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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.

C-type natriuretic peptide (CNP) is a member of the natriuretic peptide family and acts through the membrane bound guanylyl cyclase linked natriuretic peptide receptor B (NPR-B) to increase intracellular cGMP. Activation of the CNP/NPR-B pathway in pulmonary epithelium has been linked to the inhibition of amiloride-sensitive sodium absorption and to the stimulation of the cystic fibrosis transmembrane conductance regulator (CFTR). Given the importance of ion movement across the pulmonary epithelium of the fetal and newborn lung, we sought to examine the expression of CNP and NPR-B in pulmonary epithelium of the developing fetal lamb and following the transition to air breathing. Lambs were sacrificed at 100 and 136 days of gestation and at 3 days, and 4 weeks after full term delivery. Lung sections were immunostained for CNP and NPR-B. At 100 days of gestation, staining for CNP and NPR-B was absent within all pulmonary epithelium. At 136 days of gestation, prominent staining for both CNP and NPR-B was seen within alveolar type II cells, non-ciliated cells of the distal airways (Clara cells), and ciliated epithelium of the upper airways. At both 3 days and 4 weeks following birth, staining for CNP and NPR-B was absent in alveolar type II cells, ciliated bronchial epithelium and was markedly reduced in Clara cells. The presence of CNP and NPR-B within the pulmonary epithelium in the nearterm fetal period and its rapid downregulation following birth suggests that CNP may contribute to the maintenance of the fluid-filled lung through the regulation of trans-epithelial ion flux.

Exposure to inhalant organic nitrites (drugs of abuse commonly known as "poppers") has been reported to enhance tumor growth in mice, but the mechanism is not fully defined. This study examined the effect of repeated in vivo nitrite exposures on gene expression in the mouse liver and lungs using a gene array panel of 94 cancer- and angiogenesis-related genes. Using 2-fold change as a threshold criterion, repeated nitrite exposure was found to alter the expression of 65 and 23 genes in the liver and lungs, respectively. Six genes were significantly upregulated (p

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.

Endothelial nitric oxide synthase (eNOS) or NOS-III in the endothelium catalyzes production of nitric oxide (NO). Nitric oxide diffuses freely into vascular smooth muscle, where it activates soluble guanylate cyclase (sGC) to produce guanosine 3',5'-cyclic monophosphate (cGMP) and causes vasorelaxation. The NO/cGMP pathway is an important signaling pathway in the control of perinatal pulmonary circulation. An exact colocalization of NOS-III in the pulmonary endothelium and sGC in the vascular smooth muscle was demonstrated using a double immunolabeling technique. The sGC immunoreactivity was higher in resistant pulmonary vessels and veins than in conduit arteries, whereas NOS-III immunoreactivity was higher in conduit arteries than in veins. These results demonstrated anatomically in situ a paracrine role of NOS-III and sGC in the regulation of fetal pulmonary circulation and suggested a heterogeneous distribution of NOS-III and sGC within fetal ovine pulmonary vasculature. Our results provided an anatomic basis that supported previous functional studies on perinatal control of pulmonary circulation.

The nitric oxide (NO)/guanosine 3',5'-cyclic monophosphate (cGMP) pathway plays an essential role in mediating pulmonary vasodilatation during transition of the pulmonary circulation at birth. We used immunoblot analysis (Western) and semiquantitative immunohistochemistry to study the presence, distribution, and relative amounts of type I nitric oxide synthase (NOS-I). Immunoblots were performed on normal fetal sheep lungs, whereas immunohistochemistry for NOS-I was compared between lungs from normal fetal lambs vs. fetal lambs with persistent pulmonary hypertension of the newborn (PPHN) induced by ligation of the ductus arteriosus. Western blot analysis using a polyclonal antibody detected NOS-I protein in homogenates of normal fetal sheep lungs. Abundant NOS-I immunoreactivity was observed exclusively in the precapillary resistance vessels, i.e., terminal bronchiole-associated arteries (TA) and respiratory bronchiole-associated arteries (RA) in normal fetal lung. In marked contrast, immunoreactivity for NOS-I was significantly reduced in the TA and RA of hypertensive lungs. We conclude that there is a heterogeneous distribution of NOS-I in the normal fetal sheep lung, but that NOS-I staining is significantly reduced in lambs with PPHN., (Copyright 2002 Wiley-Liss, Inc.)

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.

The nitric oxide (NO)-guanosine 3',5'-cyclic monophosphate (cGMP) signaling pathway plays an important role in the pulmonary vascular transition at birth. We studied pulmonary arteries and veins isolated from normal late-gestation fetal lambs and from fetal lambs with persistent pulmonary hypertension (PPHN) following prenatal ligation of the ductus arteriosus. We additionally used double immunolabeling and immunoblot analysis to determine relative vascular contents of endothelial nitric oxide synthase (NOS-III) and soluble guanylate cyclase (sGC). Cyclic GMP content and sGC activity were significantly lower in arteries from hypertensive lambs than controls. A rank order for contents of both soluble guanylate cyclase and NOS-III was observed by both immunolabeling and immunoblotting: Control vein = Hypertensive vein > Control artery > Hypertensive artery. Our data demonstrate that the relative expression of sGC correlates well with the relative expression of NOS-III, and indicate the potential importance of soluble guanylate cyclase in the regulation of the perinatal pulmonary circulation. These data may help us understand vascular mechanisms producing PPHN, as well as patterns of response to exogenous NO.

The nitric oxide/guanosine 3',5'-cyclic monophosphate pathway plays an essential role in mediating pulmonary vasodilation at birth. Small resistance arteries in the fetal lung are vessels of major significance in the regulation of pulmonary vascular tone. The present study is to determine that type I nitric oxide synthase (NOS-I) is present in ovine fetal pulmonary vasculature and that NOS-I is distributed heterogeneously in ovine fetal pulmonary circulation. We used reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry and NOS-I immunohistochemistry to localize NOS-I in fetal sheep lungs and showed a colocalization for NADPH-d activity with NOS-I immunoreactivity. Strong NOS-I immunoreactivity was observed exclusively in the endothelium of the terminal bronchiole and respiratory bronchiole-associated arteries. As a comparison, adult sheep lung did not show positive immunoreactivity in the pulmonary endothelium. NOS-I was absent in the umbilical or systemic arteries from the ovine fetus, whereas abundant NOS-III immunoreactivity was present in these arteries. We conclude that NOS-I is present uniquely in the ovine fetal pulmonary circulation as opposed to the adult pulmonary or the fetal systemic circulation. NOS-I is distributed heterogeneously in the ovine pulmonary vasculature. We speculate that NOS-I plays an active role in the regulation of perinatal pulmonary circulation.

Background: Vascular segments in the fetal lung differ anatomically and functionally from one another. At birth, the nitric oxide (NO) pathway plays an integral role in reducing pulmonary vascular resistance through a marked vasodilation. However, the contributions of each vascular segment to this dilation are unclear. We sought to determine the distribution of soluble guanylate cyclase (sGC), the enzyme NO activates to induce vasodilation across the pulmonary vasculature., Methods: Pulmonary airspaces were expanded with freezing compound and the pulmonary arterial tree was infused with barium sulfate gelatin. Soluble guanylate cyclase was localized by immunohistochemistry across the pulmonary vasculature of four near-term fetal lambs and its immunoreaction product was assessed by a semiquantitative method. The physiologic response of fourth- and fifth-generation arteries and veins isolated from age-matched lambs to NO was measured using standard tissue bath techniques., Results: Clear differences in sGC immunostaining were present throughout the pulmonary vasculature: very weak to absent in large arteries accompanying bronchi, but intensely positive for veins. This pronounced staining for sGC in preacinar veins correlated with a 100-fold greater sensitivity to NO in veins compared to arteries of the same generation. The percentage of arteries staining positively approached 100% at the level of respiratory bronchioles and alveoli., Conclusions: These findings suggest that the increased response to NO in preacinar veins compared to that of arteries is in part due to increased sGC within venous vascular smooth muscle. Furthermore, intense staining within distal arteries implies a greater role for NO-mediated vasodilation within these segments.

The exact mechanism of how immune adjuvants function still remains largely unknown, despite their long history of use. This work reports the properties of alum and the related compounds Al(OH)3 or Al2O3. Experiments were performed in rats to determine the relative adjuvancy of silica, talc, ground glass, Al2O3, SnO2, ZrO2, hematite and magnetite. Antibody response and cell-mediated immunity (CMI) to ovalbumin (OVA) were determined and were found to be significantly enhanced by silica and talc. Antibody response to OVA was moderately enhanced by Al2O3, hematite, and magnetite, while CMI to OVA was not affected, SnO2, ZrO2, and ground glass only gave a slight adjuvant effect. The magnitude of adjuvancy appeared to correlate with the magnitude of the inflammatory response produced by each metal oxide and also correlated with their surface area. No correlation could be drawn between the hydrophilicity or hydrophobicity of the metal oxides and the magnitude of their adjuvancy.

Cytoplasmic biotin-like activity was identified in formalin-fixed and paraffin-embedded human thyroid lesions by immunostaining for biotin by use of the peroxidase-antiperoxidase method or by peroxidase-labeled streptavidin alone. The reactivity of cytoplasmic biotin-like activity was markedly enhanced both by pretreatment with trypsin and after heating by autoclaving. Of 208 thyroid lesions, 93 showed positive immunostaining for biotin with use of trypsin pretreatment. The positive incidence of cases and positive cell ratio were highest in papillary carcinoma, followed by follicular carcinoma but lowest in the lethal types of thyroid carcinoma: anaplastic carcinoma, squamous cell carcinoma and poorly differentiated insular carcinoma. We conclude that cytoplasmic biotin-like activity in the common thyroid neoplasms should be considered as an interfering factor for immunostaining with avidin-biotin combined with selected antigen retrieval methods and with in situ hybridization with biotinylated probes.

A custom-built small-animal transceiver was used for in vivo imaging of normal rat brain at 0.35 T, with the objective of identifying anatomic components by comparison of images with corresponding histologic sections. The cerebrum, cerebellum, brain stem, ventricles, hippocampus, and subarachnoid space were identified and cerebrospinal fluid (CSF) was differentiated from gray matter and white matter on coronal and transaxial magnetic resonance (MR) images. These images compare favorably with those obtained by others at higher field strengths in regard to delineating major neuroanatomic structures. It is concluded that this technique will be useful for investigating small-animal models of human neurologic disease involving morphologic and morphometric changes in gray matter, white matter, and CSF-filled spaces.

Four days of isoproterenol injections induced a marked enlargement of the rat parotid and submandibular glands reflected in significant increases in the absolute and relative wet and dry weight of the glands. The enlargement in parotid gland was attributable at least in part to cellular hypertrophy inasmuch as the average volume per cell of acinar cells increased. In contrast, the average volume of acinar cells in the submandibular gland was decreased as compared to that of control. It is likely that hyperplasia in both groups accounts in part for the enlargement. The slow calcium channel is unlikely involved in the isoproterenol-induced stimulation of the gland, inasmuch as the calcium channel antagonist did not modify the enlargement of the parotid or submandibular glands.

Magnetic resonance imaging revealed conspicuously hyperintense regions in the papillary area of kidneys of three untreated rats. When the kidneys were examined histologically, a hydronephrosis associated with the presence of bacteria was found. This study relates magnetic resonance images of an early stage of hydronephrosis to its histological picture.

Proton magnetic resonance imaging was performed on rats before induction of diabetes with streptozotocin (STZ) and at 2 and 12 days postinduction. Images revealed an increase in maximal longitudinal and axial dimensions of the kidneys at 2 days and a further increase at 12 days. Similarly, an increase in the size of the remaining kidney was seen in a rat which underwent uninephrectomy as a positive control. Two major differences were observed between the kidney undergoing compensatory hypertrophy and those developing diabetic nephropathy: (i) Expansion of the renal vasculature was seen only in images of the diabetic rat; (ii) A loss in conspicuity of the normal corticomedullary junction was seen in the T2-weighted images of the diabetic rat but not in the uninephrectomized rat. Histologic examination revealed that the medulla increased to a size greater than the cortex during diabetic nephropathy whereas the medullary volume was less than that of the cortex during compensatory hypertrophy. In vitro T1 relaxation times in cortex, outer medulla and inner medulla of kidneys from control rats were measured and compared with the same respective regions in diabetic rats. When these values were correlated with tissue water content, a linear increase in relaxation rate versus percent water content from cortex to inner medulla was found in the control kidneys, but this correlation was absent in diabetic nephropathy. These studies demonstrate that MRI is an effective noninvasive tool for studying the course of renal hypertrophy and hydration changes in the development of renal disease in STZ-induced diabetes in the rat.

When rabbits were exposed to 60% oxygen for 21 days, arterial PaO2 showed statistically insignificant changes at 6-8 days (69 +/- 5 Torr) and 14-16 days (62 +/- 5 Torr); at 21 days the PaO2 was significantly decreased (56 +/- 5.4 Torr) as compared to 79 +/- 2 Torr at 0 time of exposure. No significant alterations in PaCO2, pH, or hematocrit were present. Morphometric techniques for electron microscopy showed no qualitative or quantitative alterations in lung morphology except for a significant decrease in the volume per lung of the capillary endothelial lumen, possibly reflecting an altered recruitment of capillaries. The total capillary volume per lung however did not differ significantly between oxygen and control-treated rabbits. The physiologic finding of a decreased oxygenation after 60% oxygen exposure suggests that changes in oxygenation precede the morphologic parameters studied.

Rats were made hypertensive by implantation of a pellet of deoxycorticosterone (DOC). A low dose (1 mg/kg twice daily) of the calcium antagonist nitrendipine protects against the increase in total and ionic levels of calcium in the aorta produced by the elevated blood pressure, dissociating at least in part the hypertension from the rise in aortic calcium. Ionic (free) calcium was demonstrated in aortic smooth muscle cells by the pyroantimonate ultra-cytochemical method and the electron opaque reaction product quantitated by stereological techniques. As compared to the control group, nitrendipine did not increase the number of vesicles/micron with precipitate located adjacent to the sarcolemma. DOC however increased the number of subsarcolemmal vesicles with electron opaque precipitate and sarcoplasmic calcium. Nitrendipine administration to DOC-treated rats decreased the number of vesicles to that found in the control or nitrendipine-treated group while ionic calcium in the nitrendipine + DOC group was intermediate between the control or nitrendipine group and the DOC group. The total content of calcium measured by atomic absorption correlates with the observations of ionic calcium levels demonstrated ultracytochemically. Aortic dry weights of the DOC and DOC + nitrendipine groups were comparable and significantly greater than those in the control or nitrendipine groups.

A low dose of nitrendipine (1 mg/kg twice daily) ameliorated the percent incidence and severity of vascular lesions in the kidney and heart induced by deoxycorticosterone (DOC). Less protection was offered by administration of 1 mg/kg of the calcium antagonist once daily. A lower dose of the antagonist (0.5 mg/kg) administered twice daily produced almost no protection against myocardial scars, but the percent incidence and severity of renal tubular casts and glomerular changes were similar to those following injection of 1 mg/kg of the antagonist twice daily. DOC induced hypertrophy of the media in aorta, coronary artery and renal interlobular artery and renal arteriole. Neither 1 mg/kg once or twice daily nor 0.5 mg twice daily of calcium antagonist modified the hypertrophy of the arterial vasculature in the hypertensive DOC group. We conclude that a low dose of the calcium antagonist dissociates at least in part lesions but not hypertrophy from the increased systolic blood pressure, because the antagonist protects against vascular lesions induced by the hypertension. The antagonist likely acts on the endothelial cell of the vessels alone or combined with an effect on the vascular smooth muscle cells.

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 antihypertensive activity of nicergoline (Sermion), an adrenolytic and vasodilator drug, was tested in rats when hypertensive vascular disease was induced by administration of methylandrostenediol (MAD). Nicergoline not only counteracted the effect of MAD on the systolic blood pressure of the rats, but it also prevented the appearance of vascular lesions usually induced by this type of experimental hypertension on arterioles of several organs such as heart, kidney and brain and of the pancreatic mesenteric region. When given alone, the drug was well tolerated. Since the drug did not interfere with the modification induced by MAD on the adrenal steroidogenesis, as seen by EM studies of the zona fasciculata of the androgen-treated animals, it is likely that it may have exerted its adrenolytic effect on the peripheral vessels of the MAD-induced hypertensive rats.

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.

Intranuclear inclusions have been examined in mammotrophs of the Mongolian gerbil. No inclusions were identified in nuclei of newborn females, although inclusions were seen in the pituitary gland of young mature females. The number of inclusions in retired breeders was similar to that in young females. Estradiol benzoate increased the number of inclusions, although 10 mug/day for 5 or 14 days induced more inclusions than 5 or 100 mug for 14 days; significantly fewer inclusions were seen with the higher dose (2.81 +/- 0.10 inclusions per field for 10 mug and 2.19 +/- 0.01 inclusions for 100 mug). Significantly fewer inclusions were present at 3 and 4 weeks. The reduced number of inclusions may be attributable at least in part to cellular hypertrophy of mammotrophs which was especially prominent. Vacuolar inclusions predominated at 3 and 4 weeks and there were fewer membranous types than at previous times.

The clinical, histological, and ultrastructural aspects of a cervical paraganglioma of the vagus nerve, in a 66-year-old white man, have been discussed in detail. Ultrastructurally, the tumor chief cells contained characteristic membrane-bound and dense-cored neurosecretory granules which ranged in size from 85 millimicron to 190 millimicron. Unlike earlier ultrastructural reports, the present study showed the presence of sustentacular or supporting cells. These cells were smaller, darker, polymorphic, and were commonly located at the periphery of a single or group of chief cells. Furthermore, unlike earlier reports on vagal paragangliomas, nonmyelinated nerve fibers and an occasional axon were identified in the present fine structure study.

The zona glomerulosa of the rat has been examined at various times after hypophysectomy. There is no detectable change in the zona glomerulosa at 2 days after hypophysectomy. However by 7 days, the width of the glomerulosa became significantly larger in spite of an absolute decrease in the volume of the zone. No alternations in ultrastructure were discernible at this time in the glomerulosa, although giant mitochondria were seen in adjacent zona fasciculata cells. By 30 days there was a much wider zona glomerulosa and a restoration of the volume of the zone to that found in controls. Many zona glomerulosa cells were hypertrophic. The Golgi apparatus was enlarged and many vesicles were associated with peripheral cisternae. A few intramitochondrial tubules were observed in some mitochondria of the zona glomerulosa at 30 days.

This report describes light and ultrastructural features of a functional parathyroid gland adenoma, principally composed of transitional oxyphil cells, in a 64-yr-old hypertensive black woman. She was hospitalized for repeated episodes of headaches, lethargy, and dizzy spells. Her serum calcium level was 2.92 mmol/l and immunoassay for parathormone was 390 pg/ml. On neck exploration, the left lower parathyroid gland was found enlarged and therefore removed in toto. The serum calcium and phosphate levels returned to normal following parathyroidectomy. Microscopically, the diagnosis of functional oxyphil adenoma was made. On ultrastructural examination, the tumour was composed principally of transitional cells, occasional typical, and degenerating oxyphil cells. The predominant transitional cells were rich in mitochondria and contained multiple active Golgi complexes, stacked profiles of rough endoplasmic reticulum, and a few secretory granules. On the other hand, typical oxyphil cells were tightly packed with mitochondria at the expense of other organelles. It appeared that neoplastic oxyphil cells were chief cells transformed in response to some unknown oncogenic stimulus.

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.

Administration of Teprotide (SQ 20881), an angiotensin I-converting enzyme inhibitor for up to 10 weeks at a dose of 3 mg. per kg., subcutaneously, twice a day in the rat, effected no change in the ultrastructure of the adrenal cortex nor in the concentration of serum aldosterone. A significant increase (p less than 0.05) in renin granulation indices which was already apparent after 3 weeks of treatment with Teprotide was even more definitive after 10 weeks (p less than 0.01). Moderate renal hypertrophy was present in rats receiving the drug for 3 weeks. Findings pertaining to aldosterone production differed from those reported following acute administration of Teprotide wherein a decrease in the production of serum aldosterone and an increase in plasma renin activity was observed. It has been suggested that decreased aldosterone production following acute administration of Teprotide is a consequence of decreased stimulus of the zona glomerulosa due to diminished synthesis of angiotensin II. If this is the case, mechanisms other than angiotensin II stimulation of the zona glomerulosa must control aldosterone synthesis, perhaps through hormones of the adrenal cortex. Another possibility could be that angiotensin II synthesis may be obtained, after an interval, through an alternative pathway.

Myocardial cells from left ventricles of beating hearts of rats were fixed by immersion in an osmium tetroxide solution containing potassium pyroantimonate to study the electron-microscopic distribution of calcium, the cation being precipitated as an electron-opaque salt (calcium antimonate) by this cytochemical technique. The observed myocytes could be divided into two groups according to their contractile state, evaluated by sarcomere length measurements. In contracted cells (mean sarcomere length 1.43 microgram) the intramyofibrillar precipitate was confined to areas of I-bands bordering the A-bands, the intermyofibrillar space showing scarce content in reaction product. Relaxed cells (mean sarcomere length 1.69 microgram) presented a heavy deposition of reaction product over the sarcomeres, the electron-opaque dots being absent on the H and Z bands. The sarcotubular system and mitochondria were also clearly marked by the reaction product. This second pattern of calcium distribution has not been previously described in heart muscle cells and is interpreted as corresponding to the phase of rise of intracellular calcium which is mediated by membrane depolarization. Our results suggests that different bands of heart sarcomeres show different abilities to bind calcium. The I bands retain the cation even in cells under sustained contraction, probably due to their content in calmodulin; Z and M bands are apparently not involved in calcium sequestration, whereas the content in calcium of the A bands seems to be dependent on the contraction-relaxation cycle of heart myocytes.

The distribution of calcium in the mitochondria of the adrenal gland was studied during development of adrenal regeneration hypertension. Electron opaque precipitate (calcium antimonate) was localized predominantly in the intercristal space within mitochondria and in cisternae of smooth endoplasmic reticulum. Stereological techniques were employed to quantitate the volume per cell of precipitate. Compared to the zona glomerulosa or zona fasciculata of controls, the volume per cell of electron opaque precipitate in mitochondria of the regenerating gland was significantly reduced at 5 and 14 days after enucleation. By 21 days, the volume of mitochondrial precipitate per cell, while more than that in zona glomerulosa cells, was less than in mitochondria from control zona fasciculata cells. As a comparison, normal rats were treated with ACTH or were hypophysectomized. ACTH-treatment did not greatly increase the precipitate associated with mitochondria in the zona fasciculata. Mitochondria in the zona fasciculata of hypophysectomized rats however showed a significant reduction in precipitate per cell correlating with a significantly reduced volume of mitochondria per cell as compared to those of control zona fasciculata cells. Giant mitochondria were observed in hypophysectomized animals. Volume of precipitate per cell associated with smooth endoplasmic reticulum was increased slightly, but significantly, as compared to that in controls treated with ACTH, whereas in hypophysectomized rats, it was decreased significantly. Adrenocortical cells arising from the zona glomerulosa and sub zona glomerulosa region differentiate to zona fasciculata cells during regeneration and may have an altered capacity to concentrate calcium. Change in intramitochondrial calcium may be correlated with the reduced formation of corticosterone from its precursor, deoxycorticosterone, thereby contributing to the pathogenesis of adrenal regeneration hypertension.

The sensitivity of two strains of rat to the hypertensinogenic action of DOC was studied. Hypertensive cardiovascular disease was evident within 3 weeks of implantation of DOC pellets in sensitized female rats of the Sprague-Dawley and Long-Evans strains. Cardiac and renal hypertrophy due to DOC treatment was evident in both strains of rat. The DOC treatment also resulted in a significant decrease in absolute adrenal weight. These results, which indicate that Long-Evans rats are not resistant to DOC-induced hypertension, contrast with previous reports by others. An explanation of the discrepancy may be the use of free DOC rather than DOC acetate in the present study.

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.

At four weeks after thyroparathyroidectomy the activity of catalase in adrenal homogenates was significantly greater (2.95 +/- 0.06 mumoles 0(2)/min/mg protein) than that of controls (2.17 +/- 0.09 mumoles 0(2)/min/mg protein). Increased activity of catalase is related directly to more numerous peroxisomes in cortical cells in the zona fasciculata-zona reticularis of thyroparathyroidectomized rats. The absolute and relative volumes of adrenal gland and zona fasciculata were reduced significantly which is directly correlated with increased peroxisomal activity. A hormonally mediated mechanism reflecting alteration in ACTH, growth hormones and thyroid hormones may mediate adrenocortical atrophy and in turn increase in activity of catalase.

The neurotoxicity associated with chronic exposure to hexachlorophene (HCP) was evaluated by measuring the activity of seven cell specific marker enzymes in brain and by comparing these measurements to morphological changes analyzed by light microscopy. Animals were divided into two groups, the experimental group received HCP at a daily dose of 20 mg/kg p.o. for 53 consecutive days whereas the control group received an equivalent amount of the vehicle only. HCP produced no change in the rate of gain in body weight nor did it produce a statistically significant change in brain weight. Furthermore, no overt abnormal neurological symptoms were observed at this level of exposure to HCP. The white matter throughout the brain was extensively vacuolated in the HCP-treated rats, imparting a spongiform structure which was absent in the white matter of the control animal brains. The data obtained reveal that chronic HCP treatment produce little change in any of the neuronal marker enzymes with the exception of a significant decrease in tyrosine hydroxylase activity in the striatum. Of the nonneuronal enzymes assayed, a significant increase in non-neuronal enolase, glutamine synthetase, and 2',3'-cyclic nucleotide phosphohydrolase was observed in the sciatic nerve, hippocampus and optic nerve, respectively.

An asymptomatic renal oncocytoma was found in the upper left quadrant of an eighty-five-year-old woman during a routine physical examination. Ultrastructurally, the tumor was composed entirely of epithelial cells filled with normal and abnormal mitochondria. Selective renal angiography showed two renal arteries supplying a lobulated, highly vascular mass. The mass contained irregular and tortuous vessels without any arteriovenous shunting.

Implantation of a mammotropic tumor (MtTF4), secreting growth hormone, prolactin, and corticotropin, in female rats of Fischer F344 strain causes hypertension, vasculitis, renal and cardiac hypertrophy, and extensive renal and cardiac lesions. When rats of the same strain were implanted with the MtTF4 tumor but sodium was withheld from the diet, systolic blood pressure rose more slowly but by six weeks reached the same values recorded in the animals implanted with the tumor and allowed to consume sodium ad libitum. In the rats, on sodium deficient diet, however, the vascular damage as well as the renal and cardiac lesions were minimal or absent. Implantation of the tumor caused adrenal cortical dysfunction, and elevated levels of deoxycorticosterone were seen in the peripheral plasma of the rats of all three groups. Nonetheless, plasma deoxycorticosterone was significantly lower in rats on a sodium deficient diet as compared with those having sodium added to the diet. Light microscopic and ultrastructural studies of the adrenal glands revealed that the lack of dietary sodium largely prevented the extensive damage of the zona fasciculata cells usually seen in the tumor-bearing rats, consuming sodium ad libitum. Both hypertensive MtT tumor-bearing animals and normotensive controls on a sodium deficient diet had a conspicuous increase of renal content of renin. It is evident that hypertension may be produced in rats bearing the MtTF4 tumor even in the virtual absence of dietary sodium. It does not appear that the hypersecretion of renal renin sustains the hypertension in these rats, since high levels of this substance were seen in the kidney of normotensive controls on the same sodium deficient diet. Elevated levels of plasma DOC may possibly explain the hypertension. In addition, it is likely that the animals may also have elevated levels of glucocorticoids.

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 procedure for producing adrenal regeneration hypertension did not cause an increase in the systolic blood pressure of W/Fu animals. The regenerating adrenal gland in W/Fu animals was not restored to normal; reduced numbers of mitochondrial cristae were seen and the mitochondria were smaller in size; regeneration was complete in Sprague-Dawley rats of the Holtzman strain and there was a severe form of hypertensive, cardiovascular disease.

The effect of simultaneous injections of ACTH and methylandrostenediol (MAD), a synthetic androgen, has been studied in the rat. ACTH accelerated the production of cytoplasmic vacuoles by MAD in as much as no such structures were observed in MAD-treated animals at 4 weeks. Adrenal weight was decreased in MAD-treated animals but restored to that of controls by ACTH (MAD plus ACTH group). ACTH by itself did not, however, cause the production of any vacuolar structures nor did it cause any reparation in the number of mitochondrial cristae which were considerably reduced in numbers after MAD plus ACTH.

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.

Testosterone propionate accelerates the maturation of acinar cells in the preputial gland of female rats. The weight of the preputial gland was increased significantly and the gland contained a large amount of secretory material. Various stages of maturation of acinar cells were observed. In an early stage, cells contained predominantly rough endoplasmic reticulum. In a second stage, lipid accumulated in association with focal areas of smooth endoplasmic reticulum. Membrane-bound dense bodies similar to lysosomes and secondary lysosomes predominated in a late stage. In the final stage, acini fused with a duct, thereby liberating the cells into the ductal lumen. Cell structure then became unrecognizable.

The effect of deoxycorticosterone (DOC)-induced hypertension on the calcium content within the aorta was studied before the increase in pressure (one week) and after the pressure had reached hypertensive levels (4 weeks). The volume density of free calcium detected ultrastructurally by pyroantimonate precipitation was quantitated by stereological techniques in aortic smooth muscle cells. An increase in the volume density of electron opaque precipitate was observed in the cytoplasm at one week of DOC treatment when neither the systolic blood pressure, the thickness of the media nor volume fraction of medial smooth muscle as compared to the extracellular space was increased significantly. The total aortic calcium as measured by atomic absorption spectroscopy was not increased at one week. By 4 weeks when the rats were hypertensive, the cytoplasmic free calcium in the smooth muscle cells and the number of peripherally-located cytoplasmic vesicles with precipitate was increased significantly. Total aortic calcium was also increased significantly in the DOC-saline group but not in the DOC group drinking tap water or in the saline drinking controls. An elevation of calcium within the cytoplasm of vascular smooth muscle cells may precede the development of hypertension and play a role in the pathogenesis of the increased blood pressure, increased medial thickness and hypertrophy of the vascular smooth muscle cells.