David W. Lundgren

Gestational changes in the uterine expression of an inwardly rectifying K+ channel, ROMK.

Abstract We have examined the repertoire and relative expression levels of voltage-gated K+ channels in timed-pregnant rat uteri. These studies have revealed the gestation-specific and abundant expression of mRNA encoding an inwardly rectifying K+channel, ROMK (originally identified in renal outer medulla), within the gravid uterus. Steady-state levels of ROMK transcripts undergo dynamic gestational changes: they are undetectable in virgin uteri, reach a maximum level by Day 12 of gestation, decline thereafter until, by term, they are again undetectable. Kidney cells also express ROMK transcripts at high levels but do not undergo apparent changes during gestation. Molecular analyses (by “rapid amplification of cDNA ends, or “5′-RACE”) of the ROMK mRNAs revealed the presence of two alternative-splicing variants which are likely to arise from distinct transcription-start sites within the same gene. Polymerase chain reaction-based assessments of gravid uteri from other species revealed the expression of ROMK transcripts in the myometrium as well. Uterine expression of ROMK therefore represents a generalized phenomenon, characterized by both gestation- and tissue-specific regulation, and the transcription-regulatory mechanisms of this channel protein are potentially complex. From the biophysical properties of this channel in vitro and the observed gestational profile, we hypothesize that this channel modulates both the resting membrane potential and cellular excitability of myometrial cells, and in turn contributes to the observed contractile quiescence of the gravid uterus.

HYPOTONIC STRESS INCREASES CYCLOOXYGENASE-2 EXPRESSION AND PROSTAGLANDIN RELEASE FROM AMNION-DERIVED WISH CELLS

This report examines the effect of cell volume expansion on cyclooxygenase-2 (COX-2) mRNA expression, COX-2 protein expression, and prostaglandin E2 release from human amnion-derived WISH cells. Earle’s balanced salts solution (EBSS) with limited NaCl concentration was utilized as the induction medium. COX-2 mRNA was elevated 6-fold in cells incubated for 1 h in hypotonic EBSS. COX-2 mRNA expression was not increased when raffinose or sucrose were used to reconstitute low NaCl. Actinomycin D blocked COX-2 mRNA increase by hypotonic stress, while cycloheximide enhanced COX-2 mRNA expression. COX-2 mRNA and protein concentrations increased as a function of decreasing media osmolarity and incubation time in hypotonic EBSS. Hypotonic EBSS induced a 3-fold increase in prostaglandin E2 release. WISH cells transiently transfected with a luciferase expression vector driven by the human COX-2 promoter for the COX-2 gene show a 3-fold increase in luciferase activity when incubated in hypotonic EBSS. COX-2 mRNA levels in primary human amnion cells were also increased by hypotonic stress. This study suggests that amnion cell COX-2 gene expression is regulated by cell volume expansion and/or increased plasma membrane tension.

Neonatal glycine encephalopathy: Biochemical and neuropathologic findings

A patient with neonatal glycine encephalopathy who had severe neurologic retardation, spasticity, and seizures died at 17 years of age. Glycine concentration was markedly elevated in brain tissue, especially in the cerebellum. Neuropathologic study revealed spongy myelinopathy throughout the central nervous system and calcium oxalate crystals in the cerebellum, which are probably derived from degradation of glycine. Myelinopathy appeared to be static compared to neonatal patients. The neurologic manifestations of neonatal glycine encephalopathy are probably due to neurotransmitter abnormalities, not to myelin damage.

Gestational Changes in Uterine L-Type Calcium Channel Function and Expression in Guinea Pig

Abstract Pregnancy can influence both the resting membrane potential and the ion channel composition of the uterine myometrium. Calcium flux is essential for excitation-contraction coupling in pregnant uterus. The uterine L-type calcium channel is an important component in mediating calcium flux and is purported to play a role in parturition. This study was undertaken to characterize gestational changes in 1) the uterine contractile response to the L-type calcium channel agonist, Bay K 8644; 2) the mRNA expression of channel subunits by semiquantitative reverse transcriptase-polymerase chain reaction; and 3) estimate channel protein levels by measuring 3H-isradipine binding at the dihydropyridine binding site of the α1c subunit utilizing saturation binding methods. Sensitivity to Bay K 8644 increases beginning at 0.8 of gestation and persists through term. The change in sensitivity is coincident with an increased mRNA expression of the α1c and β2 subunits but with the least detectable amounts of isradipine binding. The expressed α1c transcript represents a novel structural variant with a 118-amino acid deletion in the III–IV linker and repeats IVS1–S3 of the protein sequence. The guinea pig uterine L-type calcium channel activity is highly regulated through gestation, but the regulation of mRNA expression may be different from regulation of protein levels, estimated by isradipine binding. The up-regulation of function, α1c subunit mRNA expression, and isradipine binding at term gestation are consistent with a role for this ion channel in parturition.

Cyclooxygenase inhibitors decrease apoptosis initiated by actinomycin D, cycloheximide, and staurosporine in amnion-derived WISH cells.

Apoptosis is a process by which external or developmental factors induce a specific series of evetns leading to cell death. Recently, apoptotic cells have been described in rat amnion membrane at late getation, suggesting apoptosis may be involved in membrane rupture. Mechanisms controlling amnion cell apoptosis are unknwon. The objective of this study was to investigate whether cyclooxygenase and prostaglandins are integral to apoptosis in amnion, as reported in intestinal epithelial cells and renal mesangial cells. Amnion-derived WISH cells underwent apoptosis in a dose- and time-dependnet manner after incubation with actinomycin D, cycloheximide, or staurosporine, as determined by cell viability, DNA fragmentation analysis, and fluorescent in situ fragmentation analysis. Cells cultured with increasing does of these agents also demonstrated concomitant increases in prostaglandin E2 outpuot. WISH cell coincubation with these agents and the cyclooxygenase inhibitors indomethacin or piroxican resulted in dose-dependent decreases in both prostaglandin E2 and apoptosis. Cultures incubated with 0.5 μg/mL actinomycin D showed 80.7% cell apoptosis after 12 hours compared with 1.1% in untreated cultures. After 24 hours incubation with actinomycin D, 0.8% of the original cell number remained attached to the plate. In cultures coincubated with 0.5 μg/mL actinomycin D and 100 μmol/L indomethacin, only 19.2%, 24.7%, and 39.3% of the cells were found to be apoptotic after 12, 24, and 48 hours in culture, respectively. Similar trends were observed after the use of cycloheximide or staurosporine in combination with indomethacin or prioxicam. These data suggest that cyclooxygenase and/or prostaglandins play a role in programmed cell death of amnion-derived WISH cells in culture.

Lactosylceramide-Induced Apoptosis in Primary Amnion Cells and Amnion-Derived WISH Cells

Objective: Amnion apoptosis is part of a programmed process of fetal membrane remodeling leading to weakening and rupture. The apoptotis agent lactosylceramide is elevated in amniotic fluid of premature infants with rupture of membranes. We have shown that apoptosis in WISH cells, induced by staurosporine, cycloheximide, or actionomycin D, can be blocked by cyclooxygenase inhibitors, suggesting a relationship between prostaglandin production and apoptosis. Cyclic adenosine monophosphate (cAMP) is known to inhibit prostaglandin release in amnion and WASH cells. This study was undertaken to determine the apoptotic potential of lactosylceramide and the effect of cyclooxygenase inhibitors and cAMP activators on lactosylceramida-induced apoptosis in primary amnion and WISH cells. Methods: Primary amnion cells and WISH cells were incubated with lactosylceramide to determine apoptosis and prostaglandin E2 (PGE2) release. Apoptosis was confirmed b agarose gel electrophoretic DNA fragmentation analysis, nuclear matrix protein (NMP), and uncleosome enzyme-linked immunosorbent assay. In some studied, cells were preincubated with cyclooxygenase inhibitors or cAMP activators. Results: Lactosylceramide induced a 20-fold increase in NMP (measure of cell death) in both cell types. Apoptosis was confirmed by the studied listed in methods. Lactosylceramide increased PGE2 release in parallel with apoptosis. Cyclooxygenase inhibitors as well as cAMP activators inhibited both PGE2 release and apoptosis. Conclusions: Lactosylceramide-induced apoptosis in both amnion and WISH cells. Parallel PGE2 release was demonstrated with apoptosis. Cyclooxygenase inhibitors and cAMP activators blocked both processes.

Spermine-Enhanced Protein Phosphorylation in Human Placenta

Abstract Polyamines are known to have a role in cell proliferation, differentiation, and protein synthesis. During pregnancy, major changes in polyamine levels occur in maternal serum, amniotic fluid, and placental tissue. Polyamine-activated phosphorylation has recently been proposed as a mechanism by which polyamines may regulate metabolic processes in target tissues. Polyamine-activated protein phosphorylation has not been studied in placenta. Homogenate membrane and cytosol fractions from human placenta were subjected to an endogenous protein phosphorylation assay using [γ-32P]ATP in the presence and absence of the polyamines, spermine and spermidine, and the diamine, putrescine. Protein phosphorylation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. When compared to basal levels, spermine (10-3 M) significantly (P < 0.001) stimulated 32P incorporation into phosphoproteins having molecular weights of 55,000 and 105,000. At this concentration spermidine and putrescine failed to stimulate phosphorylation. Half-maximal 32P incorporation was observed with 3.7 ± 1.25 × 10-4 M spermine. Polylysine enhanced the phosphorylation of phosphoproteins of the same molecular weight as those enhanced by spermine. Heparin and high Mg2+ inhibited spermine-induced phosphorylation. cAMP and Ca2+ did not stimulate phosphorylation of the spermine-dependent phosphoproteins. Spermine, however, acted as an antagonist for cAMP-dependent phosphorylation of a M r 45,000 phosphoprotein.