Oxytocin gene expression in rat uterus.

The neurohypophyseal hormone oxytocin (OT) is the most potent uterotonic agent known and is used to induce labor. Yet, endogenous circulating OT appears not to participate in the induction of labor. As shown here, the finding of OT messenger RNA and peptide in the uterus suggests a solution for this paradox. During gestation, rat uterus OT messenger RNA increased more than 150-fold and, at term, exceeded hypothalamic OT messenger RNA by 70-fold. Thus, during parturition, OT may act primarily as a local mediator and not as a circulating hormone.

Endothelin: a novel peptide in the posterior pituitary system.

Endothelin (ET), originally characterized as a 21-residue vasoconstrictor peptide from endothelial cells, is present in the porcine spinal cord and may act as a neuropeptide. Endothelin-like immunoreactivity has now been demonstrated by immunohistochemistry in the paraventricular and supraoptic nuclear neurons and their terminals in the posterior pituitary of the pig and the rat. The presence of ET in the porcine hypothalamus was confirmed by reversed-phase high-pressure liquid chromatography and radioimmunoassay. Moreover, in situ hybridization demonstrated ET messenger RNA in porcine paraventricular nuclear neurons. Endothelin-like immunoreactive products in the posterior pituitary of the rat were depleted by water deprivation, suggesting a release of ET under physiological conditions. These findings indicate that ET is synthesized in the posterior pituitary system and may be involved in neurosecretory functions.

Pulmonary and cardiac expression of preproendothelin-1 mRNA are increased in heart failure after myocardial infarction in rats. Localization of preproendothelin-1 mRNA and endothelin peptide.

OBJECTIVES: Recent reports indicate that endothelin (ET) plays an important pathophysiological role in congestive heart failure (CHF). However, existing data on local cardiopulmonary ET production are few. No studies have hitherto examined the specific anatomic localization of cardiopulmonary ET synthesis in CHF. Thus, the aims of the present study were to examine whether cardiopulmonary preproET-1 mRNA synthesis is upregulated in CHF and to determine the anatomic localization of preproET-1 mRNA and the mature peptide. METHODS: CHF was induced in rats by occluding the left coronary artery. Only animals with a left ventricular end-diastolic pressure above 15 mmHg after one week were included (n = 28). Sham-operated animals served as controls (n = 24). Hearts and lungs were examined by mRNA slot blot analyses, in situ hybridization (ISH) and immunohistochemistry (IHC). RESULTS: In CHF-rats, slot blot analyses revealed a 3.5 +/- 1.1-fold and a 6.4 +/- 0.8-fold upregulation of preproET-1 mRNA in the noninfarcted and the infarcted area of the left ventricles, respectively (p < 0.05 for both). ISH revealed that the preproET-1 mRNA was localized predominantly over the granulation tissue in the infarcted region. The ET peptide was predominantly localized to inflammatory cells and remaining cardiomyocytes in the infarcted region as determined by IHC. Lungs from CHF-rats showed a 1.5 +/- 0.1-fold upregulation of preproET-1 mRNA (p = 0.01). The most abundant preproET-1 mRNA and ET-1-like-immunoreactivity (ET-1-ir) was seen over inflammatory cells and over airway epithelial cells. Some ET-1-ir was also located to bronchial and vascular smooth muscle cells. CONCLUSION: Increased cardiopulmonary ET synthesis strongly suggest a pathophysiological role for ET in CHF.

The complementary deoxyribonucleic acid sequence, tissue distribution, and cellular localization of the rat granulin precursor.

Granulins (grns; also called epithelins) are cysteine-rich polypeptides with pleiotropic effects on epithelial cell growth in vitro. The grn/epi gene is widely expressed in epithelial cell lines, many of which respond to the gene product, raising the possibility of autocrine or paracrine regulation. In vitro the grn gene is expressed in cell types of diverse lineages, including epithelial cells, lymphoid and myeloid cells, and fibroblasts, but it is not known which cells express the gene in vivo. To understand the physiological role of the grn gene products it is necessary to know the context of grn gene expression in vivo. We have isolated the rat grn precursor complementary DNA and determined, by Northern blot analysis and in situ hybridization, the tissue distribution and cellular localization of grn gene expression. The complementary DNA predicts a 589-amino acid protein of M(r) 63,500 with seven and one-half grn repeats arranged in tandem and shows an overall identity of 75% with human progrn. The grn gene is expressed in a variety of tissues derived from all three embryonic germ layers but is most abundant in the spleen and several tissues of endocrine significance including the adrenal glands, epididymis, placenta, and ovary. Although widely expressed in tissues, gene expression is restricted to specific cell types. For example in the kidney, grn messenger RNA was detected in epithelial cells of the proximal and distal convoluted tubules and Bowman's capsule but not in medullary epithelia. In the spleen, grn messenger RNA expression was localized in lymphocytes, whereas hybridization signals were detected over scattered hepatocytes in the liver. Thus, although the grn gene is widely expressed in tissues and cell lines of many lineages in vitro, its expression in situ is restricted to hematopoietic and some epithelial cells. The restricted cell distribution suggests that the expression of the grn gene is more closely regulated in vivo than in cell cultures. Its localization to epithelial cells in situ supports an autocrine or paracrine role for these factors.

Expression of the oxytocin gene in rat placenta.

The placenta is an endocrinologically active organ and expresses an important number of polypeptide hormone genes. Although oxytocin (OT)-like immunoreactivity has been detected in placental extracts by RIA, the precise nature and origin of placental OT has remained unclear. In the present study, we examined OT gene expression in rat placental tissue at various stages of gestation using northern blot analysis, polymerase chain reaction, in situ hybridization, HPLC, and immunocytochemistry. Northern blot analysis of RNA extracted from rat placenta revealed a single type of OT gene transcript (0.66 kilobases) which differed in size from hypothalamic OT transcripts (0.75 kilobases). Deadenylation of placental and hypothalamic messenger RNA (mRNA) showed that this size difference was due to differences in poly(A) tail lengths. Polymerase chain reaction amplification of placental and hypothalamic complementary DNAs using four different exon-specific primers provided no evidence for the existence of any additional structural differences between hypothalamic and placental OT-gene transcripts. Quantitative evaluation of northern blots showed that OT mRNA abundance per microgram of total RNA was stage specific and declined by a factor of 6 from day 14 to day 21 of gestation. In contrast to the marked variation of mRNA abundance, the OT peptide content, as measured by RIA, underwent no significant change during the time period studied and varied between 0.37-0.51 ng/g wet tissue wt. Characterization of placental OT immunoreactivity by HPLC and gel filtration identified two peaks of immunoreactivity: one peak (70% of immunoreactivity) corresponded to synthetic OT; whereas the other peak (Mr 11,000, 30% of immunoreactivity) represented a noncovalent association between OT and another molecule, consistent with the formation of a neurophysin/OT complex. By in situ hybridization and immunocytochemistry, we localized OT mRNA and OT immunoreactivity to cells of the trophoblastic epithelium covering the septa of the labyrinth as well as to cytotrophoblastic elements and giant cells of the maternally derived basal zone of the placenta. Placental OT may act locally, may interact with uterine OT receptors, or may play a role in fetal development.

Uterine oxytocin gene expression. II. Induction by exogenous steroid administration.

As we have recently shown, the gene encoding the hypothalamic nonapeptide oxytocin (OT) is expressed in the rat endometrial epithelium during late pregnancy and the estrous phase of the estrous cycle. To investigate the role of ovarian steroids in the regulation of uterine OT gene expression, Silastic capsules containing estradiol or progesterone were implanted into immature ovariectomized rats. Exposure to estradiol alone for 2 days caused a significant rise in OT mRNA. Administration of progesterone alone was without effect. However, a strong synergism was observed when the two hormones were applied together; progesterone potentiated the effect of estradiol by a factor of 7. In animals treated with steroids for 4 days, the removal of either the estradiol or progesterone capsule after day 2 led to a decrease in the total amount of OT mRNA accumulation, implying that the continued action of both steroids was required to achieve maximal OT mRNA levels. Immunocytochemical analysis demonstrated that the main site of steroid-induced uterine OT gene expression is the endometrial epithelium, the same site where endogenously induced OT gene expression occurs at the end of pregnancy. The OT mRNA levels achieved after 4 days of treatment with both steroids were comparable to those achieved at estrus or during pseudopregnancy, but corresponded to less than 20% of the levels present in the uterus on day 21 of pregnancy. These data suggest that in the uterus, the synergistic action of ovarian steroids represents an important, but probably not exclusive, regulator of OT gene expression.

Effect of endocrine manipulation on anterior pituitary galanin in the rat.

Galanin is widely distributed throughout the rat neural and endocrine system. The highest concentrations are found in the anterior pituitary, and it can influence classical pituitary hormone secretion. The effects of endocrine manipulation on pituitary galanin content, mRNA, and immunostaining have been investigated in the rat. In females, medical (39 +/- 4 fmol/gland), surgical (33 +/- 2), or combined (28 +/- 6) castration resulted in a highly significant decrease in galanin content (control, 223 +/- 14; P less than 0.0001). Estrogen in physiological and pharmacological doses produced a significant increase in galanin content (368 +/- 14 and 373 +/- 13, respectively; P less than 0.01) associated with an increase in galanin mRNA content. In the male, high dose dexamethasone and thyroidectomy caused a fall in galanin content, while galanin mRNA levels showed a rise and fall, respectively. Adrenalectomy caused a rise in galanin content, while adrenalectomy and castration produced a dramatic decrease in tissue galanin content. No change in galanin mRNA was observed in these groups. Galanin immunostaining paralleled the results of tissue content in all groups examined, except in the medically castrated group, in which there was some intragroup variation in staining patterns. In normal and high-dose estrogen-treated females, galanin expression was seen mainly in lactotrophs, with a small number of somatotrophs and thyrotrophs staining. In the male, galanin expression was confined to somatotrophs and thyrotrophs. Galanin mRNA was localized at the cellular level by in situ hybridization. In the normal pituitary only scattered lactotrophs contained message, while in high-dose estrogen-treated animals the number of positive cells, mostly lactotrophs, was vastly increased. Thus, the cellular localization of galanin immunostaining varies between the sexes. Galanin peptide and mRNA levels in the pituitary are powerfully influenced by endocrine status.

Localization of immunoreactivity for calcitonin gene-related peptide in the rat anterior pituitary during ontogeny and gonadal steroid manipulations and detection of its messenger ribonucleic acid.

Localization of calcitonin gene-related peptide (CGRP) expression in the rat anterior pituitary and its changes during ontogeny and after gonadal steroid manipulations were studied by immunocytochemistry, RIA, and in situ hybridization. Colocalization studies and the combined use of immunocytochemistry and in situ hybridization revealed that CGRP immunoreactivity is localized mainly in gonadotropes and alpha- and beta-CGRP messenger RNAs were detected in CGRP-immunoreactive cells. Immunoreactivity for CGRP also was detected in nerve fibers and colocalized with substance P immunoreactivity. Cells immunoreactive to CGRP antiserum were first detected in fetal rats at gestational day 18, and the incidence considerably increased between postnatal days 5 and 14. CGRP immunoreactivity was low in control adults of both sexes and in pregnant and ovariectomized females but increased in lactating, estrogen-supplemented ovariectomized and high-dose estrogen-treated females, and in high-dose estrogen-treated and castrated males. Testosterone supplement suppressed the effect of castration on CGRP immunoreactivity in males. Quantities of extractable immunoreactive CGRP under conditions of estrogen manipulation corresponded well to the immunocytochemical findings (females: controls, 96.4 +/- 13.1 fmol/gland; ovariectomized, 107.6 +/- 19.2; high-dose estrogen-treated, 212 +/- 23.0; estrogen-supplemented ovariectomized, 680 +/- 42.1). The present study suggests that pituitary CGRP is synthesized and stored in gonadotropes, is modulated by gonadal steroids, and may have a functional link with gonadotropins.

Ontogeny of growth hormone receptors in human tissues: an immunohistochemical study.

Both experimental and clinical studies suggest that human GH (hGH) may have a functional role during early stages in human development. To determine when receptors for hGH appear in specific target cells, we carried out an immunohistochemical analysis of six tissues obtained from 20 fetuses (8.5-20 weeks fetal age [FA]), two term infants, one 11-yr-old male, and one 43-yr-old male. Liver parenchymal and bile duct cells showed specific staining from the earliest FA examined (8.5 weeks); in fetal liver, the intensity of the immunoreaction was greatest in hepatocytes surrounding the central veins and portal triads, whereas the numerous hematopoietic cells exhibited little to no immunostaining. Kidney tissues also showed positive localization at 8.5-9 weeks FA. By 13 weeks, epithelial cells of most tubules were immunoreactive, with medullary tubules showing stronger staining than those in the cortex, whereas visceral epithelial cells of glomeruli were weakly positive; metanephrogenic blastemal cells were negative. A similar immunostaining pattern was observed in midgestation and postnatal kidneys, except that glomerular staining was lost by 19 weeks FA. Fetal zone cells of fetal adrenals showed weak immunostaining beginning at 13-14 weeks; glomerulosa cells stained intensely in the postnatal adrenal. Epithelial crypt cells in both small and large intestine immunostained beginning at 19-20 weeks; a similar pattern was observed in postnatal sections. Dermal fibroblasts and skeletal muscle of abdominal skin were positive from 8.5 weeks. Epidermal cells first showed immunostaining at 13-14 weeks; germinal layer cells showed the most intense reaction until 19-20 weeks, when the staining pattern became uneven throughout the epidermal layers. Epithelial cells in hair follicles and sebaceous glands were positive from 13-14 weeks; by 19-20 weeks, these cells were the most intensely immunostained in skin sections. Lung tissue was negative from 8.5-20 weeks, as well as postnatally. Vascular endothelial and smooth muscle cells routinely stained from 8.5 weeks FA in all tissues examined. We conclude that immunoreactive hGH receptors can be identified in early fetal, as well as postnatal, tissues and that the pattern of cellular immunostaining develops gradually throughout gestation in a very tissue-specific manner.

Role for endothelin-1 in angiotensin II-mediated hypertension.

Experiments in cultured vascular smooth muscle cells have shown that angiotensin II (Ang II) stimulates expression of endothelin-1. We sought to examine role of endothelin-1 in the effects of Ang II in vivo. Ang II infusion in rats (0.7 mg/kg per day for 5 days) was associated with marked increases in vascular smooth muscle endothelin-1 levels, as assessed by immunostaining. Administration of the selective endothelin type A (ET(A)) receptor antagonist PD 155080 (50 mg/kg per day) abrogated the hypertensive response to a 5-day infusion of Ang II (0.7 mg/kg per day), as did losartan (25 mg/kg per day). ET(A) receptor blockade during Ang II-mediated hypertension was associated with marked elevations of plasma endothelin-1 levels. Ang II-mediated hypertension was associated with heightened vascular responsiveness to a variety of vasoconstrictor agents except endothelin-1. Blockade of ET(A) receptor invariably corrected this vasoconstrictor hyperresponsiveness. We conclude that some of the vascular effects of Ang II thought to be unique to this hormone are likely mediated by endothelin-1.

Immunolocalization of cyclooxygenase-2 in the macula densa of human elderly.

To gain insight into the role of prostanoids in human kidney function, we examined the distribution of cyclooxygenase (COX) 1 and COX-2 by immunofluorescence and immunohistochemistry in human kidneys from adults of various age groups. COX-1 was detected in the collecting ducts, thin loops of Henle and portions of the renal vasculature. COX-2 was detected in the renal vasculature, medullary interstitial cells, and the macula densa. In addition, COX-2 immunoreactivity was noted in afferent arteries and the macula densa of the renal cortex and was more evident in the kidneys of older adults.

The early release of interleukin-2, tumor necrosis factor-alpha and interferon-gamma after ischemia reperfusion injury in the lung allograft.

A period of cold and warm ischemia is obligatory when performing lung transplantation. Subtle ischemia-reperfusion injury induced in the course of transplantation can pass undetected or cause a short phase of reversible lung dysfunction. We hypothesized that ischemia-reperfusion injury may result in the local release of cytokines that have the capability to mediate acute lung injury early following transplantation. To test this hypothesis, 10 mongrel dogs were subjected to left lung allotransplantation. As performed in the clinical setting, donor lungs were preserved with Eurocollins solution and stored at 4 degrees C for 4 hr, which was followed by 1 hr of warm ischemia. Recipients received standard immunosuppression of cyclosporine, azathioprine, and low dose steroids. Bronchoalveolar lavage (BAL) and open lung biopsies were performed before operation and at approximately 1 hr, 4 hr, 24 hr, and 1 week after transplantation. A significant increase in BAL IL-2 levels was observed 4 hr after surgery (0 hr: 349 +/- 138 pg/ml; 4 hr: 757 +/- 284 pg/ml) (mean +/- SEM) (P < 0.05) which subsequently decreased 24 hr (320 +/- 168 pg/ml) after transplantation. BAL TNF-alpha levels were significantly increased 1 hr after transplantation (P < 0.05) (0 hr: 3.4 +/- 0.65 pg/ml; 1 hr: 13.3 +/- 8.0 pg/ml) returning to baseline after 24 hr (5.8 +/- 2.8 pg/ml). BAL IFN-gamma levels also significantly increased 1 and 4 hr after transplantation (0 hr: 7.2 +/- 2.1 pg/ml; 1 hr: 68.2 +/- 49.2 pg/ml; 4 hr: 301 +/- 131 pg/ml) (P < 0.05). This decreased back to baseline after 24 hr and 1 week (5.2 +/- 1.2 pg/ml and 9.7 +/- 7.9 pg/ml, respectively). There were no changes detected in plasma levels of cytokines. Histology showed evidence of grade 1-2 rejection after 1 week. We conclude that subjection of a lung allograft to standard periods of cold-warm ischemia will result in a temporary early elevation of IL-2, TNF-alpha, and IFN-gamma detectable only in the bronchoalveolar compartment. Such local increase in cytokines in the lung allograft may play an important role in the development of early allograft dysfunction.

Endothelin-1 immunoreactivity and mRNA in the transplanted human heart.

Endothelin-1 (ET-1) is a 21-residue peptide produced by endothelial cells and possesses a wide range of biological activities, including vasoconstriction, mitogenesis, and inotropic effects on the heart. The aim of the present study was to determine the cellular localization of ET-1 immunoreactivity and mRNA in routine endomyocardial biopsy specimens of transplanted human hearts, and to correlate the findings with the associated histological changes. Multiple-step paraffin sections of 72 biopsy samples were immunostained with antiserum to ET-1 and von Willebrand factor (factor VIII) using the avidin-biotin-peroxidase complex method. ET-1 immunoreactivity was localized to vascular and endocardial endothelial cells, as well as to cardiomyocytes. The pattern of endothelial cell immunostaining with the ET-1 antiserum was similar to that of factor VIII. Previous biopsy sites and areas of granulation tissue appeared to have greater ET-1 immunoreactivity, particularly in sections immunostained with the ET-1 antiserum. There was a significant correlation between the presence of ET-1 immunoreactivity and fibrosis or granulation tissue in the biopsy specimens (P < 0.03). There was no correlation between ET-1 immunoreactivity and the presence of cellular infiltrate, definitive rejection, or Quilty effect. In situ hybridization with radiolabeled RNA probes revealed expression of ET-1 mRNA in endothelial cells and myocytes, also in association with granulation tissue and fibrosis. No cellular reactivity was present in control sections stained with the ET-1 antiserum preadsorped with its synthetic peptide. The findings suggests a possible role for ET-1 in vascular regeneration and angiogenesis following myocardial injury.

Altered nonspecific lymphocyte cytotoxicity in bronchoalveolar lavage of lung transplant recipients: can it be useful in monitoring rejection or infection?

A reliable method for determining the adequacy of immunosuppression of the lung allograft and the absence of rejection or infection does not exist. The purpose of this study is to evaluate the potential role of bronchoalveolar lavage (BAL) in monitoring the adequacy of immunosuppression of lung allografts and in identifying the possible presence of acute rejection (AR) or infection. Thirty-one consecutive lung transplant recipients were subjected to bronchoscopy, transbronchial biopsy, and BAL either as routine surveillance or for decline in lung function (n=68 episodes: 27 normal, 17 infections, six grade IAR, 10 grade II-III AR, and eight obliterative bronchiolitis). Diagnosis was always confirmed histologically and by microbiological workup. Harvested cells underwent phenotypic analysis, and T lymphocytes were subjected to nonspecific lectin-dependent cell-mediated cytotoxicity (LDCMC) and natural killer cytotoxic assays. BAL from normal grafts predominantly contained macrophages (72.8+/-4.4%), with lower neutrophil (13.9+/-4.1%) and lymphocyte (13.2+/-2.2%) populations. Grade II-III AR and infection were associated with an increase in the percentage of neutrophils (43.3+/-8.3% and 33.2+/-3.6%, respectively, P=0.02). BAL of normal allografts contained T cells with low LDCMC (7.4+/-4.5%) and natural killer cytotoxicity (6.3+/-3.4%), whereas grade II-III AR was associated with a significant elevation in LDCMC (32.5+/-11.6%, P=0.019). Pulmonary infection, regardless of its type, was associated with significant elevation in BAL natural killer cytotoxicity (23.9+/-4.9%, P=0.033). Patients with obliterative bronchiolitis, on the other hand, had a mild elevation in the percentage of neutrophils and lymphocytes in BAL, which did not reach statistical significance. However, BAL T-cell LDCMC was significantly elevated (37.6+/-13.7%, P=0.019) compared with normal and infected allografts. We conclude that phenotypic and nonspecific cytotoxic T-cell analysis of BAL, when complimented with microbiological studies, may be useful in surveillance of lung transplant recipients and in determining whether allografts are likely to be quiescent, or possibly affected by acute/chronic rejection or infection, necessitating further definitive action.

Immunohistochemical localization of nitric oxide synthase and the oxidant peroxynitrite in lung transplant recipients with obliterative bronchiolitis.

BACKGROUND: Obliterative bronchiolitis (OB) is a disease affecting a large percentage of lung and heart-lung transplant recipients. Histologically, the disease is characterized by inflammation, cellular proliferation, and obliteration of terminal airways. METHODS: We investigated the production of inducible and constitutive nitric oxide synthases and peroxynitrite by immunohistochemistry in the lungs of control subjects (n=14) compared with those of transplant recipients with OB (n=8). RESULTS: Strong immunoreactivity for inducible nitric oxide synthase and nitrotyrosine, a marker of protein nitration by peroxynitrite, was seen in inflammatory cells, airway epithelium, and vascular endothelium of patients with OB, compared with little immunoreactivity in control lungs. Immunoreactivity for constitutive nitric oxide synthase was abundant in the airway epithelium and vascular endothelium of control lungs, however, it was decreased in airway epithelial cells and arterial endothelial cells of patients with OB. CONCLUSIONS: We conclude that increased formation of the potent oxidant peroxynitrite and decreased production of endothelial nitric oxide may contribute to the functional and morphological abnormalities of OB.

Endothelin receptor antagonist improves pulmonary hemodynamics during lung ischemia/reperfusion injury.

BACKGROUND: We have previously shown that elevated release of endothelin-1 is associated with increased pulmonary vascular resistance (PVR) immediately after reperfusion of the transplanted lung. In the present study, we investigated the effect of ET receptor blockage on pulmonary hemodynamics and function in an ex vivo lung reperfusion model after 6 hr of cold ischemia. METHODS: Eighteen rabbits were divided into three groups: no ischemia followed by 3 hr of reperfusion (group I) and 6 hr of cold ischemia followed by 3 hr of reperfusion with either blood (group II) or blood + SB209670 (mixed ETA/ETB receptor antagonist) (group III). RESULTS: Shortly after reperfusion, mean pulmonary artery pressure, PVR, and pulmonary edema were increased, and pulmonary compliance and PO2 were decreased in group II compared with group I. Treatment with SB209670 resulted in a significant decrease in mean pulmonary artery pressure, PVR, and pulmonary edema, and improvement in pulmonary compliance and PO2. CONCLUSION: The data suggest an important role for ET-1 in lung ischemia/reperfusion injury and that the use of ET receptor antagonist immediately after transplantation may provide a new therapeutic tool in the management of early graft dysfunction.

Alterations in bronchoalveolar lavage and plasma endothelin-1 levels early after lung transplantation.

A temporary increase in pulmonary vascular resistance is observed during the first 24 hr following lung allotransplantation. We hypothesized that such early vascular changes are secondary to endothelial injury by ischemia-reperfusion, and that this may be mediated by an increased pulmonary endothelin-1 production/release. To test this hypothesis, radioimmunoassay was used to analyze endothelin-1 levels in bronchoalveolar lavage and plasma taken before surgery and at 1 hr, 4 hr, 24 hr, and 1 week after transplantation. The study was carried out on 2 groups of mongrel dogs. One group was subjected to left single-lung allotransplantation and the other to autotransplantation. Endothelin-1 levels in the bronchoalveolar lavage samples from the lung allografts were significantly increased at 1 (0.70 +/- 0.18 pg/ml) and 4 (1.84 +/- 0.65 pg/ml) hr after transplantation compared with the preoperative value (0.14 +/- 0.05 pg/ml), and declined at 24 (0.85 +/- 0.84 pg/ml) hr after transplantation. Similarly, plasma endothelin-1 levels in the allografts were significantly increased at 1 (2.0 +/- 0.80 pg/ml) and 4 (2.0 +/- 0.71 pg/ml) hr after transplant when compared with preoperative levels (0.54 +/- 0.09 pg/ml). Plasma endothelin-1 levels, however, remained significantly high after 24 hr (1.4 +/- 0.4 pg/ml; P < 0.007) and decreased after 1 week after transplant (0.89 +/- 0.32 pg/ml). On the other hand, endothelin-1 levels in bronchoalveolar lavage from the autograft group remained relatively unchanged; however, plasma levels showed a significant increase at 4 hr (6.6 +/- 1.8 pg/ml) after transplantation compared with preoperative levels (2.8 +/- 0.38 pg/ml). Elevation of endothelin-1 levels early after lung transplantation may play an important role in early high pulmonary vascular resistance and temporary graft dysfunction.

Immunohistochemical characterization of inflammatory and proliferative events during chronic rejection in rat lung allografts.

BACKGROUND: Chronic rejection is assumed to be the principle cause of airway injury leading to obliterative bronchiolitis (OB) after lung transplantation (Tx). To better understand the contribution of chronic rejection in the development of OB in allografted lungs, we examined the histopathological changes and cytokine expression in inadequately immunosuppressed rat lung allografts. METHODS: Three groups of rats were studied: group I, control nontransplanted Lewis (Lew) rats (n=5); group II, syngeneic Lew-to-Lew isografts (n=25); and group III, Brown Norway-to-Lew allografts (n=25). Groups II and III received two single doses of cyclosporine on postoperative days 2-3. Transplanted animals were killed (n=5) at monthly intervals from 2 months to 6 months after Tx. Resected lungs were stained with hematoxylin and eosin, Masson's trichrome, and Van Gieson's elastin, and immunostained with antisera to interleukin (IL)-1beta, IL-8, and basic fibroblast growth factor (bFGF). The intensity of immunostaining was graded from 0 to 4 (0=no staining, 4=strong staining). RESULTS: In groups I and II, normal airways and vessels were observed. Minimal intensity and distribution of immunostaining for all markers were detected in groups I and II. Group III allografts demonstrated acute grade II-III vascular rejection with mild bronchiolar injury and inflammation at 2 months after Tx. At 6 months after Tx, all allografts demonstrated severe and diffuse chronic vascular rejection. Late airway changes consistent with OB were detected in four of five allografts, however, these lesions were expressed infrequently. Immunohistochemical findings revealed moderate to strong expression for IL-8 and bFGF over the airway epithelium, acute and chronic inflammatory cells, and fibroblasts in allografts at 2 months after Tx. Despite focal development of OB at 6 months, intensity and distribution of immunostaining significantly decreased for all three cytokine markers. CONCLUSIONS: Inadequate immunosuppression of rat lung allografts leads primarily to chronic vascular rejection but fails to induce severe and diffuse development of OB. In this animal model, cytokines IL-1beta, IL-8, and bFGF are likely to play an important role in the early inflammatory phase but not during the late proliferative events of chronic rejection.

Regional distribution of endothelin-1 and endothelin converting enzyme-1 in porcine endotoxemia.

Endothelin-1 (ET-1) levels are markedly increased in sepsis. Since ET-1 is primarily transcriptionally regulated, there should be a corresponding increase in pre-pro-endothelin-1 (ppET-1). Our objective was to determine whether ppET-1 is increased in pigs with a low systemic vascular resistance. We also examined the distribution of ET-1 and the regulation of endothelin-converting enzyme 1 (ECE-1), the rate limiting enzyme in ET-1 production. We anesthetized and ventilated 16 pigs. We measured arterial, pulmonary, and central venous pressures, as well as cardiac output. ET-1 was measured by radioimmunoassay in plasma and in multiple tissues. We infused 20 microg/kg of endotoxin over 2 h and then sacrificed the animals. ppET-1 and ECE-1 mRNA were assessed by Northern analysis. We performed immunohistochemistry for the assessment of tissue ET-1 and ECE-1. The systemic vascular resistance rose at 30 min, but fell by 120 min. Plasma ET-1 more than doubled by 2 h. However, there was no change in the concentration of ET-1 in any tissue except in the pulmonary artery. By immunohistochemistry, there was also no change in ET-1 in aorta, vena cava, heart, lung, liver, and kidney. Distribution of ECE-1 followed that of ET-1 on immunohistochemistry. There was a significant increase in ppET-1 mRNA in liver, kidney papillae, and vena cava, and a tendency for an increase in other tissues. This was paralleled by an increase in ECE-1 mRNA. In conclusion, the amount of ECE-1 mRNA and protein parallel those of ET-1. Endotoxemia is associated with a marked increase in plasma ET-1 and an increase in ppET-1 and ECE-1 mRNA in multiple tissues; however, there was no significant change in tissue ET-1 except in the pulmonary artery. The rise in plasma levels without a change in tissue levels suggests a greater release into the vasculature in sepsis than under normal conditions.

Nitric oxide and endothelin-1 in pulmonary hypertension.

BACKGROUND: We have shown previously increased expression of the potent vasoconstrictor peptide endothelin-1 (ET-1) in the pulmonary arteries of patients with pulmonary hypertension. We also demonstrated diminished expression of endothelial nitric oxide synthase, the enzyme responsible for generating nitric oxide (NO), in patients with the same disease. STUDY OBJECTIVE: To determine the expression of neuronal nitric oxide synthase (NOS-I) and endothelin-converting enzyme-1 (ECE-1) in lungs of patients with pulmonary hypertension. METHODS: Immunohistochemistry with avidin-biotin-peroxidase method. RESULTS: There was little immunostaining for NOS-I in the pulmonary arteries of normal control or diseased lungs. Moderate diffuse staining was seen in the airway epithelium and nerve bundles. Immunoreactivity for ECE-1 was seen in the airway epithelium, smooth muscle cells, and scattered macrophages of both normal and diseased lungs. Strong immunoreactivity for ECE-1 was seen in the endothelium of diseased pulmonary arteries of patients with pulmonary hypertension. CONCLUSION: We conclude that expression of NOS-I appears to be similar in normal and diseased lungs, while abundant expression of ECE-1 is present in diseased vessels, which may contribute to the pathogenesis of arteriopathy in pulmonary hypertension.