We previously reported that immunoreactive corticotropin-releasing hormone (CRH) is present in human placenta and third trimester maternal plasma, and that such material is very similar to rat CRH and the predicted structure of human CRH. We suggested that maternal plasma immunoreactive CRH may be of placental origin. To further investigate this possibility, we measured plasma immunoreactive CRH in women during pregnancy, labor, and delivery and 1 and 2 h postpartum, and in nonpregnant women. Umbilical cord plasma and placental CRH concentrations were also measured. In the first trimester of pregnancy, the mean maternal plasma level was 5.9 +/- 1.0 pg (+/- SEM)/ml (n = 24), not significantly different from that in 10 nonpregnant women (5.8 +/- 0.8 pg/ml). Plasma CRH concentrations progressively increased during pregnancy (second trimester, 35.4 +/- 5.9 pg/ml (n = 39); early third trimester (28-34 weeks), 263 +/- 41 pg/ml (n = 14); late third trimester (35-40 weeks), 800 +/- 163 pg/ml (n = 20)]. There was a significant correlation between maternal plasma CRH levels and weeks of pregnancy. Plasma CRH concentrations were further elevated (2215 +/- 329 pg/ml; n = 9). During early labor, peaked at delivery (4409 +/- 591 pg/ml; n = 28), and declined rapidly after delivery [1 h postpartum, 1042 +/- (353 pg/ml (n = 13); 2 h postpartum, 346 +/- 91 pg/ml (n = 13)]. There was a significant correlation (r = 0.562; P less than 0.01) between matched maternal plasma and placental CRH concentrations. The mean umbilical cord plasma CRH level (50.6 +/- 6.1 pg/ml; n = 28) was much lower than that in the mother at the time of delivery. Umbilical venous plasma CRH levels were significantly greater than those in simultaneously obtained umbilical arterial plasma (70.8 +/- 11.3 and 41.8 +/- 4.9 pg/ml, respectively; n = 11). There was a significant correlation (r = 0.384; P less than 0.05) between maternal and fetal CRH concentrations. Gel filtration of plasma obtained from women during the third trimester, at delivery, and early postpartum and placental extracts revealed two major peaks of immunoreactive CRH: a high mol wt peak and one at the elution position of rat CRH. In contrast, only rat CRH-sized material was detected in plasma from nonpregnant women and umbilical cord plasma. Maternal plasma immunoreactive CRH-sized material stimulated ACTH release from anterior pituitary tissue in a dose-dependent manner and was equipotent with rat CRH.(ABSTRACT TRUNCATED AT 400 WORDS)

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Immunoreactive CRH was detected in extracts of human term placentae [5.2 +/- 0.8 (+/- SE) pmol/g wet wt; n = 9]. Molecular sieve chromatography revealed three size classes of immunoreactive CRH. The major species eluted with the Kav of synthetic rat CRH; the minor species had apparent mol wt (MW) of 18,000 and 8,000. A placental CRH-(1-41)-sized peptide was isolated by fractional acetone precipitation, molecular sieve chromatography, and sequential reverse phase high performance liquid chromatography steps. This peptide had the same chromatographic behavior as did rat CRH in all high performance liquid chromatographic isolation steps as well as the same UV absorbance to immunoreactive CRH ratio after the final purification step. Purified placental CRH stimulated ACTH release from anterior pituitary tissue in a dose-dependent manner and was equipotent with synthetic rat CRH. Partial sequencing indicated that 32 amino acids of this peptide are identical to those of rat and human CRH (sequence deduced from genomic sequence), and comparative peptide mapping with rat CRH provided further evidence that the placental CRH-like peptide is very homologous if not identical to CRH. The high mol wt placental CRH fractions also were partially purified by acetone precipitation, immune affinity chromatography, and gel filtration. Neither of these materials [big form (MW, 18,000) or intermediate form (MWr, 8,000)] stimulated ACTH release from rat pituitary tissue in vitro. Limited trypsin digestion of the highest MW CRH, followed by gel filtration analysis, resulted in conversion to the smaller [8,000 MW-sized and CRH-(1-41)-sized] forms. The detection of a CRH-like peptide in placenta together with our previous demonstration of plasma immunoreactive CRH in pregnant women suggest that the placenta synthesizes and secretes CRH into the maternal circulation.

Correction. In the article "Interactions between molecules (subfactors) released by different T cell sets that yield a complete factor with biological (suppressive) activity" by Wlodzimierz Ptak, R. W. Rosenstein, and Richard K. Gershon, which appeared in number 7, April 1982, ofProc. NatL Acad. Sci. USA (79, 2375-2378 ABSTRACT Multiple molecular forms of immunoreactive corticotropin (ACTH) and ,-endorphin were present in extracts of a unicellular eukaryote (Tetrahymena pynformis). One form of immunoreactive ACTH reacted similarly with two different ACTH antisera (one specific for the 11-24 sequence and the other with determinants within sequences 1-14 and 17-39) and migrated with synthetic hACTH-(1-39) in a gel filtration system. This form also exhibited ACTH bioactivity in a dispersed rat adrenal cell bioassay system, with a mean immunoassay/bioassay ratio of 1.5. Gel filtration revealed multiple size classes of immunoreactive (-endorphin; a major peak of radioreceptor activity was detected which exhibited a Kav similar to that of authentic P-endorphin. A major portion of immunoreactive (3-endorphin-sized material exhibited retention times similar to those of synthetic human and camel 3-endorphin upon reverse-phase high-pressure liquid chromatography. These distinctive properties and specificities would seem to exclude the presence oflimited homologies with sequences present in other proteins. High molecular weight material containing both ACTH and 3-endorphin antigenic determinants was also demonstrated, suggesting, but not proving, the presence of a common precursor molecule.In recent years, classic neuropeptides have been discovered in extraneural vertebrate tissues (1), and both gut and pituitary hormones have been found in the brain (2). We Assay Methods. ACTH radioimmunoassay (4). Two antisera were used. Antiserum 7C, raised in a rabbit, contains antibody molecules directed toward determinants in the NH2-terminal region ofthe ACTH molecule. ACTH-(1-13) and human ACTH-(1-39) (hACTH) react on an equimolar basis; a-melanotropin (melanocyte-stimulating hormone) reacts in a parallel fashion at approximately 58% molar crossreactivity. Rat "pro-opiocortin" (the ACTH-lipotropin precursor molecule) purified from pituitary intermediate lobe by gel filtration and ion exchange chromatography also reacts with the antiserum on an approximately 70% molar basis, whereas a-, P3,-endorphin, -endorphin, and human 3-lipotropin do not crossreact. Four microliters ofthis antiserum is sufficient to completely immunoprecipitate 2.7 pmol ofhACTH-(1-39). The West antiserum (National Abbreviations: F3CCOOH, trifluoroacetic acid; CH3CN, acetyl nitrile; ACTH, corticotropin (adrenocorticotropic hormone); hACTH, human ACTH. ¶ To whom reprint requests should be addressed. 2086The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.Proc. Natl. Acad. Sci. USA 79 (1982) 2087 Instit...

ABSTRACr Immunoreactive and bioreactive corticotropin (ACTH-like) activities have been detected in the median eminence and remaining medial basal hypothalamus of both normal and hypophysectomized adult male rats: bioreactive ACTH (pg/100 gg of protein) 1028 in median eminence and 1289 in medial basal hypothalamus; immunoreactive ACTH (midportion MATERIALS AND METHODSThe median eminence and medial basal hypothalamus were removed from 16 control and 16 hypophysectomized (10 days postoperatively) adult male Sprague-Dawley rats as previously described (11), as were the other, larger, regions such as the cerebellum, cerebral cortex, thalamus, and hippocampus. The median eminence and medial basal hypothalamus were dissected from frozen sections of the brain; the other areas of the brain were removed from fresh tissue. The medial basal hypothalamus sample consisted of three 3-mm X 3-mm triangles of brain, each one 0.3 mm thick.Hypophysectomy was confirmed by visual inspection of the sellar area at time of sacrifice; additionally, plasma ACTH* concentrations at this time (between 0900 and 1000 hr) were less than 15 pg/ml in all the hypophysectomized animals and ranged from 27 to 153 pg/ml in the control group (1 ml is equivalent to approximately 55 ,sg of protein). Specimens of cortex, hippocampus, thalamus, and cerebellum were also obtained from normal animals (n = 5). All tissues were analyzed in pools comprised of samples from five or six animals.Immediately after removal, tissues were placed in plastic or heavily silicone-treated microhomogenizing tubes kept on ice, and were homogenized in 0.1 M HCl (0.2-0.5 ml/mg wet tImmunoassay (12) was performed on unextracted plasma by using the paradoxical binding phenomenon described by Matsukara et al. (13) and an antibody raised in our laboratory. On a molar basis, the antibody crossreacted equally with porcine ACTH (pACTH), hACTH, and ACTH1-24; a-MSH had approximately 8% crossreactivity. Over the effective range of the standard curve, ACTHI-'0, ACTH1749 (synthetic human sequence), and fl-MSH were undetectable. At a titer of 1/1500, the useful standard dose range was 0-150 pg/ml, with 2-4 pg/ml routinely being significantly different than the zero dose. pACTH 39 was used as a standard and for iodination. Most plasma specimens exhibited parallelism with the standard curve, with acceptable incubation damage to label at plasma concentrations of up to 20% of incubation volume. Plasma was assayed at dilutions of 2-10%. All specimens were assayed in duplicate at multiple dilutions. Ten pools of rat plasma (range, 18-300 pg/ml) were compared in assays with the "paradoxical" antibody and the NH2-terminal antibody provided by the National Institutes of Health (extracted plasma). Levels obtained with the latter averaged 84% of those with the paradoxical antibody, with an average coefficient of variation of 9.7% (r = 0.99; slope = 1.1). 648

alpha-Endorphin, beta-endorphin, gamma-endorphin, and N-terminal ACTH immunoreactivity were detectable in acid extracts of rat testes with concentrations of 0.07 +/- 0.01, 0.18 +/- 0.03, 0.06 +/- 0.01, and 0.33 +/- 0.08 (+/- SD) pmol/g wet wt, respectively. The forms of these immunoreactive peptides were characterized by reverse phase high performance liquid chromatography. Immunoreactive beta-endorphin was also analyzed by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results indicated that the major form of immunoreactive beta-endorphin present appears to be beta-endorphin-(1-31). No alpha-N-acetylated forms of beta-endorphin or beta-lipotropin-sized material were detected. Immunoreactive alpha- and gamma-endorphin appear to be present as alpha-endorphin and des-Tyr1-gamma-endorphin, respectively. Immunoreactive alpha MSH was present as its desacetylated form. No immunoreactive ACTH fractionating with ACTH-(1-39) or its glycosylated forms was detected. This peptide profile is most similar to that seen for proopiomelanocortin-derived peptides in the brain. The low concentrations of these peptides in rat testes suggest a paracrine function.

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Peptide and protein hormones usually considered as being of pituitary origin have been detected within the central nervous system by means of radioimmunoassay, bioassay, and immunocytochemical techniques. Intracerebral administration of some of these hormones or fragments thereof elicit behavioral responses, suggesting that they may have a physiological role similar to that described for other peptidergic neurotransmitter or neuromodulator substances. Evidence available for some of these hormones indicates that they are synthesized within the central nervous system and that their regulation may differ from that of their pituitary counterparts.