrylamide level in chips after frying were investigated. The acrylamide content in chips began to increase after 3 days of storage at 2 degrees C in response to the increase of glucose and fructose contents in the tubers. There was strong correlation between the reducing sugar content and acrylamide level, R(2)=0.873 for fructose and R(2)=0.836 for glucose. The sucrose content had less correlation with the acrylamide content because of its decrease after 4 weeks of storage at 2 degrees C, while the reducing sugar in potato tubers and the acrylamide in chips continued to increase. The contents of the four amino acids, i.e., asparatic acid, asparagine, glutamic acid and glutamine, showed no significant correlation with the acrylamide level. These results suggest that the content of reducing sugars in potato tubers determined the degree of acrylamide formation in chips. The chip color, as evaluated by L* (lightness), was correlated well with the acrylamide content.     

Cardioprotection without cardiosuppression by SEA0400, a novel inhibitor of Na+-Ca2+ exchanger, during ischemia and reperfusion in guinea-pig myocardium

The effect of SEA0400, a novel Na+-Ca2+ exchanger inhibitor, on mechanical and electrophysiological parameters of coronary-perfused guinea-pig right ventricular tissue preparation was examined during no-flow ischemia and reperfusion. Contractile force and action potential duration were decreased during no-flow ischemia, while the resting tension was increased. Upon reperfusion, transient arrhythmias were observed and contractile force returned to less than 50% of preischemic values. SEA0400 (1 microM) had no effect on the decline in contractile force during the no-flow ischemia, but abolished the rise in resting tension. SEA0400 significantly improved the recovery of contractile force after reperfusion to about 80% of the preischemic value. SEA0400 had no effect on the action potential under normal conditions and during ischemia, but significantly improved the recovery of action potential duration after reperfusion. Enhancement of the recovery of contractile force during reperfusion by SEA0400 was also observed when the drug was applied only before and during the ischemic period and when the drug was applied only during reperfusion. The present results indicate that inhibition of Na+-Ca2+ exchanger either during ischemia or during reperfusion exerts cardioprotective effects and enhances the recovery of myocardial contractile function.     

Canonical Wnt signaling and its antagonist regulate anterior-posterior axis polarization by guiding cell migration in mouse visceral endoderm

The mouse embryonic axis is initially formed with a proximal-distal orientation followed by subsequent conversion to a prospective anterior-posterior (A-P) polarity with directional migration of visceral endoderm cells. Importantly, Otx2, a homeobox gene, is essential to this developmental process. However, the genetic regulatory mechanism governing axis conversion is poorly understood. Here, defective axis conversion due to Otx2 deficiency can be rescued by expression of Dkk1, a Wnt antagonist, or following removal of one copy of the beta-catenin gene. Misexpression of a canonical Wnt ligand can also inhibit correct A-P axis rotation. Moreover, asymmetrical distribution of beta-catenin localization is impaired in the Otx2-deficient and Wnt-misexpressing visceral endoderm. Concurrently, canonical Wnt and Dkk1 function as repulsive and attractive guidance cues, respectively, in the migration of visceral endoderm cells. We propose that Wnt/beta-catenin signaling mediates A-P axis polarization by guiding cell migration toward the prospective anterior in the pregastrula mouse embryo.     

Motor discoordination in mutant mice lacking junctophilin type 3

Junctional complexes between the plasma membrane and endoplasmic reticulum (ER), often called "subsurface cisternae" or "peripheral coupling," are shared by excitable cells. These junctional membranes probably provide structural foundation for functional crosstalk between cell-surface and intracellular ionic channels. Our current studies have indicated that junctophilins (JPs) take part in the formation of junctional membrane complexes by spanning the ER membrane and interacting with the plasma membrane. Of the JP subtypes defined, JP type 3 (JP-3) is specifically expressed in neurons in the brain. It has been currently reported that triplet repeat expansions in the JP-3 gene are associated with Huntington's disease-like symptoms including motor disorder in human. To survey the physiological role of JP-3, we generated the knockout mice. The JP-3-knockout mice grew and reproduced normally, and we did not observe any morphological abnormality in the mutant brain. In the behavioral study, the mutant mice showed impaired performance specifically in balance/motor coordination tasks. Although obvious defects could not be observed in excitatory transmission among cerebellar neurons from the mutant mice, the data indicate that JP-3 plays an active role in certain neurons involved in motor coordination.     

Spontaneous recovery of the anoxic diminution in the action potential plateau of the late embryonic and neonatal chicken hearts

The action potential plateau of the embryonic chick hearts at the latest stage of development (19- to 20-day-old) was depressed initially by anoxia but recovered spontaneously under the sustained anoxic condition. A similar spontaneous recovery of the action potential plateau was observed in hearts from neonatal chicks. Propranolol (3 × 10^?6M) did not affect the spontaneous recovery of the action potential plateau, excluding the possible involvement of the release of endogenous catecholamines. Since tissue adenosine triphosphate content continued to decrease during anoxia in these hearts, it is unlikely that the anoxia-resistant metabolic pathway appeared or was enhanced to supply the energy for the rebuilding of the plateau. In a Ca²⁺-free solution the action potential plateau rapidly disappeared and did not recover spontaneously during anoxia; at this time, however, the addition of Ca²⁺ (2 mM) prolonged the plateau. An inward calcium current may play an important role in spontaneous recovery of the action potential plateau during anoxia. The duration of the action potential decreased after hatching. From these results it is suggested that the mechanism underlying the action potential plateau may somehow differ in the hearts at the stages around the time of hatching.