Guanosine 5'-triphosphate binding protein (Gi) and two additional pertussis toxin substrates associated with muscarinic receptors in rat heart myocytes: characterization and age dependency

The coupling of muscarinic receptors with G-proteins was investigated in cultured myocytes prepared from the hearts of newborn rats. The coupling was investigated in both young (5 days after plating) and aged (14 days after plating) cultures, in view of the completely different effects of 5'-guanylyl imidodiphosphate [Gpp(NH)p] on muscarinic agonist binding to homogenates from young vs aged cultures [Moscona-Amir, E., Henis, Y. I., Yechiel, E., Barenholz, Y., & Sokolovsky, M. (1986) Biochemistry 25, 8118-8124]. Pretreatment of cultures from both ages by Bordetella pertussis toxin (IAP) was found to eliminate any Gpp(NH)p effect on carbamylcholine binding. IAP by itself induced a rightward shift in the carbamylcholine competition curve in homogenates from aged cultures, but no such effect was observed in homogenates from young cultures. IAP-catalyzed [32P]ADP-ribosylation of membrane preparations from young and aged cultures revealed major differences between them. Young cultures exhibited a major IAP substrate at 40 kDa, which was also recognized by anti-alpha i antibodies, and two novel IAP substrates at 28 and 42 kDa, which were weakly ADP-ribosylated by the toxin and were not recognized with either anti-alpha i or anti-alpha o antibodies. In aged cultures, only the 40-kDa band (ribosylated to a lower degree) was detected. The parallel age-dependent changes in the three IAP substrates (28, 40, and 42 kDa) and in the interactions of the G-protein(s) with the muscarinic receptors strongly suggest close association between the two phenomena. All of these age-dependent changes in the G-protein related parameters were prevented by phosphatidylcholine-liposome treatment of the aged cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

A Ca2+-Dependent Protein Kinase Activity Associated with Serotonin Binding Protein

The endogenous phosphorylation of serotonin binding protein (SBP), a soluble protein found in central and peripheral serotonergic neurons, inhibits the binding of 5-hydroxytryptamine (5-HT, serotonin). A protein kinase activity that copurifies with SBP (SBP-kinase) was partially characterized and compared with calcium/calmodulin-dependent protein kinase II (CAM-PK II). SBP itself is not the enzyme since heating destroyed the protein kinase activity without affecting the capacity of the protein to bind [3H]5-HT. SBP-kinase and CAM-PK II kinase shared the following characteristics: (1) size of the subunits; (2) autophosphorylation in a Ca2+-dependent manner; and (3) affinity for Ca2+. In addition, both forms of protein kinase phosphorylated microtubule-associated proteins well and did not phosphorylate myosin, phosphorylase b, and casein. Phorbol esters or diacylglycerol had no effect on either of the protein kinases. However, substantial differences between SBP-kinase and CAM-PK II were observed: (1) CAM enhanced CAM-PK II activity, but had no effect on SBP-kinase; (2) synapsin I was an excellent substrate for CAM-PK II, but not for SBP-kinase; (3) 5-HT inhibited both the autophosphorylation of SBP-kinase and the phosphorylation of SBP, but had no effect on CAM-PK II. These data indicate that SBP-kinase is different from CAM-PK II. Phosphopeptide maps of SBP and SBP-kinase generated by digestion with S. aureus V8 protease are consistent with the conclusion that these proteins are distinct molecular entities. It is suggested that phosphorylation of SBP may regulate the transport of 5-HT within neurons.     

Factors involved in multiplication and survival ofEscherichia coli in lake water

The population of a strain ofEscherichia coli that was resistant to nalidixic acid and streptomycin declined rapidly in samples of sterile and nonsterile Cayuga Lake water and reached an undetectable level in nonsterile water at 24 and 72 hours when counted on eosin-methylene blue (EMB) agar and half-strength trypticase soy agar (TSA), respectively. In sterile lake water amended with 10g amino acids per ml or 0.1 M phosphate,E. coli multiplied exponentially for more than 24 hours. The addition ofRhizobium leguminosarum biovarphaseoli to unamended sterile lake water prevented the decline ofE. coli, and its addition to amended sterile lake water preventedE. coli multiplication. The cell density of this strain ofE. coli declined in the first 8 hours after its introduction into an inorganic salts solution, but the bacterium then grew extensively. This increase in abundance was not observed in the presence ofR. phaseoli, andE. coli counts on half-strength TSA remained unchanged between 8 hours and 6 days. When counted on EMB agar, the abundance of the antibiotic-resistant strain ofE. coli and a strain not selected for resistance increased in solutions containing phosphate and amino acids but declined in the presence of high densities ofR. phaseoli. Many of the cells of the antibiotic-resistantE. coli strain failed to grow on antibiotic-amended EMB agar after introduction of the organism into nonsterile or sterile lake water or into an inorganic salts solution containingR. phaseoli, although colonies appeared on TSA. The data suggest thatE. coli cells grown on rich media suffer a shock when introduced into lake water because of low hypotonicity, the indigenous competing flora, or both. This shock is prevented by either phosphate buffer or by amino acids at low concentration. The shocked bacteria formed colonies on half-strength TSA. Depending on environmental conditions, the presence of a second organism either has no effect or results in an increase or decrease inE. coli numbers.     

Interaction of Sendai virions with resealed human erythrocyte ghosts. Lateral mobility of the viral glycoproteins in the cell membrane following fusion

Two independent methods demonstrated that resealed human erythrocyte ghosts undergo Sendai virus-mediated cell-cell fusion to a much lower degree (about 4%) than intact erythrocytes, in spite of similar levels of viral envelope-cell fusion in the two preparations. Fluorescence photobleaching recovery (FPR) showed similar lateral mobilities of the viral glycoproteins following fusion with either ghosts or whole erythrocytes. It is suggested that although viral glycoprotein mobilization in the cell membrane is essential for cell-cell fusion, the target cell properties are also important; in the absence of the required cellular parameters, the mobilization may not be a sufficient condition.     

Role of lipids in age-related changes in the properties of muscarinic receptors in cultured rat heart myocytes

The effects of the culture's age and of liposome treatments on the properties of muscarinic receptors in cultured rat heart myocytes prepared from the hearts of newborn (1-3 days old) rats were investigated. In these studies we investigated the binding characteristics of antagonists and agonists to the myocyte muscarinic receptors in young (5 days after plating) vs. older (14 days after plating) cultures. Our findings demonstrate that the aging of the cells in culture is accompanied by a reduction in the muscarinic binding capacity and by alterations in the proportion of high- and low-affinity states toward muscarinic agonists, as well as by striking changes in the mode of coupling of the receptors with guanine nucleotide binding protein(s) [G protein(s)]. The above effects of the culture's age occur concomitantly with alterations in the lipid composition of the cultured myocytes (in 14-day old cultures, the phosphatidylcholine/sphingomyelin ratio is reduced, and the cholesterol level is elevated). In order to explore whether the lipid composition is involved in the mechanism that alters the properties and coupling of the muscarinic receptors, we treated aging cultures with liposomes containing egg phosphatidylcholine. This treatment resulted in 14 day old cultures with a lipid composition similar to that of young cultures, and the treated myocytes demonstrated muscarinic receptor properties similar to those of young myocyte cultures. The implications for the role of membrane lipid composition and organization in determining the properties of the muscarinic receptors and their coupling with G proteins are discussed.     

Lateral mobility and organization of phospholipids and proteins in rat myocyte membranes. Effects of aging and manipulation of lipid composition

The effects of aging and of liposome treatment on the lateral mobility of phospholipids and proteins in the plasma membrane of cultured rat heart myocytes were studied by fluorescence photobleaching recovery. Both the mobile fraction (R) and the lateral diffusion coefficient (D) of the fluorescent phospholipid N-4-nitrobenzo-2-oxa-1,3-diazolyl phosphatidylethanolamine were found to depend on the culture's age. Aged myocyte cultures (15 days old) demonstrated higher R and lower D as compared with young ones (5 days old). Treatment of aged cultures with phosphatidylcholine (PC) liposomes, which increases the PC/sphingomyelin (SM) ratio and decreases the cholesterol level, reversed the D value to the level observed in young cultures and decreased R below the value encountered in young cells. Treatments with SM liposomes (which induce cholesterol depletion without altering the PC/SM ratio) and with PC/cholesterol (1:0.9) liposomes (which increase the PC/SM ratio without cholesterol depletion) have indicated that the PC-liposome effect is due to changes in both the PC/SM ratio and in the cholesterol level. Analogous experiments on the mobility of succinyl-concanavalin A receptors yielded similar effects on R, without altering the D value. The changes in the D and R values of the markers studied are most likely initiated by the observed alterations in the myocyte lipid composition under the conditions employed. The possible involvement of changes in the organization of membrane lipids in domains in the observed phenomena is discussed.     

Cis-unsaturated fatty acids induce both lipogenesis and calcium binding in adipocytes

The addition of 0.4-3 mM of cis-unsaturated fatty acids such as oleic acid (18:1) or linoleic acid (18:2) to intact rat adipocytes stimulated lipogenesis at 37 degrees C. Saturated or trans-unsaturated fatty acids were ineffective. Fluorescence photobleaching recovery studies performed under similar conditions indicated that the cis-unsaturated fatty acids do not alter lateral mobility of either a lipid probe or a general protein marker in the plasma membrane. A high concentration (7 mM) of Ca2+, which by itself has some stimulatory effect on lipogenesis, significantly potentiated the effect of oleic acid on this insulin-like activity. Measurement of 45Ca2+ binding by fat cells has indicated that cis-unsaturated (but not saturated) fatty acids increased 12- to 20-fold the amount of Ca2+ associated with the cells. The dependence of this effect on the fatty acid concentration correlates well with the effect of the fatty acid on the induction of lipogenesis. Our results suggest that cis-unsaturated fatty acids affect membrane organization in a manner which induces a significant increase in membrane associated or intracellular Ca2+. This increase may be responsible for inducing exocytotic-like processes which facilitate translocation of glucose transport activity from storage sites to the plasma membrane and thus produce an insulin-like effect.     

Mobility modulation by local concanavalin A binding. Selectivity toward different membrane proteins

We have employed fluorescence photobleaching recovery to demonstrate selective immobilization of lymphocyte membrane proteins by localized concanavalin A (ConA) binding to the cell surface. Localized ConA binding was achieved by the binding of ConA coupled to paraformaldehyde-fixed platelets to mouse spleen lymphocytes. The effect of the localized cross-linking of ConA receptors on the lateral mobility of specific membrane proteins at regions distal to the ConA platelets was investigated. The diffusion of surface immunoglobulins and ConA receptors was inhibited above a threshold coverage (12%) of the upper lymphocyte surface by ConA platelets. In contrast, no effect was observed on the diffusion and aggregation of mouse histocompatibility antigens (H-2Kk) labeled with a fluorescent monoclonal antibody. Since the ConA modulation was shown to propagate through the cytoskeleton, these results indicate specificity in the interactions of membrane proteins with the cytoskeleton. This specificity enables a selective response of different membrane proteins to the ConA anchorage modulation.