Regional differences in gut blood flow and mucosal damage in response to ischemia and reperfusion.

Ischemia and reperfusion of the small intestine and colon in rats were produced by reversible occlusion (for 30 min and 1 or 3 h) of the superior mesenteric artery and the aorta above the inferior mesenteric artery. Despite a greater reduction of mucosal perfusion in the colon than in the small intestine with 30 min of ischemia, the depth of mucosal damage was significantly smaller in the former than in the latter. Thirty minutes of ischemia followed by 1 h of reperfusion induced an increase in polymorphonuclear leukocyte infiltration in both locations. Exacerbation of mucosal injury occurred only in the small intestine, suggesting that reperfusion injury is independent of polymorphonuclear leukocyte infiltration. Reperfusion after 1 or 3 h of ischemia did not exacerbate mucosal damage in either location. Allopurinol significantly diminished the exacerbation of injury after reperfusion in the small intestine. The protective effect of allopurinol, however, was neither associated with an improvement in perfusion nor a reduction in polymorphonuclear leukocyte infiltration. These data indicate that there is a window (30 min) of reperfusion injury in the small intestine, but there is no evidence of reperfusion injury in the colon.


Subacinar distribution of hepatocyte membrane potential response to stimulation of gluconeogenesis.

Isolated rat livers were perfused and the membrane potentials of matched periportal and pericentral hepatocytes were determined using glass microelectrodes. O2 uptake and gluconeogenesis were increased by both phenylephrine and glucagon and the extent of the increase was not affected by the direction of perfusion. With no exogenous substrate, hepatocyte membrane potentials were approximately -27 mV. No gradients were found. Substrate produced hyperpolarization in all hepatocytes, with a small but significant gradient produced. Phenylephrine-induced hyperpolarization was higher in periportal than in pericentral hepatocytes during anterograde perfusion, but reversed during retrograde perfusion. Similar effects on membrane potential were produced by phorbol myristate acetate (PMA). Glucagon hyperpolarized homogeneously during both anterograde and retrograde perfusion with no gradients across the acinus. Octanol addition during glucagon stimulation, however, resulted in heterogeneity similar to phenylephrine or PMA. Thus when hepatocytes are stimulated by substrate or hormones, the degree of hepatocyte membrane potential heterogeneity across the acinus is highly dependent on the nature of the stimulus. We propose that the differential hormone effects on hepatocyte membrane potential may be mediated at least in part by differential modulation of cell to cell communication via gap junctions.


CCK, bombesin, and carbachol stimulate c-fos, c-jun, and c-myc oncogene expression in rat pancreatic acini.

To identify possible nuclear signals mediating long-term regulation of the pancreas by gastrointestinal hormones, the expression of c-fos, c-jun, and c-myc was investigated in rat pancreatic acini. Stimulation of the acini with cholecystokinin octapeptide (CCK-8, 100 pM), bombesin (10 nM), or carbachol (10 microM), but not gastrin (100 nM), secretin (100 nM), or vasoactive intestinal peptide (10 nM) induced an increase in oncogene mRNA expression. The percent increases of c-fos, c-jun, and c-myc mRNA were 207 +/- 40, 171 +/- 26, and 46 +/- 19 (n = 5) for CCK-8; 223 +/- 71, 159 +/- 31, and 43 +/- 21 (n = 5) for bombesin; and 125 +/- 51, 123 +/- 58, and 67 +/- 19 (n = 5) for carbachol, respectively. CCK-induced increases in oncogene mRNA were rapid and transient. c-fos and c-jun mRNA levels were increased after 30 min stimulation, peaked at 1 h, and returned to basal level in 2 h. Activation of c-myc was more prolonged with levels remaining elevated for at least 3 h. The effects of CCK-8 were concentration dependent. Detectable stimulation was seen at 10 pM; maximal stimulation occurred at 10 nM and was not affected by further increase in the concentration of CCK-8. JMV-180, a high-affinity site CCK receptor agonist and low-affinity site antagonist, alone did not stimulate c-fos mRNA expression but inhibited c-fos mRNA expression induced by CCK-8. These results suggest that the interaction between CCK and the low-affinity state of the CCK receptor is responsible for oncogene activation.


Lytic effects of mixed micelles of fatty acids and bile acids.

It has been hypothesized that bile acids and fatty acids promote colon cancer. A proposed mechanism is a lytic effect of these surfactants on colonic epithelium, resulting in a compensatory proliferation of colonic cells. To investigate the first step of this hypothesis, we studied the lytic activity of fatty acids and physiological mixtures of fatty acids and bile acids. Experiments were performed in both erythrocytes and cultured Caco-2 cells, a model system for intestinal epithelium. Fatty acids with a chain length of 10 C atoms or more were lytic, and the hemolytic activity increased in the order C10:0 less than C18:0 less than C16:0 less than C12:0 less than C14:0 much less than C18:1 approximately C18:2 but was not dependent on their critical micellar concentration. Addition of a sublytic, submicellar concentration of cholate resulted in the formation of highly lytic mixed micelles. Lytic activity of these mixed micelles was closely associated with their micellar aggregation as determined in parallel incubations using a fluorescent micellar probe. With use of identical concentrations of fatty acids and mixed micelles, lysis of erythrocytes was highly correlated (r greater than 0.95) with lysis of Caco-2 cells measured by either release of the apical membrane-marker alkaline phosphatase or the cytosolic marker lactate dehydrogenase. This indicates that the cytolytic activity of these surfactants is not cell-type dependent. Addition of bile acids in concentrations corresponding with the total bile acid concentration in human fecal water resulted in an increased lytic activity of fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)


Carrier-mediated transport of tetrabromosulfonephthalein by rat liver plasma membrane vesicles.

To investigate the molecular requirements and mechanisms for the hepatic uptake of phthaleins, the transport of tetrabromosulfonephthalein (TBS) was investigated in basolateral rat liver plasma membrane vesicles. TBS uptake was electrogenic as greatly accelerated by the creation of a positive-inside membrane potential by the addition of valinomycin in the presence of an inwardly directed potassium gradient. No effect was observed when the ionophore was added in the presence of a sodium gradient. The transport occurred into an osmotic-sensitive space and was saturable with an apparent Michaelis constant of 5.32 +/- 0.56 microM and a maximal velocity of 9.23 +/- 0.25 nmol.s-1.mg protein-1 (mean +/- SD, n = 3 experiments). TBS uptake was directly related to the extra-vesicular pH, indicating the deprotonated quinoid negative-charged form of the dye as the transported species. In contrast, TBS uptake was inversely related to the intravesicular pH, suggesting that protonation inside the vesicles may act as an efficient trap in transport process. Addition of polyclonal monospecific anti-bilitranslocase antibody to liver vesicles specifically inhibited TBS uptake rate (3.27 +/- 0.17 vs. 5.82 +/- 0.61 nmol.s-1.mg protein-1, n = 3, P less than 0.001). These data indicate that TBS is electrogenically transported across the liver cell plasma membrane by bilitranslocase. They also indicate that the presence of a negative charged group on the benzenic ring of the ligand is important in accounting for the transport.


cDNA cloning and localization of a band 3-related protein from ileum.

A Cl(-)-HCO3- exchanger in the brush-border membrane mediates active Cl- absorption and regulates intracellular pH in rabbit ileum. The molecular identity of the ileal Cl(-)-HCO3- exchanger has not been established. The best-characterized plasma membrane Cl(-)-HCO3- exchanger is erythroid band 3. Structurally related proteins in nonerythroid tissues comprise an anion exchanger (AE) family. We used the polymerase chain reaction to amplify and clone a cDNA encoding an ileal band 3-related protein (B3RP) from rabbit ileal enterocytes. The composite sequence is 3,909 bp and is predicted to encode a protein of 136 kDa. The deduced amino acid sequence is 95% identical to murine renal AE2, indicating that ileal B3RP is rabbit AE2. Antisera generated against a cytoplasmic fragment of ileal B3RP recognized a 160- to 170-kDa polypeptide in the brush-border membrane, but not the basolateral membrane, of ileal crypt and villus enterocytes. This correlates with previous studies indicating that a Cl(-)-HCO3- exchange is present in brush-border but not basolateral membrane vesicles from rabbit ileal enterocytes. We conclude that ileal B3RP is a product of the AE gene family, and is present in the brush-border of ileal enterocytes, where it may mediate Cl(-)-HCO3- exchange.


Effects of nerve stimulation and zymosan on glycogenolysis in perfused livers from cold-exposed rats.

The effects of sympathetic nerve stimulation and zymosan (cell wall particles from yeast) on glycogenolysis were studied in perfused livers from rats kept for 5 and 20 days at 4 degrees C. The rate of glycogenolysis induced by nerve stimulation decreased significantly without any decrease in norepinephrine outflow during cold exposure, and the rate induced by norepinephrine did not change. By contrast, the rate of zymosan-induced glycogenolysis increased markedly during cold exposure. The rats with denervated hepatic nerves did not show the increased response to zymosan. In cold-exposed rats, both mepacrine and ibuprofen inhibited the effects of zymosan and of nerve stimulation without any inhibition of the outflow of norepinephrine. Neither inhibitor had any effect on the effects of norepinephrine. The metabolic effects of nerve stimulation and zymosan were not additive in cold-exposed rats. These results suggest that cold exposure may modulate the metabolism of arachidonic acid in Kupffer cells via hepatic nerve and decrease the eicosanoid-dependent glycogenolysis by nerve stimulation.


Development of Ca2+ homeostasis in epithelial cells from embryonic and neonatal intestine.

The fluorescent probe fura-2 was used to assay Ca2+ levels in epithelial cell suspensions from embryonic and neonatal chick duodenum. Cell preparations maintained high viability, completely hydrolyzed fura-2/AM to fura-2, retained 92% of cellular fura-2 within the cytoplasmic compartment, and gave low autofluorescence values during assay. Fura-2 leakage from loaded cells occurred at all ages, but could be corrected for in subsequent calculations of cellular Ca2+. Cytoplasmic Ca2+ concentration was 76-80 nM in cells from 14- to 16-day embryonic intestine, rose significantly to 92-98 nM at 17-20 days, and reached 209 nM at 1-day post-hatch when assayed in buffers containing 1.3 mM Ca2+. The developmental rise in cytoplasmic Ca2+ was accompanied by an enhanced ability of cells to maintain a constant Ca2+ concentration at increased levels of extracellular Ca2+ and by a highly correlated rise in alkaline phosphatase (ALP) activity. Epithelial Ca2+ subsequently decreased to the "adult" value of 133-142 nM and was constant along the crypt-villus axis of neonatal intestine. These results verify that fura-2 can be used to compare baseline cytoplasmic Ca2+ values of epithelial cells from developing intestine, reveal that significant changes in Ca2+ homeostasis occur during ontogeny, and suggest that epithelial Ca2+ may modulate ALP activity during the differentiation of embryonic enterocytes.


Sex differences in hepatic fatty acid uptake reflect a greater affinity of the transport system in females.

In this study, we examined the hypothesis that the reported sex difference in hepatic free fatty acid (FFA) uptake involves the putative FFA transport system, the plasma membrane fatty acid binding protein (FABPpm). In hepatocytes isolated from both male and female rats, initial [3H]oleate uptake velocity reflected transmembrane influx and not subsequent metabolism and was a saturable function of the unbound oleate concentration. Although Vmax values were similar (61 +/- 2 vs. 65 +/- 5 pmol.min-1.5 x 10(4) cells-1 for females and males, respectively), the apparent Km was significantly smaller in females (40 +/- 4 vs. 90 +/- 11 nM; P less than 0.05), reflecting faster influx velocities in female cells over a range of unbound oleate concentrations. The oleate efflux rate constant was also greater in females (0.280 +/- 0.014 vs. 0.198 +/- 0.020 min-1; P less than 0.05) despite their greater hepatic content of cytosolic FABP. Finally, despite the greater rates of transmembrane FFA flux in female hepatocytes, the surface expression of FABPpm was virtually identical in the two sexes (2.5 +/- 0.5 vs. 2.4 +/- 0.4 microgram/10(6) cells). Collectively, these data indicate that at FFA-to-albumin ratios occurring in vivo the plasma membrane of female hepatocytes transports oleate bidirectionally at a greater rate than that of male hepatocytes. A sex-related difference in the functional affinity of FABPpm for FFA appears the most likely explanation for the greater oleate uptake in females.


Bile salt hydrophobicity controls vesicle secretion rates and transformations in native bile.

After drainage of the bile salt pool, we infused unanesthetized bile fistula prairie dogs (Cynomys ludovicianus) intravenously with taurine-conjugated chenodeoxycholate (TCDC), cholate (TC), ursodeoxycholate (TUDC), and ursocholate (TUC) in concentrations that attained greater than 94% enrichment of biliary bile salts. With decreases in bile salt hydrophobicity, maximum steady state lecithin and to a lesser extent cholesterol secretion rates decreased in the rank order, TCDC greater than TC greater than TUDC greater than TUC. By phase analysis, TCDC-rich and TC-rich biles plotted inside their respective micellar zones, whereas TUDC-rich and TUC-rich biles plotted outside and were so-called "supersaturated" with cholesterol. Quasi-elastic light scattering and electron microscopy, when performed within 30 min of collection, revealed unilamellar vesicles in all biles. By 24 h, vesicles in TCDC-rich and TC-rich biles had dissolved into mixed micelles, whereas vesicles in TUDC-rich biles formed mixed micelles plus multilamellar liquid crystals, and vesicles in TUC-rich biles formed multilamellar liquid crystals exclusively. Because cholesterol/phospholipid molar ratios of multilamellar liquid crystals were less than or equal to 1, cholesterol monohydrate crystals did not form in these biles. We conclude that, despite drastic alterations in bile salt detergency, unilamellar vesicles are the final common pathway for lecithin and cholesterol secretion into bile. During equilibration of bile, the fate of unilamellar vesicles may be micellar, micellar plus liquid crystalline, or liquid crystalline only depending on the detergency (i.e., hydrophobic-hydrophilic balance) of the secreted bile salt.


Effects of agonists on p21ras and ras-related proteins in rat pancreatic acinar cells.

This study shows the presence of seven different low-molecular-weight GTP binding proteins (smg proteins) with molecular masses between 18 and 27 kDa in subfractions of rat pancreatic acinar cells. After stimulation of isolated intact and permeabilized pancreatic acinar cells with cholecystokinin octapeptide (CCK-OP), the diacylglycerol (DG) analogue 12-O-tetradecanoylphorbol 13-acetate (TPA), vasoactive intestinal peptide (VIP), adenosine 3',5'-cyclic monophosphate (cAMP), or guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), [alpha-32P]GTP binding to 21- to 22-kDa smg protein(s) in microsomal membranes (MM) was reduced, whereas the [alpha-32P]GTP binding to 23-kDa protein(s) was enhanced. In addition, prestimulation of permeabilized cells with GTP gamma S caused enhancement of [alpha-32P]GTP binding to a 19-kDa protein in MM [immunologically identified as the ADP-ribosylation factor (arf)]. In the presence of cytosol, direct addition of GTP gamma S to isolated MM resulted in an apparent translocation of the 19-kDa protein (arf) from the cytosol to membranes. This indicates increased association of arf with the membrane in its GTP-bound state. In CCK-OP-prestimulated acinar cells, [alpha-32P]GTP binding to plasma membrane-located 21- to 22-kDa proteins (immunologically identified as p21ras proteins) was enhanced, suggesting that there is an interrelationship between p21ras proteins and CCK receptors. Our results give evidence for a role of 19-kDa, 21- to 22-kDa, and 23-kDa smg proteins in cAMP-protein kinase A- and DG-protein kinase C-mediated stimulation of intracellular pathways in pancreatic acinar cells.


Interleukin-1 beta acts at hypothalamic sites to inhibit gastric acid secretion in rats.

It has been established that interleukin-1 beta (IL-1 beta) injected into the cerebrospinal fluid inhibits gastric acid secretion in rats. Brain sites of action of IL-1 beta were investigated in conscious rats implanted unilaterally with chronic hypothalamic cannula. Gastric acid secretion was monitored 2 h after pylorus ligation. Human recombinant IL-1 beta (10 ng) microinjected into the medial preoptic area, anterior hypothalamus, and paraventricular nucleus inhibited gastric acid secretion by 76-83%. IL-1 beta microinjected into the ventromedial hypothalamus and other hypothalamic sites outside of responsive sites had no effect. IL-1 beta inhibitory action in the medial preoptic area was dose related (0.1-10 ng), prevented by indomethacin (5 mg/kg ip), and mimicked by prostaglandin E2. These results show that IL-1 beta acts in the medial preoptic area/anterior hypothalamus and paraventricular nucleus to inhibit acid secretion in pylorus-ligated rats and that IL-1 beta action is likely to involve prostaglandin E2.


Migration of IEC-6 cells: a model for mucosal healing.

Cell migration is the principal force behind the early restitution of erosions of the mucosa of the gastrointestinal tract. Despite the importance of cell migration to healing, no attempts to study the process in culture have been reported. We have attempted to standardize conditions for migration and test the migration responses of the small intestinal epithelial crypt cell line IEC-6 in some experimental situations already well known in vivo. We found good correspondence between in culture and in vivo on the following points: 1) migration was independent of DNA synthesis; 2) DNA synthesis was not concentrated at the wound edge; and 3) inhibition of actin polymerization stopped migration altogether. In addition, the presence of an extracellular matrix maximized migration. Protein inhibitors with different modes of action inhibited cell migration to different degrees, not always commensurate with their inhibition of protein synthesis. Cell surface proteoglycans were important; hyaluronic acid had an effect, but the secretion of a migration-stimulating substance by wounded cells was equivocal. Significantly, alpha-difluoromethylornithine (DFMO), which inhibits ornithine decarboxylase and polyamine synthesis, almost totally prevented cell migration. Because DFMO also prevents healing of mucosal erosions in vivo, we believe that this model can be used, keeping in mind its spatial limitations, to study the process of cell migration involved in the early restitution of mucosal erosions.


CFU-rAM, the origin of lung macrophages, and the macrophage lineage.

Macrophage precursors and their progeny have been identified in early rat embryos with the use of a peroxidase-coupled marker, isolectin B4 of Griffonia simplicifolia (GSA I-B4). The macrophage lineage can be traced back to actively dividing GSA-positive angular cells present in the mesenchyme of late neurulas. These increase in number and establish residence successively in rudiments of the central nervous system, liver, and lungs. In organ-cultured fetal lungs they transform directly into a self-replicating population of macrophages responsive to colony-stimulating factors. The angular cell therefore can be seen as the source of lung macrophages during prenatal life. The extent to which this manner of production continues in postnatal life is unclear, but it appears that central hematopoietic tissues (bone marrow, spleen) may generate macrophages by a direct pathway from early-committed progenitors as well as indirectly through a series of intermediate stem cells. Considering the wealth of new information available from diverse studies in specialized culture environments and to a lesser extent from studies in vivo, it is time to integrate these findings into a more comprehensive theory of macrophage origin and fate than we have at present.


A novel system for the culture of human lung: lung development and the response to injury.

Existing methods of fetal lung organ culture are complicated and require special skills. With the use of a polyester-based plastic sheet, we have developed a simpler human fetal lung organ culture that is viable for 6 wk. This novel method permits the study of growth and differentiation, pulmonary surfactant secretion, and the response of human lung tissue to injury in vitro. Lung tissues, obtained from human fetuses ranging in gestational age from 14 to 18 wk, were cultured on the polyester sheet in Dulbecco's modified Eagle medium supplemented with 15% fetal calf serum and gentamicin. Microscopic study of the fetal lung before culturing revealed round epithelial tubules, lined by glycogen-rich columnar cells and a thick cellular interstitium. After 1 wk in culture, morphological examination showed the development and expansion of alveolar saccules and thinning of the interstitium; type I and II pneumocytes as well as fibroblasts and myofibroblasts were present. Lipid analysis of the tissues, 2 wk after the initiation of the culture, demonstrated a high percentage of dipalmitoyl phosphatidylcholine characteristic of pulmonary surfactant. Treatment of the organ culture with asbestos fibers induced type II cell hyperplasia, increased numbers of collagen fiber bundles within the interstitium, and the accumulation of multi-lamellated surfactant material within the alveolar lumens. We conclude that this organ culture system is suitable for studying lung growth, development, and injury in human tissue.


Role of endothelin-1 in regulating proliferation of cultured rabbit airway smooth muscle cells.

Increased expression of the potent vasoconstrictor and bronchoactive peptide, endothelin-1 (ET-1), has recently been demonstrated in airway epithelial and endothelial cells of asthmatic patients. To identify its potential role in contributing to airway smooth muscle (ASM) hyperplasia, a characteristic feature of asthmatic airways, the mitogenic action of ET-1 was investigated in cultured rabbit ASM cells. ET-1 elicited significant dose-dependent (10(-12)-10(-6) M) increases in ASM cell number, with a mean potency (i.e., -log mean effective dose) of action of 9.82-log M. ET-1 also acutely stimulated intracellular inositol 1,4,5-trisphosphate accumulation. The latter response was blocked by phospholipase C inhibition with neomycin; however, neomycin had no effect on the promitogenic action of ET-1. By contrast, the ASM cell proliferative response to ET-1 was independently inhibited by pertussis toxin, inhibitors of phospholipase A2, cyclooxygenase, and thromboxane A2 (TxA2) synthesis, as well as blockade of the TxA2 receptor. Moreover, in complementary studies, we found that administration of the stable TxA2 mimetics, carbocyclic TxA2 (CTA2) and U-46619, induced ASM cell proliferation and that ET-1 evoked the release of endogenous TxA2 from the ASM cells. Collectively, these observations provide new evidence that 1) ET-1 is a potent mitogen of ASM cells, 2) the promitogenic effect of ET-1 is associated with activation of a pertussis toxin-sensitive G protein coupled to stimulation of phospholipase A2, and 3) the latter mediates ASM cell proliferation via the release and autocrine mitogenic action of TxA2. The findings support a potential role for ET-1 in mediating the characteristic hyperplasia of ASM in asthma.


Binding and uptake of surfactant protein B by alveolar type II cells.

Surfactant protein B (SP-B, mol wt 9,000, reduced) is a low-molecular-weight hydrophobic protein found in organic extracts of lung surfactant. The interaction of iodinated bovine SP-B (125I-SP-B) and isolated rat alveolar type II cells was examined. The association of SP-B with the lung cells was time and temperature dependent; type II cells exhibited time-dependent binding (at 4 degrees C) and uptake (at 37 degrees C) of SP-B. Binding of phospholipid-poor 125I-SP-B was linearly related to the external SP-B concentration from 0.25 to 60 microgram/ml and was not inhibited by a 60-fold excess of unlabeled SP-B. However, the binding of 125I-SP-B reconstituted with bovine surfactant or with phospholipid-containing liposomes occurred through a high-affinity, saturable process and could be inhibited with unlabeled SP-B. By Scatchard analysis, half-maximum binding in the presence of surfactant occurred at 3.1 +/- 0.7 micrograms SP-B/ml. Saturable binding of SP-B reconstituted with surfactant also occurred with other cell types. The results indicate that SP-B was bound and internalized by type II cells. The apparent lack of specificity in the absence of phospholipid may have been due to the self-association of SP-B. The reconstitution of SP-B with phospholipid altered the binding of phospholipid-poor SP-B from a nonspecific process to a high-affinity process consistent with a cell surface binding site.


Kinetics of pulmonary superoxide dismutase in interleukin-1-induced oxygen-tolerant rats.

We have previously demonstrated that tracheal insufflation of interleukin-1 alpha (IL-1) enhances pulmonary Mn superoxide dismutase (Mn SOD) activity and protects rats against O2 toxicity (M. F. Tsan, C. Y. Lee, and J. E. White. J. Appl. Physiol. 71: 688-697, 1991). In this study, we investigated the kinetics of mRNA, specific (immunoreactive) proteins, and enzyme activities of pulmonary Mn SOD and Cu,Zn SOD in IL-1-induced O2-tolerant rats. At 1 day after IL-1 (5 micrograms) insufflation and O2 exposure, levels of Mn SOD mRNA and specific protein, but not enzyme activity, were markedly elevated. At 2.3 and 7 days after O2 exposure, levels of Mn SOD mRNA, specific protein, and enzyme activity were all increased in IL-1-treated animals. In contrast, in control rats at 2.3 days after O2 exposure, level of Mn SOD mRNA was markedly elevated, whereas levels of specific protein and enzyme activity were decreased. Levels of pulmonary Cu,Zn SOD mRNA, specific protein, and enzyme activity were unchanged in control and IL-1-treated rats, except that in IL-1-induced long-term O2-tolerant rats (7 days after O2 exposure), they were all increased. Since at 7 days after IL-1 insufflation, normoxia-exposed rats did not show increased levels of pulmonary Mn SOD or Cu,Zn SOD mRNA, the increased levels of pulmonary SOD seen in IL-1-induced long-term O2-tolerant rats are, at least in part, due to the effect of O2 exposure.


Alveolar type II cells synthesize hydrophobic cell-associated proteoglycans with multiple core proteins.

Type II alveolar epithelial cells interact with the extracellular matrix via cell surface receptors for matrix ligands. Cell surface proteoglycans, which are hydrophobic due to their membrane insertion domains, are one of several classes of molecules that may be receptors for matrix ligands. To analyze the hydrophobic proteoglycans synthesized by adult alveolar type II cells, we labeled these cells with 35SO4 and [3H]leucine in short-term primary cultures. Cell-associated hydrophobic proteoglycans and culture medium-derived proteoglycans were purified and characterized. Both the hydrophobic proteoglycans and medium-derived proteoglycans, which were not hydrophobic, had mainly heparan sulfate glycosaminoglycans. Analysis of core proteins of the hydrophobic proteoglycans showed three proteins, 47, 65, and 90 kDa. The 47- and 65-kDa core proteins were substituted only with heparan sulfate chains. The 90-kDa core protein was seen only after digestion with both heparitinase and chondroitin ABC lyase, suggesting it was a hybrid having both heparan sulfate and chondroitin-dermatan sulfate chains. These findings were confirmed by iodination of the core proteins. The hydrophobic cell-associated proteoglycans inserted into artificial liposomes, whereas the medium-derived molecules did not. These data document heterogeneity in core protein and glycosaminoglycan chains among hydrophobic proteoglycans synthesized in vitro by adult alveolar type II cells. These molecules may have diverse functions in regulating type II cell interaction with the extracellular matrix.


Effect of food restriction on hyperoxia-induced lung injury in preterm guinea pig.

Undernutrition may exacerbate hyperoxia-induced lung injury, a finding that may be of significance in the early clinical management of the premature human infant. Addressing this specific problem, we found that 72 h of food restriction in guinea pig pups delivered 3 days preterm increased mortality rates among pups exposed to 95% oxygen (8/18) and yet had no effect on 21% oxygen (air)-exposed pups (0/10). Reduced tolerance of hyperoxic conditions was not, however, associated with increased lung injury, assessed as pulmonary microvascular leakage. Pulmonary antioxidant enzyme activities [Cu,Zn superoxide dismutase (SOD), Mn SOD, glutathione peroxidase, and catalase] were unaltered by starvation or hyperoxia. Lung glutathione concentration was slightly decreased after food restriction, whereas hyperoxic exposure did not change either lung or bronchoalveolar lavage fluid glutathione concentrations or lung antioxidant enzyme activities. Increased susceptibility to the lethal effects of oxygen in the starved preterm guinea pig pup could not be attributed to a deficiency of pulmonary antioxidant defenses.


Stimulation of fetal rat lung cell proliferation in vitro by mechanical stretch.

On the basis of in vivo animal studies and on experiments of nature, it has been suggested that fetal breathing movements are essential for normal lung growth in utero. To study this hypothesis in vitro, we examined the effect of mechanical stretch on proliferation of fetal rat lung cells maintained in organotypic culture to provide a three-dimensional matrix. Initial studies demonstrated that stretch-mediated effects on cell division and DNA synthesis in such cultures were influenced by cell inoculation density, fetal calf serum concentration, and by the amplitude, frequency, periodicity, and duration of the applied stretch. After a 48-h exposure to an intermittent stretch pattern (5% elongation, 60 stretches/min for 15 min of each hour), cell number increased 10% (P less than 0.05), cell doubling time was reduced from 71 to 55 h (P less than 0.05), [3H]thymidine incorporation into DNA increased 61% (P less than 0.01), and the [3H]thymidine-labeling index increased 2.8-fold (P less than 0.001) compared with nonstretched controls. This effect did not appear to be mediated by prostaglandins or leukotrienes because the prostaglandin synthase inhibitors ibuprofen (2.5-50 microM) or BW 755C (5 microM), leukotriene biosynthesis inhibitors BW 755C (5 microM) or MK-886 (0.3 microM), and leukotriene D4 receptor antagonist MK-571 (0.3 microM) did not block stretch-mediated effects. We conclude that mechanical forces act directly to stimulate fetal rat lung cell growth and that these results are compatible with a significant role for fetal breathing in normal fetal lung growth.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of hypoxia and other vasoactive agents on pulmonary and cerebral artery smooth muscle cells.

Smooth muscle cells (SMC) were isolated from cat cerebral arteries and three sizes of pulmonary artery (less than 200-, 200- to 600-, and greater than 800-microns diameter) and used within 72-96 h. Change in cell length in response to hypoxia and other vasoactive agents was measured in a specially constructed cell chamber on an inverted microscope. Pulmonary artery SMC responded to hypoxia differently according to artery size. SMC from less than 200- and 200- to 600-microns-diameter pulmonary arteries shortened 18.6 +/- 4.85 and 24.2 +/- 2.70%, respectively. However, SMC from greater than 800-microns-diameter pulmonary arteries shortened 0.81 +/- 0.44%. Cerebral artery SMC plated on a flexible polydimethyl siloxane membrane showed loss of tension during exposure to hypoxia. The shortening of SMC from the 200- to 600-microns pulmonary arteries was accompanied by myosin phosphorylation. SMC from greater than 800-microns-diameter pulmonary arteries and cerebral arteries contained myosin that did not phosphorylate during hypoxia. The SMC from both artery types responded to norepinephrine, serotonin, prostaglandin F2 alpha and indomethacin and exhibited alpha-adrenergic receptor population patterns similar to those of intact arteries. The pattern of hypoxic responses exhibited by these nondedifferentiated pulmonary and cerebral artery SMC supports the idea that, at least in the cat, the hypoxic sensor is located within the SMC.


Cell volume regulation in rat thin ascending limb of Henle's loop.

Thin ascending limb cells of Henle's loop from Wistar rats were studied with in vitro microperfusion and video-optical techniques to investigate their ability in regulating cell volume during osmotic shock and to identify mechanisms of ion transport involved in the process. These cells showed a clear volume regulatory decrease (VRD) response in hyposmotic medium, but no volume regulatory increase in hyperosmotic medium. The presence of barium in the bath abolished VRD. Removal of K+ from bath and perfusate also inhibited the VRD response. Reintroduction of K+ in hyposmotic conditions reestablished cell volume regulation. Introduction of anthracene-9-COOH to the basolateral medium blocked cell volume regulatory response. Cl- removal from perfusate and bath solutions also inhibited VRD, probably because of a significant intracellular Cl- depletion. Exposure of cells to ethylene glycol-bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid in perfusate and bath solutions reduced significantly Ca2+ concentration and impaired VRD. Reintroduction of Ca2+ in hyposmotic conditions restored volume regulation. The presence of ouabain in basolateral medium also inhibited VRD. These data suggest that the following mechanisms in the basolateral membrane are involved in VRD response: K+ and Cl- conductive pathways, which might be Ca2+ dependent for activation, and an Na(+)-K(+)-adenosinetriphosphatase.


Colocalization and release of angiotensin and renin in renal cortical cells.

Angiotensin is generated within the kidney, but the precise loci for the formation of angiotensin I (ANG I) and angiotensin II (ANG II) have not been demonstrated. We performed electron microscopy immunocytochemistry in kidney sections of 10-day-old (newborn) and adult Wistar-Kyoto (WKY) rats using specific antibodies to renin, ANG I, ANG II, and angiotensinogen (AO). Renin, ANG I, ANG II, and AO were present in juxtaglomerular (JG) cells. Renin was largely confined to cytoplasmic granules; ANG I and ANG II were colocalized to these granules but also were present in the cytoplasm; AO was distributed throughout the cytoplasm. AO also was present in a renal cortical distribution in proximal tubular cells. Northern blot analysis demonstrated AO mRNA in total kidney and liver but not in renal microvessels. Using the reverse hemolytic plaque assay, we demonstrated release of ANG I and renin from individual renocortical cells of adult WKY rats. Under control conditions, the number of releasing cells was 11 +/- 1 for ANG I and 10 +/- 1 for renin. Addition of rat renin inhibitor (RI) (1 x 10(-5) M), which inhibited renin activity in the medium from 37 to 9 pg ANG I.ml-1.h-1, did not alter ANG I plaque number. Addition of rat AO increased ANG I plaque number to 17 +/- 2 (P less than 0.05). Incubation with both RI and AO prevented the increase in ANG I plaque number obtained with AO alone. Enalapril treatment (7 days; n = 5) increased the number of plaque-forming cells to 22 +/- 2 for ANG I (P less than 0.0005) and to 39 +/- 7 for renin (P less than 0.001). The results suggest an intracellular location for AO and angiotensin and release of renin and ANG I by renal cortical cells and suggest that released angiotensin is produced intracellularly and that secretion of ANG I is augmented by converting enzyme inhibition.


Extracellular ATP stimulates proliferation of cultured mesangial cells via P2-purinergic receptors.

We examined the role of the platelet product ATP in regulating replication and secretory activity of cultured rat mesangial cells (MCs). Extracellular ATP (25-100 microM) significantly increased [3H]thymidine uptake of growth-arrested MCs 2.1-fold; cell counts increased by 35.1%. Addition of ATP to MCs in combination with other platelet products, such as platelet-derived growth factor, isoform BB (100 ng/ml), and serotonin (1 microM), resulted in strong synergistic mitogenicity (up to 45.6-fold over control). As immediate signaling events following stimulation with ATP, we found increased production of inositol phosphates (3.2-fold increase for inositol bisphosphate and 1.6-fold increase for inositol trisphosphate by 30 s) and release of prostaglandin E2 (PGE2, 9.2-fold increase by 5 min). When we studied the rank order of potency of various ATP analogues for the production of inositol phosphates and PGE2, ATP, UTP, and adenosine 5'-O-(3-thio)triphosphate (ATP gamma S) were the most potent agonists. Although ATP and ATP gamma S were also strong mitogens, UTP was not. Additional inhibitor studies indicated that protein kinase C or cyclooxygenase products were not involved in the mitogenic effects of ATP. In summary, the major platelet product ATP is a potent comitogen for cultured MCs and strongly synergizes with other growth factors. The experiments with ATP analogues point to different receptors mediating mitogenesis, generation of inositol phosphates, and PGE2 production. The precise mechanism of the mitogenic action of ATP on MCs remains to be characterized.


PAH/alpha-KG countertransport stimulates PAH uptake and net secretion in isolated rabbit renal tubules.

Possible stimulation of both basolateral uptake and net transepithelial secretion of p-aminohippurate (PAH) by PAH/alpha-ketoglutarate (alpha-KG) countertransport was examined in intact perfused and nonperfused rabbit proximal S2 renal tubules. Preloading tubules with alpha-KG (100 microM) for 30 min increased [14C]-PAH rate of uptake by nonperfused tubules and rate of net secretion by perfused tubules approximately three- to sixfold. During stimulation of net secretion, intracellular [14C]PAH concentration increased to about the same extent as net secretion. Presence of Li+ (2 mM) or absence of Na+ (inhibitors of Na(+)-dicarboxylate cotransport) in bathing medium during alpha-KG preloading eliminated stimulation of PAH transport. Addition of unlabeled alpha-KG (1 mM) to bathing medium stimulated efflux of [14C]PAH across the basolateral membrane of tubules with oil-filled lumina, further supporting the concept of PAH/alpha-KG countertransport. Preloading with succinate (100 microM) also stimulated rates of [14C]PAH uptake by nonperfused tubules and net secretion by perfused tubules, but stimulation was only approximately 1.5-fold. Moreover, preloading with methyl succinate, a slowly metabolized derivative of succinate, did not stimulate [14C]PAH uptake by nonperfused tubules or net secretion by perfused tubules. Thus it seems most likely that succinate preloading stimulates PAH transport via metabolism, possibly by conversion to alpha-KG, not by direct countertransport for PAH. This study indicates for the first time in intact mammalian proximal S2 renal tubules that PAH/alpha-KG countertransport can stimulate net PAH secretion by generating an increased intracellular PAH concentration.


Basolateral Na(+)-independent Cl(-)-HCO3- exchange in primary cultures of rat IMCD cells.

The role of anion exchange in the regulation of intracellular pH (pHi) under base load and steady-state conditions was investigated in confluent monolayers of rat inner medullary collecting duct (IMCD) cells in primary culture using the pH-sensitive fluoroprobe 2,7-bis(carboxyethyl)-5(6')-carboxyfluorescein (BCECF). Recovery of pHi after imposition of a base load induced either by replacement of HCO3-/CO2 by N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) at the same extracellular pH (pHo) or deletion of Cl- from a HCO3-/CO2-buffered solution had an absolute requirement for Cl-, was Na+ independent, and was inhibited approximately 90% by 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). When pHo was decreased by lowering HCO3- concentration in the constant presence of 5% CO2, the rate of decrement in pHi was significantly blunted in the absence of Cl-. Imposition of a positive or negative diffusion potential of equal but opposite magnitude did not modify the anion exchange rate, confirming the electroneutrality of the process. Under steady-state conditions, pHi of cells bathed in a HCO3-/CO2-buffered solution was 7.33 +/- 0.06, significantly lower than that of cells bathed in a nominally HCO3-/CO2-free buffer (7.50 +/- 0.04), indicating that under physiological conditions the pathway functions as a base extruder. In studies performed on cells grown on permeable supports, the anion exchange pathway was found to be confined exclusively to the basolateral-equivalent cell surface. In summary, confluent monolayers of rat IMCD cells in primary culture possess a Na(+)-independent, DIDS-inhibitable electroneutral Cl(-)-HCO3- exchange pathway that is confined to the basolateral cell surface. The transporter is an important determinant of steady-state pHi and is the predominant mechanism whereby the cell recovers from imposed elevations in pHi.


Angiotensin II receptor subtypes in cultured rat renal mesangial cells.

The selective angiotensin (ANG II) antagonists losartan (DuP 753) and PD 123319 have been shown to bind selectively to AT1 and AT2 subtypes, respectively. To characterize ANG II receptor subtypes in mesangial cells, washed membranes were incubated with 0.1 to 0.5 nM 125I-ANG II and increasing concentrations of competitors. The inhibition of 125I-ANG II binding by losartan and PD 123319 was biphasic, and LIGAND curve-fitting analysis revealed two populations of specific binding sites. One subpopulation comprised 86% of the total and showed high affinity for ANG II and losartan, but low affinity for the AT2 antagonists PD 123319 and CGP42112A, and thus appear identical to the recently cloned AT1 subtype. The remaining 14% of the sites showed nearly 100-fold lower affinity for losartan and 10,000-fold higher affinity for PD 123319 relative to AT1 sites. However, another AT2-selective antagonist, CGP42112A, showed little affinity for these sites. Both classes of binding sites were inhibited by guanosine 5'-O-(3-thiophosphate) and pertussis toxin treatment. We propose that there are two distinct G protein-coupled ANG II receptor subtypes (AT1A and AT1B) present in renal mesangial cells.


In vitro perfusion of chinchilla thin limb segments: segmentation and osmotic water permeability.

The thin limb segments of the long loop of Henle are thought to play important roles in the urinary concentrating mechanism. In this study, we present new approaches to the identification, dissection, and in vitro perfusion of individual thin limb segments from all levels of the chinchilla renal medulla, including the deepest portions of the papilla. We have applied these techniques to the investigation of the osmotic water permeability along the chinchilla long loop of Henle. The results demonstrate that the osmotic water permeability of the thin descending limb is not uniformly high along its length, as previously thought, but that the distal 20% of the long-loop descending limb has a very low water permeability (approximately 50 microns/s). The transition to the low water permeability region of the thin descending limb is accompanied by a relatively abrupt change in morphology (increased cellularity and decreased diameter) that is readily perceptible in the perfused segments and even in the dissection dish. In contrast, the upper part of the chinchilla long-loop thin descending limb had an extremely high osmotic water permeability (greater than 2,000 microns/s) as observed in other species. Thin ascending limbs from deep in the inner medulla had water permeabilities that were indistinguishable from zero, as previously found in thin ascending limbs from near the inner-outer medullary junction. The presence of a low-water-permeability portion of the long-loop thin descending limb in chinchilla may have important implications with regard to the inner medullary concentrating process. A relatively low osmotic water permeability (397 microns/s) was also found in the deep inner medullary portion of the thin descending limb from the rat.


Dietary protein modulates intrarenal distribution of renin and its mRNA during development.

To determine whether high protein feeding throughout development affects renal growth, renal hemodynamics, and the intrarenal distribution of renin and its mRNA in the adult animal, male Wistar rats were fed diets containing either 20% protein [normal (NP), n = 12] or 40% protein [high (HP), n = 12] from weaning until studied at 6 or 12 wk of age. Kidney weight, kidney weight-to-body weight ratio, cortical DNA content, and cortical protein-to-DNA ratio were higher in HP- than in NP-fed rats at 6 and 12 wk of age. Somatic and kidney growth response to HP was blunted by angiotensin II type 1 receptor antagonist Dup 753. Glomerular filtration rate and renal plasma flow were higher in HP- than in NP-fed rats at 6 and 12 wk of age. The intrarenal distribution of renin and renin mRNA, assessed by immunocytochemistry and in situ hybridization, respectively, were markedly different between the two groups. In NP-fed rats, renin and renin mRNA were confined to a juxtaglomerular location. In HP-fed rats, renin and its mRNA extended proximally along the afferent arterioles. The percentage of visible afferent arteriolar length containing renin or renin mRNA was higher in HP-fed rats (60 +/- 3.2 and 61 +/- 3.9%, respectively) than in NP-fed rats (39 +/- 2.5 and 33 +/- 0.6%; P less than 0.05). Also, the percentage of juxtaglomerular apparatuses (JGAs) containing renin or renin mRNA was higher in HP-fed rats (80 +/- 1.6 and 72 +/- 2%, respectively), than in NP-fed rats (46 +/- 2.2 and 40 +/- 4%; P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)


Renal hemodynamic actions of lipoxins in rats: a comparative physiological study.

Interactions between either the arachidonate 5-lipoxygenase (5-LO) and 15-LO or 5-LO and 12-LO can lead to the formation of lipoxins. Although the functional significance of lipoxygenase products of arachidonic acid has been recognized increasingly in glomerular inflammation, less is known regarding the specific effects of lipoxins on renal hemodynamics. Here, we examine the renal actions of lipoxin (LX) A4, LXB4, and, the recently identified 7-cis-11-trans-LXA4, which were administered into the renal artery of the euvolemic male Munich-Wistar rat. LXA4 caused increases in renal plasma flow (RPF) and glomerular filtration rate (GFR) in a dose-dependent manner. These effects were reversed during cyclooxygenase inhibition. LXB4 decreased both RPF and GFR, and its mode of action was independent of cyclooxygenase activity. 7-cis-11-trans-LXA4 decreased RPF and GFR, which effects were abolished during systemic infusion of the leukotriene D4 receptor antagonist SKF 104353. Results indicate that each of these lipoxins displays distinct hemodynamic effects via a specific mode of action on the renal vasculature of rats. Moreover, they suggest mechanisms whereby lipoxins may participate in the changes of renal hemodynamics that occur during glomerular inflammatory processes.


Potassium conductance regulation by pH during volume regulation in rabbit proximal convoluted tubules.

When rabbit proximal convoluted tubules were microperfused in the presence of bicarbonate, a 90 mosmol hypotonic shock hyperpolarized the basolateral membrane by 5.5 +/- 1.4 mV, increased basolateral potassium selectivity (tK) from 0.30 +/- 0.02 to 0.45 +/- 0.02, and reduced the basolateral membrane resistance from 4,887 +/- 821 to 2,836 +/- 602 omega.cm. These data show that the hypotonic shock increased absolute basolateral potassium conductance. The same hypotonic shock elevated intracellular pH from 7.18 +/- 0.04 to 7.31 +/- 0.04. When bath pH was increased by 0.2 pH units (by reduction of CO2), intracellular pH rose by 0.13 +/- 0.01. In separate experiments this maneuver hyperpolarized the basolateral membrane by 5.0 +/- 0.8 mV and augmented basolateral tK from 0.58 +/- 0.06 to 0.68 +/- 0.04, suggesting that the basolateral potassium conductance is sensitive to pH changes of a magnitude similar to that evoked by a hypotonic shock. In the nominal absence of bicarbonate or presence of 0.5 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) in the bath, the hypotonic shock caused a transient intracellular acidification, suggesting involvement of basolateral bicarbonate transport in the hypotonic shock-induced alkalinization. In the absence of bicarbonate, the hypotonic shock did not increase basolateral tK or induce hyperpolarization of the basolateral membrane. We conclude that the increase in potassium conductance observed during hypotonic shock is at least partly mediated by a bicarbonate-dependent, SITS-sensitive intracellular alkalinization.


Cloning of a human kidney cDNA with similarity to the sodium-glucose cotransporter.

We have used low-stringency screening with the human intestinal Na(+)-glucose cotransporter SGLT1 to isolate a 2,271-nucleotide cDNA (Hu14) from human kidney. This clone, which encodes a 672-residue protein, is 59% identical at the amino acid level to SGLT1 and has a similar number and arrangement of predicted membrane-spanning regions. It also shares significant sequence identity with other Na(+)-coupled transporters. Northern blot analysis suggests strong expression of Hu14 in kidney, but, unlike SGLT1, no significant expression in intestine. We have been unable to demonstrate definitive transport of any of a number of substrates (including amino acids, sugars, nucleosides, and vitamins) into Hu14 cRNA-injected Xenopus oocytes, although sequence conservation makes it likely that Hu14 represents another member of the Na+ cotransporter family, possibly a second Na(+)-glucose cotransporter.


Effects of reactive oxygen species on cultured rat mesangial cells and isolated rat glomeruli.

The effects of reactive oxygen species (ROS) on cultured rat mesangial cells were studied by measuring planar cell surface area (PCSA) after incubation with xanthine plus xanthine oxidase (XXO), in the presence of superoxide dismutase (SOD; 5 micrograms/ml) or catalase (CAT; 20 micrograms/ml), or after incubation with H2O2. Myosin light chain (MLC) phosphorylation was assessed in cells prelabeled with o-[32P]phosphoric acid and incubated with H2O2, after protein separation with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A possible intermediate role for platelet-activating factor (PAF) was analyzed by preincubation of the cells with a PAF antagonist BN 52021 (BN, 5 x 10(-5) M) and by measuring PAF-specific [3H]acetate incorporation and immunoassayable PAF. XXO significantly decreased PCSA (14%), an effect abolished by CAT but not by SOD. H2O2 induced a similar effect, in a dose-dependent and time-dependent manner. MLC phosphorylation increased by 81 +/- 15% after H2O2 incubation, and this effect was blocked by BN. BN also completely blocked the effect of H2O2 on PCSA. PAF-specific [3H]acetate incorporation increased in the presence of H2O2 (from 6,886 +/- 2,030 to 58,703 +/- 16,063 counts.min-1.mg-1) as well as the immunoassayable PAF production by cells (from 0.90 +/- 0.19 to 6.71 +/- 2.27 ng/mg). These results suggest that ROS, particularly H2O2, could modulate the surface area of mesangial cells, modifying the ultrafiltration coefficient, thus explaining the decrease in glomerular filtration rate in those pathological situations characterized by an increased ROS synthesis. PAF could be involved in the genesis of these effects.


Feedback modulation of renal and hepatic erythropoietin mRNA in response to graded anemia and hypoxia.

Erythropoietin (EPO) mRNA levels were measured by ribonuclease (RNase) protection in organs from unstimulated rats and from animals after normobaric hypoxia or hemorrhagic anemia. Both liver and kidney responded to stimulation with large increases in EPO mRNA, but the response characteristic to graded stimulation was different. The liver responded poorly to mild normobaric hypoxia, accounting for only 2 +/- 1% of total EPO mRNA at 11% O2, but hepatic EPO mRNA levels increased steeply with more severe hypoxia so that at 7.5% O2 the liver contributed to 33 +/- 7% of the total. After hemorrhagic anemia, the liver also responded more strongly to more severe stimulation, but at all points it accounted for a significant proportion of total EPO mRNA, contributing 18 +/- 6% after removal of 2.5 ml (hematocrit 37.2 +/- 1.3%), increasing to 37 +/- 14% after venesection of 10.5 ml (hematocrit 15.8 +/- 0.8%). Studies of EPO mRNA in other organs confirmed that EPO production outside the liver and kidney were quantitatively insignificant in stimulated animals. However, the hypoxia-induced increases in EPO mRNA in brain, testis, and spleen suggest the existence of an oxygen-sensing mechanism at other sites.


Effects of adenosine on ion transport in rat medullary thick ascending limb.

Previously, we demonstrated that adenosine (Ado) was released by the medullary thick ascending limb (MTAL) during hypoxia. The present experiments were designed to examine the effects of Ado and adenosine analogues on net chloride (JCl) and bicarbonate (JHCO3) absorption by the isolated, perfused MTAL of the rat. Ado, 10 nM, in the presence or absence of arginine vasopressin (AVP, 10(-10) M) reduced JCl by 50%. The inhibition of Ado was reproduced with the selective A1 agonist, N-6-phenylisopropyladenine (2 nM), and was reversed by 8-cyclopentyl-1,3-dipropylxanthine, an A1-receptor antagonist. Thus the inhibition of JCl is likely mediated through A1 receptors. In contrast, Ado had no effect on (JHCO3) either in the presence or absence of AVP. Ado also had no influence on the effect of AVP to inhibit JHCO3. The lack of effect on JHCO3 suggests that the inhibition of JCl by Ado is unlikely to be mediated through changes in cellular adenosine 3',5'-cyclic monophosphate. These results support the hypothesis that Ado released into the renal medulla during hypoxia may protect the MTAL from ischemic injury by directly inhibiting NaCl absorption and reducing transport-related oxygen consumption.


Renal injury in obese Zucker rats: glomerular hemodynamic alterations and effects of enalapril.

Angiotensin-converting enzyme inhibitors may ameliorate experimental glomerular injury by either hemodynamic or nonhemodynamic mechanisms. In a long-term study, we examined the effects of 30 wk of enalapril treatment on the development of glomerular disease in obese Zucker rats (OZR). Enalapril significantly (P less than 0.05) lowered blood pressure, fasting serum cholesterol, and urine albumin excretion in OZR throughout the experimental period. At 38 wk of age, enalapril-treated OZR had a sixfold reduction in the percent glomeruli exhibiting focal glomerulosclerosis and a 20-30% reduction in kidney weight and glomerular area. A separate micropuncture study in 22- to 26-wk-old rats revealed that untreated OZR with albuminuria and increased blood pressure had elevated glomerular capillary pressure (Pgc). Enalapril-treated OZR had less albuminuria and lower blood pressure, but Pgc was not reduced. The value of the transcapillary hydraulic pressure difference (delta P) in enalapril-treated OZR was intermediate between values in untreated OZR and lean Zucker rats. Thus enalapril markedly attenuated the development of glomerular injury in OZR. The salutary effects of enalapril may have involved a reduction in delta P coupled to a nonhemodynamic action, possibly restriction of glomerular growth or lowering of serum cholesterol.


Critical role of bicarbonate in calcium release from bone.

Calcium release from cultured bone is pH dependent; net calcium flux (JCa) from bone increases with decreasing pH. At a similar decrement in pH there is greater JCa when acidosis is produced by a low medium bicarbonate concentration ([HCO3-]), a model of metabolic acidosis (Met), compared with an increased medium PCO2, a model of respiratory acidosis (Resp). To separate the role of [HCO3-] from that of pH in inducing JCa we cultured calvariae for 3 h under three different neutral (pH approximately 7.4) isohydric environments [control (Ctl), fully compensated Met (C-Met), or fully compensated Resp (C-Resp)] and two different acid (pH approximately 7.1) isohydric environments (Met or Resp). During neutral pH (Ctl, C-Met, and C-Resp) there was JCa from bone during C-Met (decreased [HCO3-]), no net flux during Ctl (normal [HCO3-]), and JCa into bone during C-Resp (increased [HCO3-]); and JCa was correlated inversely with [HCO3-] (r = -0.824, n = 36, P less than 0.001). During acid pH there was greater JCa from bone during Met (decreased [HCO3-]) than during Resp (normal [HCO3-]); and JCa was again correlated inversely with [HCO3-] (r = -0.848, n = 22, P less than 0.001). JCa from bone during Met and Resp was greater than C-Met and C-Resp, respectively. The addition of the osteoclastic inhibitor salmon calcitonin did not alter the relative JCa results. Thus at a constant pH the magnitude of JCa from cultured neonatal mouse calvariae appears dependent on the [HCO3-]; the lower the [HCO3-], the greater the calcium efflux.(ABSTRACT TRUNCATED AT 250 WORDS)


Induction of water diuresis by endothelin in rats.

Experiments were performed in anesthetized rats to examine the possibility that endothelin (ET) modifies renal epithelial function in addition to its well-established hemodynamic actions. Infusion of ET-3 at rates between 34 and 178 ng.kg-1.min-1 was in many cases followed by a rise in urine flow and a persistent decrease in urine osmolality, whereas glomerular filtration rate (GFR) did not significantly change. The extent of ET-induced diuresis was dependent on the response of GFR: in rats in which ET-3 infusion caused a marked reduction of GFR (greater than 70%) ET-induced diuresis was not seen, even though urine osmolality still fell significantly. From animal to animal, ET-induced changes of urine flow or GFR did not correlate significantly with the rate of ET-3 infusion. ET-1, another ET isopeptide, also produced water diuresis when administered in GFR-neutral doses. Urinary excretion of total solutes and of sodium was not significantly altered by ET-3. Infusion of vasopressin blunted the diuretic effect of ET-3, whereas ET-3-induced water diuresis was not measurably altered by chronic or acute treatment with a converting enzyme inhibitor or by acute inhibition of prostaglandin synthesis. Induction of water diuresis was not secondary to an inhibition of vasopressin secretion since it could be demonstrated in homozygous Brattleboro rats in which antidiuresis was produced by the infusion of vasopressin at a rate of 200 microU.kg-1.min-1. These data suggest that ET may be an inhibitory modulator of the hydrosmotic action of vasopressin at the level of the renal collecting duct.


Enhanced intrarenal angiotensin II generation in response to obstruction of the pig ureter.

The effect of acute unilateral complete ureteral obstruction (UUO) on renal hemodynamics has been examined in a pig model. Pigs were operated on under general anesthesia, electromagnetic flow probes were inserted around renal arteries, and catheters were implanted in renal veins and aorta for blood sampling. The influence of 15 h of obstruction on the physiological variation in immunoreactive angiotensin II (ANG II) was measured together with detailed analysis of the renal handling of ANG II in anesthetized animals. Pelvic pressure rose significantly within 2 h to a maximum of 59.9 +/- 2.9 cmH2O (n = 10). The ipsilateral renal blood flow (RBF) was reduced by 30% from 319 +/- 35 to 241 +/- 42 ml/min (P = 0.001), whereas contralateral RBF did not change. A temporary significant increase in mean aortic blood pressure was seen during the first 2 h after UUO. Renal vascular resistance increased by 44% in the obstructed kidney from 37 +/- 3 to 55 +/- 9 mmHg.ml-1.min.g during the course of obstruction. Concomitantly, the plasma concentration of immunoreactive ANG II increased from all three sample sites by 8-15 times. This increase was highly significant compared with the sham-operated group. The results show that the pig kidney responds to UUO by a net secretion of ANG II from the ipsilateral kidney. Our observations on the ANG II renal secretion rate suggest that part of the immunoreactive ANG II in the renal venous effluent originated de novo from intrarenal generation.


Effect of cyclooxygenase inhibition on renal blood flow autoregulation in SHR.

The effect of acute and chronic indomethacin treatment on renal blood flow (RBF) autoregulation was studied in 10- and 40-wk-old spontaneously hypertensive rats (SHR). RBF autoregulation was substantially reduced in 40-wk-old SHR both during acute and chronic indomethacin treatment, whereas no effect was seen in the young SHR. The pressure range of autoregulation was 169 +/- 9 to 130 +/- 5 mmHg in the untreated 40-wk-old SHR, and 154 +/- 14 to 146 +/- 6 mmHg in indomethacin-treated 40-wk-old SHR (P less than 0.001). Indomethacin treatment had no effect on control RBF, mean arterial pressure, or renal vascular resistance in the 40-wk-old SHR. After removal of the renal nerves, RBF autoregulation during indomethacin treatment was restored in 40-wk-old SHR. The pressure range of RBF autoregulation was 158 +/- 7 to 142 +/- 7 mmHg in sham-operated animals, significantly different from the denervated 40-wk-old SHR, where RBF was autoregulated from 150 +/- 5 to 118 +/- 6 mmHg (P less than 0.01) during indomethacin treatment. The afferent arteriolar diameter (DAA) was studied by the microsphere method in 10-wk-old SHR and in untreated and indomethacin-treated 40-wk-old SHR. DAA was significantly greater in 40-wk-old compared with 10-wk-old SHR (22.1 +/- 0.4 vs. 17.9 +/- 0.5 microns) (P less than 0.01), whereas indomethacin treatment in 40-wk-old SHR did not influence the DAA significantly (21.5 +/- 0.3 microns, P greater than 0.10).(ABSTRACT TRUNCATED AT 250 WORDS)


Beta 2-microglobulin induces calcium efflux from cultured neonatal mouse calvariae.

beta 2-Microglobulin (beta 2M) polymerizes to form amyloid fibrils that deposit and cause destructive bone lesions in patients on chronic dialytic therapy. beta 2 M is mitogenic to osteoblasts; however, its effect on bone mineralization is unknown. To determine whether beta 2M causes bone demineralization, neonatal mouse calvariae were incubated with and without beta 2M, and net calcium flux was calculated. Following a 48-h but not 3- or 24-h incubation, beta 2M (10(-8)-10(-6) M) induced a net calcium efflux. The efflux was similar to that observed with 10(-10) M parathyroid hormone (PTH) but less than that observed with 10(-8 M PTH. Devitalizing the calvariae resulted in a net calcium influx that was unaffected by the addition of beta 2M, indicating a cell-mediated phenomenon. The release of beta-glucuronidase, an osteoclast enzyme, increased after a 48-h but not a 24-h incubation with beta 2M. Calcitonin, an osteoclast inhibitor, blocked the beta 2M-induced calcium efflux and beta-glucuronidase release, suggesting osteoclast involvement. Thus beta 2M induces a dose- and time-dependent, cell-mediated calcium efflux from neonatal mouse calvariae that involves osteoclast stimulation.


Clearance receptor and neutral endopeptidase-mediated metabolism of atrial natriuretic factor.

A novel small linear C-atrial natriuretic factor receptor ligand [C-ANF-(11-15)] and phosphoramidon (PHO) were used to determine the effects of C-ANF receptor blockade alone, or in combination with inhibition of neutral endopeptidase (NEP), on the pharmacokinetics and metabolism of ANF in the rat. C-ANF-(11-15) infusion decreased apparent volume of distribution (Vss) and metabolic clearance rate (MCR) of administered 125I-ANF-(1-28) to one-third of their control values, whereas PHO alone was without effect on these parameters. In combination with C-ANF-(11-15), however, PHO further decreased MCR of 125I-ANF-(1-28) and increased plasma half time by more than threefold. High-performance liquid chromatography analysis revealed that C-ANF-(11-15) inhibited the delayed appearance of free 125I and [125I]monoiodotyrosine but had no effect on the small proportion of NEP metabolites in plasma. The combination of C-ANF-(11-15) and PHO further delayed the appearance of small metabolites, abolished the appearance of NEP metabolites, and markedly prolonged the permanence of intact 125I-ANF-(1-28) in plasma. The results demonstrate that C-ANF receptor blockade by C-ANF-(11-15) impairs clearance and metabolism of ANF, an effect which is synergistically potentiated by concomitant inhibition of NEP. C-ANF-(11-15) alone or in combination with NEP inhibitors may be a potentially useful therapeutic tool in the treatment of cardiovascular and renal diseases.


Calorie restriction decreases microalbuminuria associated with aging in barrier-raised Fischer 344 rats.

Renal function as a sensitive biomarker of aging has been studied in specific pathogen-free (SPF) Fischer 344 rats (n = 211), and results are presented according to animal age (5, 8, 12, 18, 24 mo), sex, and diet (ad libitum vs. 40% calorie restriction). Plasma creatinine concentration, endogenous creatinine clearance, total protein excretion, and albumin excretion were measured. Kidney histology was evaluated by light microscopy. In both calorie-restricted and ad libitum-fed animals, kidney weight (KW) and body weight (BW) showed parallel changes with age. The KW-to-BW ratio was unaffected by age in all groups. There was no alteration in plasma creatinine concentration as a function of age or diet. In these SPF animals there was also no change in glomerular filtration rate with age. In animals fed ad libitum, albumin and protein excretion increased with age (females: 0.39 +/- 0.05 at 5 mo vs. 7.4 +/- 2.6 mg protein.24 h-1.g KW-1 at 24 mo; males: 4.1 +/- 0.6 at 5 mo vs. 15 +/- 3 mg protein.24 h-1.g KW-1 at 24 mo). The higher protein excretion rate in all males at 5 mo reflected the excretion of sex-dependent low-molecular-weight proteins that commenced with sexual maturation. Calorie restriction prevented the age-dependent increase in total protein excretion. Kidney histopathology was positively correlated with total protein and albumin excretion. Microalbuminuria preceded the development of lesions detectable by light microscopy. These observations support the concept that microalbuminuria in this model is a sensitive and early biomarker of nephropathy that can be monitored easily and noninvasively.


Three-dimensional reconstructed glomerular capillary network: blood flow distribution and local filtration.

We developed a mathematical model to simulate blood flow and filtration in individual capillary segments of a glomerular network reconstructed from a normal Munich-Wistar (MW) rat. Three-dimensional geometric reconstruction was obtained by semithin serial sections (1 micron) of one glomerulus after perfusion fixation of kidney. Photomicrographs of each section were digitized and processed, using a computer-based image-analysis system, to derive the topological organization of the capillary network and mean diameter and length of individual capillary segments. Blood flow rate in capillary segments was calculated using a theoretical model that considers apparent viscosity of blood in small capillaries as a function of local rheological parameters, partition of cells at bifurcations, and local filtration dependent on transmembrane hydraulic and oncotic pressure gradients along the network. In accord with previous observations, the topological organization of the capillary network disclosed a three-lobular structure. The ultrafiltration coefficient (Kf) calculated for the euvolemic MW rat with the present network approach was compared with that derived from a theoretical model that assumes identical capillaries in parallel. The latter model is shown to underestimate Kf, particularly under conditions in which filtration pressure equilibrium is approached. Calculation of local blood flow and filtration along the network indicates a heterogeneous distribution of these parameters and that some parts of the capillary network operate at filtration pressure equilibrium even if the overall network operates at filtration disequilibrium.


Cellular mechanisms involved in the vascular myogenic response.

By definition, the myogenic response is the contraction of a blood vessel that occurs when intravascular pressure is elevated and, conversely, the vasodilation that follows a reduction in pressure. Over the last several decades numerous investigators have demonstrated the importance of the myogenic response in the local regulations of blood flow, capillary pressure, and in the generation of basal vascular tone. Despite the considerable information obtained from these investigations, information about the cellular mechanisms that underlie this response has been slow to accumulate. Because of the physiological significance of the myogenic response, its mechanistic basis represents an important subject for research. Currently, there are several broad hypotheses concerning the sequence of events that couple changes in intravascular pressure or stretch with alterations in vascular smooth muscle activation. These hypotheses include 1) altered membrane properties leading to activation of ion channels; 2) modulation of biochemical cell-signaling pathways within vascular smooth muscle; 3) length-dependent changes in contractile protein function; and 4) endothelial-dependent modulation of vascular smooth muscle tone. This review summarizes current work relative to each of these hypotheses and describes a possible sequence of events to account for myogenic activation of vascular smooth muscle.


Effects of a novel prostaglandin, 8-epi-PGF2 alpha, in rabbit lung in situ.

We determined the effects of 8-epiprostaglandin (PG) F2 alpha, a noncyclooxygenase free radical-catalyzed product of arachidonic acid, on pulmonary vascular tone, its potency, and its mechanism of action. 8-Epi-PGF2 alpha (0.5-20 micrograms) was injected into the pulmonary artery (PA) catheter of 10 rabbits whose lungs were perfused in situ with Krebs-Henseleit buffer solution with 3% bovine serum albumin. PA pressure increased from a baseline of 13.5 +/- 0.6 to 25.6 +/- 2.0 cmH2O with 20 micrograms 8-epi-PGF2 alpha. 8-Epi-PGF2 alpha caused a rapid rise in PA pressure followed by a gradual decline over 40-60 min to baseline levels. Double vascular occlusion revealed a twofold increase in arterial resistance at peak rise in PA pressure. The rise in PA pressure with 20 micrograms 8-epi-PGF2 alpha was fivefold greater than with 20 micrograms of the cyclooxygenase-derived prostaglandin PGF2 alpha. The PA pressure response to 8-epi-PGF2 alpha was not altered by either cyclooxygenase block-ade with 150 microM meclofenamate or alpha-receptor blockade with 70 microM phentolamine, but was fully prevented by 40 microM SQ 29548, a thromboxane receptor antagonist. We conclude that in rabbits 8-epi-PGF2 alpha is a potent vasoconstrictor of the pulmonary vasculature, which appears to be due to the activation of SQ 29548-responsive thromboxane receptors.


Salt intake and angiotensin II alter microvessel density in the cremaster muscle of normal rats.

This study investigated the effect of salt intake and angiotensin II (ANG II) levels on microvessel density (MVD). Rats with indwelling arterial and venous catheters were placed on either a high-salt (HS; 4%) or a low-salt diet (LS; 0.4%) for 2 or 4 wk, and blood pressure, heart rate, and plasma renin activity were measured. Plasma ANG II was fixed at normal levels in half of the rats on HS by continuous intravenous infusion of ANG II (5 ng.kg-1.min-1). Samples of cremaster muscle were examined histologically to determine MVD. No difference in MVD was observed between HS and LS groups after 2 wk. After 4 wk on HS, MVD was reduced (22.4%, P less than 0.05) compared with the LS group. In rats fed HS, ANG II infusion induced a significant dose-dependent increase in MVD from 85.11 +/- 3.34 to 98.94 +/- 4.62 (ANG II, 5 ng.kg-1.min-1) and to 107.60 +/- 7.00 (ANG II, 10 ng.kg-1.min-1) (P less than 0.05), with no change in blood pressure. Maintenance of ANG II levels for 4 wk blocked the rarefaction due to salt. These results suggest that the decrease in MVD due to salt could be the result of a dietary-induced fall in plasma ANG II levels.


Tumor necrosis factor challenges in canines: patterns of cardiovascular dysfunction.

Three groups of conscious canines were given different intravenous doses of human recombinant tumor necrosis factor (TNF) over 1 h, and the resulting cardiovascular abnormalities were examined for 10 days. As TNF dose increased [0 (controls), 30, 60, and 120 micrograms/kg body wt], the number of deaths increased (P less than 0.025; 0 of 6, 1 of 8, 4 of 8, number of deaths in each group, respectively). In all three groups receiving TNF, the mean left ventricular ejection fraction (LVEF) at 2 h after infusion decreased (P less than 0.003) compared with controls. The group receiving the highest dose of TNF (120 micrograms/kg body wt) had the greatest decrease (P less than 0.05) in LVEF from 0 to 2 h. At 8 h, all three groups receiving TNF had similar LVEF. In these three groups, other multiple measures of LV function at 8 h showed significant and similar decreases in cardiac contractility compared with controls. From 24 to 240 h, however, the time required for cardiac performance (LVEF) to return to normal was dose dependent (30 less than 60 less than 120 micrograms/kg body wt; P less than 0.05). Canines receiving the lowest dose of TNF had near normal cardiac function (LVEF) at 24 h, whereas canines receiving the highest dose had persistent cardiac abnormalities at 240 h. Thus, at 8 h, the severity of cardiac dysfunction is independent of TNF dose, but the rate of onset and the duration of cardiac abnormality are markedly dependent of dose.(ABSTRACT TRUNCATED AT 250 WORDS)


Bioassay of endothelium-derived relaxing factor in diabetic rat aorta.

The bioassay technique was utilized to quantitate endothelium-derived relaxing factor (EDRF) released from perfused donor segments of control and diabetic rat aorta. In the presence of indomethacin, perfusates of donor segments with endothelium were allowed to superfuse recipient detector rings of normal rat aorta without endothelium. Under basal conditions, relaxations of the bioassay rings to perfusates of control and diabetic donor segments were similar. Perfusion of donor segments with acetylcholine produced relaxation of bioassay rings, which was decreased from endothelial perfusion of diabetic donor segments. These relaxations were inhibited by addition of methylene blue to the detector ring or by perfusion of donor segments with nitro-L-arginine. Infusion of superoxide dismutase (SOD) at a site proximal to the donor segment normalized relaxations induced by acetylcholine addition to diabetic donors. In contrast, infusion of SOD distal to the donor had no effect on acetylcholine-stimulated relaxations of detector rings from control donors while attenuating, paradoxically, the relaxations of detector rings from diabetic donors. These results suggest that diabetic rat aortas release similar levels of EDRF in response to acetylcholine, but the action of EDRF arising from diabetic donors is attenuated by enhanced release of oxygen-derived free radicals, which limits EDRF-mediated relaxation of vascular smooth muscle.


Increased oxyhemoglobin affinity by carbamylation: coronary autoregulation and O2 transport.

Hemoglobin carbamylation with sodium cyanate was used to study myocardial O2 delivery during increased oxyhemoglobin affinity. A group of control dogs was compared with a group of dogs, in which their hemoglobin had been carbamylated by daily ingestion of sodium cyanate over a period of 6-8 wk. The O2 tension at 50% saturation of hemoglobin (P50) averaged 23 mmHg in the carbamylated group compared with an average P50 of 31 mmHg in the control group. The left main coronary artery was cannulated and perfused via an extracorporeal, pressure-controlled reservoir. Coronary pressure, coronary flow, and blood gas measurements were made at coronary perfusion pressures spanning the autoregulatory range. The increased oxyhemoglobin affinity produced no significant difference in convective O2 transport to the coronary capillaries, although the carbamylated group had a significantly higher arterial O2 content and significantly lower coronary flow. The autoregulatory process was not significantly altered by carbamylation-induced shifts in the O2 dissociation curve. Despite a significantly lower coronary sinus PO2 in the carbamylated group, the increase in oxyhemoglobin affinity was associated with significantly lower O2 extractions in the carbamylated group. Significant extraction reserve was present in both the control and carbamylated groups as demonstrated by the fact that O2 extraction could still increase well above control levels when perfusion pressure was lowered. We conclude that there is no significant vasodilatory compensation for moderate increases in oxyhemoglobin affinity, despite the continued presence of autoregulatory vasodilatory reserve.


Recovery of arterial pressure control after partial baroreceptor denervation in awake rabbits.

We examined recovery of control of heart rate (HR) and total peripheral resistance (TPR) by arterial baroreceptors after bilateral carotid sinus and aortic denervation or unilateral carotid sinus and aortic denervation in conscious rabbits. In one group of animals, HR responses to changes in mean arterial pressure (MAP) after injection of nitroglycerin or phenylephrine were measured in control studies and at 2, 5, 10, and 15 days after partial baroreceptor denervation. All denervation procedures increased MAP and HR at 2 and 5 days after denervation. Reflex sensitivity decreased to 57-67% of control on day 2 after denervation. HR responses recovered by day 10 after bilateral aortic or carotid sinus denervation; however, recovery following unilateral denervation was less complete. In a second group of animals, studied after implantation of aortic flowmeters, TPR changes following reduction in cardiac output by inferior vena caval occlusion were 49% of control responses on day 2 after denervation and returned close to control level on day 5. Controls of HR and TPR recovered substantially and were not significantly different from control 10 days after partial denervation. Recovery apparently occurred through the remaining arterial baroreceptors, possibly due to central reorganization of reflex pathways.


Coronary microvascular response to endothelin is dependent on vessel diameter and route of administration.

Endothelin is a 21-amino acid peptide originally isolated from vascular endothelial cells. In the present study, we examined the effect of topical and intracoronary administration of endothelin-1 on the coronary microcirculation and the effect of inhibition of cyclooxygenase on the microvascular response to intracoronary endothelin. In anesthetized dogs (n = 39), the coronary microcirculation was visualized using stroboscopic epi-illumination synchronized to the cardiac cycle. Topical application of endothelin [(5 x 10(-9) to 10(-8) M] constricted all arteries and arterioles with the degree of constriction inversely related to vessel size. Coronary veins and venules did not constrict to endothelin. Topical application of EDTA (10 mg/ml) reversed the constriction to endothelin in arterioles of all sizes. In contrast, intracoronary administration of endothelin (10(-8) to 10(-7) M) produced dilation of small arterioles (less than 130 microns) and no response of large arterioles (greater than 130 microns). The response of small arterioles to intracoronary endothelin was not altered by inhibition of cyclooxygenase with indomethacin (5 mg/kg); however, large arterioles constricted. Thus the coronary microvascular response to endothelin is dependent on the route of administration. Constriction of arteries and arterioles of all sizes to endothelin is dependent on extracellular calcium. Vasodilator prostaglandins may be released in response to intracoronary administration of endothelin predominantly in larger vessels. Thus the differential response to endothelin with topical and intracoronary administration may reflect a diffusional barrier of the endothelium or release of endothelium-derived relaxing factor and prostaglandins in response to endothelin.


Norepinephrine increases the economy of pressure development in isolated canine hearts.

To test for oxygen wasting by norepinephrine (NE) without relying on normalization by measures of performance such as the pressure-volume area, myocardial oxygen consumption (MVO2) was determined for isovolumic beats at five different left ventricular (LV) end-diastolic volumes (EDV) in nine isolated cross-perfused canine hearts in each of three states: a basal anesthetic state (B); after depression with halothane (H); and after adding NE to increase contractility back to the B state (H+NE). The end-diastolic and peak systolic pressure-volume lines were identical for B and H+NE. The R2 for a linear regression of MVO2 per beat for B vs. H+NE for beats originating at the same EDV and developing similar (within 10%) peak isovolumic pressures for all hearts was 0.85. The slope and intercept were 0.83 and 0.01, which are significantly less than one (P less than 0.001) and greater than zero (P less than 0.001), respectively. These data suggest that NE increases both the economy of pressure development as well as activation energy of an isovolumically contracting LV.


Dexamethasone-induced differentiation of atrial myocytes in culture.

Atrial and ventricular myocytes from fetal and newborn rats were cultured in medium supplemented with fetal or newborn calf serum with and without glucocorticoid. Myocyte morphology was examined by light and electron microscopy, and the amount of stored and secreted atrial natriuretic peptide (ANP) was measured. Without dexamethasone, neonatal atrial myocytes cultured for 7 days contained myofibrils organized into sarcomeres and numerous endocrine granules containing immunostainable ANP. Secretion of immunoreactive ANP reached a peak between days 7 and 9 of culture. Myocytes from fetal rats secreted ANP but contained few endocrine granules, and myofilaments were poorly organized. By contrast, the addition of dexamethasone (1 nM-1 microM) to the culture medium of newborn myocytes promoted development of numerous endocrine storage granules, mitochondria, and myofibrils with prominent Z-bands. Dexamethasone also increased the cellular content of ANP and ANP-specific mRNA in both atrial and ventricular myocytes. In the presence of dexamethasone myocytes maintained their structural integrity for periods of at least 45 days.


Fluorochemical emulsion APE-LM substantially improves cardiac preservation.

We determined the efficacy of a novel fluorochemical emulsion for long-term hypothermic preservation of hearts. Rat hearts were preserved for 12 h at 12 degrees C with use of continuous low-pressure coronary perfusion with one of three oxygenated media (n = 6 hearts/groups): an "extracellular" crystalloid solution; APE-LM, a novel fluorochemical emulsion of perfluoroperhydrophenanthrene in egg yolk phospholipid; and FC-43, the Fluosol-43 (Oxypherol) fluorochemical emulsion of perfluorotributylamine in Pluronic F68. The emulsion media contained the same components as the crystalloid medium. All three media contained 0.5% albumin. An isolated working heart perfusion system was used to quantify the function of preserved hearts and controls (fresh hearts, n = 6). The APE-LM-preserved hearts were not significantly different from control hearts in contractile function, output, and energetics during a 4-h 37 degrees C reperfusion period. The control and APE-LM-preserved hearts had significantly better performance than crystalloid- and FC-43-preserved hearts. All preserved hearts gained fluid during preservation. The edema of APE-LM-preserved hearts, but not that of the other two preserved groups, was reversed during 37 degrees C reperfusion. These data provide the first evidence that a unique fluorochemical emulsion improves long-term preservation of cardiac tissue and produces significantly better recovery of cardiac function after preservation. This salutary effect was specifically associated with APE-LM emulsion and may result from its high O2 capacity, its biologically compatible emulsifier, and its superior physical properties, which include very small emulsion particle size (0.1-0.15 micron), low viscosity, and minimal toxicity.


Topical arachidonic acid restores pial arteriolar dilation to hypercapnia of postischemic newborn pig brain.

In the newborn pig, cerebral vasodilator responses to hypercapnia are lost after cerebral ischemia. We examined the effect of topical application of arachidonic acid (30 micrograms/ml, 20 min) to the postischemic piglet brain on subsequent pial arteriolar dilated in response to hypercapnia (10% CO2 ventilation) and topical isoproterenol (10(-6), 10(-7) M). After 20 min cerebral ischemia, pial arterioles did not dilate to hypercapnia but responded to isoproterenol in a fashion similar to before ischemia. Treatment with arachidonic acid after ischemia restored pial arteriolar dilation to hypercapnia. Hypercapnia caused an increase in cortical periarachnoid concentration of 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha) before but not after ischemia. After postischemic treatment with arachidonic acid, the increase in cortical periarachnoid fluid 6-keto-PGF1 alpha during hypercapnia was restored. Therefore, topical application of arachidonic acid to cerebral vessels restores cerebral prostanoid synthesis and pial arteriolar dilation in response to hypercapnia that has been abolished by ischemia.


EDRF plays central role in collateral flow after arterial occlusion in rabbit ear.

An in vitro model has been developed in which the acute development of collateral perfusion of a segment of rabbit central ear artery, isolated between ligatures, is assessed by X-ray microangiography. Collateral perfusion was quantified by normalizing the volume of the segment filled with respect to its preocclusion control. The influence of endothelium-derived relaxing factor (EDRF) activity on perfusion was examined by using 100 microM NG-nitro-L-arginine methyl ester (L-NAME), a potent inhibitor of nitric oxide synthesis. Filling of the isolated segment after occlusion was time dependent, being 21.6 +/- 4.2% after 2 min and 46.6 +/- 5.3% after 90 min. This acute development of collateral flow was reversed by addition of L-NAME 60 min after ligation, after which filling was reduced to 17.8 +/- 3.8%. When L-NAME was added before ligation, filling of the segment was 15.6 +/- 5.9% at 2 min and 14.8 +/- 7.4% at 90 min, so that the time-dependent component of collateral flow development was abolished. The inhibitory effects of L-NAME on collateral perfusion were reversed by an excess of L-arginine. These findings indicate that EDRF plays a central role in the development and maintenance of collateral flow.


Differential effects of WEB 2086 and SRI 63-441 on TNF-alpha-induced alterations in cardiopulmonary function.

We hypothesized that platelet-activating factor (PAF) and cyclooxygenase products might be important mediators of the cardiopulmonary effects induced by tumor necrosis factor (TNF-alpha) in anesthetized pigs. A 6-h infusion of human recombinant TNF-alpha caused hypoxemia, leukopenia, thrombocytopenia, decreased cardiac index (CI), increased pulmonary vascular resistance (PVR) and increased mean pulmonary arterial (Ppa) and intratracheal (Pt) pressures. Administration of the PAF receptor antagonist SRI 63-441 or indomethacin blocked the early (0.25-0.5 h) and attenuated the later increases in PVR and Ppa; indomethacin also attenuated the increase in Pt and hypoxemia associated with TNF-alpha infusion. WEB 2086 did not attenuate the TNF-alpha-induced alterations in CI, PVR, Pt, or PaO2. The in vivo specificity of SRI 63-441 and WEB 2086 was tested by infusing exogenous PAF, prostaglandin (PG) F2 alpha, U-46619 [thromboxane (Tx)A2 receptor mimetic], or arachidonic acid (AA) before and during administration of SRI 63-441 or WEB 2086. Both antagonists blocked the cardiopulmonary effects induced by exogenous PAF. SRI 63-441, but not WEB 2086, significantly attenuated the increased PVR caused by PGF2 alpha, U-46619, and AA. We conclude that SRI 63-441 is a less specific PAF receptor antagonist in vivo compared with WEB 2086 and that cyclooxygenase products, but not PAF, contribute significantly to the cardiopulmonary responses induced by exogenously infused TNF-alpha in pigs.


Beneficial effects of SPM-5185, a cysteine-containing NO donor in myocardial ischemia-reperfusion.

Intravenous administration of SPM-5185 [N-nitratopivaloyl-S-(N'-acetylalanyl)-cysteine ethyl ester], a cysteine-containing nitric oxide (NO) donor, or SPM-5267 [pivaloyl-S-(N'-acetylalanyl)-cysteine ethyl ester], an analogue of SPM-5185 that lacks the NO moiety, was studied in a feline myocardial ischemia-reperfusion model. Administration of SPM-5185 (1 mg/kg), followed by a 2-mg.kg-1.h-1 infusion starting 10 min before reperfusion, resulted in significant protection 4.5 h postreperfusion. In the myocardial ischemia (MI)+SPM-5267 group, 38 +/- 4% of the area at risk was necrotic, whereas the necrotic area/area at risk was only 7 +/- 2% in the MI+SPM-5185 group (P less than 0.01). Moreover, SPM-5185 treatment markedly attenuated the endothelial dysfunction observed in the left anterior descending coronary artery after reperfusion by 50%. These beneficial effects occurred despite the absence of a significant change in myocardial oxygen demand, as measured by the pressure-rate index. In vitro experiments demonstrated that SMP-5185, but not SPM-5267, decreased adherence of neutrophils to the coronary vascular endothelium and decreased production of superoxide radicals. Therefore, a likely mechanism of the observed cardioprotection by SPM-5185 involves attenuation of polymorphonuclear leukocyte-induced endothelial dysfunction.


Aging- and training-induced alterations in collagen characteristics of rat left ventricle and papillary muscle.

We evaluated the single and interactive effects of aging and exercise training on selected parameters of myocardial interstitium in both the left ventricle (LV) and LV papillary muscle of female Fischer 344 specific pathogen-free rats. Ten weeks of treadmill running resulted in significant LV hypertrophy as well as elevated plantaris muscle citrate synthase activity in both young adult (5-mo-old) and senescent (23-mo-old) trained animals (YT, young trained; OT, old trained) compared with age-matched sedentary controls (YC, young control; OC, old control). Proline and hydroxyproline pools were significantly higher (both P less than 0.05) in 23-mo-old vs. 5-mo-old papillary muscles. Degree of maturation (nonreducible cross-linking) of LV collagen was evaluated by measurement of hydroxylysylpyridinoline concentration ([HP]). In a comparison of YC with OC rats, ventricular [HP] increased approximately fivefold from 0.059 +/- 0.007 to 0.285 +/- 0.018 (SE) mol HP/mol collagen (P less than 0.001). Whereas training had no effect on ventricular [HP] in young adult rats, it significantly reduced LV collagen cross-linking in OT rats (0.131 +/- 0.027) so that HP values in this group were less than one-half of those observed in OC rats. Because both collagen concentration and degree of cross-linking are thought to affect muscle stiffness characteristics, we conclude that the observed changes should be considered in any explanation for aging- and training-induced alterations in LV and papillary muscle contractile indexes.


Factors involved in left ventricular dysfunction after massive sympathetic activation.

We sought to determine whether catecholamines are responsible for the depressed left ventricular (LV) function that follows massive sympathetic nervous system (SNS) activation and whether the additional myocardial energy demands of SNS-induced hypertension contribute to this disorder. An intracisternal injection of veratrine was used to intensely activate the SNS of anesthetized rabbits, and 150 min later, LV function was evaluated in vitro using established techniques. To assess catecholamine involvement, rabbits were pretreated with phentolamine, propranolol, or saline prior to SNS activation. Control animals received veratrine intravenously. In separate experiments, angiotensin II (ANG II) was administered to rabbits to produce hemodynamic and plasma catecholamine profiles comparable to that produced by intense SNS activity. LV function of hearts after either massive SNS activation or ANG II administration was significantly diminished compared with control (P less than 0.01) and could be prevented by pretreatment with the catecholamine antagonists. LV function was also not diminished in another group of animals in which arterial pressure was maintained near baseline throughout the SNS discharge, thus suggesting that the increased myocardial energy demand associated with the development of arterial hypertension contributes to the LV dysfunction. We conclude that toxic concentrations of catecholamines are responsible for SNS-induced LV dysfunction and that hypertension, most likely because of its ability to increase myocardial energy demand, is one of the important events that leads to depressed cardiac function.


Saline diuresis and natriuresis in unanesthetized dogs: a missing atrial factor?

Recent studies in our laboratory indicated that a blunted (40-50%) renal excretory response to isotonic intravenous saline loads occurred in conscious, renal-denervated dogs after 70% of the atrial mass was removed. The blunted responses could not be explained by differences in the responses of arterial pressure, renal nerve activity, or by measured changes of plasma immunoreactive atrial natriuretic peptide (iANP), arginine vasopressin (AVP), plasma renin activity (PRA), or aldosterone (Aldo). The present study was designed to determine whether the central nervous system (CNS) was the source of an unidentified substance, which could account for the blunting of the urine excretory response seen in the atrial-resected dogs. Renal denervation was performed in all dogs to eliminate alterations in efferent renal sympathetic nerve activity derived from reflexes activated during volume expansion. Cardiac denervation (CDX) was used to eliminate sensory cardiac afferent nerve activity to the CNS. A group of five renal-denervated dogs was given an isotonic volume load (400 ml/30 min) before and after complete CDX. Plasma AVP was fixed at normal plasma levels of 3 pg/ml by continuous intravenous infusion. Na and H2O excretion were not different in renal-denervated dogs compared with combined renal and cardiac denervation during the 5 h after the saline load. Plasma AVP and Aldo were unchanged with the volume loads, although PRA rose gradually over the 5 h after the saline loads. Plasma iANP increased transiently in the combined renal and cardiac-denervated state rising from a control of 65-120 pg/ml at the end of the load period.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of aging on baroreflex regulation of sympathetic activity in humans.

Arterial baroreflexes contribute importantly to blood pressure regulation through their influence on parasympathetic outflow to the sinus node and sympathetic outflow to the peripheral circulation. Baroreflex control of heart rate is known to be diminished in older individuals. Whether advancing age is associated with a parallel attenuation in baroreflex control of sympathetic outflow to the peripheral circulation has not been studied in humans. To provide such information, we made direct measurements of muscle sympathetic nerve activity (MSNA) in healthy males who ranged in age from 18 to 71 yr. The subjects were arbitrarily divided into three groups: younger (18-34 yr; n = 35), middle aged (35-50 yr; n = 15), and older (51-71 yr; n = 16). Although basal levels of MSNA were higher in older subjects than in younger and middle-aged subjects, the gains of baroreflex control of MSNA were the same in the older, middle-aged, and younger subjects (-4.6 +/- 0.6, -4.8 +/- 0.9, -5.1 +/- 0.5 U/mmHg, P greater than 0.10). In contrast, the gains of baroreflex control of cardiac intervals were attenuated in the older and middle-aged subjects compared with the younger subjects (9.8 +/- 1.2, 13.6 +/- 1.4, 21.7 +/- 1.3 ms/mmHg, P less than 0.05). Our data indicate that although the parasympathetic component of the arterial baroreflex becomes impaired with advancing age, the sympathetic component can be well maintained in healthy individuals even into the seventh decade.