Inactivation kinetics and pharmacology distinguish two calcium currents in mouse pancreatic B-cells

Summary Voltage-dependent calcium currents were studied in cultured adult mouse pancreatic B-cells using the whole-cell voltage-clamp technique. When calcium currents were elicited with 10-sec depolarizing command pulses, the time course of inactivation was well fit by the sum of two exponentials. The more rapidlyinactivating component had a time constant of 75±5 msec at 0 mV and displayed both calcium influx- and voltage-dependent inactivation, while the more slowly-inanctivating component had a time constant of 2750±280 msec at 0 mV and inactivated primarily via voltage. The fast component was subject to greater steady-state inactivation at holding potentials between –100 and –40 mV and activated at a lower voltage threshold. This component was also significantly reduced by nimodipine (0.5 m) when a holding potential of –100 mV was used, whereas the slow component was unaffected. In contrast, the slow component was greatly increased by replacing external calcium with barium, while the fast component was unchanged. Cadmium (1–10 m) displayed a voltage-dependent block of calcium currents consistent with a greater effect on the high-threshold, more-slowly inactivating component. Taken together, the data suggest that cultured mouse B-cells, as with other insulin-secreting cells we have studied, possess at least two distinct calcium currents. The physiological significance of two calcium currents having distinct kinetic and steady-state inactivation characteristics for B-cell burst firing and insulin secretion is discussed.     

The synovial lining of the rabbit knee: A scanning electron microscopy study of specimens reinforced structurally with tannic acid

Summary Medial and lateral synovial linings of the rabbit knee, structurally reinforced with tannic acid during fixation, were studied in the scanning electron microscope. Low-resolution micrographs revealed, in both linings, gross architecture of four types: accordion-like, lobe-like, fatty areolar, and flattened areas. In high resolution, both cellular and acellular surfaces were recorded. A novel, bubble-like appearance, of unknown nature and origin, accounted for 70% of both linings. No definite correlation between anatomical location, gross type, or microarchitectural pattern was noted.     

Met-enkephalin effects on associations formed in utero

Rat fetuses exposed to an odor stimulus and an aversive stimulus in utero showed an aversion to the odor when tested 16 days postnatally. Fetuses that also received 80 μg/kg Met-enkephalin showed a greater aversion to the odor stimulus than those subjects that did not receive the peptide. The difference between these groups was marginally significant. Control subjects did not show an aversion. But, subjects exposed to the odor and Met-enkephalin without the aversive stimulus, when tested, showed a significant preference for the odor over other control groups. These data show that associative learning in rat fetuses at 20 days of gestation may be enhanced by administration of Met-enkephalin.     

Effects of high concentrations of secretagogues on the morphology and secretory activity of the pancreas: A role for microfilaments

Summary Cholecystokinin (CCK) and acetylcholine, at concentrations greater than those required for maximal pancreatic enzyme secretion, elicit a submaximal secretory response. The mechanism for this secretagogue-induced unresponsiveness is unknown. Using isolated pancreatic acini of the mouse, we now find that high concentrations of secretagogues also induce a profound alteration in acinar morphology, characterized by the formation of spherical protrusions on the basal surface of the cells. Since both the determination of cell shape and exocytosis may involve calcium and contractile proteins, we used a calcium-free medium and cytochalasin B (CB) to evaluate the importance of a contractile mechanism in the secretory and morphological effects of high concentrations of CCK-octapeptide (CCK₈). Incubation in a calcium-free medium partially blocked CCK-induced unresponsiveness, but brought about dissociation of the acini. CB at a concentration of 3 g/ml caused the disappearance of apical microfilaments and, most strikingly, completely prevented the morphological alteration induced by CCK₈. Furthermore, CB converted the biphasic dose-response curve for CCK₈-induced amylase release to a monophasic shape, such that the amylase release stimulated by a high concentration of CCK₈ (10 nM) was augmented. It is concluded, therefore, that a contractile process involving microfilaments may mediate secretagogue-induced unresponsiveness in pancreatic acinar cells.     

The role of RNA primer in discontinuous DNA replication in isolated nuclei from HeLa cells

RNA-primed discontinuous DNA synthesis was studied in an in vitro system consisting of washed nuclei from synchronized S-phase HeLa cells. A new technique proved useful for the purification of short nascent fragments of DNA (Okazaki fragments). Mercurated dCTP was substituted for dCTP in the DNA synthesis reaction. Short nascent pieces (4–6 S) of mercurated DNA were found to bind preferentially to sulfhydryl-agarose, and could be eluted with mercaptoethanol. The isolated fragments were assayed for the presence of covalently linked RNA by the spleen exonuclease method described by Kurosawa et al. (Kurosawa, Y., Ogawa, T., Hirose, S., Okazaki, T. and Okazaki, R. (1975) J. Mol. Biol. 96, 653–664). Following a 30 s incubation with [³H]TTP in the absence of added ribonucleotides, approximately 20% of the nascent strands synthesized in washed nuclear preparations had RNA attached. These RNA primers either preexisted in the nuclei or were formed from endogenous ribonucleotides. The 5′ ends of the primers appeared to be largely in a phosphorylated state. In the absence of added ribonucleotides, these RNA-DNA linkages disappeared within 2 min, whereas if ribonucleotides were added, the number of RNA primers increased to 40% and remained at this level for greater than 2 min. To obtain maximal levels of RNA primer, the addition of all three of the ribonucleotides, rCTP, rGTP and rUTP (0.1 mM), as well as high levels of rATP (5 mM) was required. Addition of ribonucleotides also markedly enhanced the amount of nascent DNA fragments synthesized. However, in the absence of added ribonucleotides, after RNA primers had disappeared, nascent DNA fragments were still initiated at a significant rate. These results suggest that RNA primers play an important role in the initiation of Okazaki fragments but that synthesis can also be initiated by alternative mechanisms. An important role for ATP in RNA primer synthesis is suggested.     

Selective release of extrahepatic lipoprotein lipase by polymetaphosphate

We have studied the lipase released into the circulation by polymetaphosphate injection into rats. Lipase release was in proportion to the dose injected. The post-polymetaphosphate lipase was almost completely inhibited by high salt concentrations or by addition of protamine sulfate to the assay system suggesting that this compound released lipoprotein lipase and not hepatic triglyceride lipase. The lipases released by polymetaphosphate and by heparin were compared using a heparin-sepharose affinity column technique which separates lipoprotein lipase from hepatic triglyceride lipase. While heparin released both lipoprotein lipase and hepatic triglyceride lipase, polymetaphosphate released almost exclusively lipoprotein lipase. Other experiments showed that neither polymetaphosphate nor heparin inhibited the hepatic lipase when added to the assay. These results suggest that lipoprotein lipase may be released by the negative charge on these high-charge polymers while hepatic triglyceride lipase release may require the specific sugar configuration of heparin.     

Flavonoid chemistry of arceuthobium (viscaceae)

Flavonoid compounds from 36 of the 38 known taxa of the genusArceuthobium (dwarf mistletoes) were examined. The flavonoid chemistry of the genus is rather uniform, all taxa producing 3-O-glycosides of the flavonols quercetin and myricetin. No infraspecific chemical variation was encountered, and in those instances where subspecific taxa are recognized, their chemistry was uniform. At the subgeneric level, members of subgenusArceuthobium synthesize primarily glucosides, whereas galactosides are more common in subgenusVaginata. In two of the four Old World species of subgenusArceuthobium (A. juniperi- procerae andA. oxycedri) only myricetin 3-O-glucoside was detected. There are no absolute flavonoid differences between subgenera, sections, or series. On the other hand, flavonoids are useful in several instances at the species level. In several cases, chemical data lend support to the recognition of species which in the past have been considered doubtfully distinct on the basis of morphology.     

Homology between ricin and Ricinus communie agglutinin: Amino terminal sequence analysis and protein synthesis inhibition studies

The existence of three forms of ricin and two forms of the Ricinus communis agglutinin (RCA) was established using cation exchange chromatography, isoelectric focusing, and polyacrylamide gel electrophoresis. The preparation of the RCA we obtained was 60–75 times more potent than ricin in the agglutination of erythrocytes, but was about 4% as effective as an inhibitor of cell-free protein synthesis. When reduced with 2-mercaptoethanol, the RCA was activated 3000-fold as an inhibitor of cell-free protein synthesis, whereas ricin was activated about 600-fold by the same treatment. A mixture of the RCA A chains was about one-fifth as effective as the ricin A chain in the inhibition of cell-free protein synthesis. The purified polypeptide subunits of the castor bean lectins were subjected to automated Edman degradation. The sequence for 17 of the first 19 residues of the agglutinin A chain was determined. The first seven residues of the ricin A chain were determined and they are identical with those of the RCA A chain. Nineteen turns of Edman degradation on the RCA B chain resulted in the identification of 18 amino acids. The sequence determined for the first 17 residues of the ricin B chain was identical with that of the RCA B chain. It is likely that the identity of the ricin/RCA A and B chain sequences extends further along the polypeptide chains than the sequences we have determined. The similar structural and catalytic potentials of the RCA and ricin suggest that they bear a precursor-product relationship.     

Control of acetylcholine receptor mobility and distribution in cultured muscle membranes. A fluorescence study

The molecular control of the distribution and motion of acetylcholine receptors in the plasma membrane of developing rat myotubes in primary cell culture was investigated by fluorescence techniques. Acetylcholine receptors were marked with tetramethylrhodamine-labeled α-bungarotoxin and lateral molecular motion in the membrane was measured by the fluorescence photobleaching recovery technique. Three types of experiments are discussed: (I) The effect of enzymatic cleavages, drugs, cross-linkers, and physiological alterations on the lateral motion of acetylcholine receptors and on the characteristic distribution of acetylcholine receptors into patch and diffuse areas. (II) Observation of the distribution and/or motion of fluorescence-labeled concanavalin A receptors, lipid probes, cell surface protein, and stained cholinesterase in acetylcholine receptor patch and diffuse areas. (III) The effect of a protein synthesis inhibitor and electrical stimulation on membrane incorporation of new acetylcholine receptors.Some of the main conclusions are: (a) acetylcholine receptor lateral motion is inhibited by concanavalin A plant lectin and by anti-α-bungarotoxin antibody, but marginally enhanced by treatment with a local anesthetic; (b) patches are stabilized by an immobile cellular structure consisting of molecules other than the acetylcholine receptors themselves; (c) this structure is highly selective for acetylcholine receptors and not for other cell membrane components; (d) acetylcholine receptor patch integrity and diffuse area motion are independent of direct metabolic energy requirements and are sensitive to electrical excitation of myotube; (e) lipid molecules can move laterally in both acetylcholine receptor patches and diffuse areas; and (f) acetylcholine receptor lateral motion in diffuse areas and immobility in patch areas are not altered by specific agents which are known to affect extrinsic cell surface proteins, or cytoplasmic microfilaments and microtubules.