Intracellular Ca2+ concentration and the antidiuretic hormone-induced increase in water permeability: effects of ionophore A23187 and quinidine

The hydroosmotic responses induced by oxytocin and 8-bromo-cyclic AMP, in frog and toad urinary bladders, were recorded minute by minute. 3HHO and 45Ca unidirectional fluxes as well as prostaglandin B2 liberation were also measured. It was observed that: (1) Addition of the calcium ionophore A23187 or quinidine to the serosal bath inhibited the response to oxytocin, but not to 8-bromo-cyclic AMP, while increasing prostaglandin E1 liberation into the serosal but not into the mucosal bath. (2) Addition of A23187 to the mucosal bath induced a transient and temperature-dependent inhibition of the response elicited by 8-bromo-cyclic AMP. The time-course of this reduction in water permeability and its sensitivity to medium temperature were similar to those observed after the withdrawal of agonist, but clearly different of those observed after intracellular acidification. (3) The hydroosmotic response was also transitorily inhibited when the Ca2+ concentration was step-changed in the mucosal bath. (4) When added to the mucosal or to the serosal baths, the ionophore increased either the apical or the laterobasal Ca2+ permeabilities. It is concluded that manipulation of intracellular Ca2+ interferes with the hydroosmotic response at two different levels. (1) A first target point located 'pre-cyclic-AMP production'. This effect would be mediated by prostaglandin liberation. (2) A second target point located after cyclic AMP production and before the 'temperature-dependent rate-limiting step'. This effect is probably related to the mechanism controlling the insertion and removal of water channels.     

Detection by ELISA of Two Tobamoviruses in Orchids Using Monoclonal Antibodies

Five monoclonal antibodies (McAbs) were raised to the tobamovirus, odontoglossum ringspot virus (ORSV). All five McAbs reacted with the virus in double antibody sandwich (DAS) ELISA but not in an ELISA using virus-coated plates. All the McAbs recognized a panel of ORSV strains and isolates, although one of the antibodies reacted better with some isolates and another reacted less with certain isolates than with type ORSV. It was possible to use the same McAbs both as coating and as biotinylated antibody in DAS-ELISA. None of the five McAbs was able to bind to orchid strains of tobacco mosaic virus (TMV). In order to detect strains of both viruses, ORSV and TMV, in infected orchids it was necessary to include also McAbs raised against TMV in the immunoassays. The use of a mixed polyclonal-monoclonal antibody DAS-ELISA system is advocated for detecting both tobamoviruses in orchids.     

Synthesis of rhodanese in Hep 3B cells

Summary The biosynthesis of rhodanese was studied in human hepatoma cell lines by immunoblotting and pulselabeling experiments using polyclonal antibodies raised against the bovine liver enzyme. Rhodanese, partially purified from human liver, showed an apparent molecular weight of 33,000 daltons, coincident with that of rhodanese from Hep 3B cells. After pulse labeling of Hep 3B cells both at 37°C and 25°C, rhodanese in the cytosol fraction exhibited the same molecular weight as the enzyme isolated from the particulate fraction containing mitochondria. Moreover, newly synthesized rhodanese from total Hep 3B RNA translation products showed the same electrophoretic mobility as rhodanese from Hep 3B cells. These results suggest that rhodanese, unlike most mitochondrial proteins, is not synthesized as a higher molecular weight precursor.     

Development of tool use in a macaque and a gorilla

The development of the capacity to use a stick as a tool was tested in a macaque (Macaca fuscata) and a gorilla (Gorilla gorilla gorilla) infants that had previously shown to be able to use strings and supports as dragging tools. Subjects were tested between 15 and 38 months of age. Different levels of competence between the subjects emerged over testing. The macaque developed a stereotyped strategy to cope with the problem, only getting random successes, whilst the gorilla developed a flexible strategy and revealed to be able to mentally represent the solution of the problem. In fact, when not successful using the stick, the gorilla thought out an alternative strategy, choosing and adapting a new object to use it as a tool.     

Analysis of X-chromosome inactivation and presumptive expression of the Wiskott-Aldrich syndrome (WAS) gene in hematopoietic cell lineages of a thrombocytopenic carrier female of WAS

Summary We report on a thrombocytopenic female belonging to a pedigree with the Wiskott-Aldrich syndrome (WAS). Restriction fragment length polymorphism (RFLP) analysis with probe M27, closely linked to the WAS gene, demonstrated that she is a carrier of WAS. Both small-sized and normal-sized platelets were present, suggesting that, unlike the vast majority of WAS carriers, she does not manifest nonrandom X-chromosome inactivation in the thrombopoietic cell lineage. Study of X-chromosome inactivation by means of RFLP and methylation analysis demonstrated that the pattern of X-chromosome inactivation was nonrandom in T lymphocytes, but random in granulocytes. While this is the first complete report on the occurrence of thrombocytopenia in a carrier female of WAS as the result of atypical lyonization, it also suggests that expression of the WAS gene occurs at (or extends up to) a later stage than the multipotent stem cell along the hematopoietic differentiation pathway.     

Substance P as a transglutaminase substrate: identification of the reaction products by fast atom bombardment mass spectrometry

Substance P was found to be an effective acyl donor substrate of transglutaminase in vitro, the reaction products having been examined by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fast atom bombardment mass spectrometry. Electrophoretic experiments showed that Substance P incorporated 14C-labeled polyamines when incubated with purified guinea pig liver transglutaminase and Ca2+. Extensive use of fast atom bombardment mass spectrometry allowed to establish that: i) a 1:1 adduct Substance P-spermine is formed; ii) only a single glutamine residue out of two, i.e. Gln-5, acts as acyl donor, iii) the single lysine residue of the neuropeptide is unable to act as acyl acceptor. A direct analytical methodology to detect transglutaminase reaction products is described.     

Hydrochlorothiazide enhances the apical Cl− backflux in rabbit gallbladder epithelium: Radiochemical analysis

Hydrochlorothiazide (HCTZ) was shown to inhibit the transepithelial NaCl transport and the apical Na⁺-Cl^? symport and to depolarize the apical membrane potential in the rabbit gallbladder epithelium. The depolarization was likely related to the opening of a Cl^? conductance. To better understand whether an apical Cl^? leak is involved in the mechanism of action of HCTZ, the transapical Cl^? backflux was measured radiochemically by the washout technique. The gallbladder wall, pretreated with pronase on the serosal side to homogenize the subepithelium, was loaded with ³⁶Cl^? on the luminal side; mucosal and serosal ³⁶Cl^? effluxes (J _m , J _s ) were then measured every 2 min. The pretreatment with pronase did not alter the membrane potentials and the selectivity of the epithelium. Under control conditions and the tissue in steady-state, J _m and J _s time courses were each described by two exponential decays (A,B); the rate constants, k ^A and k ^B , were 0.71 ±0.03 and 0.16±0.01 min^?1, respectively, and correspondingly the half-times (t ₁ ^2A , t ₁ ^2B ) were 1.01±0.05 and 5.00±0.44 min (n=10); these parameters were not significantly different for J _m and J _s time courses. J _s was always greater than J _m (J _s /J _m =2.02±0.22 and 1.43 ±0.17 for A and B decays). Under SCN^? treatment in steady-state conditions, both J _m and J _s time courses were described by only one exponential decay, the component B being abolished. Moreover t ₁ ^2A was similar to that predictable for the subepithelium. It follows that it is the component B which exits the epithelial compartment. Based on the intracellular specific activity and ³⁶Cl^? J _m ^B at 0 min time of the washout experiment, the cell-lumen Cl^? backflux in steady-state was calculated to be equal to about 2 μmol cm^?2hr^?1, in agreement with the value indirectly computable by other techniques. The experimental model was well responsive to different external challenges (increases in media osmolalities; luminal treatment with nystatin). HCTZ (2.5 · 10^?4 m) largely increased ³⁶Cl^? J _m ^B . The increase was abolished by luminal treatment with 10^?4 m SITS, which not only brought back the efflux time courses to the ones observed under control conditions but even increased J _s /J _m of the cellular component, an indication of a reduced J _m ^B . It is concluded that HCTZ opens an apical, SITS-sensitive Cl^? leak, which contributes to dissipate the intracellular Cl^? accumulation and to inhibit the NaCl transepithelial transport. Moreover, the drug is likely to reduce the basal electroneutral Cl^? backflux supported by Na⁺-Cl^? cotransport, in agreement with the inhibition of the cotransport itself.     

Hydrochlorothiazide action on the apical Cl−, Ca2+ and K+ conductances in rabbit gallbladder epithelium. Presence of an apamin-sensitive,Ca2+-activated K+ conductance

In the rabbit gallbladder epithelium, hydrochlorothiazide (HCTZ) was shown to inhibit the transepithelial NaCl transport and the apical Na⁺-Cl^– symport, to depolarize the apical membrane potential and to enhance the cell-to-lumen Cl^– backflux (radiochemically measured), this increase being SITS-sensitive. To better investigate the causes of the depolarization and the Cl^– backflux increase, cells were punctured with conventional microelectrodes on the luminal side (incubation in bicarbonate-free saline at 27°C) and the apical membrane potential (V _m) was studied either with prolonged single impalements or with a set of short multiple impalements. The maximal depolarization was of 3–4 mV and was reached with 2.5 × 10^–4 m HCTZ. It was significantly enhanced by reducing luminal Cl^– concentration to 30 mm; it was abolished by SCN^–, furosemide, SITS; it was insensitive to DPC. SITS converted the depolarization into a hyperpolarization of about 4 mV; this latter was apamin, nifedipine and verapamil sensitive. It was concluded that HCTZ concomitantly opens apical Cl^– and (probably) Ca²⁺ conductances and, indirectly, a Ca²⁺-sensitive, apamin inhibitable K⁺ conductance: since the intracellular Cl^– activity is maintained above the value predicted at the electrochemical equilibrium, the opening of the apical Cl^– conductance depolarizes V _mand enhances Cl^– backflux. In the presence of apamin or verapamil, to avoid the hyperpolarizing effects due to HCTZ, the depolarization elicited by this drug was fully developed (7–10 mV) and proved to be Ca²⁺ insensitive. On this basis and measuring the transepithelial resistance and the apical/basolateral resistance ratio, the Cl^– conductance opened by HCTZ has been estimated and the Cl^– backflux increase calculated: it proved to be in the order of that observed radiochemically. The importance of this Cl^– leak to the lumen in the overall inhibition of the transepithelial NaCl transport by HCTZ has been evaluated.This research was supported by Ministero dell'Università e della Ricerca Scientifica e Tecnologica, Rome, Italy. We are very grateful to prof. G. Meyer and dr. G. Bottà for helpful discussion and criticism.