Mechanism of uterine vascular refractoriness to endothelin-1 in pregnant sheep.

Endothelin-1 (ET-1) is a potent vasoconstrictor and produces marked pressor responses when given systemically. Studies in sheep have demonstrated that during pregnancy the uterine vasculature is refractory to exogenously administered ET-1. We hypothesize that this pregnancy-dependent refractoriness is due to an upregulation of local uterine metabolism of ET-1 and/or ET(B) receptors and/or downregulation of local uterine ET(A) receptors. To investigate these possibilities, 21 nonpregnant and 17 pregnant sheep were used. Dose-response curves to intravenous infusion of ET-1 and phenylephrine were generated for pregnant and nonpregnant sheep. ET-1 infused systemically demonstrated vasoconstriction in the systemic and renal vasculature of pregnant and nonpregnant animals and vasoconstriction in the uterine vasculature of nonpregnant animals. The pregnant animals showed no uterine vascular response to ET-1. In contrast, phenylephrine showed vasoconstriction in the systemic, renal, and uterine circulations in both pregnant and nonpregnant sheep. After experimentation, the animals were euthanized, and tissues were harvested for Western blot and activity analysis of neutral endopeptidase (NEP) or RT-PCR analysis of endothelin-converting enzyme (ECE) and ET(A) and ET(B) receptors. The content and activity of NEP in the uterine and renal vasculature of pregnant and nonpregnant animals were similar. RT-PCR demonstrated the presence of ECE in the uterine vasculature of pregnant and nonpregnant sheep. ET(A) receptor mRNA was significantly reduced in pregnant compared with nonpregnant sheep, whereas ET(B) receptor mRNA remained unchanged. We conclude that the uterine vascular refractoriness seen in the pregnant sheep is due to a downregulation of ET(A) receptors.

Estrogen receptor beta is the predominant isoform expressed in the brain of adult and fetal sheep.

OBJECTIVE: The objective of this study was to examine the expression of estrogen receptors alpha and beta in the cerebral cortex of the adult and fetal sheep. STUDY DESIGN: A reverse transcriptase-polymerase chain reaction-based approach was used to examine the expression of ovine estrogen receptor alpha and estrogen receptor beta in the cerebral cortex of 4 adult and 2 fetal sheep. RESULTS: Estrogen receptor beta was expressed in the 4 adult and 2 fetal brain samples. Estrogen receptor alpha expression was seen in only 1 adult brain and 1 fetal brain. CONCLUSION: Estrogen receptor beta is the predominant isoform expressed in the cerebral cortex of both adult and fetal sheep. These data may have implications for the many important actions of estrogen in the adult and developing ovine brain.

Evaluation of leukemia inhibitory factor as a marker of ectopic pregnancy.

OBJECTIVE: Our purpose was to determine the utility of measuring serum leukemia inhibitory factor, a cytokine expressed in the process of pregnancy implantation, for the diagnosis of ectopic pregnancy. STUDY DESIGN: Serum samples from 40 patients with positive serum quantitative beta-human chorionic gonadotropin levels were used for leukemia inhibitory factor determination. The serum leukemia inhibitory factor concentration was determined by enzyme-linked immunosorbent assay in the following 4 groups: (1) normal intrauterine pregnancies, (2) threatened abortions, (3) spontaneous abortions, and (4) ectopic pregnancies. RESULTS: All patients had detectable concentrations of leukemia inhibitory factor in serum, ranging from 2.44 to 8.25 pg/mL. Mean leukemia inhibitory factor concentrations for ectopic pregnancy were significantly lower (P <.05) than those of both the spontaneous abortion and threatened abortion groups by 1-way analysis of variance. When a cutoff point of serum leukemia inhibitory factor <6.2 pg/mL is assigned as diagnostic of ectopic pregnancy, leukemia inhibitory factor in patients with ectopic pregnancies versus all other groups predicted ectopic pregnancy with a sensitivity of 73%, specificity of 72%, positive predictive value of 50%, and negative predictive value of 88%. CONCLUSION: Serum leukemia inhibitory factor concentration is lowest in patients with ectopic pregnancy. A cutoff point of 6.2 pg/mL maximizes the sensitivity and specificity of the test; however, it is not sufficiently discriminatory to be used clinically for the diagnosis of ectopic pregnancy.

[Sequencing and cloning of human estrogen beta receptor (hERbeta) cDNA in human granulosa].

OBJECTIVE: To analyze the nucleotide sequence of cDNA and deduce the amino acid sequence of human estrogen receptor (hERbeta) in human granulosa cells. METHODS: Granulosa cells were prepared from the ovary of IVF-ET cases by Percoll technique with Dulbecco's modified eagle medium. RNA was extracted with the TRIzol reagent kit, and mRNA was purified with oligo-(dT)-cellulose in a sterile dispocolumn. cDNA was synthesized by RT-PCR using the SuperScript(TM) II RT kit. Amplified products were cloned into the pGEM-T vector and transfected into E. coli XL1-Blue. The nucleotide sequences were determined by repeated sequencing of both strands of alkaline-denatural plasmid DNA using the Sequenase Version 2.0 DNA sequencing kit. The obtained DNA sequence was compiled and analyzed using DNAMAN computer programs. RESULTS: Amplified cDNA of hER beta in human granulosa cells was composed of 1 495 bp, containing a 1 431 bp open reading frame. The predicted ER beta protein consisted of 477 amino acids. The predicted ER protein included 4 function domains: A/B, C, D, and E/F domains. Among these domains, C domain, richly containing cysteine, was the DNA-binding domain (DBD), and E/F domain was the ligand-binding domain (LBD). CONCLUSION: Detection of ERbeta in the ovary granulosa cells played an important role in explaining the self-endocrine function of estrogen.

Properties of three COOH-terminal splice variants of a human cardiac L-type Ca2+-channel alpha1-subunit.

There is growing evidence for diversity of cardiac-type (class C) voltage-dependent calcium-channel alpha1-subunits arising from the alternative splicing of a primary transcript. In this study, we show the existence of carboxy-terminal variability in the human cardiac alpha1-gene by genomic cloning. We found that the genomic DNA segment encoding the COOH-terminal tail of the protein is composed of nine invariable and two alternative exons. The alternative utilization of these latter two exons gives rise to the formation of three message variants for this region. Reverse transcription followed by polymerase chain reaction and radioanalytic quantitation of the reverse transcription-polymerase chain reaction products showed significant variations in the distribution of these isoforms (hHt alpha1, rHt alpha1, fHt alpha1) in distinct parts of the heart, the aorta, and fibroblasts. Expression of the three alpha1-isoforms in Xenopus oocytes or in HEK-293 cells and analysis of the kinetics and voltage dependence of the induced calcium-channel currents revealed only insignificant differences in the behavior of these isoforms. When the alpha1-isoforms were coexpressed with a human beta-subunit, no alpha1-specific divergences were observed, but the effects of beta-subunit coexpression on alpha1-isoform biophysical properties were confirmed. The differential abundance of the three isoforms and the influence of an accessory subunit are of potential physiological significance.

Peptide V: a VGF-derived neuropeptide purified from bovine posterior pituitary.

The objective of this study was to purify PRL-releasing factor (PRF) from the bovine posterior pituitary (PP) and determine its structure. Five hundred bovine PPs were acid extracted and fractionated using gel filtration chromatography followed by semipreparative and analytical HPLC. PRF activity was determined by an in vitro bioassay. After six chromatographic steps, a single peak with PRF activity was resolved. As determined by mass spectrometry and microsequencing, this peak contained a major peptide composed of 30 amino acids with a mol wt of 3708K. A synthetic peptide was then produced by solid-phase synthesis. When tested both in vivo and in vitro, the synthetic peptide lacked PRF activity. Further HPLC fractionation under different conditions resolved the synthetic peptide from a highly purified PRF activity. This indicated that the isolated peptide was coincidentally eluted with PRF during the purification. The major isolated peptide has 94% identity with a sequence at the C-terminus of a rat protein named VGF. VGF is a nerve growth factor-inducible protein that has been identified in PC12 cells and is localized in selected sites throughout the central nervous system. The isolated peptide has an Arg-Arg cleavage site at its junction within the VGF protein. Based on this information, we named this substance Peptide V (VGF-derived peptide). We postulate that Peptide V is: 1) a natural cleavage product of the VGF protein; 2) produced and processed either in the hypothalamus or within the pituitary proper, and 3) a releasable peptide that fulfills one or more endocrine functions.

Changes in the expression of the L-type voltage-dependent calcium channel during pregnancy and parturition in the rat.

The L-type voltage-dependent calcium channel (L-VDCC) is assumed to be a critical component of excitation-contraction coupling in smooth muscle. Using pregnant rat myometrium, we examined the hypothesis that parturition is associated with significant changes in the expression of the alpha 1 subunit of the L-VDCC at the mRNA or protein level. The binding of radiolabeled dihydropyridine, which correlates with the total number of calcium channels in the membrane, was increased by 14 days' gestation, in comparison to that in nonpregnant controls. The elevation in binding capacity persisted through labor and fell postpartum. Northern and RNA dot-blot analysis demonstrated the highest level of expression on Days 20 and 21, with a 3- to 10-fold decrease during parturition. We believe these studies are most consistent with a one-day lag time between mRNA and protein expression, and generally support a modest increase in L-VDCC expression in pregnancy and labor. Reverse transcriptase polymerase chain reaction was used to examine changes in isoform expression in Motif IV, a region of the alpha 1 subunit known to be alternatively spliced. These studies revealed the presence of multiple isoforms in rat myometrium, with a predominance of IVS3B. Interestingly, a marked increase in the ratio of S3B:S3A was noted at parturition. In summary, these data demonstrate that the number of L-type calcium channels, although increased in pregnancy, do not change prior to, or with the onset of, myometrial contraction. Intriguingly, mRNA expression was markedly decreased at parturition. The change in isoform expression during labor is of unknown, but potential, physiologic significance.

Evidence of a corticotropin-releasing hormone pulse generator in the macaque hypothalamus.

The secretion of hormones from the hypothalamic-pituitary axis is, in general, characterized by an episodic pattern of release. In the adrenal axis, ACTH and cortisol levels in peripheral blood display irregularly pulsatile ultradian patterns that are superimposed on the well characterized circadian rhythm. While it is generally accepted that CRH is released from the hypothalamus in a similar manner, very few studies have actually examined the temporal release of CRH. To examine the temporal release of CRH directly, we have established an in vitro perifusion system using the hemisectioned macaque hypothalamus. Perifusate samples were collected at 10-min intervals for 20 h and assayed for CRH by RIA. In control animals, a very regular, pulsatile pattern of hormone release was present, with a pulse interval of 90 +/- 11 min. Although this interval closely approximates the average pulse interval of ACTH and cortisol in the human, the regular pattern revealed in our study has not been demonstrated previously in the adrenal axis in vivo and suggests that factors outside the hypothalamus play a major role in controlling adrenal hormone levels. When hypothalami were perifused with dexamethasone added to the culture medium, no change in pulsatile activity was detected, indicating that a site outside of the hypothalamus may function as the primary center of feedback inhibition by adrenal glucocorticoids in the central nervous system. Because the very regular pulses of CRH that we observed bear striking similarity to the circhoral pulses of GnRH, we speculate that CRH may play a more subordinate role in regulating the adrenal axis and that other releasing factors and/or feedback effects at the pituitary level may be more important in the generation of the irregularly pulsatile, circadian patterns of ACTH and cortisol seen in peripheral blood.