Target size analysis of neurotensin receptors

The neurotensin receptors from rat brain synaptosomal membranes differed in subunit structure from those in rat gastric fundus smooth muscle plasma membranes when studied following photoaffinity labelling or after exposure to cross-linking reagents. However, these two receptors were similar in their recognition properties. In this study we compared the target size of the two receptors by radiation inactivation and observed that the receptors in both tissues had similar target sizes (mean values 103,000 and 108,000 daltons). This suggests that the differences in size observed in biochemical studies may reflect changes occurring during the isolation procedures, or, on the other hand, there might be inherent difference in the subunit structure of these receptors.     

Carboxylmethylation of Calmodulin in Cultured Pituitary Cells

We have used fast protein liquid chromatography (FPLC) and reverse-phase HPLC to rapidly resolve carboxylmethylated proteins in cultured pituitary GH3 cells. This procedure preserves labile carboxylmethyl esters, which are lost under the usual procedures employed for protein fractionation. GH3 cells were incubated with [methyl-3H]-methionine in culture and incorporation of label into the soluble fraction, total cell protein, and protein carboxylmethyl esters was determined; protein carboxylmethyl ester formation was shown to be resistant to cycloheximide. Fractionation of protein carboxylmethyl esters from GH3 cells by gel permeation FPLC, anion-exchange FPLC, and reverse-phase HPLC in the presence of calcium and in the presence of EGTA identified two proteins that are major substrates for protein carboxylmethyltransferase and indicated that one of these proteins is calmodulin. Similar results were obtained when a cytosolic fraction from GH3 cells was incubated with S-adenosyl-L-[methyl-3H]methionine. These results indicate that rapid chromatography at low temperature and low pH is useful for the analysis of eucaryotic carboxylmethylated proteins and that contrary to reports obtained in other systems, calmodulin is carboxylmethylated in intact pituitary cells.     

Cortical activity in vertebrate eggs. I: The activation waves

We present a physical model for the propagation of chemical and mechanical waves on the surface of vertebrate eggs. As a first step we analyzed the propagation of the calcium wave observed to sweep over the surface of the Medaka egg (Gilkey et al., 1978). It has been assumed that this wave is driven by a mechanism of calcium-stimulated-calcium-release. By formulating this hypothesis mathematically we can use the observed wavefront data to obtain a map of cortical reactivity. This map indicates a gradient of reactivity along the egg: highest in the animal hemisphere and tapering off towards the vegetal hemisphere. The cortex of Xenopus eggs is also capable of propagating a calcium wave (Busa & Nuccitelli, 1985). At about the same time a wave of expansion followed by a wave of contraction sweeps across the egg surface (Takeichi et al., 1984). We have proposed a mechanism for this wave pair based on the physical chemistry of actomyosin gels. The calcium wave activates solation factors which sever some of the actin chains which leads to an osmotic swelling of the gel. Calcium also activates the contractile machinery of the actomyosin system which causes the gel to contract. The contraction lags the swelling because of the nature of the kinetics: solation and swelling is a more rapid process than contraction. By writing the equations for gel expansion and contraction we can mimic the mechanical and chemical wave propagation by a computer simulation. If the model is correct this provides a method for using the waves as a diagnostic of the mechanochemical properties of the egg cortex.     

The effect of muscarinic and beta-adrenergic blockade on cysteamine-induced gastrin secretion by the isolated perfused rat stomach

Cysteamine-induced duodenal ulceration in rats is accompanied by increased circulating gastrin. Although cysteamine appears to exert a direct action on the gastrin cell some groups have provided evidence for an involvement of the autonomic nervous system. The current experiments were performed to determine whether beta-adrenergic or cholinergic (muscarinic) pathways are involved in the acute effect of cysteamine on gastrin secretion in the isolated perfused rat stomach. Cysteamine (1 mM) increased gastrin (IRG) secretion to a maximum ranging between 100% and 192% above basal. A cysteamine concentration of 5mM resulted in peak levels ranging between 150% and 1050% above basal. Addition of atropine or propranalol did not influence the responses obtained. The present results, therefore, do not support a role for either cholinergic or beta-adrenergic pathways in cysteamine-induced gastrin release at the level of the stomach and suggest that in vivo such autonomic effects are mediated extrinsically.     

High-performance liquid chromatography of type-III heptocarboxylic porphyrinogen isomers.

A reversed-phase h.p.l.c. system is described for the separation of the four type-III heptacarboxylic porphyrinogen isomers. The effects of buffer concentration, pH and type and proportion of organic modifier in the mobile phase on retention and resolution of isomers were studied. Optimum separation on an ODS-Hypersil column was by elution with a ternary mobile phase of acetonitrile, methanol and 1 M-ammonium acetate, pH 5.16 (7:3:90, by vol.). Isomer identification was based on a comparison of their retention times with those of authentic standards, and was further confirmed by h.p.l.c. analysis of the characteristic mixture of three pentacarboxylic porphyrins formed after partial decarboxylation of individual isomers in 0.3 M-HCl at 160 degrees C.     

A photoaffinity label for the thromboxane A2/prostaglandin H2 receptor in human blood platelets

A photoactive iodoarylazide derivative (I-APA-PhN3) of the competitive thromboxane A2/prostaglandin H2 (TXA2/PGH2) antagonist 13-azaprostanoic acid is evaluated. Upon photoactivation, the compound was found to inhibit specifically and irreversibly human platelet aggregation induced by the TXA2/PGH2 mimetic U46619. In receptor-binding studies using [3H]U46619, I-APA-PhN3 exhibited an IC50 of 300 nM for inhibition of U46619 binding. Photoactivation of I-APA-PhN3 resulted in an irreversible 58% reduction in specific binding of U46619. This compound and its corresponding ratio-iodinated form will prove to be useful tools for the isolation and purification of the TXA2/PGH2-binding protein in human platelets.     

Evidence that arginyl-glycyl-aspartate peptides and fibrinogen gamma chain peptides share a common binding site on platelets

Synthetic peptides corresponding to the carboxyl terminus of the fibrinogen gamma chain inhibit the binding of fibrinogen, fibronectin, and von Willebrand factor to platelets, yet the active decapeptide sequence has only been found in fibrinogen to date. In contrast, all three proteins contain Arg-Gly-Asp sequences, and peptides containing Arg-Gly-Asp are potent inhibitors of their binding to activated platelets. We have analyzed the relationship between these peptide sets by direct binding assays. H12 (gamma 400-411) inhibited the binding of an Arg-Gly-Asp-containing peptide to platelets with similar dose response to inhibition of fibronectin binding. We have previously reported that GPIIb-IIIa binds to immobilized Arg-Gly-Asp peptides and can be eluted by Arg-Gly-Asp-containing peptides in solution. Both H12 and L10 (gamma 402-411) completely eluted GPIIb-IIIa bound to immobilized Arg-Gly-Asp peptides. Conversely, when GPIIb-IIIa was bound to immobilized L10, either L10 or an Arg-Gly-Asp peptide could elute it. Peptide specificity was established by the failure of Gly-Arg-Gly-Glu-Ser-Pro or acetylated L10 to elute GPIIb-IIIa from the immobilized peptides. These results indicate that the two peptide sets interact with the same receptor which contains GPIIb-IIIa.     

Trifluoperazine Activates and Releases Latent ATP-Generating Enzymes Associated with the Synaptic Plasma Membrane

Neurone-specific enolase (NSE) and the brain form of creatine phosphokinase (CPK-BB) were previously found to be present in rat synaptosomal plasma membranes (SPM) using two-dimensional gel (2-D gel) and peptide analysis; enzymatic activities of these and of pyruvate kinase (PK), all involved in ATP generation, were shown to be "cryptic" unless the SPM were treated with Triton X-100. We now show that enzymatic activation also occurs when the SPM are treated with trifluoperazine (TFP). TFP activation occurred even when the enzymes were membrane associated, showing that solubilization was not responsible for "unmasking" the enzyme activities. When TFP treatment was performed at alkaline instead of neutral pH, NSE and CPK-BB were released as well as PK, nonneuronal enolase, and aldolase which were identified by 2-D gel and tryptic peptide analysis. Other proteins released included calmodulin, actin, and the 70-kilodalton heat-shock cognate protein. Tubulin, synapsin I, and a 35-kilodalton basic protein were largely unaffected. The latter was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase on the basis of 2-D gel and peptide analyses and subsequent partial sequencing of a rat brain cDNA coding for the same protein. TFP treatment is thus useful for activating latent enzymes as well as for distinguishing enzymes that have a different disposition on the membrane.     

Characterization by cytofluorometry of the different types of adenohypophyseal cells in the rat

The different antehypophysical cell types which synthetize and release somatotroph (GH), corticothroph (ACTH), gonadotroph (LH-FSH) and lactotroph (PRL) hormones were analysed. The experiments were performed on hypophyses from five groups of animals: adult males, 14 days-old female, adult females, gestating adult females and lactating adult females. The cells were analysed by immunofluorescence using flow cytometry. For each of the hormones studied, there was a characteristic spectral distribution of cells. The evolution of cell size and granular content with respect to sex and physiological state of each group was studied by the analysis of diffused light. Small, slightly granular cells represented 50% of the cell population in males and 14 day-old females but only 8% in gestating or lactating females. The study of the cell cycle showed the presence of dividing cells in the population of large, granular cells from gestating and from lactating females. No features of cell division were observed in the population of small, slightly granular cells. This study indicates the potential value of multiparametric analysis in the separation of pure sub-populations of antehypophysial cells.