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A E Johnson

Publications and source records attributed to A E Johnson.

At least 19 recordsLinked to original sources
PubMed1992

Binding of factor VIIIa and factor VIII to factor IXa on phospholipid vesicles.

The activation of factor X by factor IXa (fIXa) in the presence of phosphatidylcholine-phosphatidylserine (PCPS) vesicles is markedly accelerated by thrombin-activated factor VIII (fVIIIa). The interaction between highly purified fVIIIa and fIXa in this complex was studied fluorometrically at 25 degrees C by using a derivative of D-phenylalanyl-prolyl-arginyl-fIXa which was modified at the active site with fluorescein-5-maleimide (Fl-M-FPR-fIXa). Titration of Fl-M-FPR-fIXa with fVIIIa at fixed PCPS resulted in a large, saturable increase in anisotropy (delta r = 0.09). The titration data were fit to a model assuming a reversible equilibrium between fVIIIa and fIXa, resulting in an apparent dissociation constant of 2 nM and a stoichiometry of 1 mol of fVIIIa/mol of Fl-M-FPR-fIXa. The initial velocity of factor X activation was measured under identical conditions except that active fIXa and factor X were included, which yielded binding parameters similar to those determined fluorometrically. Thus, the fluorescence method accurately reflects complex formation between fVIIIa and fIXa on the phospholipid surface, and the fVIIIa-fIXa interaction is not influenced by the presence of the substrate, factor X. Addition of fVIII to Fl-M-FPR-fIXa and PCPS produced a small, saturable increase in anisotropy (delta r = 0.03), followed by a larger increase (delta r = 0.07) upon addition of thrombin to activate fVIII. Thus, fVIII binds fIXa, but proteolytic modification of fVIII must occur before the complete fVIIIa-dependent structural change in the active site of fIXa, as reflected in the anisotropy change, occurs

Animals↗
PubMed1992

The active site of factor IXa is located far above the membrane surface and its conformation is altered upon association with factor VIIIa. A fluorescence study.

The topography of membrane-bound blood coagulation factor IXa (fIXa) and the nature of its interaction with its cofactor, factor VIIIa (fVIIIa), were examined using fluorescent derivatives of fIXa. A fluorescein dye was covalently attached to the active-site histidine of fIXa via a D-Phe-Pro-Arg tripeptide tether to form Fl-A-FPR-fIXa; similarly, a 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) dye was covalently attached via Glu-Gly-Arg to form DEGR-fIXa. When either Fl-A-FPR-fIXa or DEGR-fIXa was titrated with phosphatidylcholine-phosphatidylserine vesicles containing octadecylrhodamine in the presence of Ca2+, fluorescence energy transfer was observed. Assuming a random orientation of dyes, the distance of closest approach between the donor dyes in the active sites of the membrane-bound enzymes and the acceptor dyes at the membrane surface was found to be 89 +/- 3 A for Fl-A-FPR-fIXa and 73 +/- 4 A for DEGR-fIXa. Although the exact distance remains uncertain, it is clear that the active site of fIXa is positioned more than 70 A above the surface, and hence that the elongated fIXa molecule projects approximately perpendicularly from the surface when bound to the membrane. The binding of fVIIIa to membrane-bound Fl-A-FPR-fIXa or DEGR-fIXa did not alter the location of the active site relative to the membrane surface, but did alter both the emission intensity and anisotropy of the fluorescein and dansyl probes and hence their environments. Cofactor stimulation of fIXa activity therefore appears to be mediated, at least in part, by a conformational change in the active site that occurs when fVIIIa binds to the enzyme on the phospholipid surface.

Animals↗
PubMed1992

Fluorescence-detected assembly of the signal recognition particle: binding of the two SRP protein heterodimers to SRP RNA is noncooperative.

Protein-RNA and protein-protein interactions involved in the assembly of the signal recognition particle (SRP) were examined using fluorescence spectroscopy. Fluorescein was covalently attached to the 3'-terminal ribose of SRP RNA following periodate oxidation, and the resulting SRP RNA-Fl was reconstituted into a fluorescent SRP species that was functional in promoting translocation of secretory proteins across the membrane of the endoplasmic reticulum. Each of the two protein heterodimers purified from SRP elicited a substantial change in fluorescein emission upon association with the modified RNA. The binding of SRP9/14 to singly-labeled SRP RNA-Fl increased fluorescein emission intensity by 41% at pH 7.5 and decreased its anisotropy from 0.18 to 0.16. The binding of SRP68/72 increased the fluorescein anisotropy from 0.18 to 0.23 but did not alter the emission intensity of SRP RNA-Fl. These fluorescence changes did not result from a direct interaction between the dye and protein because the fluorescein remained accessible to both iodide ions and fluorescein-specific antibodies in the complexes. The spectral changes were elicited by specific SRP RNA-protein interactions, since (i) the SRP9/14- and SRP68/72-dependent changes were unique, (ii) an excess of unlabeled SRP RNA, but not of tRNA, blocked the fluorescence changes, and (iii) no emission changes were observed when SRP RNA-Fl was titrated with other RNA-binding proteins. Each heterodimer bound tightly to the RNA, since the Kd values determined spectroscopically and at equilibrium for the SRP9/14 and the SRP68/72 complexes with SRP RNA-Fl were less than 0.1 and 7 +/- 3 nM, respectively. The binding affinity of SRP68/72 for SRP RNA-Fl was unaffected by the presence of SRP9/14, and hence the binding of the heterodimers to SRP RNA is noncooperative in the absence of SRP54 and SRP19. The SRP protein heterodimers therefore associate randomly and independently with SRP RNA to form domains in the particle that are distinct both structurally and functionally. Any cooperativity in SRP assembly would have to be mediated by SRP54 and/or SRP19.

Animals↗
PubMed1992

The regulation of oxytocin receptor binding in the ventromedial hypothalamic nucleus by gonadal steroids.

In the previous sections, data were presented on the regulation of OT receptor binding by gonadal hormones. However, it is important to emphasize that gonadal steroids and E2 in particular also regulate other facets of OT transmission. For example, OT peptide levels as indicated by mRNA levels and OT immunoreactivity are enhanced by gonadal steroids. Similarly, OT release is also influenced by circulating steroids. By focusing on data collected in female rats and combining these different factors, the following relationships emerge. Within the ventromedial hypothalamus, OT peptide levels are virtually undetectable and OT receptor binding is negligible in the absence of E2. As circulating levels of E2 rise, increases in OT immunoreactivity and OT-receptor binding in the ventromedial hypothalamus and in the VMN itself can be detected. Under these conditions, OT receptor binding in and around the vl-VMN is markedly increased, rendering cells in the vl-VMN responsive to OT stimulation. Significantly, this increased responsivity is most evident at low concentrations of OT. With further increases in E2, P4 is released from the ovary, resulting in a number of biochemical changes in the central nervous system, including a potentiation of the effects of E2 on hypothalamic OT-receptor binding. Within a few hours after the initiation of P4 release, sexual receptivity can be elicited. At this time, sexual contact by the male enhances OT-peptide levels in the ventromedial hypothalamus of steroid-primed female rats, which in turn potentiates the display of sexual receptivity. The model outlined above not only summarizes a large portion of the data on steroid regulation of hypothalamic OT receptors, but more importantly, serves to highlight areas that require additional study. One important question that remains to be resolved is the source of OT receptors in the ventromedial hypothalamus. While the lesion studies discussed above indicate that the majority of fibers in the ventrolateral hypothalamus originate in the VMN, definitive results can only be obtained with more direct methods. For example, following the eventual cloning and sequencing of the OT receptor, studies on the distribution and regulation of OT receptor mRNA could be conducted to determine the hypothalamic cells that synthesize OT receptors. Complementary experiments that employ an immunohistological approach in combination with electron microscopy could be used to visualize the region(s) of the cell that react to OT receptor antibodies. Additional studies are also required to determine the role of steroid-modulated OT receptor binding in the hypothalamus of male rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

The fifth and sixth growth factor-like domains of thrombomodulin bind to the anion-binding exosite of thrombin and alter its specificity.

The domain of thrombomodulin that binds to the anion-binding exosite of thrombin was identified by comparing the binding of fragments of thrombomodulin to thrombin with that of Hirugen, a 12-residue peptide of hirudin that is known to bind to the anion-binding exosite of thrombin. Three soluble fragments of thrombomodulin, containing (i) the six repeated growth factor-like domains of thrombomodulin (GF1-6), (ii) one-half of the second through the sixth growth factor-like repeats (GF2.5-6), or (iii) the fifth and sixth such domains (GF5-6), were examined. Hirugen was a competitive inhibitor for either GF1-6 or GF2.5-6 stimulation of thrombin activation of protein C. GF5-6, which binds to thrombin without altering its ability to activate protein C, competed with fluorescein-labeled Hirugen for binding to thrombin. Therefore, all three thrombomodulin fragments, each of which lacked the chondroitin sulfate moiety, competed with Hirugen for binding to thrombin. To determine whether GF5-6 and Hirugen were binding to overlapping sites on thrombin or were interfering allosterically with each other's binding to thrombin, the effects of each thrombomodulin fragment and of Hirugen on the active site conformation of thrombin were compared using two different approaches: fluorescence-detected changes in the structure of the active site and the hydrolysis of chromogenic substrates. The GF5-6 and Hirugen peptides affected these measures of active site conformation very similarly, and hence GF5-6 and Hirugen contact residues on the surface of thrombin that allosterically alter the active site structure to a similar extent. Full-length thrombomodulin and GF1-6 alter the active site structure to comparable extents, but the amidolytic activity of thrombin complexed to thrombomodulin or GF1-6 differs significantly from that of thrombin complexed to GF5-6 or Hirugen. Taken together, these results indicate that the GF5-6 domain of thrombomodulin binds to the anion-binding exosite of thrombin. Furthermore, the binding of GF5-6 to the anion-binding exosite alters thrombin specificity, as evidenced by GF5-6-dependent changes in both the kcat and Km of synthetic substrate hydrolysis by thrombin. The contact sites on thrombin for the GF4 domain and the chondroitin sulfate moiety of thrombomodulin are still unknown.

Amino Acid Sequence↗
PubMed1992

A model for the aminoacyl-tRNA binding site of eukaryotic elongation factor 1 alpha.

Eukaryotic elongation factor 1 alpha (EF-1 alpha) binds all the aminoacyl-tRNAs except the initiator tRNA in a GTP-dependent manner. While the GTP binding site is delineated by the three GTP binding consensus elements, less is known about the aminoacyl-tRNA binding sites. In order to better understand this site, we have initiated cross-linking and protease mapping studies of the EF-1 alpha-GTP-aminoacyl-tRNA complex. Two different chemical cross-linking reagents, trans-diaminedichloroplatinum(II) and diepoxybutane, were used to cross-link four different aminoacyl-tRNA species to EF-1 alpha. A series of peptides were obtained, located predominantly in domains II and III. The ability of aminoacyl-tRNA to protect protease digestion sites was also monitored, and domain II was found to be protected from digestion by aminoacyl-tRNA. Last, an aminoacyl-tRNA analog with a reactive group on the aminoacyl side chain, N epsilon-bromoacetyl-Lys-tRNA, was cross-linked to EF-1 alpha. This reagent cross-liked to histidine 296 in a GTP-dependent manner and thus localizes the aminoacyl group adjacent to domain II. A model is developed for aminoacyl-tRNA binding to EF-1 alpha based on its similarity to the prokaryotic factor EF-Tu, for which an x-ray crystal structure is available.

Amino Acid Sequence↗
PubMed1992

Sex differences in the regulation of oxytocin receptors by ovarian steroids in the ventromedial hypothalamus of the rat.

The facilitation of sexual receptivity by oxytocin (OT) in female rats is related to the regulation of oxytocin receptors (OTR) by ovarian steroids in the ventromedial nuclei (VMN) of the hypothalamus. In a previous study, we have shown that estradiol benzoate (EB) causes a twofold increase in OTR binding in the VMN. Progesterone (P) then modulates levels of the estrogen-induced OTR and increases the area occupied by the receptors by acting on the neuronal membrane. In the present study, we compared the effects of EB and P on OTR binding between males and females. In both sexes, EB increased the density of OTR and the area covered by the receptors at the level of the medial and caudal VMN. In estrogen-primed females, P further increased OTR levels in the medial VMN and the area covered by OTR at the level of the caudal VMN. By contrast, P did not modulate OTR binding in estrogen-primed males. Thus, the behavioral insensitivity of male rats to ovarian hormones, in particular to P, may be related to sex differences affecting the modulation of OTR binding.

Animals↗
PubMed1991

Projections from the ventromedial nucleus of the hypothalamus contain oxytocin binding sites.

Experiments were conducted to determine the source of steroid-dependent oxytocin (OT) receptors that surround the ventrolateral portion of the ventromedial hypothalamic nucleus (vl-VMN). Ovariectomized rats received sham or unilateral electrolytic lesions of the vl-VMN. Three days later and for the next 4 days, animals were injected with 10 micrograms of estradiol benzoate (EB). OT receptors were labelled with 125I-d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT and binding was assessed with autoradiographic methods. Lesions of the vl-VMN reduced OT receptor binding in the area surrounding the nucleus by over 80% indicating that the majority of OT receptors in the ventromedial hypothalamus are located on fibers originating within the VMN.

Angiotensin Receptor Antagonists↗
PubMed1991

Proteolytic formation of either of the two prothrombin activation intermediates results in formation of a hirugen-binding site.

Hirugen, a synthetic dodecapeptide corresponding to the carboxyl-terminal amino acids 53-64 of hirudin, binds within a deep groove in thrombin that contains a cationic region referred to as the anion-binding exosite. This region is important in many of the binary interactions of thrombin with macromolecular substrates and cofactors. Fluorescein-labeled hirugen was used to probe which steps in the prothrombin activation process generate this anion-binding exosite. Two activation cleavage sites exist in bovine prothrombin. Cleavage at Arg274-Thr275 releases the activation fragments to generate the thrombin precursor, prethrombin 2. Cleavage of prothrombin within a disulfide loop at Arg323-Ile324 leads to formation of meizothrombin with no loss of peptide material but with formation of amidolytic activity. Cleavage of the same bond in prethrombin 2 generates thrombin. Hirugen, labeled at the amino terminus with fluorescein isothiocyanate, does not bind to prothrombin but does bind to thrombin (Kd = 9.6 +/- 1.2 x 10(-8) M), prethrombin 2 (Kd = 1.3 +/- 0.1 x 10(-7) M), thrombin-fragment-2 complex (Kd = 1.1 +/- 0.2 x 10(-6) M), and meizothrombin (Kd = 1.6 +/- 0.5 x 10(-8) M). Prothrombin fragment-2 and hirugen both bind independently to thrombin. A ternary complex can form with hirugen and fragment-2 and either thrombin or prethrombin 2, suggesting that fragment-2 and hirugen bind to discrete sites. Hirugen also alters the active site conformation of thrombin as detected by modulation of synthetic substrate hydrolytic activity. These studies suggest that conformational changes, rather than alleviating steric hindrance, are responsible for the formation of the hirugen-binding site during prothrombin activation. Furthermore, this conformational change can be effected by the cleavage of either of the two bonds required for activation of prothrombin.

Amino Acid Sequence↗
PubMed1991

The active site of thrombin is altered upon binding to thrombomodulin. Two distinct structural changes are detected by fluorescence, but only one correlates with protein C activation.

The association of thrombin with thrombomodulin, a non-enzymatic endothelial cell surface receptor, alters the substrate specificity of thrombin. Complex formation converts thrombin from a procoagulant to an anticoagulant enzyme. Structure-function analysis of this change in specificity is facilitated by the availability of two soluble proteolytic derivatives of thrombomodulin, one consisting of the six repeated growth factor-like domains of thrombomodulin (GF1-6) and the other containing only the fifth and sixth such domains (GF5-6). Both derivatives can bind to thrombin and block fibrinogen clotting activity, though only the larger GF1-6 can stimulate the activation of protein C. To ascertain whether the substrate specificity change from fibrinogen to protein C is accompanied by structural changes in the active site of the enzyme, fluorescent dyes were positioned at different locations within the active site. A 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) dye was covalently attached to the active site serine to form dansyl-thrombin, while either a fluorescein or an anilinonaphthalene-6-sulfonic acid (ANS) dye was attached covalently to the active site histidine of thrombin via a D-Phe-Pro-Arg linkage. The environment of the dansyl dye was altered in a similar fashion when either GF1-6 or GF5-6 bound to thrombin, since a similar reduction in dansyl emission intensity was elicited by these two thrombomodulin derivatives (25 and 32%, respectively). These spectral changes, and all others in this study, were saturable and reached a maximum when the ratio of thrombomodulin derivative to thrombin was close to 1. The environments of the fluorescein and ANS dyes were also altered when GF1-6 bound to thrombin because binding resulted in emission intensity changes of -13% and +18%, respectively. In contrast, no fluorescence changes were observed when the fluorescein and ANS thrombin derivatives were titrated with GF5-6. Thus, the structure of the active site was altered by thrombomodulin both immediately adjacent to the active site serine and also more than 15 A away from it. However, the structural change far from Ser-195 was only elicited by thrombomodulin species that stimulate thrombin-dependent activation of protein C.

Amino Acid Sequence↗
PubMed1991

Neutrophil recognition requires a Ca(2+)-induced conformational change in the lectin domain of GMP-140.

GMP-140, a receptor for myeloid cells that is expressed on surfaces of thrombin-activated platelets and endothelial cells, is a member of the selectin family of adhesion molecules that regulate leukocyte interactions with the blood vessel wall. Each selectin contains an N-terminal domain homologous to Ca(2+)-dependent lectins and mediates cell-cell contact by binding to oligosaccharide ligands in a Ca(2+)-dependent manner. The mechanisms by which Ca2+ promotes selectin-dependent cellular interactions have not been defined. We demonstrate that purified GMP-140 contains two high affinity binding sites for Ca2+ as measured by equilibrium dialysis (Kd = 22 +/- 2 microM). Occupancy of these sites by Ca2+ alters the conformation of the protein as detected by a reduction in intrinsic fluorescence emission intensity (Kd = 4.8 +/- 0.2 microM). This Ca(2+)-dependent conformational change exposes an epitope spanning residues 19-34 of the lectin domain that is recognized by a monoclonal antibody capable of blocking neutrophil adhesion to GMP-140 (half-maximal antibody binding at approximately 20 microM Ca2+). Furthermore, a synthetic peptide encoding this epitope, CQNRYTDLVAIQNKNE, inhibits neutrophil binding to GMP-140. Mg2+ also alters the conformation of the protein, but not in a manner that will support leukocyte recognition in the absence of Ca2+. There is a strong correlation between the Ca2+ levels required for neutrophil adhesion to GMP-140, for occupancy of the two Ca(2+)-binding sites, for the fluorescence-detected conformational change, and for exposure of the antibody epitope in the lectin domain. We conclude that binding of Ca2+ to high affinity sites on GMP-140 modulates the conformation of the lectin domain in a manner that is essential for leukocyte recognition.

Amino Acid Sequence↗
PubMed1991

Effects of hydroxyurea on hemoglobin F and water content in the red blood cells of dogs and of patients with sickle cell anemia.

A rationale for clinical trials of hydroxyurea (HU) treatment in sickle cell disease is that the agent increases red blood cell (RBC) fetal hemoglobin content. However, an additional effect of HU is to raise the mean corpuscular volume (MCV). To investigate the action of HU in a species that makes no electrophoretically distinguishable fetal hemoglobin, we treated dogs with the drug and compared their response to that of five patients with sickle cell anemia. Both dogs and patients had an increase in MCV, but the effect of HU treatment on the mean corpuscular hemoglobin concentration (MCHC), density, and water content of the RBCs differed in the two species. The dog RBCs became low in MCHC, high in ion and water content, and low in mean density. Thus, HU can raise MCV and lower MCHC without influencing fetal hemoglobin synthesis. A different pattern was seen in the sickle cell patients during HU treatment. Although the MCV of their RBCs increased, there was no change in MCHC, ion content, or mean density. A notable change in the sickle cell patients' blood was that two subpopulations of cells were nearly eliminated during HU treatment; the hypodense reticulocyte fraction and the hyperdense fraction that contains irreversibly sickled cells. These findings lead us to suggest that trials of HU in sickle cell disease must recognize the possibility that any beneficial effect of this agent might be due not only to an increase in hemoglobin F alone, but perhaps also to the associated increase in MCV or the altered RBC density profile.

Administration, Oral↗
PubMed1991

A nascent membrane protein is located adjacent to ER membrane proteins throughout its integration and translation.

The immediate environment of nascent membrane proteins undergoing integration into the ER membrane was investigated by photocrosslinking. Nascent polypeptides of different lengths, each containing a single IgM transmembrane sequence that functions either as a stop-transfer or a signal-anchor sequence, were synthesized by in vitro translation of truncated mRNAs in the presence of N epsilon-(5-azido-2-nitrobenzoyl)-Lys-tRNA, signal recognition particle, and microsomal membranes. This yielded nascent chains with photoreactive probes at one end of the transmembrane sequence where two lysine residues are located. When irradiated, these nascent chains reacted covalently with several ER proteins. One prominent crosslinking target was a glycoprotein similar in size to a protein termed mp39, shown previously to be situated adjacent to a secretory protein during its translocation across the ER membrane (Krieg, U. C., A. E. Johnson, and P. Walter. 1989. J. Cell Biol. 109:2033-2043; Wiedmann, M., D. Goerlich, E. Hartmann, T. V. Kurzchalia, and T. A. Rapoport. 1989. FEBS (Fed. Eur. Biochem. Soc.) Lett. 257:263-268) and likely to be identical to a protein previously designated the signal sequence receptor (Wiedmann, M., T. V. Kurzchalia, E. Hartmann, and T. A. Rapoport. 1987. Nature (Lond.). 328:830-833). Changing the orientation of the transmembrane domain in the bilayer, or making the transmembrane domain the first topogenic sequence in the nascent chain instead of the second, did not significantly alter the identities of the ER proteins that were the primary crosslinking targets. Furthermore, the nascent chains crosslinked to the mp39-like glycoprotein and other microsomal proteins even after the cytoplasmic tail of the nascent chain had been lengthened by nearly 100 amino acids beyond the stop-transfer sequence. Yet when the nascent chain was allowed to terminate normally, the major photocrosslinks were no longer observed, including in particular that to the mp39-like glycoprotein. These results show that the transmembrane segment of a nascent membrane protein is located adjacent to the mp39-like glycoprotein and other ER proteins during the integration process, and that at least a portion of the nascent chain remains in close proximity to these ER proteins until translation has been completed.

Endoplasmic Reticulum↗
PubMed1991

Case report: splenic infarction and acute splenic sequestration in adults with hemoglobin SC disease.

While acute splenic sequestration and splenic infarction are commonly observed in infants and young children with sickle cell anemia, they are rarely experienced by adult hemoglobin S homozygotes because the recurrent splenic infarction that takes place during childhood is typically followed by scarring, atrophy, and splenic fibrosis. Both acute splenic sequestration and splenic infarction do remain relatively common in adults with the other sickle hemoglobinopathies. These episodes are almost certainly a consequence of the persistently enlarged and distensible spleens that often remain present in these conditions. In this report, the authors describe two adult patients with hemoglobin SC disease: one who developed acute splenic sequestration and one with splenic infarction. In neither case was there a history of recent air travel or exposure to altitude. The clinical course of these two syndromes is presented, and the hematologic, radiologic, and pathologic manifestations are discussed. Because they can sometimes be difficult to distinguish from one another, and because a failure to identify acute splenic sequestration can be catastrophic, these two entities must be included in the differential diagnosis for any hemoglobin SC patient who present with an unexplained fall in hemoglobin, left upper quadrant pain, unexplained fever, or symptomatic splenomegaly.

Acute Disease↗
PubMed1991

Initiation of the protein C pathway.

The protein C activation system provides an interesting model for the control of coagulation. Expression of the critical receptor appears to be under the control of inflammatory mediators. Open questions of considerable importance relate to the physiological significance of these observations. Preliminary evidence is emerging that thrombomodulin is down-regulated in patients and animals with inflammation, but it remains to be determined if the loss of thrombomodulin causes the thrombotic complications or occurs in response to these complications.

Animals↗
PubMed1991

Electrophysiological actions of oxytocin on hypothalamic neurons in vitro: neuropharmacological characterization and effects of ovarian steroids.

Oxytocin (OT) neurotransmission in the brain has a facilitatory effect on sexual receptivity in rats. This effect of OT is dependent on priming by ovarian steroids, estrogen and progesterone. These steroids modulate OT binding in specific brain nuclei, including the ventrolateral portion of the ventromedial hypothalamic nucleus (vlVMN). In the present study, single-unit activity was recorded from the vlVMN in hypothalamic slices to characterize the electrophysiological actions of OT. To examine the effects of ovarian steroids on OT actions, we used brain slices prepared from ovariectomized rats either treated with estrogen or not, and some slices were treated with progesterone in vitro. OT had little modulatory action on neuronal responses to other agents, but affected the activity of large numbers of vlVMN units. Of those neurons affected, 94% responded with excitation. This predominant stimulatory action of OT is consistent with its lordosis-facilitating effect, because increases in the activity of VMN neurons are generally associated with the facilitation of lordosis. Pharmacological analyses with selective OT agonists and antagonists as well as structurally related peptides showed that the excitatory action of OT is mediated by OT receptors. Estradiol modulated several aspects of OT transmission. First, it increased neuronal responsiveness to OT, especially at the lowest concentration used (0.2 nM). In addition, it caused neuronal responses to OT to correlate significantly with responses to acetylcholine and norepinephrine, which also can act on the ventromedial hypothalamus to facilitate lordosis. Finally, estradiol enhanced the excitability of laterally projecting neurons, which have been implicated in lordosis. In estrogen-pretreated slices, addition of progesterone in vitro caused little further effect on responses of individual neurons to exogenous OT. Altogether, the present electrophysiological findings are consistent with the hypothesis that estrogen potentiates OT action by increasing functional OT receptors preferentially in lordosis-relevant neurons, thereby enabling OT to efficiently facilitate female reproductive behavior.

Acetylcholine↗