C-type natriuretic peptide system in fetal ovine pulmonary vasculature.

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.

Pulmonary hypertension alters soluble guanylate cyclase activity and expression in pulmonary arteries isolated from fetal lambs.

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.

Heterogeneous distribution of type I nitric oxide synthase in pulmonary vasculature of ovine fetus.

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.

Heterogeneous distribution of soluble guanylate cyclase in the pulmonary vasculature of the fetal lamb.

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.

Mechanisms of adjuvancy: I--Metal oxides as adjuvants.

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 interferes with immunohistochemical analysis of thyroid lesions: a comparison of antigen retrieval methods.

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.

Pre- versus post-ventilatory surfactant treatment in surfactant-deficient preterm lambs.

Twenty lambs at 127 days' gestation (term is 145 days) were randomly assigned to receive Infasurf (Calf Lung Surfactant Extract, ONY Inc., Amherst, NY) as an intratracheal bolus (3 mliter kg-1) either into a fluid-filled lung before ventilation (n = 10), or after ventilation for 5 min (n = 10). All lambs were surfactant-deficient by analysis of lung liquid obtained before surfactant administration. Lambs were then mechanically ventilated for 4 h. Oxygenation for the lambs given surfactant before ventilation did not change during the experiment; a/A pO2 was 0.50 +/- 0.13 at 1 h and 0.52 +/- 0.17 at 4 h. For the lambs given surfactant after initial ventilation, oxygenation decreased over time; a/A pO2 decreased from 0.48 +/- 0.23 at 1 h to 0.37 +/- 0.22 at 4 h (P < 0.05). Compliance, as calculated from the Ventilator Efficiency Index (VEI), improved over time in both groups, but was always significantly higher for lambs given surfactant before ventilation (P = 0.03). Histologic examination of the lungs revealed no differences between the groups; no evidence of epithelial denudation or hyaline membrane formation was seen in either group. Thus, ventilation of surfactant-deficient newborn lambs for 5 min before surfactant administration results in significantly decreased lung function when compared with surfactant administration before ventilation. These differences in lung function are not dependent on a histopathologic injury to the lung. It is possible that unevenness of deposition of the surfactant in an air-filled lung, compared to more uniform deposition in a fluid-filled unventilated lung, produces these differences.

MR imaging of normal rat brain at 0.35 T and correlated histology.

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.

Effect of nitrendipine, a calcium antagonist, on cell volume in rat salivary glands after isoproterenol stimulation.

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 and histopathology of hydronephrosis in the rat.

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.

Magnetic resonance imaging (MRI) and pathophysiology of the rat kidney in streptozotocin-induced diabetes.

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.

Quantitative ultrastructural study of the rabbit lung: exposure to 60% oxygen for 21 days.

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.

Effect of nitrendipine, a calcium antagonist, on the distribution of calcium in aortic smooth muscle cells of deoxycorticosterone-hypertensive rats. A quantitative ultracytochemical study.

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.

Effect of nitrendipine on cardiac and renal lesions and arterial hypertrophy. Protective effect of a low dose of calcium antagonist in deoxycorticosterone-induced hypertensive rats.

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.

Primary cultures of rabbit renal proximal tubule cells: I. Growth and biochemical characteristics.

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.

Ligating the ductus arteriosus before birth remodels the pulmonary vasculature of the lamb.

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)

Cell specific enzyme markers as indicators of neurotoxicity: effects of acute exposure to methylmercury.

In order to assess the sensitivity of several cell specific enzyme markers (tyrosine hydroxylase (TH), glutamic acid decarboxylase, choline acetyltransferase, glutamine synthetase (GS), neuron specific and non-neuronal enolase and 2',3'-cyclic nucleotide phosphohydrolase (CNP] as indices of neurotoxicity, changes in their activities were monitored after rats were treated with two doses of the neurotoxic agent, methylmercury chloride (MMC). Comparisons were also made of any histopathological changes occurring in the tissues examined. At the low dose rate (3.36 mg Hg/kg, po, for 14 days), the rats exhibited less body weight gain compared to untreated animals. No change in either the neuronal or noneuronal enzyme markers was observed in brain but a significant increase in the myelin marker, CNP, and total enolase activity was seen in the optic nerve. Morphological evaluation by light microscopy indicated no discernible neuronal lesions in MMC-exposed animals. At the higher MMC dose (7.05 mg Hg/kg, po, for 7 days), there was about a 20% loss in the body weight of treated animals and partial hind limb paralysis was observed. Of all the neuronal marker enzymes examined, only TH was found to be decreased in the striatum. The proliferating astroglial marker, GS, was elevated only in the cerebellum. CNP was found to be decreased in both the optic and sciatic nerve. As in the lower dose group no pathological changes were observed at the light microscopic level in the brain of MMC-treated rats. These data suggest that of the cell specific marker enzymes studied, GS in the cerebellum and TH in the striatum may be useful biochemical markers for the neurotoxic action of MMC.

Effect of chronic exposure to hexachlorophene on rat brain cell specific marker enzymes.

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.

Direct correlation between calcium content and blood pressure: a study of aortic coarctation in rats.

The calcium content of aorta was measured by atomic absorption after coarctation in the rat. At 7 and 14 days, the calcium content was elevated on the proximal side of the coarctation, where pressure was increased significantly. On the distal, low pressure side of the aortic coarctation, calcium was reduced significantly. There is a direct correlation between the blood pressure and the content of calcium (r = 0.69, P less than 0.001). The width of the aortic media on the high pressure side was increased significantly at 7 and 14 days after coarctation, whereas no significant changes in width were present on the low pressure side of the constriction. We conclude that pressure regulates the aortic calcium content, likely acting through a local effect.

Effect of short-term exposure to hexachlorophene on rat brain cell specific marker enzymes.

Seven cell specific marker enzymes in brain and optic nerve and morphological evaluation by light microscopy were used to characterize the neurotoxicity associated with exposure of rats to hexachlorophene (HCP; 40 mg/kg/day, po, for 9 days). In vitro exposure to HCP at concentrations up to 100 microM had no direct inhibitory effect on the marker enzymes, validating their use in evaluating brain function in vivo. Rats exhibited a reduction in body weight gain, weakness, and ataxia of the hind limbs by the ninth day of HCP exposure. At 24 hr following the last day of exposure to HCP, the activities of the three neuron specific enzymes, glutamic acid decarboxylase, tyrosine hydroxylase, and choline acetyltransferase, in rat brain were unchanged from those of the vehicle-treated control group. Of the two astroglial enzyme markers measured, a small but significant increase was observed in the activity of nonneuronal enolase in the cerebellum and glutamine synthetase in the hippocampus of HCP-treated rats. The optic nerve appeared to be the most sensitive tissue in that the activity of both the astroglial marker, nonneuronal enolase, and the myelin marker, 2',3'-cyclic nucleotide phosphohydrolase, was significantly decreased following HCP exposure. This decrease in enzyme activity is consistent with the histological observations demonstrating extensive vacuolization and edema in the optic nerve after exposure to HCP.