Polarised expression of human intestinal N-benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase (human meprin) alpha and beta subunits in Madin-Darby canine kidney cells.

N-Benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase (PPH, human meprin), is a peptidase found in the microvillus membrane of human small intestinal epithelial cells. PPH belongs to the astacin family of zinc-metalloendopeptidases and is a protein complex composed of two glycosylated subunits, alpha and beta. The present report describes the cloning of the complete beta subunit and the remaining N2-terminal end of the alpha subunit for analysis of their primary structures in addition to the examination of their biogenesis in transfected cell cultures. The complete open reading frame of the PPH beta cDNA translates into 700 amino acid residues compared with 746 residues for the PPH alpha cDNA. The primary structure of beta and alpha subunits are 44% identical and 61% similar. As predicted from their primary structure, the two subunits of PPH have identical modular structures; starting at the N2-terminus both contain a signal peptide, a propeptide, a protease domain containing the astacin signature, a meprin A5 protein tyrosine phospatase mu (MAM) and a meprin and TRAF homology domain (MATH) domain, an epidermal growth factor(EGF)-like domain, a putative transmembrane anchor domain and a short cytosolic tail. Pulse/chase labelling and immuno-Gold electronmicroscopy of recombinant PPH beta and alpha subunits expressed in transfected Madin-Darby canine kidney (MDCK) cells show that post-translational processing and transport of the two subunits are very different. When expressed alone, the beta subunit acquired complex glycan residues, readily formed homodimers and was transported to the plasma membrane. Small amounts of PPH beta were found in the culture medium. In contrast, the cell-bound alpha subunit, when expressed alone, remained primarily in the high-mannose form, was aggregated and not expressed at the cell surface. However, the bulk of mostly endo-beta-N-acetylglucosaminidase H-resistant alpha subunit was found in the filtered culture medium. The proteolytic event that leads to the formation of this soluble transport-competent form occurs in the endoplasmic reticulum (ER). Coexpression of the alpha subunit with the beta subunit allowed the localisation of the alpha subunit to the plasma membrane. These studies indicate that assembly of the two subunits of PPH is required for the localisation of the alpha subunit to the plasma membrane. In contrast to rodent meprin, both PPH subunits are apically secreted from MDCK cells.

The central oxytocin pulse generator: a pacemaker for the ovarian cycle.

During luteolysis in sheep, episodic pulses of oxytocin (OT), contributed by the neurohypophysis and the corpus luteum (CL), stimulate uterine luteolytic pulses of prostaglandin (PG) F2 alpha via endometrial OT receptors. To distinguish relative contributions of neurohypophysial and luteal OT, ovariectomized sheep were given estradiol-17 beta (E) and progesterone (P) to stimulate levels during the cycle. In intact sheep, luteectomy was performed to exclude the CL as a source of OT and to initiate P withdrawal. In ovariectomized sheep, E (1 microgram/h) for 12 to 36 h) superimposed on basal E(0.05 microgram/h), caused a series of 4 to 6 episodes of high frequency pulses of OT, each episode lasting 1 to 2 h at intervals of 3 h, and commencing at 24 h. Withdrawal of P (500 micrograms/h), superimposed on basal E in ovariectomized sheep, or luteectomy in intact sheep, evoked similar episodes of high frequency pulses of OT beginning at 24 h. We conclude that (1) an increase in E levels, or the return of E action following P withdrawal, causes intermittent increases in the frequency of the central OT pulse generator. (2) high frequency pulses of OT initiate subluteolytic levels of uterine PGF2 alpha which trigger a supplemental release of luteal OT; (3) luteal OT amplifies the secretion of uterine PGF2 alpha which initiates luteolysis and causes more luteal OT to be secreted; and (4) in addition to the established hypothalamic-anterior pituitary-gonadal axis for initiating the ovarian cycle (via the gonadotrophins), there is now evidence for a hypothalamic-posterior pituitary-gonadal axis for terminating the ovarian cycle (via OT).

Identification of functional high and low affinity states of the prostaglandin F2 alpha receptor in the ovine corpus luteumin vivo and their role in hormone pulsatility.

Equivocal evidence has accumulated for the presence of high and low affinity receptors for PGF(2α) in the corpus luteum based on binding affinities of(3)H-PGF(2α) to cell membranes or separated whole cells. Some studies report only high affinity sites, while others report the occurrence of both high and low affinity sites. We have previously demonstrated, using subluteolytic levels of PGF(2α), the existence of functional high affinity luteal PGF(2α) receptors which show desensitization and recovery after 6 to 9 h. The present study, using direct intra-arterial infusions of PGF(2α) into the autotransplanted ovary in conscious sheep, was designed to probe for the existence of functional high and low affinity states of the PGF(2α) receptor in the corpus luteumin vivo. Subluteolytic and luteolytic concentrations of PGF(2α) (100 pg/min and 2500 pg/min, respectively) were infused sequentially, each for 2 h, into the ovary during the luteal phase (n=7 sheep). The same low and high concentrations of the inactive metabolite of PGF(2α) (PGFM) were given over the same time periods as negative controls (n=4 sheep). During the 2 h intra-arterial infusion of 100 pg/min of PGF(2α) the secretion rate of oxytocin increased (P<0.01) while the secretion rate of progesterone was unaffected. In contrast, during the 2 h intra-arterial infusion of 2500 pg/min of PGF(2α), secretion rate of oxytocin increased (P<0.01) and secretion rate of progesterone now began to decline (P<0.05). During the 2 h infusions of identical concent-rations of PGFM, the secretion rate of oxytocin and progesterone remained unchanged. These results indicate the existence of functional high and low affinity states of the PGF(2α) receptor within the ovine corpus luteumin vivo.

Characterization of a membrane-bound metalloendoprotease of rat C6 glioblastoma cells.

C6 rat glioblastoma cells are able to attach to and to spread on culture dishes which are coated with purified central nervous system myelin, in contrast to normal astrocytes, fibroblasts or neurons which adhere poorly and are unable to spread on this substrate. The metalloprotease blockers o-phenanthroline and a newly developed oligopeptide could specifically inhibit C6 cell spreading on central nervous system myelin, suggesting a crucial role for a metalloprotease. Here we characterize this metalloproteolytic activity of C6 cells using a peptide degradation assay with the iodinated tetrapeptide carbobenzoxy-Phe-Ala-Phe-125I-Tyr-amide as a substrate. Purified, salt-washed C6 plasma membranes cleaved the peptide between alanine and phenylalanine, an effect which is strongly inhibited by o-phenanthroline, but not by thiol-blocking agents or aspartic and serine protease inhibitors. The metalloendoprotease is highly sensitive to phosphoramidon but insensitive to thiorphan. The enzyme is tightly bound to the plasma membrane but not G protein-phosphatidylinositol linked. It can be solubilized in part by the detergents 3-(3-cholamidopropyldimethylamino)-1-propanesulfonate or Triton X-114. Gel filtration chromatography using the Triton X-114-solubilized proteins or the proteins removed by a short trypsin treatment revealed a molecular weight range for the C6 enzyme of 60,000-100,000. Polymerase chain reaction with primers corresponding to endopeptidase 24.11 or to the highly conserved motif of the "astacin family" showed that both enzymes were not detectable in the C6 glioblastoma cells.

Cloning of the PABA peptide hydrolase alpha subunit (PPH alpha) from human small intestine and its expression in COS-1 cells.

PABA peptide hydrolase (PPH) from human enterocytes is comprised of two subunits, alpha and beta. PPH alpha is over 70% identical to meprin, a protease isolated from mouse and rat kidney. The enzyme shows a modular organization in that it contains an astacin protease domain, an adhesive domain, an EGF-like domain, an a putative C-terminal membrane spanning domain. Expression of a chimeric meprin-PPH alpha cDNA in COS-1 cells led to the synthesis of immature, transport-incompetent homodimers. In addition, complex glycosylated forms were detected in the culture medium, suggesting that the enzyme is secreted after proteolytic removal of the membrane anchor.

Expression of the alpha subunit of PABA peptide hydrolase (EC 3.4.24.18) in MDCK cells. Synthesis and secretion of an enzymatically inactive homodimer.

In this paper, we report the expression of PPH alpha in the polarized cell line MDCK (Madin Darby canine kidney). In these cells, the enzyme was synthesized in an inactive proform, which upon treatment with trypsin was activated. The enzyme isolated from cell extracts was core-glycosylated and appeared to be retained in the ER as a homodimer. No PPH alpha was detectable on the surface of intact cells by immunofluorescence. However, a complex glycosylated soluble but inactive form was present in the culture medium, suggesting that proteolytic removal of the C-terminal membrane anchoring peptide leads to the secretion of PPH alpha.

Hormonal regulation of endometrial prostaglandin F2 alpha production during the luteal phase of the rhesus monkey.

To study the hormonal regulation of prostaglandin (PG) production by the endometrium during the luteal phase of the primate menstrual cycle, the standard artificial menstrual cycle (SAMC) of the rhesus monkey was manipulated (MAMC) such that in one group of monkeys, there was an absence of the mid-cycle peak of estradiol-17 beta (E), but normal luteal phase progesterone (P). In the second group, there was a mid-cycle peak of E, but no luteal phase P. The accumulation of PGF2 alpha in tissue culture medium from explants of endometrium obtained on cycle Day 14 or 23 of the MAMC was compared to the accumulation of PGF2 alpha from explants on cycle Day 14 or 23 of the SAMC (expressed as mean ng +/- SEM/mg/24 h). Omission of the mid-cycle E peak in the MAMC did not alter endometrial PGF2 alpha production in vitro on cycle Day 14, compared to the SAMC; whereas, on cycle Day 23 PGF2 alpha, production was reduced (35.1 +/- 6.4 ng/mg/24 h), compared to that in the SAMC (53.8 +/- 10.3 ng/mg/24 h; p = 0.06). Omission of P during the MAMC resulted in higher PGF2 alpha production in vitro on cycle Day 14 (p < 0.01) and lower PGF2 alpha production on cycle Day 23 (p = 0.05), compared to that in the SAMC on these days.(ABSTRACT TRUNCATED AT 250 WORDS)

Presence of lipocortins I and IV, but not II and VI, in human platelets.

The present investigation revealed the presence of lipocortins I and IV, but not lipocortins II and VI, in human platelets. Lipocortin I was found in the Triton-soluble fraction of both resting and thrombin-activated platelets and was not covalently bound to skeletal components. Without detergents, when resting platelets were lysed and fractionated in the absence of Ca2+, lipocortin I was found only in the cytosolic fraction, whereas, in the presence of Ca2+, lipocortin I was associated only with the crude particulate and not with the membrane nor the cytosolic fractions.

Hormonal regulation of prostaglandin production by rhesus monkey endometrium.

Although there have been numerous studies on the production of prostaglandins (PGs) by human endometrium in vitro during the menstrual cycle, considerable variation exists in the levels reported during the proliferative vs. the secretory phase. Such variation may be due in part to the difficulty in obtaining endometrium from a precisely known hormonal environment and in part to the use of the different culture systems employed. The aim of the present study was to develop a non-human primate model in which precisely dated endometrial tissue could be obtained reliably. Moreover, PG levels in the endometrium of the rhesus monkey or other primates have not previously been reported during the artificial menstrual cycle. An important objective in establishing such a model was to permit future manipulations of the cycle in vivo [e.g. by omitting the midcycle estradiol (E) peak] to further dissect specific roles of E and progesterone (P) in regulating PG synthesis during the menstrual cycle. Ovariectomized rhesus monkeys were maintained on a standard artificial menstrual cycle via the insertion and removal of Silastic capsules containing E or P. Samples of endometrium (approximately 50 mg) were obtained by hysterotomy under sterile conditions at predetermined stages of separate menstrual cycles: day 9 (midproliferative; n = 5), day 13 (E peak; n = 3), day 14 (1 day post-E peak; n = 5), and day 23 (midsecretory; n = 8). Measurement of the primary PGs in unextracted medium by RIA over 4 days of organ culture indicated PGF2 alpha greater than 6-keto-PGF1 alpha greater than PGE2 greater than thromboxane-B2, PGD2 greater than leukotrienes. PGF2 alpha, the most abundant PG produced on the first day of culture, was low on day 9 and increased dramatically on day 13 (P less than 0.01). On day 14, PGF2 alpha levels fell significantly only 1 day post-E peak (P less than 0.01), while on day 23, after exposure to P in vivo, PGF2 alpha was 10-fold higher (P less than 0.01) than on cycle days 9 and 14. The other PGs measured showed a lower but similar profile at the cycle stages examined. Physiological concentrations of P (5.0 ng/mL) added to cycle day 23 cultures in both the absence and presence of low or high E markedly inhibited the high levels of PGs found in day 23 cultures (P less than 0.01).(ABSTRACT TRUNCATED AT 400 WORDS)

Prostaglandin F2 alpha-stimulated release of ovarian oxytocin in the sheep in vivo: threshold and dose dependency.

To determine the threshold of prostaglandin F2 alpha (PGF2 alpha)-stimulated oxytocin secretion from the ovine corpus luteum, low levels of PGF2 alpha (5-100 pg/min) were infused into the ovarian arterial blood supply of sheep with ovarian autotransplants. PGF2 alpha was infused for six sequential 10-min periods at hourly intervals, 6, 12, or 24 days after estrus (n = 3 for each day). Each cycle day was studied during a separate cycle. Oxytocin and progesterone in ovarian venous and carotid arterial plasma was measured by radioimmunoassay, and secretion rates were determined (venous-arterial concentration x plasma flow). In animals treated on Day 6, 5 pg/min PGF2 alpha caused a significant release of oxytocin (p less than 0.01), whereas in animals treated on Day 12, this threshold was 40 pg/min (p less than 0.05). In animals treated on Day 24, the threshold for oxytocin release was greater than 100 pg/min. PGF2 alpha did not significantly change ovarian blood flow or progesterone secretion rate on any day (p greater than 0.05). To determine residual luteal oxytocin after each threshold experiment, 5 mg PGF2 alpha was given i.m. to all animals. Significantly more oxytocin was released by Day 6 than by Day 12 and Day 24 corpora lutea, and by Day 12 than by Day 24 corpora lutea (1.2 micrograms, 0.7 microgram, and 0.3 microgram, respectively; p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)