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Background: At birth, the release of surfactant from alveolar type II cells (ATIIs) is stimulated by increased activity of the beta-adrenergic/adenylyl cyclase/cyclic 3'-5' adenosine monophosphate-signaling cascade. Atrial natriuretic peptide (ANP) stimulates surfactant secretion through natriuretic peptide receptor A (NPR-A). ANP inhibits adenylyl cyclase activity through its binding to NPR-C. We wished to further understand the role of the NPR-C in perinatal transition. Methods: We studied ATII expression of NPR-C in fetal and newborn sheep using immunohistochemistry, and surfactant secretion in isolated ATIIs by measuring 3 [H] choline release into the media. Results: ANP induced surfactant secretion, and, at higher doses, it inhibits the stimulatory effect of the secretagogue terbutaline. ATII NPR-C expression decreased significantly after birth. Premature delivery also markedly decreased ANP and NPR-C in ATIIs. Co-incubation of terbutaline (10 -4  M) with ANP (10 -6  M) significantly decreased 3 [H] choline release from isolated newborn ATII cells when compared with terbutaline alone; this inhibitory effect was mimicked by the specific NPR-C agonist, C-ANP (10 -10  M). Conclusion: ANP may act as an important epithelial-derived inhibitor of surfactant release in the fetal lung, and downregulation of ANP and NPR-C following birth may sensitize ATII cells to the effects of circulating catecholamines, thus facilitating surfactant secretion.

During the transition at birth to air breathing, regulation of surfactant release from alveolar type II (ATII) cells is critical. Atrial natriuretic peptide (ANP) stimulates natriuretic peptide receptor-A (NPR-A) and increases intracellular cGMP. We examined the changes in ANP and NPR-A in respiratory epithelium during the perinatal period using immunohistochemistry and studied the effect of ANP on surfactant release from ATII cells isolated from fetal and newborn lambs. NPR-A mRNA was detected in the fetal lung by Northern Blot and RT-PCR. At 100 d gestation (term 145 d), ANP staining was absent and NPR-A staining was weak in cuboidal epithelial cells. ANP and NPR-A staining was prominent in ATII cells at 136 d gestation and was undetectable postnatally. ANP stimulated (maximal effect at 10(-10)M) surfactant release from both late gestation fetal and neonatal ATII cells. Protein kinase G inhibition significantly blocked this release. We conclude that ANP stimulates surfactant release in isolated perinatal ATII cells by a cGMP-dependent mechanism. ANP and NPR-A expression in ATII cells is greatest in late gestation and declines sharply postnatally. We speculate that increased activity of the ANP/NPR-A pathway in late gestation may prime the surfactant system, preparing the lung for air breathing.

Tubules may arise during branching morphogenesis through several mechanisms including wrapping, budding, cavitation and cord hollowing. In this report we present evidence that is consistent with renal proximal tubule formation through a process of cord hollowing (a process that requires the concomitant establishment of apicobasal polarity and lumen formation). Pockets of lumen filled with Lucifer Yellow were observed within developing cords of rabbit renal proximal tubule cells in matrigel. The observation of Lucifer Yellow accumulation suggests functional polarization. In the renal proximal tubule Lucifer Yellow is initially transported intracellularly by means of a basolaterally oriented p-aminohippurate transport system, followed by apical secretion into the lumen of the nephron. Consistent with such polarization in developing tubules, Triticum vulgare was observed to bind to the lumenal membranes within pockets of Lucifer Yellow-filled lumens. As this lectin binds apically in the rabbit renal proximal tubule, T. vulgare binding is indicative of the emergence of an apical domain before the formation of a contiguous lumen. Both epidermal growth factor and hepatocyte growth factor stimulated the formation of transporting tubules. The stimulatory effect of both epidermal growth factor and hepatocyte growth factor on tubulogenesis was inhibited by PD98059, a mitogen activated protein kinase kinase inhibitor, rather than by wortmannin, an inhibitor of phosphoinositide 3-kinase. Nevertheless, Lucifer Yellow-filled lumens were observed in tubules that formed in the presence of PD98059 as well as with wortmannin, indicating that these drugs did not prevent the process of cavitation. By contrast, rapamycin, an inhibitor of the mammalian target of rapamycin, prevented the process of cavitation without affecting the frequency of formation of developing cords. Multicellular cysts were observed to form in 8-bromocyclic AMP-treated cultures. As these cysts did not similarly accumulate Lucifer Yellow lumenally, it is very likely that processes other than organic anion accumulation are involved in the process of cystogenesis, including the Na,K-ATPase.

C-type natriuretic peptide (CNP) is a recently described endothelium-derived relaxing factor. CNP relaxes vascular smooth muscle and inhibits smooth muscle proliferation by binding to natriuretic peptide receptor (NPR) type B (NPR-B) and producing cGMP. Lung parenchyma and fifth-generation pulmonary arteries (PA) and veins (PV) were isolated from late-gestation fetal lambs. All three types of NPR mRNA were detected in PA and PV by RT-PCR. CNP and NPR-B immunostaining was positive in pulmonary vascular endothelium and medial smooth muscle. CNP concentration-response curves of PA and PV were compared with those of atrial natriuretic peptide (ANP) by use of standard tissue bath techniques. CNP relaxed PV significantly better than PA. ANP relaxed PA and PV equally, but ANP relaxed PA significantly better than CNP. Pretreating PA and PV with natriuretic peptide receptor blocker (HS-142-1) or cGMP-dependent protein kinase inhibitor Rp-beta-phenyl-1- N2-etheno-8-bromoguanosine 3',5'-cyclic monophosphorothionate significantly inhibited the CNP relaxation response, indicating that the response was mediated through the NPR-cGMP pathway. We conclude that CNP is important in mediating pulmonary venous tone in the fetus.

Rabbits were exposed to 100% oxygen or to air at one atmosphere. No alterations were observed in the lung of rabbits breathing air for up to 66 hours or 100% oxygen for 24 hours; after 48 hours, inflammatory cells, chiefly neutrophils, were located in the interstitium of the lung. By 66 hours of oxygen, the number of inflammatory cells in the interstitial space was greater than at 48 hours. At 72 hours, alveolar space in focal areas of the lung was filled with edema fluid containing a lightly flocculent material, and more densely staining fibrin. In experiments for the study of alveolar permeability, cytochrome C was instilled through the tracheobronchial tree into alveoli and demonstrated ultracytochemically by its peroxidase activity. No electron-opaque reaction product was observed in control rabbits or in those breathing oxygen for 24 hours, indicating that the tracer did not leave the alveolar space. However, after 48 hours of the breathing of 100% oxygen, electron-opaque reaction product was localized to the basal lamina of alveolar capillaries in focal areas, whereas in other alveolar capillaries there was no reaction product in the basal lamina. Vesicles filled with reaction product were observed in Type 1 pneumocytes and in alveolar capillary endothelial cells within capillary loops having increased electron density in the basal lamina. After 66 hours of the breathing of 100% oxygen, virtually all alveolar capillaries showed electron-opaque reaction product in the basal lamina and in vesicles within Type 1 cells and capillary endothelial cells. Increased permeability of Type 1 pneumocytes appears as an early manifestation of oxygen-induced changes in the lung preceding pulmonary edema. The presence of numerous inflammatory cells in the interstitium and in alveolar capillaries may play some part in the pathogenesis of the oxygen-induced increase in alveolar permeability.

Hypertension was induced in young rats by latex encapsulation of both kidneys. By the fourth week, 85% of the renal-encapsulated (RE) rats became hypertensive. Varying degrees of cardiovascular involvement were evident in the moderately to severely hypertensive rats. The level of systolic blood pressure was directly correlated with the width and the volume of zona glomerulosa of the adrenal cortex. Electron microscopy combined with morphometric-stereologic techniques was employed to quantitate change in the adrenal cortex. The cells of both zona glomerulosa and zona fasciculata of RE rats showed significant increases in the volume of the cell, nucleus, smooth endoplasmic reticulum, and lipid droplets; only in the zona glomerulosa cells was the increase in surface area of the smooth endoplasmic reticulum statistically significant. It is suggested that these structural changes associated with renal-encapsulation hypertension are related at least in part to stress of the hypertensive cardiovascular disease.

The zona reticularis of 70- and 95-week-old female Wistar-Kyoto (WKY) and spontaneously hypertensive (SH) rats was studied by quantitative stereologic techniques. The zona reticularis in the adrenal gland of 70-week-old SH rats was virtually identical to that of 70-week-old WKY rats. In both SH and WKY rats at 95 weeks of age, however, there was hypertrophy of smooth endoplasmic reticulum reflected quantitatively in increased volume and surface area, as compared with that of WKY rats or SH rats at 70 weeks of age. Ninety-five-week-old WKY rats had a significantly greater mitochondrial volume/cell and surface area of mitochondrial membranes than SH rats. Inclusion of lipid droplets within mitochondria was seen in zona reticularis cells from SH and WKY rats at 70 weeks of age; mitochondria-lipid-droplet association was more frequent at 95 weeks of age. The volume of lipofuscin per cell was significantly greater in WKY than in SH rats at 95 weeks of age. Thus, by quantitative techniques, one can see that the zona reticularis of 95-week-old SH rats differs from that of WKY rats, principally in the presence of smaller cells with a smaller surface area of mitochondrial cristae and a reduced volume of mitochondria, lipofuscin, and lipid droplets.

Thyroparathyroidectomy (TPX) prevents adrenal regeneration hypertension (ARH) in female rats and concomitantly inhibits regeneration of the adrenal cortex. Removal of the thyroid gland plays the major role in preventing ARH inasmuch as parathyroidectomized adrenal-enucleated (PX-AE) rats became hypertensive, whereas thyroparathyroidectomized adrenal-enucleated rats (TPX-AE + PT) did not. Inhibition of adrenocortical regneration by TPX is reflected by a significant decrease in adrenal weight, volume of cortical parenchymal tissue per gland, and average cell volume at three weeks, compared with the regenerating adrenal gland in adrenal-enucleated thyroid-parathyroid-intact (AE) rats. Mitochondria in TPX-AE rats resembled closely those from zona fasciculata cells of a normal adrenal gland; stereologic techniques for electron microscopic examination confirmed that mitochondrial volume/cell and surface area of total mitochondrial membranes/cell (outer/inner membranes plus cristae) of adrenocortical cells from TPX-AE rats did not differ significantly from those of AE animals. The surface area of mitochondrial cristae of TPX-AE rats, however, was significantly greater than that of AE rats, whereas the surface area of the inner/outer mitochondrial membrane of the TPX-AE group was decreased significantly as compared with that of the AE group. The diameter of mitochondria in TPX-AE rats was larger than in the AE group, although the number of mitochondria/cell was significantly less in TPX-AE rats than in AE rats. Although TPX had no significant effect on the levels of DOC or corticosterone in the serum of quiescent AE rats as compared with TPX-AE rats, the rise in DOC in the serum after ether stress was blunted in the TPX-AE group as compared with that in the AE group. The rise in corticosterone in the TPX-AE group was comparable to that of the AE animals. Thus, partial inhibition of adrenal regeneration in TPX-AE rats in combination with a blunted rise in DOC levels in response to stress may well contribute to the prevention of ARH.

Alterations occur predominantly in cells of the zona reticularis in retired breeder Mongllian gerbils and golden hamsters. In gerbils, the size and number of lipid droplets increased and numerous large lipid-lysosomal complexes were observed in zona reticularis parenchymal cells or in macrophages, which were more numerous than in younger animals. Abundant lipofuchsin pigment was observed in zona reticularis parenchymal cells, and mitochondria contained electron-opaque inclusions. In the hamster, lipid droplets were present in inner zona reticularis cells and there was an increased amount of pigment within macrophages. Some of the alterations in gerbils and hamsters may at least in part reflect a stimulation of zona reticularis cells in retired breeders, attributable to increased prolactin secretion.

The systolic blood pressures of rats that underwent parathyroidectomies and uninephrectomies reached hypertensive levels after implantation of deoxycorticosterone (DOC) pellets and were compared to those in rats with intact parathyroids bearing 20-mg or 50-mg pellets of DOC. Parathyroidectomy, however, ameliorated the incidence and severity of cardiac and renal lesions induced by DOC. The beneficial effect of parathyroidectomy on vascular lesions may well be attributable at least in part to a reduced level of calcium in the serum or to the absence of parathyroid hormone, which is involved directly in the regulation of calcium transport and influx into the cell. Parathyroidectomy significantly reduced the compensatory renal hypertrophy and splenomegaly induced by DOC, although cardiac hypertrophy and hepatomegaly induced by DOC were not affected by parathyroidectomy.

The effect of verapamil, a calcium antagonist, was studied in rats treated with deoxycorticosterone (DOC). DOC induced hypertensive cardiovascular disease with accompanying gross and microscopic lesions in heart and kidney. Verapamil administered in the drinking fluid (1% sodium chloride) prevented hypertension and significantly ameliorated the incidence and severity of cardiovascular lesions. With exception of the spleen, verapamil did not prevent renal or myocardial hypertrophy in rats treated with DOC in spite of prevention of hypertension. The level of verapamil in the serum of animals consuming verapamil (0.37 +/- 0.16 microgram/ml) was less than that of the DOC-verapamil group (0.89 +/- 0.16 microgram/ml), although the difference was not significant. These results confirm the efficacy of verapamil in reducing blood pressure and in ameliorating vascular lesions.

The adrenal cortex of spontaneously hypertensive rats (SHR) has been examined by quantitative morphologic techniques for electron microscopy. The volume and surface area of smooth endoplasmic reticulum and the volume of Golgi apparatuses in zona glomerulosa cells of SHR was significantly greater than those of Wistar-Kyoto strain (W/KY) normotensive controls; the volume of lipid droplets and nucleus was significantly less in SHR than in W/KY animals. A stimulation of the zona glomerulosa in SHR may well be attributable to the elevation in systolic blood pressure. A distinct lipid-free subglomerulosa was observed in the adrenal gland of W/KY rats; the cell volume was similar to that of the zona glomerulosa although the cells showed a significantly greater volume of mitochondria and surface area of mitochondrial membranes and greater volume of smooth endoplasmic reticulum and lysosomes. In the zona fasciculata, cell volume, volumes and surface area of mitochondria and smooth endoplasmic reticulum, and volume of lipid droplets were significantly lower in SHR than in W/KY rats. The volume of the Golgi apparatus was greater in SHR than in W/KY rats. Glycogen particles were observed in focal areas of some zona fasciculata cells. The adrenal cortex of another strain of normotensive Wistar rat (W/CFN) was compared with that of the W/KY and SHR. Although the relative adrenal weights of SHR and W/KY animals were identical, the weight of that in W/CFN was significantly smaller. The volume of the zona glomerulosa of SHR was significantly greater than that of W/KY although the volume of the zona glomerulosa in W/CFN was significantly greater than the other two groups. The volume of nucleus and lipid droplets of zona glomerulosa in W/KY was significantly greater than that in the S/CFN; the volume of the cell, mitochondria, smooth endoplasmic reticulum, lipid droplets, and lysosomes, and the surface area of smooth endoplasmic reticulum and mitochondrial membranes of W/KY animals was significantly greater than those of W/CFN animals. It is concluded that the W/CFN rat is not an appropriate control for spontaneously hypertensive rats.

Before the usefulness of a new in vitro model can be ascertained, the model must be properly defined and characterized. This study presents the growth rate and biochemical characteristics of rabbit renal proximal tubule cells in primary culture over a 2-wk culture period. When grown in a hormonally defined, antibiotic-free medium these cells form confluent monolayer cultures within 7 d after plating. Multicellular dome formation, an indicator of transepithelial solute transport, was expressed after confluent cultures were formed. The activity of the cytosolic enzyme, lactate dehydrogenase, and the lysosomal enzyme, N-acetyl-glucosaminidase, increased 14- and 2-fold during the first 8 d of culture, respectively. In contrast, the activity of a brush border enzyme, alkaline phosphatase, decreased 85% within the first 8 d of culture. Release of these enzyme markers into the culture medium, which are routinely used to measure cytotoxicity, stabilized after 8 d in culture. The ratio of cellular protein to DNA changed according to the state of cellular growth. Values rose from 0.035 mg protein/micrograms DNA in preconfluent cultures to 0.059 mg protein/micrograms DNA in confluent cultures. These results document the characteristics of a primary proximal tubule cell culture system for future studies in in vitro toxicology.

The clinical syndrome of persistent pulmonary hypertension of the newborn includes a developmentally abnormal pulmonary microvasculature which contains excessive amounts of muscle and which cannot adapt to air breathing in the perinatal period. Surgical ligation of the ductus arteriosus of the fetal lamb has produced a physiologic model of pulmonary hypertension of the newborn. The aim of the present investigation is to determine whether surgical ligation of the ductus arteriosus in fetal sheep produces anatomic changes in the pulmonary blood vessels. The pulmonary vasculature of seven neonatal lambs that underwent surgical ligation of the ductus arteriosus from 6 to 17 d before birth was compared to that of five control lambs with a patent ductus arteriosus without fetal surgery and three control lambs with a patent ductus arteriosus that underwent sham surgery. Quantitative microscopic analysis of the barium gelatin-filled peripheral pulmonary vascular bed revealed an increase in the proportion of partially and fully muscularized pulmonary arteries at the level of the terminal bronchiole and within the acinus (p less than 0.0001). This finding demonstrates that medial muscle develops in areas of the distal pulmonary vascular bed where it is normally absent. Periadventitial fibrosis surrounding intraacinar pulmonary arteries was also present. No change in the number of small intraacinar arteries was detected. This structural remodeling of the peripheral pulmonary vascular bed was initiated in utero by ductus arteriosus occlusion.(ABSTRACT TRUNCATED AT 250 WORDS)

All cortical cells appeared alike in fetal gerbils of both sexes though glomerulosa cells were distinguishable at the time of birth. Fenestrated cisternae of rough endoplasmic reticulum were observed in all cortical cells of fetal gerbils, but smooth endoplasmic reticulum predominated. Concentric whorls of rough endoplasmic reticulum first appeared in adrenocortical cells at 42 days of age. The whorls developed within cells having abundant smooth endoplasmic reticulum and which were located in the region between the zona fasciculata and zona reticularis. Cells with whorls possibly serve as reserve cells which transform into actively secreting steroid cells in times of stress.

The ultrastructure of the adrenal cortex was studied by quantitative stereologic techniques in LAF1 mice bearing a transplantable, ACTH-secreting pituitary tumor (AtT). The tumor stimulated a significant increase in volume and surface area of smooth endoplasmic reticulum and mitochondrial membranes in all three zones of the adrenal cortex. Concomitantly, plasma corticosterone was increased significantly in tumor-bearing animals. The AtT was an adenoma of the chromophobe type. Continuity of the nuclear membrane with the rough endoplasmic reticulum was readily observed in tumor parenchymal cells.

The purpose of this study was to test the hypothesis that bleomycin administration enhances the toxic effects of oxygen on the respiratory system. Twenty-one rabbits, with no evidence of respiratory disease, received intravenous injections of 45 units of bleomycin (Blenoxane), twice a week, for a total dose of 300 units. Fifteen rabbits received an equal volume of saline and served as controls. Treatment with bleomycin resulted in failure to thrive, weight loss, and 30% mortality from nonpulmonary causes, as indicated by the lack of respiratory distress or cyanosis, during or shortly after the injection period. The remainder of the animals were allowed to recover for 21 days following the last injection. At that time, no differences were found between the experimental and the control groups with respect to arterial blood gases, total lung capacity, compliance, and hydroxyproline content. Histologic examination of lung tissue revealed normal lung architecture. When exposed to 100% O2, bleomycin-treated rabbits developed arterial hypoxemia and died from respiratory failure at the same rate as the controls. It was concluded that pretreatment of healthy rabbits with 300 units of intravenous bleomycin did not result in the development of significant amounts of lung fibrosis or enhance the toxic effects of oxygen on the respiratory system.

Female, uninephrectomized rats injected with methylandrostenediol became hypertensive by 4 weeks of treatment. After cessation of treatment, blood pressure continued to rise during the first week but gradually decreased below the borderline of hypertension by 5 weeks. After methylandrostenediol treatment for 4 weeks and after 1 week of recovery, adrenocortical fine structure exhibited a number of abnormalities including reduced numbers of mitochondrial cristae, hypertrophic smooth endoplasmic reticulum, microfilaments and large lipid vacuoles within zona reticularis cells. During an intermediate period of recovery (2 weeks), mitochondrial cristae increased in number, although animals remained hypertensive. After 2 weeks, all of the ultrastructural abnormalities seen at early and intermediate periods of recovery were fully reversed, concomitant with a return of systolic blood pressure to below hypertensive levels.

Two morphologically distinct gonadotrophs have been identified in the anterior pituitary of rats bearing the Snell 494 tumor. Type 1 gonadotrophs first showed ultrastructural alterations at 4 weeks after implantation of the tumor. Numerous large 300 to 600 mmu granules appeared in type 1 cells. The opacity of the granules was variable, and they sometimes contained non-membrane-bounded spherical inclusions. These granules were dispersed among more electron-opaque, 200-mmu granules usually seen in gonadotrophs. A third granule approximately 500 to 600 mmu in diameter was apparently a lysosome-derived structure. Type 2 gonadotrophs first showed changes after 4 weeks, but the alterations were especially prominent by 8 to 10 weeks. No large granules characteristic of type 1 cells were seen in type 2 cells. By 8 to 10 weeks, very few 200-mmu granules were observed in the cytoplasm of type 2 cells. The endoplasmic reticulum was dilated in most type 2 cells; frequently, the cisternae were dilated considerably, forming large lake-like areas. Hypertrophic rough endoplasmic reticulum and the presence of intracisternal granules within cisternae of the rough reticulum suggest that type 2 cells were stimulated by the tumor. Changes in gonadotrophs were undoubtedly produced by the large amount of corticosterone secreted by the tumor. The corticosterone secreted by the tumor. The corticosterone may well act directly on the hypothalamo-hypophyseal axis or it could act indirectly on the ovary; ovarian steroids would then exert a feedback effect. The ovary was atrophic and did not contain maturing Graafian follicles or corpora lutea. In somatotrophs, the number of secretory granules increased, probably reflecting suppression of hormonal release by corticosterone.

Administration of 10 mg of methylandrostenediol for 10 weeks to uninephrectomized, salt drinking, female Sprague Dawley rats caused severe hypertension with extensive renal and cardiovascular damage. The hypertension was accompanied by increased consumption of sodium, high sodium levels in peripheral plasma, decreased weight of the pituitary, thymus, adrenals and ovaries and decreased content of renal renin. Methylandrostenediol treatment also produced impairment of normal adrenal steroidogenesis, reflected in elevated production in vitro of 11-deoxycorticosterone during incubation of adrenal gland homogenates with (14)C-progesterone. Such increased production of deoxycorticosterone is probably responsible for the development of the hypertensive disease. If the methylandrostenediol-treated animals were kept alive for 12 additional weeks after suspension of the treatment with the androgen, the hypertension, as well as the high sodium consumption, high plasma sodium concentrations and low levels of renal renin, persisted to the end of the experiment. The cardiovascular and renal lesions in these animals, killed 12 weeks after suspension of the androgen administration, were similar to those seen in the rats receiving methylandrostenediol but killed at the tenth week of the treatment. Suspension of methylandrostenediol administration, however, resulted in a return to normal weight of the pituitary, thymus, adrenals and ovaries within 12 weeks. Normal amounts of deoxycorticosterone were formed in vitro by the adrenal glands of these rats and the return to normal structure was also confirmed by a electron microscopic study. Thus, contrary to a previous experiment where methylandrostenediol was given for a shorter period of time and the hypertension was reversible, it was shown in this study that metacorticoid hypertension is induced by methylandrostenediol administration, as it is with treatment with deoxycorticosterone. Since adrenal steroidogenesis returned to normal, some other mechanisms must be involved in maintaining the hypertension. It is very likely that these factors are consequent to the extensive and irreversible renal and cardiovascular damage.