Binding of 17beta-estradiol by variants of alpha-fetoprotein in rat amniotic fluid.

Two variants of alpha-fetoprotein in rat amniotic fluid were separated by their different affinity for concanavalin A-Sepharose, which selectively binds alpha-D-manno-pyranosides and alpha-D-glucopyranosides. Both forms had the same mobility upon polyacrylamide gel electrophoresis. The binding of 17beta-estradiol per mg of alpha-fetoprotein, determined both immunologically and electrophoretically, was the same for both variants. These results indicate that a specific carbohydrate portion of the molecule is not necessary for steroid binding.

The ACTH-releasing hormone of the hypothalamus requires a co-factor.

Gel filtration of an extract of rat median eminence tissue in 0.1 N HCl on Sephadex G-25 separated two peaks, both with feeble ACTH-releasing activity. Full activity of the extract was regained when both peaks were recombined. This observation suggests that the ACTH-releasing hormone of the hypothalamus requires a co-factor for activity.

Transmembrane potentials and steroidogenesis in normal and neoplastic human adrenocortical tissue.

Trans-membrane potentials and steroidogenesis were measured in superfused slices of non-tumor and neoplastic human adrenocortical tissue. Non-tumor tissue was obtained at the time for renal transplant or from tissue removed along with tumors. Non-tumor human adrenocortical tissue had electrophysiological and steroidogenic properties similar to those of the rat and rabbit. In normal medium ACTH stimulated steroidogenesis but had no effect on the membrane potential. In K+-free medium, the cells hyperpolarized, and subsequent addition of ACTH caused depolarization. Trans-membrane potentials of adrenocortical tumors were lower than those of non-tumor cells. Ommission of K+ from the medium caused hyperpolairzation of the tumor cells, but the trans-membrane potentials did not reach the values of hyperpolarized non-tumor cells. ACTH, added to the K+-free medium, caused little or no change in membrane potential of tumor cells except in one case of a virilizing adenoma, which responded very much like non-tumor tissue. Except for the virilizing adenoma, tumor tissue slices produced little or no detectable fluorogenic steroid, even in the presence of large amounts of ACTH or cyclic AMP. The virilizing adenoma responded with increased steroidogensis to ACTH and cyclic AMP.

Specific arginine vasopressin binding in particulate membrane from rat aorta.

Arginine vasopressin (AVP) has been shown to have direct pressor effects on vascular smooth muscle. We have characterized a specific binding site for AVP in rat aorta membranes. We identified a specific binding site for AVP with a Kd of 1.6 nM and Bmax of 48 pM/mg protein. The time course, pH dependence, and temperature dependence were consistent with those found for other peptide receptors. Analogues of AVP competed with tritium labelled AVP for binding to the aortic vascular receptor in direct proportion to their pressor activities.

Characterization of 3H-AVP binding sites in particulate preparations of rat brain.

Specific binding sites for vasopressin (AVP) were located in subcellular particulate fractions of rat brain with tritiated vasopressin of high specific activity, 22.5 Ci/mmol. Rat brain tissue was dissected, placed in cold 0.32 M sucrose containing proteolytic inhibitors, homogenized and fractionated into a crude nuclear fraction (1K pellet), crude mitochondrial fractions (12K pellet), and plasma membranes and microsomes (100K pellet). Specific binding of vasopressin was found in the 12K and 100K pellets in the presence of a divalent metal ion with Ni greater than Co greater than Mg greater than Mn greater than no metal ion at pH 7.4 in 50 mM Tris-Maleate buffer. Maximum specific binding of 16 nM AVP was located in the 100K anterior cortex fraction which bound 350 fmoles/mg protein; striatum, midbrain/thalamus, cerebellum, and medulla oblongata and pons bound specifically about 200 fmoles/mg protein and frontal poles and parietal cortex about 100 fmoles/mg protein in the 100K pellet. In all of the brain regions studied, except hippocampus and septum, the 100K pellet bound specifically 2 to 4 times more 3H-AVP than the 12K pellet. In the hippocampus with 16 nM AVP, the 12K pellet bound specifically 150 fmoles/mg protein; the septum, 75 fmoles/mg protein. Little or no binding to the 100K pellet was present in these regions. Bound AVP could be dissociated rapidly from the membranes by the addition of EDTA. The 12K hippocampal pellet was further fractionated into myelin, mitochondria, and synaptosomes; purification was confirmed by marker enzyme assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein phosphorylation in rat hippocampal synaptic plasma membranes in response to neurohypophyseal peptides.

The effect of arginine vasopressin (AVP) on protein phosphorylation in rat hippocampal synaptic plasma membranes (SPM) was examined. With a crude SPM preparation, AVP (10(-8)-10(-5) M) stimulated phosphorylation of a number of proteins which included a brain-specific protein of 48 kDa called B50 or protein F1, which is thought to be related to synaptic plasticity. Equimolar levels of oxytocin also stimulated B50/F1 phosphorylation. AVP and oxytocin at higher concentrations (10(-4)-10(-3) M) reduced SPM protein phosphorylation. When SPM was treated with both AVP and oxytocin, the effects were not additive; on the other hand, the effects of the phorbol ester (TPA) and AVP were additive. With SPM, partially purified by sucrose density centrifugation, only the inhibitory effect of AVP on B50/F1 phosphorylation was seen. These results suggest that AVP and oxytocin stimulation of B50/F1 phosphorylation requires cellular factors which are removed from SPM during membrane purification. In contrast, the inhibitory mechanism triggered by AVP and oxytocin appears to be associated with, or an integral part of, the synaptic membrane itself. Because the effects on membrane protein phosphorylation with maximal amounts of AVP and oxytocin were not additive, they must bind to the same sites on the membrane. This conclusion is supported by the additivity of the effects of AVP and phorbol ester, since the phorbol ester can act directly on the kinase and does not require a membrane recognition site.

Arginine vasopressin receptors in pig cerebral microvessels, cerebral cortex and hippocampus.

We tested for the presence of arginine vasopressin (AVP) receptors in pig cerebral microvessels, cerebral cortex and hippocampus by specific binding methods with [3H]AVP as the ligand. The specific binding of [3H]AVP to all preparations was saturable and Scatchard analysis indicated a single class of high affinity binding sites (dissociation constant of 1-2 nM). Maximal binding capacity in cerebral microvessels was about 60% that of the cerebral cortex; and there were no apparent differences in the maximal binding capacity between cerebral cortex and hippocampus. These findings suggest the existence of AVP receptor sites in cerebral microvessels and support the hypothesis that AVP has a role in the control of the brain microcirculation.

Properties of the specific binding site for arginine vasopressin in rat hippocampal synaptic membranes.

In a previous paper (Pearlmutter, A. F., Constantini, M. G., and Loeser, B. (1983) Peptides 4, 335-341), we have shown that saturable, high-affinity binding sites for [3H]arginine vasopressin (AVP) are located in rat brain membrane preparations. Binding was dependent upon the presence of Ni2+ and could be dissociated by EDTA. In the hippocampus, [3H]AVP binding could be localized to synaptic membranes. In this paper, we characterize in more depth the specificity of [3H]AVP binding to a crude hippocampal synaptic preparation and the metabolism of [3H]AVP in our synaptic preparation. By means of HPLC analysis we demonstrate that the radioactive material specifically bound to hippocampal synaptic membranes is intact [3H]AVP. The ability of analogues of AVP to displace the high-affinity, specific binding of [3H]AVP parallels closely the potency of these analogues to inhibit the extinction of avoidance behavior. In the presence of membrane and Ni2+, [3H]AVP has a half-life of 7 h. In the absence of Ni2+, the half-life of [3H]AVP is 1.2 h. Fractionation by high-pressure liquid chromatography of the supernatant from the incubation media not containing Ni2+ yields three peaks of radioactivity. Analysis of the biological activity of the [3H]AVP peak and the two non-AVP peaks which represent breakdown products show the following: (a) the [3H]AVP peak (52%) and peak III (8%) bind to fresh membranes and (b) peak II (40%) has no binding activity. Although Ni2+, Co2+, benzamidine, and phenanthroline can prevent [3H]AVP degradation, only Ni2+ and, to a much lesser extent, Co2+, can potentiate specific [3H]AVP binding. The results show that AVP-specific binding has properties which parallel its biological activity in behavioral assays; that, ultimately, proteolysis by membrane-bound peptidases inactivates AVP; and that Ni2+ acts both by preventing AVP breakdown and by potentiating specific binding.

Thermodynamics and kinetics of bovine neurophysins binding to small peptide analogues of oxytocin and vasopressin.

Thermodynamic binding constants for the interactions of mononitrated neurophysins with oxytocin, vasopressin, and peptide analogues of the hormones were determined by using a spectrophotometric titration technique. The data were fit to a binding model which included all known interactions in these systems. From an examination of the free energies for the binding reaction, we concluded that residues 1-3 contribute 84% of the binding energy for formation of the neurophysin dimer mono complex and 79% for the formation of the bis complex. Rate constants for complex formation and dissociation with native bovine neurophysin were determined by using temperature-jump relaxation. The association rate constants for neurophysin dimer binding to oxytocin, vasopressin, and the peptide analogues were all in the range of 1.3 X 10(6) M-1 s-1 for mono complexation and 1.5 X 10(6) M-1 s-1 for bis complexation. Thus, formation rate constants are identical for both mono and bis complexation, and no significant differences exist between formation constants for hormones and peptides. On the other hand, a clear distinction in dissociation rate constants is apparent when one compares the hormones (kr = 4 to 16 s-1) with the peptide analogues (kr = 54 to 182 s-1). There is rougly a tenfold increase in overall dissociation rate constant when one compares the peptides to the hormones. From these data, we conclude that the rate-determining step in the association reaction involves the first two or three residues on the hormone. After the initial binding takes place, only with intact hormone, i.e., oxytocin or vasopressin, can additional bonding interactions in the complex take place. These additional interactions are reflected in the slower off-rate of the hormone complexes relative to the peptide complexes.

Stopped-flow investigation of nitrated bovine neurophysin monomer binding to oxytocin.

By use of stopped-flow kinetic data, we have measured the kinetics of mononitrated neurophysin I monomer binding to oxytocin. The association constant was 1.3(+/-0.3) x 10(5) M-1s-1 and the dissociation rate constant was 2.0(+/-0.5)s-1 for protonated oxytocin binding. Both rates are significantly slower than those observed for neurophysin dimer. These data suggest that the binding process by which the monomer binds oxytocin is not identical to that of dimer.

Interaction of bovine neurophysin with oxytocin and vasopressin measured by temperature-jump relaxation.

The interaction between bovine neurophysins I and II and oxytocin and vasopressin was measured using temperature-jump relaxation. A single relaxation time in the 10 to 90 ms range was noted for each solution. This time depended upon the concentration of both neurophysin and hormone and increased with increasing pH. The formation rate constants (+/- SE) for the interaction of neurophysin I dimer with the protonated form of oxytocin and vasopressin at pH 7.4 in 0.1 M KNO3 and 25 degrees C were 2.8 (+/- 0.4) x 10(6) and 2.3 (+/- 0.5) x 10(6) M-1 s-1, respectively; the dissociation rate constants were 11 and 15 s-1, respectively.For neurophysin II dimer, formation rate constants were 6.0 (+/- 0.2) x 10(6) and 2.4 (+/- 0.3) x 10(6) M-1 s-1; dissociation rate constants were 24 and 16 s-1 for oxytocin and vasopressin, respectively. Formation rate constants for the interaction of neurophysin monomer with protinated hormone were approximatley an order of magniment with circular dichorism and pH titration studies which indicate that this interaction occurs between a negatively charged carboxyl group on the neurophysin and a protonated alpha-amino group on the hormone. Our results indicate that oxytocin and vasopressin bind with greater affinity to the neurophysin dimer than the monomer and that the binding of oxytocin and vasopressin in neurophysin dimer than the monomer and that the binding of oxytocin and vasopressin in neurophysin dimer at pH 7.4 and concentrations between 10(-4) and 10(-5) M is nearly identical for each hormone.

Effects of arginine vasopressin on protein phosphorylation in rat hippocampal synaptic membranes.

Our laboratory has reported previously the characteristics of specific AVP binding to rat hippocampal synaptic membranes (SPM) in the presence of Ni2+ [Costantini MG, Pearlmutter AF: J Biol Chem 259: 11739-11745, 1984]. We extended our investigation to determine the effects of Ni2+, (AVP), and AVP analogs on SPM protein phosphorylation. Ni2+ (5 mM) caused a dramatic reduction in phosphorylation of most SPM phosphoproteins. The most prominent protein which is phosphorylated in SPM has a molecular weight of 48 kilodaltons (KDa) and has been named B50 or F1; this protein shows altered phosphorylation in vitro in response to long-term potentiation in vivo as well as changes induced by exposure of SPM to ACTH (1-24), dopamine, and somatostatin. AVP and related peptides reduced phosphorylation of this pre-synaptic phosphoprotein in the following order of potency: AVP = oxytocin greater than DG-AVP greater than dDAVP greater than d(CH2)5Tyr(Me)AVP = [pGlu4,Cyt6]AVP-(4-9). Except for the pressor antagonist d(CH2)5Tyr(Me)AVP, this corresponds to their relative efficacy in displacing 3H-AVP from high-affinity specific binding sites on rat hippocampal synaptic membranes. Ni2+ did not alter the degree of inhibition caused by the peptides. When SPM were treated with AVP after the attainment of maximum 32P incorporation, AVP inhibited dephosphorylation over a 30-min period. Our results show that AVP can alter both phosphorylation and dephosphorylation of hippocampal SPM phosphoproteins in vitro; the direction of these effects depends upon experimental conditions. Since B50/F1 is known to be a substrate for protein kinase C, AVP may act by inhibition of protein kinase C activity, either directly or indirectly.

Perinatal thiouracil exposure depresses corticotropin-releasing factor activity in 15 day old rats.

Although previous in vivo studies have shown thiouracil to delay maturation of the hypothalamus-pituitary-adrenal (HPA) axis response to stress, the nature of the developmental deficit was not determined. By in vitro methods we determined which HPA components are influenced by thiouracil-induced hypothyroidism in 15 day old rats. Our results indicate that adrenal response to ACTH stimulation and adenohypophysial ACTH content were not significantly modified by thiouracil exposure. On the other hand, the corticotropin-releasing factor-like activity of median eminence extracts was severely depressed by thiouracil-induced hypothyroidism. Thus, the delayed maturation of functional capacity of the central nervous system caused by hypothyroidism includes synthesis of biologically active corticotropin-releasing factor.

The human platelet vasopressin receptor and its intracellular messengers: key role of divalent cations.

The effects of divalent cations on human platelet vasopressin receptor binding characteristics and effects of receptor occupancy on endogenous protein phosphorylation were investigated. Binding of vasopressin to its receptor is modulated by both the nature and the concentration of ions. Whatever the cation present, guanosine 5'-triphosphate or 5' guanylylimidodiphosphate do not alter the receptor binding characteristics. In the presence of extracellular calcium, vasopressin stimulates the phosphorylation of a 45,000-dalton protein and to a lesser degree of a 20,000-dalton protein following a pattern observed with thrombin and 12-O-tetradecanoylphorbol-13-acetate, a phorbol ester. Phosphorylation is also stimulated by a V1 vascular agonist, but not V2 renal agonists, and is more potently blocked by a V1 vascular antagonist than by a V2 renal antagonist. These results suggest that human platelets bear typical V1 vascular vasopressin receptors which stimulate the phosphorylation of specific substrates of protein kinase C and myosin light-chain kinase.

Properties of CRF from normal and Brattleboro rat median eminence.

Most of the corticotrophin-releasing factor (CRF) activity of normal rat median eminence (ME) extract binds to a neurophysin affinity column. The bound material contains the large and small factors, which we have previously demonstrated to be required together for full activity. Most of the CRF activity of Brattleboro rat ME extract, which contains as much CRF activity as the ME extract of a normal rat, does not bind to a neurophysin affinity column. The CRF activity of Brattleboro rat ME extract resides entirely in a large molecule as determined by Sephadex G-25 chromatography. The different properties of Brattleboro and normal rat CRF suggest that the CRF activity in the Brattleboro rat may result from a substance which is different from that in a normal rat.

Characterization of oxytocin receptors in the uterus and mammary gland.

High affinity binding sites for 3[H] oxytocin have been demonstrated in particulate fractions from rat uterus and oviduct, myometrium from the sow, ewe and human, ewe endometrium, and mammary gland from the lactating rat. The binding activity has been localized to enriched plasma membrane fractions from the rat uterus and mammary gland; cells isolated from the mammary gland also bind oxytocin. The apparent dissociation constant (Kd) for the interaction of oxytocin with its binding sites in a variety of tissue preparations is in the nanomolar range. The concentration of oxytocin eliciting half-maximal contraction of the rat isolated uterus corresponds to the apparent Kd of oxytocin interaction with uterine particulate fractions. Binding is specific with respect to the target tissue or cell, as well as to the ligand. The affinity of binding sites for oxytocin analogues corresponds generally to their potencies as agonists or antagonists. Factors that affect the binding of oxytocin affect the biological response in the same way. For example, certain divalent metal ions, which increase oxytocin binding activity, enhance the sensitivity of the contractile response of the uterus and mammary gland to oxytocin. Estrogen administration, which increases the uterine binding of oxytocin, increases the sensitivity of the myometrium to oxytocin. The myometrium binds the most oxytocin at estrus and is most sensitive to oxtocin at that time. The dgree of stimulation by oxytocin of prostaglandin F2alpha synthesis by ewe endometrium is paralleled by an increased concentration of oxytocin binding sites. The marked increase in sensitivity to oxytocin of the rat uterus occurring on the day of parturition also is reflected by the amount of oxytocin bound by the uterus. Because of the many correlations between oxytocin binding and bioactivity, it appears that oxytocin binding sites on the plasma membrane of target cells constitute the recognition part of oxytocin receptors.