Intraoperative oliguria predicts acute kidney injury after major abdominal surgery.

Background: The threshold of intraoperative urine output below which the risk of acute kidney injury (AKI) increases is unclear. The aim of this retrospective cohort study was to investigate the relationship between intraoperative urine output during major abdominal surgery and the development of postoperative AKI and to identify an optimal threshold for predicting the differential risk of AKI. Methods: Perioperative data were collected retrospectively on 3560 patients undergoing major abdominal surgery (liver, colorectal, gastric, pancreatic, or oesophageal resection) at Kyoto University Hospital. We evaluated the relationship between intraoperative urine output and the development of postoperative AKI as defined by recent guidelines. Logistic regression analysis was performed to adjust for patient and operative variables, and the minimum P -value approach was used to determine the threshold of intraoperative urine output that independently altered the risk of AKI. Results: The overall incidence of AKI in the study population was 6.3%. Using the minimum P -value approach, a threshold of 0.3 ml kg -1 h -1 was identified, below which there was an increased risk of AKI (adjusted odds ratio, 2.65; 95% confidence interval, 1.77-3.97; P <0.001). The addition of oliguria <0.3 ml kg -1 h -1 to a model with conventional risk factors significantly improved risk stratification for AKI (net reclassification improvement, 0.159; 95% confidence interval, 0.049-0.270; P =0.005). Conclusions: Among patients undergoing major abdominal surgery, intraoperative oliguria <0.3 ml kg -1 h -1 was significantly associated with increased risk of postoperative AKI.

Cytokine profile during the clinical course of toxic shock syndrome.

Toxic shock syndrome (TSS) is an acute febrile disease with multiple organ involvement caused by massive and rapid release of cytokines induced by staphylococcal exotoxins. However, the precise cytokine profile is still undefined in clinical cases. We measured serum cytokine concentrations in a patient who developed TSS after a caesarean section. Measurements were taken on admission and several times during the course of the disease. Methicillin-resistant Staphylococcus aureus producing TSS toxin-1 and staphylococcal enterotoxin C was detected in the lochia and venous blood. Serum interleukin (IL)-6 level was markedly increased on admission, and IL-10, tumour necrosis factor-alpha, and interferon-gamma levels were also raised. These cytokine levels rapidly returned to normal levels. In contrast, IL-1beta and IL-2 were below the analytical sensitivity threshold throughout the course. Our data and other previous case reports indicate that a marked increase in IL-6 concentration could be a clinical marker of TSS onset.

Spectral changes upon subunit association in valency hybrid hemoglobins.

The absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of valency hybrid hemoglobins and their constituents (alpha + and beta chains for alpha 2+beta 2, alpha and beta + chains for alpha 2 beta 2+: + denotes ferric heme) were measured in the Soret region for F-, H2O, N3- and CN- derivatives. Absorption and MCD spectra of valency hybrid hemoglobins were very similar to the arithmetic mean of respective spectra of their corresponding component chains in all derivatives. The Soret MCD intensity around 408 nm for various complexes of valency hybrid hemoglobins seems to reflect the spin state of ferric chains. Upon ferric and deoxy ferrous subunit association to make the deoxy valency hybrid hemoglobins, only the high-spin forms bound with F- and H2O of alpha 2+beta 2 displayed a blue shift in the peak position around 430 nm and those of alpha 2 beta 2+ an increase in intensity around 430 nm. The blue shift and the increase in intensity were considered to be caused by the structural changes in deoxy beta chains of alpha 2+beta 2 and deoxy alpha chains of alpha beta 2+, respectively. These spectral changes were interpreted on the basis of their oxygen-equilibrium properties. In contrast to absorption and MCD spectra, the CD spectra of valency hybrid hemoglobins were markedly different from the simple addition of those of their component chains in all derivatives examined. The large part of CD spectral changes upon subunit association were interpreted as changes in the heme vicinity accompanied by formation of the alpha 1 beta 1 subunit contact.

Different effects of subunit association upon absorption and circular dichroism spectra of methemoglobin.

Absorption and circular dichroism (CD) spectra of the Soret band, assigned as a pi-pi transition of the porphyrin pi-electron system, showed a great difference between alpha and beta subunits in the ferric state (alpha +, beta +). The nonequivalence of the spectra between alpha + and beta + subunits partly originates from the difference in the strength of the bond between heme iron and the proximal histidine. The peak positions for absorption and CD spectra of the ferric derivatives associated with F-, H2O, N-3 and CN- of the isolated subunits qualitatively correlate with the spin state of the ferric heme. The Soret absorption spectra obtained by simple addition of those for alpha + and beta + subunits are very similar to those for methemoglobin A (metHb A). On the other hand, the arithmetic means for the Soret CD spectra of alpha + and beta + subunits are different from those for metHb A. These differences were not observed between the Soret CD spectra of alpha 1 beta 1 dimer, which is predominantly present in metHb Hirose (beta 37Trp-Ser), and those of tetrameric metHb A. Therefore, the interaction between alpha 1 and beta 1 subunits to make the alpha 1 beta 1 dimer may strongly affect the CD spectral properties of alpha + and beta + subunits. The effect of the interaction between two homogeneous dimers, alpha 1 beta 1 and alpha 2 beta 2, forming a tetramer, on the Soret CD spectral properties, if any, is very small compared with that between alpha 1 and beta 1 subunits.

Assessment of the alpha 1 beta 2 contact structure of valency hybrid hemoglobins by ultraviolet difference spectra.

We have developed a rapid and useful method for purification of valency hybrid hemoglobins (alpha 2+ beta 2 and alpha 2 beta 2+: + denotes ferric heme) from a hemoglobin solution oxidized partially with ferricyanide by preparative high-performance liquid chromatography. This method does not involve the separation of hemoglobin subunits and the reconstitution of ferric and partner ferrous subunits. Using the valency hybrid hemoglobins thus prepared, the effect of the ferric spin state on the alpha 1 beta 2 subunit boundary structure was investigated by measuring the ultraviolet difference absorption spectra between the deoxy and the oxy valency hybrids associated with various ferric ligands (fluoride, aquo, azide and cyanide). All derivatives of both alpha 2+ beta 2 and alpha 2 beta 2+ showed the difference spectra characteristic of R-T quaternary structural transition. However, the magnitude of the difference spectral peak observed near 288 nm was larger for high-spin derivatives than for low-spin ones. The magnitude of the peak for the valency hybrid hemoglobin was closely correlated with the difference in the free energy of oxygen binding between the R and T states. Since the R state of high-spin hybrids is considered to be identical to that of low-spin hybrids, we concluded from these results that the alpha 1 beta 2 subunit boundary structure plays an important role in regulating the oxygen affinity of deoxy T state.

Tryptophan fluorescence of human hemoglobin. II. Effect of inositol hexaphosphate on the T-R transition.

Fluorescence spectra of tryptophan residues of human hemoglobin in the absence and presence of inositol hexaphosphate were measured at room temperature. The tryptophan fluorescence intensity of deoxy HbA was observed to decrease in accordance with the binding with inositol hexaphosphate. The fluorescence intensity of HbA, Hb Kempsey (beta 99 Asp-Asn), Hb Chesapeake (alpha 92 Arg-Leu) and NES-des-Arg Hb (des-141 alpha Arg and beta 93 Cys-N-ethylsuccinimide derivative) in the presence of inositol hexaphosphate exhibits a considerable decrease in the deoxy to oxy transition, while no or slight fluorescence intensity change was observed in the deoxy to oxy transition of Hb Kempsey and NES-des-Arg Hb in the absence of inositol hexaphosphate. The tryptophan fluorescence behavior suggest that the inositol hexaphosphate-induced structural change in these hemoglobins is attributable to the formation of a different T type of structure from that of the normal T-R transition.

Captopril therapy following percutaneous transluminal angioplasty for bilateral renal artery stenosis.

To establish an optimal approach for the patients with bilateral atherosclerotic renal artery stenosis, combined treatment involving percutaneous transluminal renal angioplasty and oral captopril was applied. Five patients were examined for effects of the combined treatment on blood pressure and renal function. After percutaneous transluminal renal angioplasty on one renal artery, the blood pressure (mean +/- SD) fell from 210/118 +/- 26/8 to 176/104 +/- 11/9 mm Hg without any deterioration of renal function. This reduced blood pressure was further lowered to 155/92 +/- 6/3 mm Hg by adding captopril therapy. This level of blood pressure has been maintained for an average of 37 months. Significant increases in serum creatinine concentration were not observed (124 +/- 27 vs 141 +/- 44 mumol/L), and renal size has been sustained. These results indicate that combined treatment with percutaneous transluminal renal angioplasty and captopril is effective in reducing the blood pressure and preserving renal function as an approach for the patients with bilateral atherosclerotic renal artery stenosis.

Role of endogenous angiotensin II in the control of vasopressin secretion during hypovolemia and hypotension in conscious rabbits.

In order to investigate the physiological role of angiotensin II (ANG II) in the control of vasopressin (VP) secretion, the VP responses to hypotension induced by hemorrhage (20 ml/kg, n = 10) or nitroprusside infusion (1-10 micrograms/kg.min, n = 9) were studied with or without blockade of ANG II formation by the converting enzyme inhibitor captopril in conscious rabbits. Administration of captopril (5 mg/kg, iv) caused a small decrease in mean arterial pressure but did not enhance the hypotensive response to subsequent hemorrhage or nitroprusside infusion. The renin response to both stimuli was enhanced by captopril, whereas the increase in plasma ANG II concentration was attenuated. Plasma VP (PAVP) concentration increased during hemorrhage (2.0 +/- 0.2-113.6 +/- 47.7 pg/ml, P less than 0.01) and nitroprusside infusion (2.1 +/- 0.3-5.1 +/- 1.0 pg/ml, P less than 0.01). Captopril did not change basal plasma PAVP, nor did it attenuate the VP responses to hemorrhage or nitroprusside. Indeed, captopril tended to enhance the VP responses to hemorrhage (2.3 +/- 0.3-147.1 +/- 65.9 pg/ml) and nitroprusside infusion (1.9 +/- 0.2-15.4 +/- 6.0 pg/ml). The relationship between log PAVP and mean arterial pressure during hemorrhage and nitroprusside infusion in the presence of captopril was not different than in the absence of captopril. These results indicate that in conscious rabbits, the renin-angiotensin system does not contribute to the increase in VP secretion during hypotension induced by hemorrhage or nitroprusside infusion.

Immunohistochemical demonstration of thyrotropin (TSH)-receptor in normal and diseased human thyroid tissues using monoclonal antibody against recombinant human TSH-receptor protein.

We performed an immunohistochemical analysis of TSH-receptor in normal and diseased human thyroid tissues using a monoclonal antibody (T3-356) against the C terminal region of human TSH receptor. In normal human thyroid tissues, a positive staining was observed exclusively along the basal cell surface of the flattened follicular cells. In the tissues from adenomatous nodules, adenomas, and papillary carcinomas, a positive staining was also found along the basal cell surface of the follicular cells. In addition, a considerable cytoplasmic staining was observed. The apical and lateral cell surfaces of the follicular cells showed no staining. The foci of squamous cell metaplasia of papillary carcinomas, anaplastic carcinoma, and medullary carcinoma did not show a positive staining. In Graves' thyroids, the positive staining was also observed along the basal cell surface of the follicular cells. The staining was obviously intense in the Graves' thyroids, and the most intense staining was noted in the foci of papillary projection of the columnar follicular cells. These findings indicate that TSH receptor is preserved essentially in the basal cell surface of the thyroid follicular cells in neoplastic conditions and that the amount of TSH receptor protein is increased in Graves' thyroid.

Appearance of 1-2 Mbp giant DNA fragments as an early common response leading to cell death induced by various substances that cause oxidative stress.

The effects of oxidative stress on double strand DNA breakage were examined in T-24 human bladder tumor cells using various active oxygen producing agents such as hydrogen peroxide (H2O2), bleomycin (BLM), neocarzinostatin (NCS), and x-ray irradiation. Analysis of the DNA by pulsed-field gel electrophoresis (PFGE) revealed that discrete giant DNA fragments of 1-2 Mbp and 200-800 kbp had accumulated in the nuclei of the treated cells. The 1-2 Mbp giant DNA fragments were first observed 2 h after the T-24 cells were exposed to the active oxygen producing agents, or irradiated with x-ray. The appearance and the amounts of 1-2 Mbp and 200-800 kbp giant DNA fragments seemed to depend on the concentration and the type of reagents used or the dose of x-ray. Following the accumulation of giant DNA fragments, another type of DNA fragmentation was detected and DNA fragments smaller than 100 kbp accumulated in the nuclei of the cells irradiated with x-ray or treated with NCS. In addition, DNA ladder formation, which is characteristic of apoptosis, was observed. The giant DNA fragments appeared to arise as a consequence of double-stranded DNA breakage, which occurred earlier than cell lysis, as assessed by 51Cr release. These findings indicate that the formation of giant DNA fragments is a specific characteristic of cells responding to oxidative stress, and it may be an initial event that leads to cell death.

Active oxygen-mediated chromosomal 1-2 Mbp giant DNA fragmentation into internucleosomal DNA fragmentation in apoptosis of glioma cells induced by glutamate.

C6 glioma cells treated with 10 mM glutamate reduced intracellular GSH to one-seventh of the initial level, and induced cytolysis accompanied by apoptosis. The treated cells produced extracellular H2O2. The cytolysis of the C6 cells induced by glutamate was prevented by antioxidants such as N-acetylcysteine (NAC), ascorbic acid (ASC), catalase, and NaN3, iron chelators such as deferoxamine and 1,10-phenanthroline, and oxygen radical scavengers such as 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) and alpha-phenyl-tert-butyl nitrone (PBN). The effect of these antioxidants, iron chelators, and oxygen radical scavengers on the cytolysis of C6 cells was dependent on the dose and the intracellular GSH level. Furthermore, 1-2 Mbp chromosomal DNA (giant DNA) fragments were observed during cytolysis. The giant DNA fragments were further cleaved into smaller DNA fragments of 200-800 kbp, and then to fragments of less than 300 kbp in size including chromosomal ladder DNA fragments. Such serial chromosomal DNA degradations induced by glutamate were also inhibited by addition of these antioxidants, iron chelators, and oxygen radical scavengers. These findings suggest that glutamate induces GSH depletion, and consequently, apoptosis through endogenously produced active oxygen species in C6 glioma cells and that the apoptosis is accompanied by 1-2 Mbp giant DNA fragmentation prior to the internucleosomal DNA fragmentation.

Leukotoxin (9, 10-epoxy-12-octadecenoate) impairs energy and redox state of isolated perfused rat lung.

We investigated the perturbation of energy balance and redox state in leukotoxin (9, 10-epoxy-12octadecenoate) (Lx)- and endothelin-1 (ET-1)-induced lung injury, using isolated perfused rat lungs. To examine any relationship between these parameters, intracellular levels of adenine nucleotides, pyridine coenzymes and glutathione were determined by reversed-phase high-performance liquid chromatography (HPLC) in the freeze-dried tissues of isolated rat lungs. The tissue samples were perfused with a physiological salt solution containing either Lx only, Lx plus NG-monomethyl-L-arginine (L-NMMA), Lx plus NG-monomethyl-D-arginine (D-NMMA), Lx plus superoxide dismutase (SOD) or ET-1 only. In isolated perfused lung tissue, 10 mol of Lx caused permeability-increased lung injury, and 10 nM of ET-1, which caused a comparable increase in wet lung weight, evoked pulmonary capillary hypertensive lung injury. Lx-injured lungs showed decreases in the contents of ATP, NADPH, NADH, reduced glutathione (GSH), (2ATP + ADP)/2(ATP + ADP + AMP) ratio (energy charge) and NADH/NAD+ ratio, and increased the contents of ADP and AMP compared with the vehicle control and ET-1-injured lungs. Such effects of Lx were significantly attenuated by pretreatment with 0.4 mM L-NMMA or 500 units/ml of SOD, but not with 0.4 mM D-NMMA. On the other hand, the ET-1-injured lung evidenced decreased tissue GSH. These findings indicate that Lx shifted the lung redox state toward oxidation and that Lx-induced lung injury was involved in the imbalance of the energy and redox state via production of nitric oxide and/or superoxide anion.

Role of nitric oxide and superoxide anion in leukotoxin-, 9,10-epoxy-12-octadecenoate-induced mitochondrial dysfunction.

The present study was carried out to explore the involvement of nitric oxide (NO) and superoxide anion (O2.-) in Leukotoxin (Lx)-induced suppression of mitochondrial respiration. Glutamate- and succinate-dependent oxygen consumption and cytochrome c oxidase activity were assayed. Lx-induced mitochondrial damage was significantly attenuated by the pretreatment of lung with 4 x 10(-4) M NG-monomethyl-L-arginine (L-NMMA) or 500 units/ml superoxide dismutase (SOD) in ex vivo. However, L-NMMA plus SOD pretreatment showed no additive effect on the recovery of mitochondrial functions. The same assay was performed after the exposure of intact mitochondria to NO containing solution (1.25 x 10(-5) M) or 0.1 mM KO2/18-Crown-6 solution, which generated O2.-(6.4 x 10(-5) M). NO, but not O2.-, significantly inhibited the respiration of isolated mitochondria in vitro. Thus, there were great discrepancies in the involvement of NO and O2.- between ex vivo and in vitro system. Together with the previous reports, these facts suggested that the mechanisms by which NO and O2.- probably from vascular constituent cells inhibit mitochondrial respiration function of isolated perfused rat lung may not be simply due to their direct reactions with mitochondrial electron transport chain components, but may rely on the formation of peroxynitrite, and/or peroxynitrite-derived oxidants.

The activation of eNOS by copper ion (Cu2+) in human pulmonary arterial endothelial cells (HPAEC).

We have previously reported that Cu2+ endothelium-dependently dilated rat pulmonary arterial rings in a few minutes by increasing NO production via constitutive endothelial nitric oxide synthase (eNOS) activation in rat pulmonary arterial endothelial cells (Eur. J. Pharmacol., 1997). In the present study, using cultured human pulmonary arterial endothelial cells (HPAEC), we assessed the effects of divalent cations (Cu2+, Mn2+, Zn2+, and Fe2+) on NOS activity in crude cell extracts and intact cells. NO synthase activity was measured by monitoring the conversion of L-[14C] arginine to L-[14C] citrulline. The NOS enzyme in crude HPAEC extract showed similar characteristics to previously reported eNOS from other sources. All the divalent cations tested suppressed the NOS activity in crude cell extract by about 50% at 1 x 10(-4) M, but only Cu2+ from 10(-6) M increased eNOS activation dose dependently with a significant elevation in whole-cell assay. Extracellular Ca2+ was prerequisite to the eNOS activation by Cu2+ in intact cells. Furthermore, we measured NO production determined as NOx (NO, .NO2-, and .NO3-) from HPAEC using NO chemiluminescence analyzer. HPAEC monolayers were treated with either buffer alone, Cu2+ (10(-4) M) or thapsigargin (10(-6) M). The amount of .NOx increased from 10.93 (pmoml/ml/10(6) cells) to 41.27 (pmol/ml/10(6) cells) by thapsigargin (10(-6) M) and to 45.24 (pmol/ml/10(6) cells) by Cu2+ (10(-4) M). The increase in NOx by Cu2+ was inhibited by L-NMMA. These results indicated that Cu2+, but not Mn2+, Zn2+, and Fe2+, causes the activation of eNOS, while Cu2+, Mn2+, Zn2+, and Fe2+ directly suppressed eNOS activity extracted from HPAEC. Further, our study showed that extracellular Ca2+ was essential for eNOS activation by Cu2+.

The renal antihypertensive effect of angiotensin I converting enzyme inhibitors.

Intrarenal administration of angiotensin I converting enzyme (ACE) inhibitors carried out in norepinephrine- (NE; 2-4 micrograms/kg per min) or in angiotensin II- (ANG II; 60-90 ng/kg per min) induced acute hypertension in conscious unrestrained rabbits. Intrarenal administration of captopril (5 mg/kg) and MK-422 (1 mg/kg) caused no significant effect when injected intravenously. However, it showed a prompt and marked depressor effect in NE- but not in ANG II-induced hypertension. This effect was not observed after intrarenal infusion of saralasin (2 and 10 micrograms/kg per min) in NE-induced hypertension. While pretreatment with aprotinin or indomethacin failed to inhibit the depressor action, 2-bromoethylamine hydrobromide (BEA), which is known to induce necrosis of the renal papilla, produced complete abolition of the depressor effect of an intrarenal injection of MK-422 in NE-induced hypertension. These results indicate that the kidney plays an important role in the depressor action of ACE inhibitors in NE- but not in ANG II-induced acute hypertension, and that this effect may be related to the potentiation of antihypertensive renomedullary lipids rather than the inhibition of the renin-angiotensin system or the potentiation of bradykinin or prostaglandins.

Relationship between flushing pressure and nitric oxide production in preserved lungs.

BACKGROUND: Nitric oxide (NO) is considered to be one of the endogenous inhibitory factors of ischemic reperfusion injury. In this study, the NO-producing ability of the preserved lung, flushed at various pulmonary artery pressures (flushing pressure), was studied during reperfusion using an ex vivo rabbit lung perfusion model. METHODS: The lungs were flushed with 200 ml of preservation solution with flushing pressures adjusted to 15, 15, 20, and 25 mmHg for groups 1, 2, 3, and 4, respectively (n=5 in each group). In the control group (group 1), the heart-lung block was harvested after flushing and the lungs were assessed without preservation. In the other groups, the harvested blocks were preserved at 8 degrees C for 24 hr and reperfused with homologous blood for pulmonary functional assessment. Pulmonary function was assessed by measuring mean airway pressure, mean pulmonary arterial pressure, partial oxygen tension of pulmonary venous effluent blood, and pulmonary wet-dry weight ratio. The sequential changes in the concentration of NO-related substances (NO-RS) in the serum of reperfused blood were also measured by chemiluminescence. RESULTS: During reperfusion, biphasic increases in NO-RS were observed in all groups. In groups 3 and 4, the increases in NO-RS were significantly lower than those of groups 1 and 2, and pulmonary function deteriorated. CONCLUSION: These data suggest that in order to maintain the endogenous NO-producing ability of preserved lung, the flushing pressure must be less than 20 mmHg.

The activation of nitric oxide synthase by copper ion is mediated by intracellular Ca2+ mobilization in human pulmonary arterial endothelial cells.

The aim of the study was to elucidate the vasodilatory mechanism due to Cu2+ by assessing nitric oxide (NO) production as determined by NOx (NO, NO2-, and NO3-) that is released from human pulmonary arterial endothelial cell (HPAEC) monolayers using a NO chemiluminescence analyzer, and also to assess Ca2+ movement using 45Ca and fura 2 in HPAEC. Cu2+ (10(-6)-10(-4) M) significantly increased NO production in a dose-dependent manner when extracellular Ca2+ was present. 45Ca influx into the adherent cells was dose-dependently enhanced by Cu(2+) (10(-6)-10(-4) M), but not by Mn(2+), Zn(2+) or Fe(2+). [Ca2+]i, measured by monitoring the fluorescence changes of fura 2, was significantly elevated in the presence of Cu2+. The increase in [Ca2+]i induced by Cu2+ was inhibited by either diethyldithiocarbamate (DDC) or the depletion of extracellular Ca2+. The dihydropyridine receptor agonist, BayK8644, significantly attenuated the Cu2+-induced increase in [Ca2+]i in a dose dependent manner and nitrendipine or nifedipine, the dihydropyridine receptor antagonists, dose-dependently inhibited a Cu2+-induced increase in [Ca2+]i. These results suggest that Cu2+ activates eNOS through the mechanism of [Ca2+]i elevation due to Ca2+ influx into HPAEC and that the Cu2+-induced [Ca2+]i elevation in HPAEC is likely due to activation of the dihydropyridine-like receptors.

Effect of ovarian steroid hormones and the presence of the fetus on oxytocin gene expression in the uterus.

Oxytocin (OT) is a neurohypophysial hormone with potent stimulating activity of the pregnant uterus, but its physiological role in parturition is still unclear. Recently, OT was found to be synthesized in the pregnant uterus, indicating that OT originating from the uterus, not from the posterior pituitary gland, may trigger the onset of labour. In order to define the factors responsible for the induction of uterine OT, the effect of ovarian steroid hormones and conceptus on the induction of OT mRNA in the rat uterus was examined by Northern and dot blot hybridization analysis. OT mRNA in the uterus started to increase on day 14 of pregnancy and showed very high levels at the time of parturition. Uterine OT mRNA was not altered by any steroid treatment, oestradiol-17 beta (0.2 microgram), progesterone (4 mg) or both in combination, for 6 days. The gravid horn of the uterus had 3.6-fold as much OT mRNA as the non-gravid horn on day 21 of pregnancy in hemipregnant rats with one ligated oviduct. The ovarian steroid hormones could not induce accumulation of OT mRNA in the uterus of ovariectomized rats, at least under the conditions used, but the presence of a conceptus may be critical for the very high levels of OT mRNA.

Changes in oxytocin receptor mRNA in the rat uterus measured by competitive reverse transcription-polymerase chain reaction.

Large changes in the responsiveness of target organs to oxytocin are thought to originate from alteration of the number of oxytocin receptors (OTR). To elucidate the molecular mechanisms regulating the synthesis of the OTR, we developed a competitive reverse transcription-PCR protocol to measure OTR mRNA. We synthesized cRNA comprising a small stuffer introduced into the target mRNA. Using this cRNA as an internal standard, we made a quantitative estimation of OTR mRNA. Application of this method to the rat uterus revealed that the mean levels of OTR mRNA remained unchanged until 1030-1100 h on day 21 of pregnancy, increased significantly after 2200-2230 h on the same day and declined rapidly after parturition. A similar rapid increase in uterine OTR mRNA content was observed in rats given prostaglandin on day 18, inducing premature delivery on day 19 of pregnancy. All parturient rats had higher OTR mRNA levels regardless of whether parturition was spontaneous or prostaglandin induced. However, in a few rats, OTR mRNA remained as low as that observed during mid pregnancy even on day 22 of gestation, the expected day of parturition in about 70% of the rats in our colony. A similar increase in uterine OTR mRNA content to that observed at parturition was induced by oestrogen treatment for 3 days in ovariectomized virgin rats, but concomitant injection of progesterone did not influence the effect of oestrogen. The present results revealed that the large increase of uterine OTR at the peripartum period is accompanied by an increase in OTR mRNA content that may be brought about, at least in part, by increased oestrogen secretion following luteolysis.