Proadrenomedullin N-Terminal 20 Peptide (PAMP), an Endogenous Anticholinergic Peptide: Its Exocytotic Secretion and Inhibition of Catecholamine Secretion in Adrenal Medulla

Abstract: In cultured bovine adrenal medullary cells, stimulation of nicotinic receptors by carbachol evoked the Ca²⁺-dependent exocytotic cosecretion of proadrenomedullin N-terminal 20 peptide (PAMP) (EC₅₀ = 50.1 µ M ) and catecholamines (EC₅₀ = 63.0 µ M ), with the molar ratio of PAMP/catecholamines secreted being equal to the ratio in the cells. Addition of PAMP[1–20]NH₂ inhibited carbachol-induced ²²Na⁺ influx via nicotinic receptors (IC₅₀ = 2.5 µ M ) in a noncompetitive manner and thereby reduced carbachol-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels (IC₅₀ = 1.0 µ M ) and catecholamine secretion (IC₅₀ = 1.6 µ M ). It did not alter high K⁺-induced ⁴⁵Ca²⁺ influx via voltage-dependent Ca²⁺ channels or veratridine-induced ²²Na⁺ influx via voltage-dependent Na⁺ channels. PAMP seems to be a novel antinicotinic peptide cosecreted with catecholamines by a Ca²⁺-dependent exocytosis in response to nicotinic receptor stimulation.     

Mercurial-sensitive water transport in barley roots

An isolated barley root was partitioned into the apical and basal part across the partition wall of the double-chamber osmometer. Transroot water movement was induced by subjecting the apical part to a sorbitol solution, while the basal part with the cut end was in artificial pond water. The rate of transroot osmosis was first low but enhanced by two means, infilitration of roots by pressurization and repetition of osmosis. Both effects acted additively. The radial hydraulic conductivity (Lp_r) was calculated by dividing the initial flow rate with the surface area of the apical part of the root, to which sorbitol was applied, and the osmotic gradient between the apical and basal part of the root. Lp_r which was first 0.02–0.04 pm s⁻¹ Pa⁻¹ increased up to 0.25–0.4 pm s⁻¹ Pa⁻¹ after enhancement. Enhancement is assumed to be caused by an increase of the area of the plasma membrane which is avallable to osmotic water movement. The increased Lp_r is in the same order of magnitude as the hydraulic conductivity (Lp) of epidermal and cortical cells of barley roots obtained by Steudie and Jeschke (1983). HgCl₂, a potent inhibitor of water channels, suppressed Lp_r of non-infiltrated and infiltrated roots down to 17% and 8% of control values, respectively. A high sensitivity of Lp_r to HgCl₂ suggests that water channels constitute the most conductive pathway for osmotic radial water movement in barley roots.     

Establishment and characterization of a new,spontaneously immortalized,pancreatic ductal cell line from the Syrian golden hamster

Spontaneously immortal pancreatic cell lines are not available. By use of a defined culture medium, such a line (TAKA-1) was established from the Syrian golden hamster. Cytological, cytogenetic, molecular biological, enzymatic and receptor patterns as well as antigenicity were studied and were compared with those of the normal hamster pancreatic ductal cells in vivo. TAKA-1 cells grew exponentially in a monolayer on collagen gel in a defined medium but did not proliferate in soft agar. Ultrastructurally, the cells closely resembled the normal hamster pancreatic ductal cells. Similarities and dissimilarities were found between the normal ductal cells and TAKA-1 cells. Similarities included the presence of cytokeratin, carbonic anhydrase and some tumor-associated antigens. However, unlike the normal ductal cells, TAKA-1 cells expressed blood group A angigen and anti-vimentin, showed affinity to selected lectins, and an abnormality of chromosome 3, which is suggested to be associated with immortality. Moreover, unlike the hamster pancreatic ductal cancer cells but like the normal hamster pancreatic ductal cells, TAKA-1 cells did not have a c-Ki-ras mutation. EGF, TGF- and secretin, but not CCK or GRP, bound to the TAKA-1 cells. TAKA-1 cells produced TGF-, and their growth was stimulated by exogenous EGF in serum-free medium. This cell line presents a suitable model for biologic and pathologic study of the hamster pancreatic ductal cells in vitro.     

Electron microscopic study of endogenous peroxidase activity in human liver macrophages

We evaluated the conditions of fixation for ultrastructurally demonstrating the endogenous peroxidase (PO) activity of macrophages in biopsied human liver. The application of microwaving and immersion fixation with tannic acid and aldehydes allowed excellent visualization of PO activity in the nuclear envelope (NE), rough endoplasmic reticulum (rER), and cytoplasmic granules (CG), with good preservation of cellular ultrastructures. The macrophages with PO activity showed one of the following five patterns of PO localization: positive in both the NE and rER but negative in the CG (type 1); negative in both the NE and rER but positive in the CG (type 2); negative in the NE but positive in both the rER and CG (type 3); positive in all three (type 4); PO negative (type 5). The type 1 cells resembled typical Kupffer cells, type 2 cells monocytes, and type 3 and 4 cells the exudate-resident macrophages considered to be a transitional form between exudate and resident macrophages. Type 5 cells may also be a transitional form between the exudate and resident macrophage, or an end-stage macrophage derived from exudate macrophages which have lost their PO activity. Tannic-acid-aldehyde immersion fixation with microwaving may be a useful method in the study of the PO activities of macrophages in biopsied human liver specimens.     

Stimulation of prolyl hydroxylase activity by chelating agents.

The activity of purified prolyl hydroxylase was enhanced several fold by addition of some chelating agents to the assay medium. Chelating agents could be classified into three groups. The chelating agents of Group I such as α, α′-dipyridyl were inactive until they reached equimolar concentration with ferrous ion in the assay mixture. The Group II agents, EDTA, diethylenetriaminepentaacetic acid, etc., stimulated the enzymatic activity 1.5- to 3-fold at equimolar concentration with ferrous ion. But the agents of both groups precipitously inhibited the enzymatic activity at concentrations greater than ferrous ion. On the other hand, Group III chelating agents, such as nitrilotriacetic acid, enhanced the enzymatic activity 5- to 10-fold at concentrations greater than ferrous ion. Nucleoside triphosphates, which also stimulate the enzymatic activity several fold and whose optimal concentrations are 1–3 × 10^?m, may be analogous to nitrilotriacetic acid of Group III.     

Studies on factors in sweet potato root which induce spore germination of Ceratocystis fimbriata

Spore germination of Ceratocystis fimbriata was studied in termsof host-parasite specificity. The sweet potato, coffee and cacaostrains of Ceratocystis fimbriata germinated well in a fractionof sweet potato root water extract which had been passed througha column of cation exchange resin. The results showed that germinationof these strains was independent of exogenous cations. On theother hand, the prune, oak, taro and almond strains requiredfor germination both the absorbed and unabsorbed fractions ofsweet potato root water extract which were separated from eachother with a cation exchange resin column. Divalent cationssuch as Ca²⁺ Mg²⁺, Mn²⁺ and Zn²⁺ were identified as the activeprinciples in the absorbed fraction and Ca²⁺ showed the highestinductive activity for spore germination in the presence ofthe unabsorbed fraction. The active principle(s) in the unabsorbedfraction has not yet been identified. There was no relationshipbetween the Ca²⁺ and Mg²⁺ contents of the spores and the requirementof exogenous Ca²⁺ for germination. Ca²⁺ appeared to functionas a trigger of spore germination, not as a normal nutrient.These results suggest that the divalent cations such as Ca²⁺and Mg²⁺ in sweet potato contribute to the establishment ofhost-parasite specificity of this system. (Received August 10, 1977; )     

Differential agglutination of various strains of Ceratocystis fimbriata, black rot fungus, by concanavalin A

Germinated spores of various strains of Ceratocystis fimbriatawere agglutinated differentially by concanavalin A. -Methyl-D-mannosideand -methyl-D-glucoside, specific hapten sugars of concanavalinA, inhibited agglutination of the fungus by concanavalin A.These results support the assumption that differences existamong various strains of C. fimbriata in carbohydrate constituentsor locations on the cell surfaces. (Received March 7, 1978; )     

Photosynthetic activities of a thermophilic blue-green alga

Photosynthetic activities of a thermophilic blue-green alga,a species of Synechococcus, were studied with special referenceto its growth at high temperatures. A rapid algal growth occurredin the temperature range between 50 and 60?C, showing the maximumrate, six doublings per day, at about 57?C. Photosynthetic oxygenevolution and methyl viologen photoreduction in the cells werealso active at high temperatures and the optimum temperaturesfor these activities agreed with that of the algal growth. Thegrowth and photosynthetic activities were very low at room temperatureand irreversibly inactivated at temperatures above 60?C. The thylakoid membranes isolated from the alga were also photochemicallyactive at high temperatures. The membranes mediated ferricyanidephotoreduction coupled with a stoichiometric oxygen evolutionat a rate comparable to that of photosynthetic oxygen evolutionin the cells. The optimum temperature for the reaction was ashigh as 50?C. The membranes also showed a photosystem I-mediatedreaction at high temperatures. These observations indicate thatthe thylakoid membranes are intrinsically thermophilic in thisorganism. Thus the growth of the alga at high temperatures canbe well correlated to thermophilic properties of the photosyntheticapparatus. (Received February 20, 1978; )     

Effects of dibromothymoquinone and bathophenanthroline on flash-induced cytochrome f oxidation in spinach chloroplasts

The effects of several electron transport inhibitors on themagnitude and kinetics of cytochrome f oxidation induced byflash illumination were studied in the - and -band regions.On the flash excitation only a fraction of cytochrome f presentin the chloroplasts was oxidized with a half time of 0.1 to0.3 msec and then reduced with a half time of 10 to 25 msec. Dibromothymoquinone (DBMIB) at concentrations which severelysuppressed the reduction of cytochrome f approximately doubledthe magnitude of cytochrome f oxidation caused by a flash, mainlyby inducing an additional slow oxidation of cytochrome f witha half time longer than 1 msec. Enhancement of the cytochromef oxidation was also observed in the presence of bathophenanthroline.Such enhanced oxidation in duced by the two inhibitors was largelydiminished with the addition of reduced 2,6-dichlorophenolindophenolwhich accelerated cytochrome f reduction. In contrast, the inhibitionof cytochrome f reduction by 3-(3,4-dichlorophenyl)-1,1-dimethylurea(DCMU) was not associated with an increase in the magnitudeof cytochrome f oxidation. However, addition of DBMIB to theDCMU-poisoned chloroplasts enhanced cytochrome f oxidation,suggesting that this is related to a block of the electron transportbetween plastoquinone and cytochrome f. The results are explainedby assuming the occurrence of an electron carrier between plastoquinoneand cytochrome f. (Received May 10, 1978; )     

Oxidation and reduction of plastoquinone by photosynthetic and respiratory electron transport in a cyanobacterium Synechococcus sp.

The I-D dip, an early transient of the fluorescence induction, was examined as a means to monitor redox changes of plastoquinone in cells of a cyanobacterium, Synechococcus sp. That the occurrence of the dip depends upon the reduced state of the plastoquinone pool was indicated by observations that 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone and 3-(3,4-dichlorophenyl)-1,1-dimethylurea did not affect the initial rise to I but abolished the subsequent decline from I to D and that illumination of the cells with light 1, prior to fluorescence measurements, eliminated the transient. The I-D dip was prominent in freshly harvested cells containing abundant endogenous substrates, disappeared slowly as the cells were starved by aeration but reappeared on addition of fructose to the starved cells in the dark. The dip that had been induced by a brief illumination of the starved cells with light 2 was rapidly diminished in the dark and KCN inhibited the dark decay of the transient. The results indicate that plastoquinone is reduced with endogenous as well as exogenous substrates and oxidized by a KCN-sensitive oxidase in the dark, thus providing strong support for the view that plastoquinone of photosynthetic electron transport also functions in respiration. In addition, the occurrence of a cyclic pathway of electrons from Photosystem I to plastoquinone, possibly via ferredoxin or NADP, was suggested. Several lines of evidence indicate that, under a strong light 2, Photosystem I-dependent oxidation of plastoquinone predominates over Photosystem II-dependent reduction of the quinone in the cyanobacterium which contains Photosystem I more abundantly than Photosystem II.