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Biomedical subjects

M Trueba

Publications and source records attributed to M Trueba.

At least 19 recordsLinked to original sources
PubMed1998

Vasopressin regulates adrenal functions by acting through different vasopressin receptor subtypes.

In mammals, vasopressin is known to be synthesized in the hypothalamus and released in the blood stream at the pituitary level. This neuropeptide is also synthesized and secreted by the adrenal medulla in many species including human. Moreover, agents like acetylcholine and corticotropin releasing factor stimulates its basal secretion. V1a vasopressin receptors are present in the adrenal cortex and are involved in steroids secretion (aldosterone in the zona glomerulosa and glucocorticoids in the zona fasciculata of some species). These receptors are coupled to phospholipase C beta and to dihydropyridine-sensitive calcium channels via heterotrimeric G proteins differing by their sensitivities to pertussis toxin. The adrenal medulla, from many species, exhibits V1a vasopressin receptors. In rat adrenal medulla, functional V1b vasopressin receptors could also be characterized. These receptors stimulate catecholamines secretion via activation of phospholipase C beta and subsequent mobilization of intracellular calcium. The adrenal medulla secretes AVP and exhibits functional vasopressin receptors. The adrenal cortex also possesses functional vasopressin receptors and is in contact with adrenal medulla via "medullary rays". We may thus reasonably conclude that AVP physiologically regulates adrenal gland functions via autocrine/paracrine mechanisms.

Adrenal Cortex↗
PubMed1997

Characterization of specific corticosterone binding sites in adrenal cortex plasma membrane and their localization by autoradiographic studies.

Specific corticosterone binding to calf adrenal cortex plasma membrane was measured using the biologically active radioligand [3H]corticosterone. Corticosterone binding was found to be time-dependent, saturable and reversible, and was reduced by more than 70% when membranes were pretreated with proteases. The population of corticosterone binding sites in calf adrenal cortex plasma membrane was homogeneous and displayed the following characteristics: equilibrium dissociation constant Kd = 77 +/- 8 nM and maximum specific binding capacity Bmax = 70,378 +/- 6,385 fmol/mg protein. The relative affinities of several structural analogues of steroids were deduced from competition assays. From these experiments we can conclude that the plasma membrane binding site characterized is selective for corticosterone and progesterone derivatives, and different from nuclear glucocorticoid, mineralocorticoid, estrogen and progestin receptors. Likewise, this corticosterone binding site is independent of mineralocorticoid and Na+, K(+)-ATPase digitalis receptors. From autoradiographic studies we suggest these corticosterone binding sites are located in the whole adrenal cortex.

Adrenal Cortex↗
PubMed1996

Purification of a cortisol binding protein from hepatic plasma membrane.

A cortisol binding protein from rat liver plasma membranes has been solubilized in active form by using the zwitterionic detergent CHAPS. Two types of binding sites have been characterised in both native and solubilized membranes. The first is of high affinity and low binding capacity (12 nM; 946 fmol/mg) and the other one is of low affinity and high capacity of binding (344 nM; 12677 fmol/mg) for solubilized membranes. The purified material retained a binding activity comparable to that displayed by the original membrane. The specific binding activity was enriched about 12700-fold, with an 8% yield. Analysis of the purified preparation on sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed two protein subunits with molecular mass of 52000 and 57000 Da. The new cortisol-specific binding membrane protein could be related to the nongenomic effects previously described for this hormone.

Animals↗
PubMed1996

Thrombin-stimulated phospholipase C activity is inhibited without visible delay by a rapid increase in the cyclic GMP levels induced by sodium nitroprusside.

Different drugs that elevate the cGMP levels inhibit the agonist-induced platelet activation. The mechanisms of action of cGMP probably include inhibition of both phospholipase C and the increase in intracellular Ca2+ concentration, and these effects seem to be mediated by cGMP-dependent protein kinases. However, in most studies, cells were preincubated with nitrovasodilators before stimulation. The effect of the preincubation with sodium nitroprusside before stimulation or the simultaneous addition of sodium nitroprusside and thrombin has been compared. The simultaneous addition of sodium nitroprusside and thrombin was able to inhibit without any significant delay the platelet aggregation. This rapid effect was correlated with an inhibition of both the maximum increase in intracellular Ca2+ concentration and the phospholipase C activity. Also, the simultaneous addition of sodium nitroprusside and thrombin clearly accelerated the decline in the Ca2+ signal, which was not observed in platelets preincubated with sodium nitroprusside. The rapid inhibition induced by sodium nitroprusside was correlated with a rapid and significant increase in the cGMP levels and reversed when platelets were pretreated with methylene blue. The inhibitor of cAMP-dependent protein kinase Rp-8-(4-chlorophenylthio)-adenosine-3',5'-cyclic monophosphorothioate was able to abolish nearly completely the inhibitory effect induced by sodium nitroprusside independent of the protocol used. Thus, the rapid inhibition induced by sodium nitroprusside seems to be induced by a rapid phosphorylation-dependent mechanism. In addition, both cGMP- and cAMP-dependent protein kinase seem to be involved; however, the cAMP-dependent protein kinase seems to be more important.

Calcium↗
PubMed1995

Vasopressin stimulates steroid secretion in human adrenal glands: comparison with angiotensin-II effect.

Autoradiographic experiments using iodinated vasopressin analog revealed the presence of specific vasopressin-binding sites in the human adrenal cortex (zona glomerulosa and zona fasciculata). These receptors exhibited a good affinity for arginine vasopressin (3.3 nM), with classical V1a pharmacology and densities of 65 and 135 fmol/mg protein-enriched membranes from zona glomerulosa and fasciculata, respectively. Vasopressin receptors present in both glomerulosa and fasciculata cell-enriched primary cultures were coupled to phospholipase C (ED50, 0.9 and 1.8 nM; maximal stimulation, 4.3- and 5.8-fold, respectively). Vasopressin also stimulated an increase in intracellular calcium through at least two distinct mechanisms: the mobilization of intracellular pools via vasopressin-stimulated inositol phosphate accumulation and the activation of calcium influx. In glomerulosa cell-enriched primary cultures, vasopressin increased aldosterone secretion (ED50, 0.4 nM; maximal stimulation, 2.5-fold) and was found to be as potent as angiotensin-II in stimulating aldosterone secretion, phosphoinositide turnover, and calcium mobilization. In fasciculata cells, vasopressin and angiotensin-II were also able to stimulate cortisol secretion and inositol phosphate accumulation. Moreover, perifusion experiments demonstrated that vasopressin was released from the adrenal medulla. Together, these results indicate that vasopressin can be considered a potent paracrine modulator of adrenal steroid secretion in man.

Adrenal Glands↗
PubMed1993

Ni2+ impairs thrombin-induced signal transduction by acting on the agonist and/or receptor in human platelets.

We have investigated the effect of NiCl2 on platelet activation induced by thrombin, phorbol 12-myristate 13-acetate, and calcium ionophores. Besides blocking Ca2+ influx, NiCl2 inhibited platelet aggregation, intracellular Ca2+ mobilization, and phospholipase C activation induced by thrombin in a dose-dependent manner. In contrast to ionomycin, NiCl2 completely blocked the platelet aggregation and intracellular Ca2+ mobilization induced by A23187. A23187 was not able to translocate Ni2+ across the plasma membrane. Ni2+ also inhibited phorbol myristate acetate-induced platelet aggregation. The results with staurosporine and low NiCl2 concentrations are in agreement in that increases in intracellular Ca2+ concentration and protein kinase C activation are necessary for full platelet activation mediated by thrombin.

Alkaloids↗
PubMed1992

Characterization by photoaffinity labeling of a steroid binding protein in rat liver plasma membrane.

The mechanism of steroid uptake by the cell remains controversial. [3H]R5020 was utilized to characterize by photoaffinity labeling the steroid binding site in plasma membrane. This binding was saturable, reversible and had one type of binding site (Kd = 33 +/- 4 nM, Bmax = 32 +/- 2 pmol/mg). [3H]R5020 could be prevented from binding by a variety of steroids (cortisol, progesterone, deoxycorticosterone, and levonorgestrel); estradiol did not have affinity for this binding site. The kinetics of R5020 photoactivation was time dependent and saturable. SDS-PAGE showed a specific band which corresponded to a 53-kDa peptide. The sucrose density gradient analysis has revealed the existence of a protein with a sedimentation coefficient of 3.6 +/- 0.2 S. This polypeptide shows different characteristics than cytosolic steroid receptor or serum steroid binding proteins. This binding protein could correspond to the steroid binding site previously found in the plasma membrane.

Affinity Labels↗
PubMed1992

Intracellular Ca2+ mobilization and not calcium influx promotes phorbol ester-stimulated thromboxane A2 synthesis in human platelets.

Phorbol esters, potent activators of protein kinase C (PKC), greatly enhance the release of arachidonic acid and its metabolites (TXA2, HETES, HHT) by Ca2+ ionophores in human platelets. In this paper, we report the relationship between intracellular Ca2+ mobilization and external calcium influx into platelets and the ability of PMA plus A23187 to promote thromboxane A2 (TXA2) synthesis. The enhanced levels of TXA2 due to the synergistic stimulation of the platelets with A23187 and phorbol esters are not affected significantly by the presence of external Ca2+ or the calcium-chelator EGTA. PKC inhibitors, staurosporine and sphingosine, abolished phorbol myristate acetate (PMA) potentiation of TXA2 production which strongly supports the role of PKC in the synergism. Platelet aggregation is more sensitive to PMA and external calcium than TXA2 formation. PMA increased TXA2 production as much as 4-fold at low ionophore concentrations. The A23187-induced rise in [Ca2+]i was reduced by pretreatment of human platelets with phorbol esters, both in the presence and absence of EGTA, and staurosporine reversed this inhibitory effect. These results indicate that the synergistic stimulation of TXA2 production by A23187 and phorbol esters is promoted by intracellular Ca2+ mobilization and not by external calcium influx. Our data also suggest that PKC is involved in the regulation of Ca2+ mobilization from some specific intracellular stores and that PKC may also stimulate the Ca(2+)-dependent phospholipase A2 at suboptimal Ca2+i concentrations.

Alkaloids↗
PubMed1991

Steroid hormone specifically binds to rat kidney plasma membrane.

A high-affinity and low-capacity corticosterone specific binding was detected in the purified plasma membrane preparation from rat kidney using an in vitro steroid hormone binding assay. The specific-bound hormone was efficiently distinguished from the irreversible-bound hormone with 10 microM corticosterone. Under standardized conditions of pH 7.4 at 2 degrees C and 30 min incubation time, the binding was saturable and showed Kd = 13 +/- 3 nM and Bmax = 616 +/- 34 fmol/mg of protein. Competitive binding studies with analogue steroids indicated that corticosterone binding to kidney plasma membrane is hormone-specific. Results indicated that the possible nongenomic effects of steroids could be mediated by their interaction with plasma membrane.

Animals↗
PubMed1991

Specific binding sites for corticosterone in isolated cells and plasma membrane from rat liver.

The specific binding of [3H]corticosterone to hepatocytes is a nonsaturable, reversible and temperature-dependent process. The binding to liver purified plasma membrane fraction is also specific, reversible and temperature dependent but it is saturable. Two types of independent and equivalent binding sites have been determined from hepatocytes. One of them has high affinity and low binding capacity (KD = 8.8 nM and Bmax = 1477 fmol/mg protein) and the other one has low affinity and high binding capacity (KD = 91 nM and Bmax = 9015 fmol/mg). In plasma membrane only one type of binding site has been characterized (KD = 11.2 nM and Bmax = 1982 fmol/mg). As it can be deduced from displacement data obtained in hepatocytes and plasma membrane the high affinity binding sites are different from the glucocorticoid, progesterone nuclear receptors and the Na+,K(+)-ATPase digitalis receptor. Probably it is of the same nature that the one determinate of [3H]cortisol and [3H]corticosterone in mouse liver plasma membrane. Beta- and alpha-adrenergic antagonists as propranolol and phentolamine did not affect [3H]corticosterone binding to hepatocytes and plasma membranes; therefore, these binding sites are independent of adrenergic receptors. The binding sites in hepatocytes and plasma membranes are not exclusive for corticosterone but other steroids are also bound with very different affinities.

Animals↗
PubMed1991

Dual effects of ATP on phosphatidylinositol breakdown in rat hepatocyte membranes.

The mechanisms whereby adenosine-5'-triphosphate (ATP) regulates the inositol phospholipid-signalling system were studied in rat hepatocytes. Intact hepatocytes respond to extracellular ATP, adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S), ADP and weakly to guanosine-5'-triphosphate (GTP), but not to other purine nucleotides (GDP or AMP). This is consistent with the idea that a P2 purinergic receptor is coupled to the phosphatidylinositol metabolism in these cells. Partially purified plasma membranes prepared from myo-[3H]inositol prelabelled hepatocytes exhibit a phosphatidylinositol-4,5-bisphosphate phospholipase C activity sensitive to ATP, ATP gamma S and guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S). Moreover the GTP gamma S effect is greatly enhanced by ATP and ATP gamma S. These potentiating effects differ according to the adenylnucleotide considered. ATP produces (1) an increase in the GTP gamma S-PLC sensitivity, (2) a potentiation of the phospholipase C (PLC) response induced by maximal dose of GTP gamma S, and (3) an increase in the inositol lipids pools. At variance, ATP gamma S, a nonhydrolysable analogue of ATP, only increases the PLC-sensitivity towards GTP gamma S. These results may signify that ATP stimulates inositol phosphate accumulation via at least two distinct mechanisms (i) a direct activation of a P2 purinergic receptor coupled to a PLC via a GTP binding protein and (ii) a stimulation of the phosphatidylinositol (PI) and phosphatidylinositol-4-phosphate (PIP) kinases which increased the pool of phospholipase C substrates.

Adenosine Triphosphate↗
PubMed1990

Binding of progesterone to specific sites in isolated hepatic cells and purified plasma membrane fraction.

Specific binding for progesterone has been determined in rat hepatocytes and mouse liver purified plasma membranes. The binding is saturable, reversible and temperature dependent. Two types of binding sites have been characterized in hepatocytes. The first is of high affinity and low binding capacity and the other one is of low affinity and high capacity of binding. In plasma membranes one type of specific binding site only exists. These high affinity sites are different from nuclear progesterone receptor, nuclear glucocorticoid receptor, digitalis receptor of Na+, K(+)-ATPase, transcortine and from corticoid binding sites determined previously in plasma membrane. We also have observed that specific progesterone binding to hepatocytes and plasma membrane is independent from the alpha and beta adrenergic receptors and from P-site adenosine receptor.

Animals↗
PubMed1990

Calcium-activated, phospholipid-dependent protein kinase activity in calf platelets.

Protein kinase C (PKC) has been widely studied from different tissues of mammals. Human platelets display higher levels of PKC activity, if compared with other sources. The PKC activity from calf platelets crude extract was determined in the presence of various protease inhibitors such as PMSF, Leupeptin or Trypsin inhibitro, and the Ca(2+)-chelators EGTA and EDTA. The free calcium requirement was 0.25 mM, calculated with the help of the Solgas-water computer program, which represents 1 mM CaCl2, in these assay conditions. Optimum PKC activity was obtained at 4 min in the presence of PS plus DAG or TPA, using H1 type III-S histone as substrate. Phospholipid-interacting drugs, such as trifluoperazine, chlorpromazine and tetracaine, inhibited the PKC activity in a dose-dependent manner. Triton X-100, a non-ionic detergent, which is usually employed to solubilize the membrane fraction, in different translocation assays, inhibited PKC activity at concentrations higher than 0.01%. In these conditions, non-proteolytic PKC activity from calf platelets was easily determined, and shares similar activity levels with those described in human platelets.

Animals↗
PubMed1989

Evidence for the presence of specific binding sites for corticoids in mouse liver plasma membranes.

The specific binding of [3H]cortisol to plasma membranes purified from mouse liver, studied by the ultrafiltration method, shows the existence of specific binding sites for cortisol. The kinetic parameters of this binding are KD = 4.4 nM and Bmax = 685 fmol/mg protein in presence of 1 microM of corticosterone. With respect to the binding of 4 nM [3H]cortisol to the membrane, the affinities of the steroids decreased in the following order: deoxycorticosterone greater than corticosterone greater than progesterone greater than cortisol greater than prednisolone greater than testosterone greater than 20 beta-hydroxyprogesterone greater than cortisone. Estradiol, dexamethasone, ouabain and triamcinolone acetonide do not have affinity for this binding site. Neither Ca2+ nor Mg2+ affected the binding of [3H]cortisol to the plasma membranes. Likewise, the presence of agonists and antagonists of alpha and beta-adrenergic receptors did not modify the binding of [3H]cortisol. The results suggest that the plasma membrane binding site characterized is more specific for corticoids and is different from nuclear glucocorticoid and progesterone receptors.

Adrenal Cortex Hormones↗
PubMed1989

Characterization of specific binding sites for corticosterone in mouse liver plasma membrane.

The specific binding of [3H]corticosterone to mouse liver purified plasma membrane fractions is a saturable, reversible, and temperature-dependent process. Only one type of independent and equivalent binding sites has been determined in plasma membrane (Kd = 4.1 nM and Bmax = 3368 fmol/mg). As can be deduced from displacement data obtained in plasma membrane, the high-affinity binding site is different from nuclear glucocorticoid, nuclear progesterone, and Na+, K(+)-ATPase digitalis receptors. Probably this corticosterone binding site or receptor is the same one determined previously for [3H]cortisol in mouse liver plasma membrane. Such beta- and alpha-adrenergic antagonists as propranolol and phentolamine did not affect [3H]corticosterone binding to plasma membranes; therefore, this binding site is independent of these receptors. The binding sites in plasma membranes are not exclusive for corticosterone, but other steroids are also bound with very different affinities.

Animals↗
PubMed1988

Glycogen phosphorylase activation by progesterone in liver.

Glycogen phosphorylase activity is increased before protein synthesis activation by progesterone. This effect is not blocked by antibiotics (actinomycin D and cycloheximide) that are known to inhibit mRNA or protein synthesis. At times similar to those of phosphorylase activation, cAMP are not enhanced, as would be expected considering the classical glycogenolytic cascade, but depleted with respect to control values. A little earlier, cGMP levels are significantly increased.

Animals↗