Intracellular mechanism of action of sympathetic hepatic nerves on glucose and lactate balance in perfused rat liver

In rat liver perfused in situ stimulation of the nerve plexus around the hepatic artery and the portal vein caused an increase in glucose output and a shift from lactate uptake to output. The effects of nerve stimulation on some key enzymes, metabolites and effectors of carbohydrate metabolism were determined and compared to the actions of glucagon, which led to an increase not only of glucose output but also of lactate uptake. 1. Nerve stimulation caused an enhancement of the activity of glycogen phosphorylase a to 300% and a decrease of the activity of glycogen synthase I to 40%, while it left the activity of pyruvate kinase unaltered. Glucagon, similarly to nerve action, led to a strong increase of glycogen phosphorylase and to a decrease of glycogen synthase; yet in contrast to the nerve effect it lowered pyruvate kinase activity clearly. 2. Nerve stimulation increased the levels of glucose 6-phosphate and of fructose 6-phosphate to 200% and 170%, respectively; glucagon enhanced the levels to about 400% and 230%, respectively. The levels of ATP and ADP were not altered, those of AMP were increased slightly by nerve stimulation. 3. Nerve stimulation enhanced the levels of the effectors fructose 2,6-bisphosphate and cyclic AMP only slightly to 140% and 125%, respectively; glucagon lowered the level of fructose 2,6-bisphosphate to 15% and increased the level of cyclic AMP to 300%. 4. In calcium-free perfusions the metabolic responses to nerve stimulation showed normal kinetics, if calcium was re-added 3 min before, but delayed kinetics, if it was re-added 2 min after the onset of the stimulus. The delay may be due to the time required to refill intracellular calcium stores. The hemodynamic alterations dependent on extracellular calcium were normal in both cases. The activation of glycogen phosphorylase, the inhibition of glycogen synthase and the increase of glucose 6-phosphate can well explain the enhancement of glucose output following nerve stimulation. The unaltered activity of pyruvate kinase and the marginal increase of fructose 2,6-bisphosphate cannot be the cause of the nerve-stimulation-dependent shift from lactate uptake to output. The very slight increase of the level of cyclic AMP after nerve stimulation cannot elicit the observed activation of glycogen phosphorylase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of protein kinase C by cyclic adenosine 3':5'-monophosphate and a tumor promoter in skeletal myoblasts

The specific activity of protein kinase C in rat skeletal myoblasts decreased when they were exposed for very short periods to isoproterenol, forskolin, dibutyryl cyclic AMP (Bt2cAMP), or the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). In the presence of Bt2cAMP or forskolin only the cytosolic but not the membrane-bound kinase activity was found to decrease. Treatment with TPA, however, led to a decrease in the activity of the enzyme both in the cytosolic as well as the membrane fractions. The effects observed in vivo could be duplicated in crude extracts of myoblasts incubated with cAMP analogues or TPA. In the presence of ATP, protein kinase C activity decreased considerably in crude cytosolic fractions treated with the cAMP analogues, but a requirement for ATP was not evident for the decrease in activity brought about by TPA. For the cAMP analogues the decrease in protein kinase C was also prevented by incubation of the extracts with an inhibitor of cAMP-dependent protein kinase. The regulation of protein kinase C by Bt2cAMP (but not by TPA) was altered in Rous sarcoma virus-transformed myoblasts. It is considered likely that a component affected by cAMP (probably a substrate for cAMP-dependent protein kinase) participates in the regulation of protein kinase C activity, and it is altered in unknown ways in transformed myoblasts.     

Day-night changes in the levels of adenine nucleotides,phosphoenolpyruvate and inorganic pyrophosphate in leaves of plants having Crassulacean acid metabolism

The levels of phosphorylated compounds studied during the dark period of Crassulacean acid metabolism (CAM) in Kalanchoë leaves showed increases for ATP and pyrophosphate and decreases for ADP, AMP and phosphenolpyruvate; levels of inorganic phosphate remained constant. Changes in adenylate levels and the correlated nocturnal increase in adenylate-energycharge were closely related to changes in malate levels. The increase in ATP levels was much inhibited in CO₂-free air and stimulated after induction of CAM in short-day-treated plants of K. blossfeldiana cv. Tom Thumb. Changes in levels of phosphoenolpyruvate and pyrophosphate were independent of the presence of CO₂. The results show the operation of complex regulatory mechanisms in the energy metabolism of CAM plants during nocturnal malic-acid accumulation.Abbreviations CAM Crassulacean acid metabolism - FW fresh weight - OAA oxaloacetic acia - PEP phosphoenol pyruvate - PP_i pyrophosphate     

Idiotypic determinants of monoclonal antibodies that bind to 3-fucosyllactosamine

Many monoclonal antibodies that react with the lacto-N-fucopentaose III (LNF III) antigenic determinant, Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc, have been described recently. The terminal trisaccharide of this determinant, fucosyllactosamine, is present on glycolipids and glycoproteins and on the surface of granulocytes, monocytes, and other cells. To study the structural and genetic diversity of these antibodies, syngeneic anti-idiotypic monoclonal antibodies were produced in BALB/c mice against PMN 6, a monoclonal antibody directed against this sequence. Anti-idiotypic antibodies 6B1 and 6C4 reacted with 50% of a panel of 20 anti-LNF III monoclonal antibodies, whereas 6A3 reacted strongly only with PMN 6. This indicates that the determinants recognized by 6C4 and 6B1 represent major cross-reactive idiotopes of this family of antibodies. The binding of idiotypic antibodies to a glycolipid bearing this antigenic determinant was completely inhibited by the three anti-idiotypic antibodies, 6A3, 6B1, and 6C4. The idiotopes could be demonstrated on the heavy chain of the monoclonal antibodies by an antibody transfer technique when mild reducing conditions were employed, but a high concentration of reducing agent destroyed the idiotypic determinants. This suggests that the anti-idiotypic antibodies recognize conformational structures expressed on the heavy chain molecules. The binding of 18 monoclonal antibodies to two glycolipid antigens and to a fucosyllactosamine-bovine serum albumin conjugate was compared. Antibodies that possessed the 6C4 cross-reactive idiotope bound to fucosyllactosamine-bovine serum albumin more weakly than idiotype-negative antibodies (p = 0.001). This suggests that the 6C4-positive antibodies might represent germline structures.     

Immunochemical comparison of cardiac glycoside-sensitive (lamb) and -insensitive (rat) kidney (Na+ + K+)-ATPase

The immunological cross-reactivity of the ouabain-sensitive lamb kidney and the ouabain-insensitive rat kidney (Na+ + K+)-ATPase (EC 3.6.1.37) was examined using polyclonal and monoclonal antibodies. Studies using rabbit antisera prepared against both the lamb kidney and rat kidney holoenzymes showed the existence of substantial antigenic differences as well as similarities between the holoenzymes and the respective denatured alpha and beta subunits of these two enzymes. Quantitation of the extent of cross-reactivity using holoenzyme-directed antibodies showed a 40-60% cross-reactivity. In addition, rabbit antisera monospecific to the purified, denatured alpha and beta subunits of the lamb kidney enzyme showed about a 50% cross-reactivity towards the respective subunit of the rat enzyme. In contrast to the cross-reactivity observed using the polyclonal antibodies, six monoclonal antibodies specific for the alpha subunit of the lamb holoenzyme exhibited no cross-reactivity with the rat holoenzyme. Four of these monoclonal antibodies, however, showed substantial cross-reactivity with rat alpha subunit as resolved by SDS-polyacrylamide gel electrophoresis. A fifth antibody did not bind to the denatured alpha subunit of either the lamb or the rat enzyme. Another monoclonal antibody (M7-PB-E9), which is specific for an epitope previously implicated in the regulation of both ATP and ouabain binding to (Na+ + K+)-ATPase (Ball, W.J., Jr. (1984) Biochemistry 2275-2281) was found to bind to the denatured lamb alpha but not to the rat alpha. This antibody has identified a region of the lamb alpha that has an altered amino acid sequence in the ouabain-insensitive rat enzyme. These immunological studies indicate that there are substantial antigenic differences between the lamb and rat kidney (Na+ + K+)-ATPases. The majority of these antigenic differences appear to be due to variations in the tertiary structures rather than to variations in the primary structures of the alpha subunits.     

The respiratory chain of the facultative thermophile,Bacillus coagulans

Two oxidases were found to be present in membranes from the facultative thermophile Bacillus coagulans grown at 55°C, compared to one in cells grown at 37°C. Cytochrome spectra and inhibitors of the respiratory chain identified them as cytochrome oxidases aa ₃ and d. Both were present in membranes from 55°C grown cells, but only cytochrome oxidase aa ₃ was found in membranes from 37°C grown cells. The presence of cytochrome d in 55°C grown cultures was found to be due to decreased oxygen tension and not to the high growth temperature. This was confirmed by (a) induction of cytochrome d at 37°C under conditions of oxygen limitation and (b) its repression at 55°C under conditions of high aeration and its subsequent induction on lowering the dissolved oxygen concentration in chemostat cultures. Two cytochromes b (_max 558 and _max 562) were present in both 37°C and 55°C grown cells. Results from the inhibition of substrate oxidation by membranes suggested different pathways of electron transport by the respiratory chain.     

Ethyl-3,4-dihydroxybenzoate inhibits myoblast differentiation: evidence for an essential role of collagen

To study the role of (pro)collagen synthesis in the differentiation of rat L6 skeletal myoblasts, a specific inhibitor of collagen synthesis, ethyl-3,4-dihydroxybenzoate (DHB), was utilized. It is shown that DHB reversibly inhibits both morphological and biochemical differentiation of myoblasts, if it is added to the culture medium before the cell alignment stage. The inhibition is alleviated partially by ascorbate, which along with alpha-ketoglutarate serves as cofactor for the enzyme, prolyl hydroxylase. DHB drastically decreases the secretion of procollagen despite an increase in the levels of the mRNA for pro alpha 1(I) and pro alpha 2(I) chains. Probably, the procollagen chains produced in the presence of DHB, being underhydroxylated, are unable to fold into triple helices and are consequently degraded in situ. Along with the inhibition of procollagen synthesis, DHB also decreases markedly the production of a collagen-binding glycoprotein (gp46) present in the ER. The results suggest that procollagen production and/or processing is needed as an early event in the differentiation pathway of myoblasts.     

A Val-25-to-Ile substitution in the envelope precursor polyprotein, gPr80env, is responsible for the temperature sensitivity, inefficient processing of gPr80env, and neurovirulence of ts1, a mutant of Moloney murine leukemia virus TB.

ts1 is a neurovirulent spontaneous temperature-sensitive mutant of Moloney murine leukemia virus TB which causes hindlimb paralysis in mice. Previously, it had been shown that the temperature-sensitive defect resided in the env gene. At the restrictive temperature, the envelope precursor polyprotein, gPr80env, is inefficiently processed intracellularly into two cleavage products, gp70 and Prp15E. This inefficient processing of gPr80env is correlated with neurovirulence. In this study, it was shown that a single amino acid substitution, Val-25----Ile in gPr80env, is responsible for the temperature sensitivity, inefficient processing of gPr80env at the restrictive temperature, and neurovirulence of ts1. At the restrictive temperature, a steady-state level of nonprocessed, endoglycosidase H-sensitive gPr80env remained in the endoplasmic reticulum of cells infected by ts1, but no endoglycosidase H-resistant gPr80env and only trace amounts of gp70 were detected in the infected cells. Since the host cell-encoded processing protease resides in the cis cisternae of the Golgi apparatus, inefficient processing of gPr80env at the restrictive temperature is most likely due to inefficient transport of gPr80env from the endoplasmic reticulum to the cis cisternae of the Golgi apparatus rather than due to misfolded gPr80env being a poor substrate for the processing protease at the restrictive temperature.     

Galanin-, neuropeptide Y- and enkephalin-like immunoreactivities in catecholamine-storing paraganglia of the fetal guinea pig and newborn pig

Summary The occurrence and distribution of several neuropeptides and transmitter enzymes have been investigated by means of indirect immunofluorescence histochemistry in preaortal and carotid body-like paraganglia of the fetal guinea pig and the newborn pig. Preaortal paraganglia from the celiac and inferior mesenteric ganglion regions in fetal guinea pigs showed cell bodies immunoreactive (IR) for tyrosine hydroxylase (TH), dopamine -hydroxylase (DBH), neuropeptide Y (NPY), galanin (GAL) and metenkephalin (ENK). Almost all cells were IR for TH and DBH, whereas NPY-like immunoreactivity (-LI), GAL-LI and ENK-LI occurred less frequently. Direct double-labeling revealed the coexistence of NPY/GAL, NPY/ENK and GAL/ENK in paraganglion cells from the celiac and inferior mesenteric region. Nerve fibers and terminals were IR for ENK; fibers IR for calcitonin-gene-related peptide (CGRP) were present in the inferior mesenteric ganglion region. Preaortal paraganglia cells from the newborn pig showed TH-LI, DBH-LI, GAL-LI and ENK-LI, the distribution pattern being similar to that seen in the guinea pig; however, NPY-LI was absent. Carotid-body-like paraganglia from the newborn pig showed cell bodies IR to TH, GAL and ENK. Few cells were seen with DBH-LI. A rich supply of nerve fibers with CGRP-LI was present; some fibers exhibited ENK-LI and CCK-LI. In the adjacent superior cervical ganglion, ganglion cell bodies showed immunoreactivity to TH, DBH and NPY. A small number of cells were positive for GAL, CGRP and vasoactive intestinal polypeptide (VIP). Physiological activation of the paraganglia, leading to release or increase in catecholamines, may also change the content of the neuropeptides present in the paraganglia.