A neurochemical study of rat brain maldevelopment induced by MAM treatment at different stages of gestation

The regional levels of several cell marker proteins in the brain and the ability of operant discrimination learning on a multiple fixed ratio (FR), fixed interval (FI) schedule were determined in rats with microencephaly induced by prenatal treatment with methylazoxymethanol (MAM), an antimitotic agent, on the 11 th to 13 th days (Group A) or on the 15 th day (Group B) of gestation. The cell marker proteins were determined with a sensitive enzyme immunoassay. Neuron-specific enolase (NSE; gamma gamma-enolase) had a significantly lowered level in the neocortex anterior in Group A. Non-neuronal enolase (NNE; alpha alpha-enolase) was significantly reduced in the superior colliculus, lateral geniculate body and optic nerve, but increased 1.5 fold in the retina in Group A. S-100b protein, a marker of astroglial cells, showed no significant change. As for the learning performance, the Group B animals showed an elevated behavioral activity and made evident discrimination between the FI and FR schedule. But Group A animals had prolonged FR components requiring responses to light on, and their spontaneous activity counts recorded by Automex showed an inhibition of behavior in light environments. These findings suggest a causative role of some developmental abnormality in the central visual system, indicated by the aberrant cell marker levels, in the disturbed learning ability of the Group A animals.     

ATP-Dependent Ca2+ Transport in Tonoplast Vesicles from Apple Fruit

Protoplasts and vacuoles were isolated from immature apple fruit(Malus pumila Mill. cv. Golden Delicious). ATP-stimulated Ca²⁺uptake was identified in both protoplast vesicles and tonoplastvesicles. The apparent K_m for Ca²⁺ of the tonoplast transportsystem was 43.4 µM. The pH optima were 7.2 and 6.7 forCa²⁺ transport by protoplast and tonoplast vesicles, respectively.Ca²⁺ transport in tonoplast vesicles was strongly inhibitedby the calmodulin antagonists fluphenazine and N-(6-aminohexyl)-5-chloro-l-naphthalensulfonamidehydrochloride (W-7), while N-aminohexyl)-l-naphthalensulfonamidehydrochloride (W-5) was relatively ineffective. Addition ofexogenous calmodulin stimulated transport by 35%. Ca²⁺ uptakewas inhibited by vanadate, but not by the ionophores carbonylcyanidem-chlorophenyl hydrazone (CCCP) or valinomycin. The resultsindicate that apple tonoplasts have a Ca²⁺ transport systemthat is driven by the direct hydrolysis of ATP, and may be calmodulindependent. ¹Present address: Morioka Branch, Fruit Tree Research Station,Ministry of Agriculture, Forestry and Fisheries, Shimokuriyagawa,Morioka 020-01, Japan. To whom reprint requests should be addressed. (Received October 18, 1985; Accepted January 29, 1986)     

Bimodal length frequency distribution in 0+aged masu salmon,Oncorhynchus masou,in a natural stream of southern Hokkaido

The frequency distribution of the fork length of 0⁺ aged masu salmon,Oncorhynchus masou, changed from unimodal to bimodal distribution in autumn of the years from 1982 to 1984 in the Mogusa River of southern Hokkaido, Japan. The bimodal distribution consists of two (upper and lower) modal groups. These two groups resulted from a difference in growth rate of 0⁺ aged individuals in autumn. Fish belonging to the upper modal group are assumed to be potential 1⁺ smolts. Whether 0⁺ aged parr transform into smolt or remain as parr in the following spring may be related to the growth rate of fish in the first autumn.     

Specific binding sites for atrial natriuretic peptide (ANP) in cultured mesenchymal nonmyocardial cells from rat heart

Specific binding sites for atrial natriuretic peptide (ANP) were studied in cultured mesenchymal nonmyocardial cells (NMC) from rat heart. Binding study using 125I-labeled synthetic rat (r) ANP revealed the presence of a single class of high-affinity binding sites for rANP in cultured NMCs derived from both atria and ventricles; the apparent dissociation constant (Kd) was approximately 0.2 - 0.3 nM and the number of maximal binding sites was approximately 190,000 - 300,000 sites/cell. rANP significantly stimulated intracellular cGMP formation of cardiac NMCs in a dose-dependent manner (1.6 X 10(-8) M - 3.2 X 10(-7) M). rANP had no effect on synthesis of prostaglandin I2 by cultured cardiac NMCs. The physiological significance of ANP action on cardiac tissue remains to be determined.     

Glutaraldehyde pretreatment blocks phospholipase A2 modulation of adrenergic receptors

Treatment of rat cerebral cortical membranes with phospholipase A2 affects, in a parallel fashion, beta-, alpha 1- and alpha 2-adrenergic receptor binding, but not the affinity of these receptors for their respective ligands. Pretreatment of membranes with 0.1 percent glutaraldehyde blocks the effects of phospholipase A2 on adrenergic receptor binding. The results support the hypothesis that desensitization or "masking" of adrenergic receptors may involve changes in membrane lipid composition. Furthermore, glutaraldehyde may prove a useful tool in the investigation of the dynamic roles of lipids in receptor function and more specifically, their regulation and coupling to physiological events.     

Cytoplasmic Ca2+ has No Effect on the Excitability of Chara Plasmalemma

Internodal cells of Chara australis were subjected to two consecutiveintracellular perfusions with a Ca²⁺-free EGTA medium whichdisintegrated the tonoplast within about 10 minutes and thenwith a Ca²⁺-buffered medium. All perfusion media usually contained1 mM ATP. To stop the electrogenic pump, the internode was depletedof intracellular ATP. The excitability of the plasmalemma wasnot significantly influenced by intracellular free Ca²⁺ concentrationsup to 10^–4 M. To trigger action potentials, minimum currentdensities of 1 to 2 µA cm^–2 had to be applied atall tested Ca²⁺ concentrations. In the absence of cytoplasmicATP, excitability was completely lost at all Ca²⁺ concentrations. ¹ Present address: Botanisches Institut der Universit?t Bonn,Venusbergweg 22, D-5300 Bonn, FRG. (Received September 22, 1984; Accepted March 6, 1985)     

Ionic Activity Gradients across the Surface Membrane of Cytoplasmic Droplets Prepared from Chara australis

The membrane potential and the ionic activity gradients of K⁺and Cl^– across the surface membrane of cytoplasmic dropletsprepared from Chara australis internodal cells, were measuredin high and low ionic strength bathing solutions using liquidion exchange microelectrodes selective for K⁺ and Cl^–.Our results indicate that K⁺ is close to electrochemical equilibriumwhereas Cl^– is not. ¹ Present address: ICI Japan, Palace Hotel Annex Building, Marunouchi,Tokyo, Japan.     

Stimulatory effect of epidermal growth factor on prostaglandin E2 production in mouse fibrosarcoma cell line (HSDM1 C1)

Using HSDM1 C1 cell line derived from the mouse fibrosarcoma which synthesizes and secretes prostaglandin (PG) E2, specific binding sites for epidermal growth factor (EGF), a potent growth stimulator of many tissues, and its effect on PGE2 production by cultured tumor cells were studied. HSDM1 C1 cell line possessed specific, high-affinity receptors for EGF: Kd (5.5 X 10(-10 M) and binding capacity (17,650 sites/cell). EGF significantly stimulated PGE2 production in HSDM1 C1 line cultured in serum-free medium for 24 h in a dose-dependent manner; a 2.5-fold increase over control was induced by as little as 0.1 ng/ml and the maximal effect (3.5-fold increase) by 1 ng/ml. Its stimulatory effect on PGE2 production was completely blocked by indomethacin, an inhibitor of PG biosynthesis. These data suggest that EGF may be involved in modulation of synthesis and/or secretion of PGE2, a potent bone-resorbing factor, by the tumors which may partly contribute to hypercalcemia in certain types of neoplasms.     

Concomitant suppression of plasma ACTH- and beta-endorphin-like immunoreactivity by cyproheptadine, naloxone, and somatostatin in the ectopic ACTH syndrome

Release of plasma ACTH- and beta-endorphin (beta-EP)-like immunoreactivity (LI) was studied in vivo in a patient with an ectopic ACTH-producing malignant thymoma. Administration of lysin vasopressin stimulated concomitant release of plasma ACTH- and beta-EP-LI. Administration of cyproheptadine, naloxone, and somatostatin significantly suppressed plasma levels of ACTH- and beta-EP-LI, while saline infusion did not. Gel exclusion chromatography of the plasma extracts revealed that ACTH-LI consisted of two components, large and small molecular weight form, while beta-EP-LI consisted of three components, large molecular weight, beta-lipotropin-, and beta-EP-sized form; each of these components was incompletely suppressed by somatostatin infusion. It is suggested that certain tumors may have acquired aberrant multiple receptors during malignant transformation which may lead to the paradoxical hormone response as demonstrated in this case.     

Dependence on Ca2+ of the activities of phosphatidylinositol 4,5-bisphosphate phosphodiesterase and inositol 1,4,5-trisphosphate phosphatase in smooth muscles of the porcine coronary artery.

The activities of phosphatidylinositol 4,5-bisphosphate (PIP2) phosphodiesterase (PDE) and inositol 1,4,5,-trisphosphate (IP3) phosphatase in the particulate and cytosol fractions prepared from porcine coronary artery smooth muscles were examined using 32P-labelled PIP2 and IP3 as substrates, respectively. The activity of PIP2 PDE, as assessed from the production of IP3, in the cytosol fraction was about 10-fold higher than that in the particulate fraction. In the absence of MgCl2, the activity of PIP2 PDE in both fractions showed no causal relation to the free Ca2+ concentration in the physiological range of 10(-7)-10(-5) M, but was enhanced remarkably by 10(-4) M free Ca2+. The addition of 1 mM-MgCl2 to the assay medium markedly inhibited the activity of PIP2 PDE in both fractions in the presence of free Ca2+ (10(-8)-10(-5) M). In the absence of MgCl2, 10(-5)M-acetylcholine (ACh) produced IP3, and this action was blocked by 3 X 10(-6) M-atropine. The ACh-induced activation of PIP2 PDE ceased in the presence of 1 mM-MgCl2; however, the reactivation occurring on the addition of 10 microM-guanosine 5'-[gamma-thio]triphosphate did not depend on the free Ca2+ concentrations (10(-7)-10(-5)M). The activities of IP3 phosphatase, determined from decrease in the amount of IP3 in the particulate and cytosol fractions, had much the same potency in both fractions. The activity of IP3 phosphatase in the cytosol fraction was enhanced by MgCl2 in a concentration-dependent manner, the maximal value occurring at 1 mM-MgCl2, and was also enhanced in the presence of physiological concentrations of free Ca2+ (10(-7)-10(-6) M). These findings suggest that the activation of PIP2 PDE which occurs with application of ACh in the presence of guanine nucleotides and 1 mM-MgCl2 is independent of the free Ca2+ concentration, and that the hydrolysis of IP3 by phosphatase increases, depending on the concentration of free Ca2+.