Metabolic effects of salbutamol: comparison of aerosol and intravenous administration.

The effects of intravenous salbutamol (4 mug/kg) were compared with those of aerosol salbutamol (200 mug) in 10 asthmatic patients in a double-blind placebo-controlled study. Both methods of administration produced equal bronchodilatation. Intravenous salbutamol caused significant increases in plasma insulin and glucose levels and a fall in serum potassium concentration in addition to tachycardia and tremor, whereas aerosol salbutamol produced only a small transient increase in the plasma glucose level. The initially raised non-esterified fatty acid levels decreased significantly after aerosol and placebo but not after intravenous salbutamol.     

Cytokinin metabolism in nondividing and auxin-induced dividing explants ofHelianthus tuberosus L. Tuber tissue

Aqueous solutions of auxin (indole-3-acetic acid,-naphthalene acetic acid, or 2,4-dichlorophenoxyacetic acid) were active in inducing DNA synthesis and mitosis in prewashed tissue explants of mature Jerusalem artichoke tubers. Explants did not respond in this way to aqueous solutions of cytokinin (zeatin, zeatin riboside, 6-benzylaminopurine, or kinetin). The metabolism of [8-³H]zeatin riboside (ZR) was studied in non-dividing and auxin-induced synchronously dividing explants over the first 36 h of culture. ZR was taken up rapidly and to the same extent by both tissues. Sequential analysis of tissue extracts by thin-layer and high-performance-liquid chromatography identified zeatin nucleotide(s) (ZN), O-glucosyl zeatin riboside (OGZR), adenosine, and adenine nucleotide(s) (AN) as the principal metabolites in both tissues. The proportion of radio-activity due to ZR declined steadily and OGZR accumulated steadily at similar rates in both tissues. ZN was the major metabolite in both tissues at 12 h; thereafter ZN continued to accumulate in nondividing tissue, but its level declined in dividing tissue, and a corresponding increase in the levels of AN and adenosine was observed. These treatment differences in cytokinin metabolism were apparent at least 6 h before the onset of mitosis.     

Serine utilization as a precursor of phosphatidylserine and alkenyl-(plasmenyl)-, alkyl-, and acylethanolamine phosphoglycerides in cultured glioma cells

In several tissues and cell lines, serine utilized for phosphatidylserine (PS) synthesis is an eventual precursor of the base moiety of ethanolamine phosphoglycerides (PE). We investigated the biosynthesis and decarboxylation of PS in cultured C6 glioma cells, with particular attention to 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (plasmenylethanolamine) biosynthesis. Incorporation of [3H]serine into PS reached a maximum within 4-8 h, and label in nonplasmenylethanolamine phosphoglyceride (NP-PE) and plasmenylethanolamine was maximal by 12-24 h and 48 h, respectively. After 8 h, label in PS decreased even though 40-60% of initial label remained in the culture medium. Serial additions of fresh [3H]serine restored PS synthesis to higher levels of labeled PS accumulation followed by a subsequent decrease in 4-8 h. High performance liquid chromatographic analyses confirmed that medium serine was depleted by 8 h, and thereafter metabolites, including acetate and formate, accounted for radioactivity in the medium. The rapid but transient appearance of labeled glycine and ATP inside the cells indicated conversion of serine by hydroxymethyltransferase. 78-85% of label from serine was in headgroup of PS or of PE formed by decarboxylation. A precursor-product relationship was suggested for label from [3H]serine appearing in the headgroup of diacyl, alkylacyl, and alkenylacyl subclasses of PE. By 48 h, a constant specific activity, ratio of approximately 1:1 was reached between plasmenylethanolamine and NP-PE, similar to the molar distribution of these lipids. In contrast, equilibrium was not achieved in cells incubated with [1,2-14C]ethanolamine; plasmenylethanolamine had 2-fold greater specific activity than labeled NP-PE by 72-96 h. These observations indicate that in cultured glioma cells 1) serine serves as a precursor of the head group of PS and of both plasmenyl and non-plasmenyl species of PE; 2) exchange of headgroup between NP-PE and plasmenylethanolamine may involve different donor pools of PE depending on whether the headgroup originates with exogenous serine or ethanolamine; 3) serine is rapidly converted to other metabolites, which limits exogenous serine as a direct phospholipid precursor.     

Physiologic expression of two superantigens in the BDF1 mouse.

The majority of endogenous superantigens in the mouse (including the Mls loci) is encoded by mouse mammary tumor proviruses (Mtv) carried in the germline. To understand the differences between the highly stimulatory viral superantigens such as Mls-1a (encoded by Mtv-7), which have biologic activity in vivo and in vitro, and the poorly stimulatory viral superantigens such as Etc-1 (encoded by Mtv-9), which are active only in vivo, the physiologic expression of each Ag was studied in the Mtv-7+ (Mls-1a+), Mtv-9+ (Etc-1+) C57BL/6 x DBA/2 F1 (BDF1) mouse. Using the T cell hybridomas, 1BVB11.40 (anti Etc-1) and 18bbm.19 (anti Mls-1a), we found that similar to Mls-1a, B cells from the spleen and from the thymus present the Etc-1 superantigen, whereas macrophages and dendritic cells do not. Small, resting B cells present the Mls-1a and Etc-1 superantigens poorly; however, the same cells treated with LPS or IL-4 are at least eightfold more efficient in the presentation of these gene products. Furthermore, the effects of LPS and IL-4 are synergistic, but this synergy is not fully explained by the enhancement of I-A and I-E expression. The depletion of IgM+ B cells from neonatal BDF1 mice prevents the clonal deletion of V beta 5+ and 11+ (Etc-1-reactive) cells but not the deletion of V beta 6+ and 8.1+ (Mls-1a reactive) T cells. Despite the persistence of Mls-1a-mediated clonal deletion in B cell-depleted BDF1 mice, these results taken together, argue that the highly stimulatory Mls-1a gene product and the weakly stimulatory Etc-1 gene product are expressed on similar cell types and that their presentation is regulated in a similar way by agents active with B lymphocytes. It is argued that the differences between the highly stimulatory and weakly stimulatory superantigens reflect differences in avidity between the relevant V beta domain and its class II MHC protein/superantigenic ligand.     

Predominant T cell receptor gene elements in TNP-specific cytotoxic T cells.

H-2b class I-restricted, TNP-specific CTL clones were obtained by limiting dilution cloning of either short term polyclonal CTL lines or spleen cells of TNP-immunized mice directly ex vivo. Sequence analyses of mRNA coding for TCR alpha- and beta-chains of 11 clones derived from CTL lines from individual C57BL/6 mice revealed that all of them expressed unique but clearly nonrandom receptor structures. Five alpha-chains (45%) employed V alpha 10 gene elements, and four of those (36%) were associated with J beta 2.6-expressing beta-chains. The alpha-chains from these four TCR, moreover, contained an acidic amino acid in position 93 of their N or J region-determined sequences. Clones isolated directly from spleen cells carried these types of receptors at lower frequency, 27% V alpha 10 and 19% J beta 2.6, indicating that bulk in vitro cultivation on Ag leads to selection for these particular receptors. However, even in TNP-specific CTL cloned directly ex vivo, V alpha 10 usage was increased about fivefold over that in Ag-independently activated T cells in H-2b mice (4 to 5%). The selection for V alpha 10/J beta 2.6-expressing cells was obtained repeatedly in other TNP-specific CTL lines from C57BL/6 mice but not in FITC-specific CTL from the same strain or in TNP-specific CTL lines from B10.BR (H-2k) or B10.D2 (H-2d) mice. We conclude from this (a) that the selection for V alpha 10/J beta 2.6+ T cells is driven by the complementarity of these receptors to a combination of TNP and MHC epitopes and (b) that predominant receptor structures reflect the existence of a surprisingly limited number of "T cell-relevant" hapten determinants on the surface of covalently TNP-modified cells.     

Thymic stromal cells in culture. 2. Binding of normal thymocytes to a cloned thymic stromal cell line.

Thymic stromal cell line TS-9 was found to selectively bind a subpopulation of normal murine thymocytes. Selective binding allowed the isolation and phenotypic characterization of the adherent and nonadherent subpopulations of thymocytes. Flow cytometric analysis of fluorescently labeled thymocytes revealed that the adherent and nonadherent populations differ in maturity, with the adherent population enriched in immature thymocytes of the PNAhi, Thy-1hi, CD3-/lo, and CD4+/CD8+ double positive surface phenotype. A quantitative microwell assay was developed to measure the binding of thymocytes to TS-9. Thymocytes labeled with vital DNA stain Hoechst 33342 were allowed to bind to TS-9 in microwells and the intense fluorescence of this label was readily detected with a scanning fluorometer. The binding was trypsin-sensitive and hyaluronidase and PI-PLC resistant. The binding was also temperature dependent and sensitive to cytochalasin B. A panel of monoclonal antibodies to cell surface antigens including CD2, LFA-I/ICAM-I, and Thy-1 was screened in a quantitative binding assay for their ability to inhibit the binding of thymocytes to TS-9. The binding was partially inhibited by the C3C12 monoclonal antibody which recognizes the recently identified and apparently unique gp23,gp45 complex expressed on murine stromal cells.     

Role of conserved threonine and tyrosine residues in acetylcholine binding and muscarinic receptor activation. A study with m3 muscarinic receptor point mutants.

Structure-function relationship studies of the m3 muscarinic acetylcholine receptor have recently identified a series of threonine and tyrosine residues (all located within the hydrophobic receptor core) that are critically involved in acetylcholine binding (Wess, J., Gdula, D., and Brann, M.R. (1991) EMBO J. 10, 3729-3734). To gain further insight into the functional roles of these amino acids, the agonist binding properties of six rat m3 muscarinic receptor point mutants, in which the critical threonine and tyrosine residues had been individually replaced by alanine and phenylalanine, respectively, were studied in greater detail following their transient expression in COS-7 cells. The binding profiles of a series of acetylcholine derivatives suggest that the altered threonine and tyrosine residues are primarily involved in the interaction of the acetylcholine ester moiety with the receptor protein. The two m3 receptor point mutants, Thr234----Ala and Tyr506----Phe, which showed the most pronounced decreases in acetylcholine binding affinities (approximately 40-60-fold as compared with the wild-type receptor), were stably expressed in CHO cells for further functional analysis. Both mutant receptors were found to be severely impaired in their ability to stimulate agonist-dependent phosphatidylinositol hydrolysis. Consistent with this observation, acetylcholine binding to the two mutant receptors was not significantly affected by addition of the GTP analog Gpp(NH)p (5'-guanylyl imidodiphosphate). Our data suggest that Thr234 and Tyr506 (located within transmembrane domains V and VI, respectively), which are conserved among all muscarinic receptors (m1-m5), may play an important role in agonist-induced muscarinic receptor activation.     

Regulation of principal cell pH by Na/H exchange in rabbit cortical collecting tubule

Summary Changes in intracellular pH (pH _i ) were measured using the pH indicator, BCECF, in principal cells from split opened cortical collecting tubules (CCTs) derived from rabbits maintained on a normal diet. This monolayer preparation has the advantage of allowing us to visualize the morphological differences in the two major cell types in this nephron segment under transmitted light. The visual identification of the cell types was verified using emission measurements taken from single principal and intercalated cells in the opened tubule which had been exposed to fluorescein isothiocyanate (FITC)-labeled peanut lectin. We confirmed the existence of an amiloride-sensitive Na/H exchange process activated during intracellular acidosis in principal cells. In addition, the exchanger was active under basal conditions and over a wide range of pH _i . Because the exchanger was active under basal conditions we tested the hypothesis that changes in intracellular Na (Na _i ) would alter pH _i in a predictable way. Maneuvers designed to alter Na _i were without significant effects within a 10-min time frame. Specifically, addition of 100 m ouabain to increase Na _i or exposure of the tubules to 10^–5 m amiloride to decrease luminal Na entry and reduce Na _i did not have an effect on pH _i . In some experiments we did observe however, after a 30-min exposure to ouabain, a small decrease in pH _i . These results suggest that Na/H exchange is a major regulator of pH _i in principal cells. However, regulation of Na transport by changes in pH _i in principal cells of rabbit CCT via the activity of a Na/H exchanger do not seem to contribute to the feedback control of Na transport.This work was supported by U.S. Public Health Service grants DK27847 to L.G. Palmer and DK11489 to E.E. Windhager.     

The selection of an adjuvant emulsion for polyclonal antibody production using a low-molecular-weight antigen in rabbits.

Although Freund's adjuvant has been used for decades as an immune enhancer in rabbits, adverse physiologic side effects have prompted the search for more suitable alternatives. We used osteocalcin, a bovine bone protein (M.W. 5,800), as the test antigen to evaluate four adjuvant regimens: a) primary inoculation with complete Freund's adjuvant (CFA) followed by three boosts with incomplete Freund's adjuvant (IFA), b) four serial inoculations with RIBI MPL+TDM+CWS adjuvant, c) four serial inoculations with TiterMax #R-1, and d) primary inoculation (only) with TiterMax #R-1. The antibody yield associated with the CFA/IFA regimen (mean OD = 2.152) was at least sixfold that of either TiterMax (mean OD = 0.358) or RIBI (mean OD = 0.239) multiple injection regimens. No antibody response was observed after the single injection of TiterMax antigen emulsion. Maximal antibody production occurred rapidly in response to Freund's adjuvant (day 31) as compared with TiterMax (day 74) and RIBI (day 66).