Philip J. Lowry

A placental clock controlling the length of human pregnancy

We report the existence of a ‘placental clock’, which is active from an early stage in human pregnancy and determines the length of gestation and the timing of parturition and delivery. Using a prospective, longitudinal cohort study of 485 pregnant women we have demonstrated that placental secretion of corticotropin-releasing hormone (CRH) is a marker of this process and that measurement of the maternal plasma CRH concentration as early as 16–20 weeks of gestation identifies groups of women who are destined to experience normal term, preterm or post-term delivery. Further, we report that the exponential rise in maternal plasma CRH concentrations with advancing pregnancy is associated with a concomitant fall in concentrations of the specific CRH binding protein in late pregnancy, leading to a rapid increase in circulating levels of bioavailable CRH at a time that coincides with the onset of parturition, suggesting that CRH may act directly as a trigger for parturition in humans.

Corticotropin Releasing Factor (CRF) Binding Protein: A Novel Regulator of CRF and Related Peptides

A 37-kDa corticotropin releasing factor (CRF) binding protein (CRF-BP) was purified from human plasma by repeated affinity purification and subsequently sequenced and cloned. The human and rat CRF-BP cDNAs encode proteins of 322 amino acids with one putative signal sequence, one N-glycosylation site, and 10 conserved cysteines. Human CRF-BP binds human CRF with high affinity but has low affinity for the ovine peptide. In contrast, sheep CRF-BP binds human and ovine CRF with high affinity. The CRF-BP gene consists of seven exons and six introns and is located on chromosome 13 and loci 5q of the mouse and human genomes, respectively. CRF-BP inhibits the adrenocorticotrophic hormone (ACTH) releasing properties of CRF in vitro. CRF-BP dimerizes after binding CRF and clears the peptide from blood. This clearance mechanism protects the maternal pituitary gland from elevated plasma CRF levels found during the third trimester of human pregnancy. CRF-BP is expressed in the brains of all species so far tested but is uniquely expressed in human liver and placenta. In brain, CRF-BP is membrane associated and is predominantly expressed in the cerebral cortex and subcortical limbic structures. In some brain areas CRF-BP colocalizes with CRF and CRF receptors. The protein is also present in pituitary corticotropes, where it is under positive glucocorticoid control, and is likely to locally modulate CRF-induced ACTH secretion. The ligand requirements of the CRF receptor and the CRF-BP can be distinguished in that central human CRF fragments, such as CRF (6-33) and CRF (9-33), have high affinity for CRF-BP but low affinity for the CRF receptor. The binding proteins ability to inhibit CRF-induced ACTH secretion can be reversed by CRF (6-33) and CRF (9-33), suggesting that ligand inhibitors may have utility in elevating free CRF levels in disease states associated with decreased CRF. Thus, by controlling the amount of free CRF which activates CRF receptors, it is likely that the CRF-BP is an important modulator of CRF both in the CNS and in the periphery.

Characterization of the endokinins: Human tachykinins with cardiovascular activity

We report four human tachykinins, endokinins A, B, C, and D (EKA–D), encoded from a single tachykinin precursor 4 gene that generates four mRNAs (α, β, γ, and δ). Tachykinin 4 gene expression was detected primarily in adrenal gland and in the placenta, where, like neurokinin B, significant amounts of EKB-like immunoreactivity were detected. EKA/B 10-mers displayed equivalent affinity for the three tachykinin receptors as substance P (SP), whereas a 32-mer N-terminal extended form of EKB was significantly more potent than EKA/B or SP. EKC/D, which possess a previously uncharacterized tachykinin motif, FQGLL-NH2, displayed low potency. EKA/B displayed identical hemodynamic effects to SP in rats, causing short-lived falls in mean arterial blood pressure associated with tachycardia, mesenteric vasoconstriction, and marked hindquarter vasodilatation. Thus, EKA/B could be the endocrine/paracrine agonists at peripheral SP receptors and there may be as yet an unidentified receptor(s) for EKC/D.

Characterization of a Serine Protease that Cleaves Pro-γ-Melanotropin at the Adrenal to Stimulate Growth

The adrenal gland requires stimuli from peptides derived from the ACTH precursor, pro-opiomelanocortin (POMC), to maintain its tonic state. Studies have proposed that a specific postsecretional cleavage of the nonmitogenic N-terminal 16 kDa fragment, also known as pro-gamma-melanotropin (pro-gamma-MSH), is required, releasing shorter fragments that promote adrenal growth. Here, we provide evidence for this hypothesis by the cloning and characterization of a serine protease that is upregulated during growth of the adrenal cortex. It is expressed exclusively in the outer adrenal cortex, the site of cell proliferation, and in the Y1 adrenal cell line. We also show that it is required for growth of Y1 cells, remains bound to the cell surface, and cleaves its substrate, pro-gamma-MSH, at a specific bond.

A regulatory role for neurokinin B in placental physiology and pre-eclampsia.

Tachykinin dogma has assumed, so far, that neurokinin B (NKB) is a neuropeptide that is not produced in any peripheral tissue even though its endogenous receptor, NK3, has been found in a number of locations throughout the human body. We have found an abundant source of peripheral NKB in the human and rat placenta. In this review we describe the discovery of NKB in the placenta and examine its possible role in placental physiology and pre-eclampsia (PE). Excessive secretion of placental NKB into the maternal circulation during the third trimester of pregnancy has been found in women suffering from PE. This may provide the key to the cause of the multiple and complex symptoms associated with this potentially life-threatening illness. We also reveal the structural organisation of the human NKB gene for the first time as well as discussing putative mechanisms for its control.

Growth Hormone Releasing Factor (GRF) Immunoreactivity in Human and Rat Gastrointestinal Tract and Pancreas

Rabbit antisera were raised against a synthetic growth hormone releasing factor, which was originally isolated from a human pancreatic endocrine tumor (hpGRF-44). The antisera obtained showed no significant cross-reactivity with a variety of neurohormonal peptides. In addition to its occurrence in the human, but not in the rat, hypothalamus, hpGRF-44-like immunoreactivity was identified in human gastric antrum and human as well as rat pancreatic islets, using an indirect immunoperoxidase technique. Staining of serial sections and double staining revealed that in the gastric antrum the immunoreactivity was largely confined to gastrin (G) cells, whereas in pancreatic islets polypeptide (pp) cells were reactive. The physiological significance of these findings remains to be established.

Heterogeneous Immunocytochemical Reactivities of oCRF-41-Like Material in the Human Hypothalamus, Pituitary and Gastrointestinal Tract

Tissue specimens of human hypothalami, pituitaries and gastrointestinal tract were studied with an indirect immunoperoxidase technique using 6 different rabbit ovine corticotrophin-releasing factor (oCRF-41) antisera. All these antisera detected oCRF-41-like immunoreactivity in parvocellular neurones of the paraventricular nucleus of the hypothalamus and their nerve terminals adjacent to portal capillaries in the infundibular stem and posterior pituitary. 1 antiserum recognised oCRF-41-like immunoreactivity in the growth hormone cells of the anterior pituitary. 3 other antisera recognised oCRF-41-like immunoreactivity in mucosal cells of the gastric antrum. Pre-treatment of the antisera with sauvagine did not affect the immunostaining of the hypothalamic and gastric cells, but quenched the immunostaining of growth hormone cells in the anterior pituitary. It is concluded that (1) in human hypothalami a CRF is synthesized that is structurally very similar to oCRF-41; (2) in growth hormone cells of the anterior pituitary oCRF-41-like immunoreactive material can be detected which shares antigenic determinants with sauvagine; (3) in mucosal cells of the gastric antrum oCRF-41-like immunoreactivity is present that is comparable but probably not identical to hypothalamic CRF and is not sauvagine-like.

Characterization of a sheep brain corticotropin releasing factor binding protein.

We report here the identification, purification and cDNA cloning of a corticotropin releasing factor (CRF) binding protein(s) (CRF-BP) from sheep brain. Native sheep and rat brain CRF-BP and recombinant rat CRF-BP were shown to be N-glycosylated. Two membrane associated forms of brain CRF-BPs of 33 and 35 kDa were purified from sheep brain homogenates after solubilization in the presence of detergent. N-Terminal sequence analysis revealed that the 35 kDa protein is proteolytically cleaved near the N-terminus giving rise to an 18 amino acid peptide and a 33 kDa CRF-BP. Both the purified 33 and 35 kDa ovine CRF-BPs could be specifically cross linked to ovine [125I]CRF and human [125I]CRF. In contrast, recombinant rat CRF-BP can only be cross-linked to human [125I]CRF. A 1.7 kb cDNA clone (Basil 7) encoding an open reading frame for a 324 amino acid CRF-BP precursor was cloned from a sheep brain lambda gtlO cDNA library and was shown to have 85% and 87% amino acid homology to the rat and human proteins, respectively. Competitive binding analysis of the recombinant sheep CRF-BP (Basil 7) expressed in CHO cells revealed that it binds human and ovine CRF with high affinity. However, the recombinant sheep CRF-BP (Basil 7) had approximately 50-fold higher affinity for human CRF than for the ovine peptide. These data present the first biochemical proof that CRF-BP is in the brain and provides evidence for the existence of different forms of CRF-BP which have evolved across species to regulate CRF.

Assessment of Corticotropin-Releasing Factor, Vasopressin and Somatostatin Secretion by Fetal Hypothalamic Neurons in Culture

The concomitant release of corticotropin-releasing factor (CRF), vasopressin (AVP) and somatostatin (SRIF) has been followed from primary cultures of rat hypothalamic neurons. 18-day-old fetal rat hypothalami were dissociated enzymatically and mechanically, then plated and maintained in a serum-containing medium at a density of 2.5 x 10(6) cells per dish (equivalent to 3 hypothalami). Cultured neurons remained viable for up to 6 weeks, and peptide release was followed by immuno-assay between days 14 and 39 in culture. The incubation media were concentrated on C4 and C8 silica columns to facilitate detection of CRF and AVP. Peptide release was measured at various times up to 4 h, at which point it was still increasing. To optimise measurements, taking into account peptide degradation, a 1-hour incubation period was chosen for further studies. Release of CRF, AVP and SRIF by 56 mM K+ or 10 microM veratridine was statistically significantly greater than basal (p less than 0.01) and was Ca2-dependent. For CRF and AVP, stimulated release increased considerably with the age of culture, whereas SRIF release was steadier. Basal release for all 3 peptides did not fluctuate greatly over this period. Basal and stimulated release of the peptides continued over at least 5 successive 1-hour periods. At day 35 of culture, the peptide content was still increasing in a pattern which paralleled the increasing content in hypothalami freshly removed from age-matched rats. In conclusion, we have demonstrated a development of CRF, AVP and SRIF production by neurons over extended periods in culture as assessed by their peptide content and increasing responses to depolarizing stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of a novel mammalian post-translational modification, phosphocholine, on placental secretory polypeptides

Placental neurokinin B appears to be post-translationally modified by phosphocholine (PC) attached to the aspartyl side chain at residue 4 of the mature peptide. Corticotrophin releasing factor (CRF) was found to be expressed by the rat placenta with the main secreted forms being phosphocholinated proCRF+/− one or two polysaccharide moieties. A combination of high-pressure liquid chromatography (HPLC) and two-site immunometric analysis suggested that PC was also attached to the placental precursors of adrenocorticotrophin, hemokinin, activin and follistatin. However, the fully processed forms of rat placental activin and CRF were free of PC. Formerly, the parasitic filarial nematodes have used PC as a post-translational modification, attached via the polysaccharide moiety of certain secretory glycoproteins to attenuate the host immune system allowing parasite survival, but it is the PC group itself which endows the carrier with the biological activity. The fact that treatment of proCRF peptides with phospholipase C but not endoglycosidase destroyed PC immunoreactivity suggested a simpler mode of attachment of PC to placental peptides than that used by nematodes. Thus, it is possible that by analogy the placenta uses its secreted phosphocholinated hormones to modulate the mothers immune system and help protect the placenta from rejection.