Effects of cytochalasin B on the response of toad urinary bladder to vasopressin.

A combined physiological and morphological study of the effects of cytochalasin B (CB) on the toad urinary bladder has been carried out. CB inhibits the hydro-osmotic response to vasopressin without altering basal water permeability or diffusion, or the increase in (3)H(2)O diffusion observed after hormone addition. Although CB increases [(22)Na]-, [(36)Cl]-, and [(14)C]urea fluxes, and decreases transepithelial potential, no alteration in basal short-circuit current, the vasopressin-induced increase in this parameter, or [(14)C]inulin permeability occurs. In the absence of hormone, CB does not markedly alter the structure of the toad bladder. However, in the presence of vasopressin, CB induces the formation of large intracellular vacuoles. These results suggest a possible coupling of solute and water movement across the tissue.

Oxygen-sensitive stages of the cell cycle of human diploid cells.

We had established that growth of human diploid WI-38 cells is reversibly inhibited by elevated partial pressures of oxygen (PO2) and we were interested in determining where in the cell cycle growth was delayed. A technique combining cytospectrophotometry and autoradiography was used to determine cell cycle parameters. Confluent cells that were subcultivated and exposed to a PO2 of 365 +/- 8 mm Hg were delayed primarily after DNA synthesis but before metaphase. At a PO2 of 590 +/- 35 mm Hg, most cells did not initiate DNA synthesis, and the few that did, failed to complete the process. When exponentially growing cells that had already begun DNA synthesis were exposed to a PO2 of 590 p 35 mm Hg, they accumulated after completing DNA synthesis but before initiating mitosis. The rate at which (3H)thymidine was incorporated into DNA was inversely correlated with oxygen tension (PO2 of 135--590 mm Hg). These results suggest that the process most sensitive to oxygen causes cells to be delayed after DNA synthesis but before metaphase. Slightly higher PO2's were needed to inhibit the initiation of DNA synthesis. Further, the rate of DNA synthesis is decreased by elevated oxygen tensions.

The effect of oxygen and vitamin E on the lifespan of human diploid cells in vitro.

Human diploid cells (WI-38) were serially subcultivated at partial pressures of oxygen (Po2) ranging from 5.6 mm Hg to 608 mm Hg. At a Po2 of 5.6 mm Hg, the number of doublings to phase out was less than that of control cells at a Po2 of 137 mm Hg. Cultures grown at Po2's of 24, 49, or 137 mm Hg grew at the same rate and phased out after a similar number of population doublings. Population lifespan was markedly shortened by chronic exposure to elevated Po2's, a phenomenon that was, in part, reversible. d-1-alpha-Tocopherol (10 microgram/ml or 100 microgram/ml) homogenized into the medium at each weekly subcultivation did not extend the lifespan of cells at reduced, ambient, or elevated oxygen tensions. These results indicate that neither oxygen toxicity nor free radical reactions play a significant role in limiting the lifespan of WI-38 cells grown in vitro under ambient oxygen tensions (Po2 137 mm Hg).

Somatostatin: occurrence in urinary bladder epithelium and renal tubules of the toad, Bufo marinus.

Immunohistochemical techniques were used to detect immunoreactive somatostatin-like material in toad urinary bladder epithelium and in kidney distal tubules and collecting ducts. This material has immunological and chromatographic properties identical to those of synthetic cyclic somatostatin. The occurrence of somatostatin-like material in antidiuretic hormone-sensitive portions of the renal urinary system suggests a local regulatory or paracrine role for somatostatin.

Immunofluorescent localization of cyclic AMP in toad urinary bladder: possible intercellular transfer.

By use of an immunofluorescent cytochemical staining technique, adenosine 3',5'-monophosphate (cyclic AMP) has been localized in toad bladder epithelial cells. Within 2 minutes after addition of vasopressin, staining intensity increases in both mitochondria-rich and granular cells. This finding, taken together with the precise anatomical relation between these two epithelial cell types and the observation that after separation of the two cell types vasopressin stimulates cyclic AMP accumulation in only mitochondria-rich cells, suggests that cyclic AMP may be transferred from mitochrondria-rich to granular cells as part of the response of the toad urinary bladder to vasopressin.

Role of prostaglandin E2 in mediating the effects of pH on the hydroosmotic response to vasopressin in the toad urinary bladder.

Acidosis inhibits the hydroosmotic response to vasopressin. Since prostaglandins are known to modulate vasopressin-stimulated water flow we investigated the role of endogenous prostaglandin E2(PGE2) production in the pH-dependent response of the toad urinary bladder to vasopressin. Graded acidification of the serosal medial resulted in a progressive decline in vasopressin-stimulated water flow from 26.6 +/- 0.5 mg/min at pH 8.4 to 1.7 +/- 0.6 at pH 6.9. In these bladders basal PGE2 synthesis increased from 5.09 +/- 0.51 pmol/min per g hemibladder at pH 8.4 to 18.8 +/- 2.8 at pH 6.9. The addition of that concentration of PGE2 produced by the bladder at pH 7.4 (4 nM) to bladders at pH 8.4 resulted in 62-71% of the inhibition usually seen at pH 7.4; these data suggest that basal PGE2 production per se and not other products of prostaglandin synthesis or other pH-dependent events is responsible for the effect of acidosis. Preincubation with prostaglandin synthesis inhibitors reversed in major part the effect of serosal acidification on the response to submaximal concentrations of vasopressin and completely abolished the effect of pH on near maximal concentrations of the hormone. An increase in PGE2 synthesis after vasopressin was not seen at any pH. These studies establish that increased basal PGE2 synthesis plays a critical role in the pH dependence of the hydroosmotic response to vasopressin and demonstrate that factors that modulate the response to vasopressin may exert this effect by changing the basal rate of prostaglandin synthesis.

Hormonal control of the renal conversion of 25-hydroxycholecalciferol to 1,25-dihydroxycholecalciferol.

Isolated renal tubules from vitamin D-deficient chicks catalyse the in vitro conversion of 25-hydroxycholecalciferol to 1,25-dihydroxycholecalciferol. This conversion is stimulated by 5 x 10(-10) M bovine parathyroid hormone, or by 10(-6) M cyclic AMP. It is inhibited by 10(-9) M porcine calcitonin. It is concluded that these hormonal controls of the synthesis of the renal hormone 1,25-dihydroxycholecalciferol are of particular physiological significance in coordinating the activities of the various organs involved in extracellular calcium homeostasis.

Skeletal muscle calcium metabolism and contractile force in vitamin D-deficient chicks.

The myopathy associated with vitamin D deficiency has not been well characterized, and it is not known if weakness is a result of a specific effect of vitamin D deficiency on skeletal muscle. Chicks were raised from hatching on a vitamin D-deficient diet, and by 3 wk of age were hypocalcemic and appeared weak. Tension generated by triceps surae during repetitive stimulation of posterior tibial nerve was significantly less than that developed by chicks given vitamin D(3) supplements (309 g tension/g wet weight of triceps surae, SD 60, for vitamin D-deficient chicks; 470, SD 77, for vitamin D(3)-treated chicks, P < 0.01). Histochemical and electron microscopic examination of skeletal muscles of these chicks showed no abnormalities, and there were no electrophysiologic evidences of motor nerve or neuromuscular junction dysfunction. The concentration of ATP in skeletal muscle of the vitamin D-deficient chicks (5.75 mumol/g wet weight, SD 0.17) was not significantly different from that in vitamin D-treated chicks (5.60, SD 0.50). There was no correlation between strength and serum calcium, serum inorganic phosphate, or skeletal muscle inorganic phosphate. Relaxation of tension after tetanic stimulation was slowed in the vitamin D-deficient chicks (20.6 ms, SD 1.7, vs. 15.4, SD 1.3, in vitamin D-treated chicks and 15.3, SD 1.0, in normal control chicks), and in vitro (45)Ca(++) transport by sarcoplasmic reticulum from the vitamin D-deficient chicks was reduced. Calcium content of mitochondria prepared from leg muscles of vitamin D-deficient chicks (24 nmol/mg mitochondrial protein, SD 6) was considerably lower than that of mitochondria from normal control chicks (45, SD 8) or from chicks treated with vitamin D for 2 wk or more (66-100, depending upon level and duration of therapy). Treatment of the vitamin D-deficient chicks from hatching with sufficient dietary calcium to produce hypercalcemia did not significantly raise skeletal muscle mitochondrial calcium content (31 nmol/mg mitochondrial protein, SD 7) and did not prevent weakness. These studies demonstrate objective weakness as a result of myopathy in vitamin D-deficient chicks, and provide evidence that vitamin D deficiency has effects on skeletal muscle calcium metabolism not secondary to altered plasma concentrations of calcium and phosphate.

t-Butyl hydroperoxide alters fatty acid incorporation into erythrocyte membrane phospholipid.

Because the ability of cells to replace oxidized fatty acids in membrane phospholipids via deacylation and reacylation in situ may be an important determinant of the ability of cells to tolerate oxidative stress, incorporation of exogenous fatty acid into phospholipid by human erythrocytes has been examined following exposure of the cells to t-butyl hydroperoxide. Exposure of human erythrocytes to t-butyl hydroperoxide (0.5-1.0 mM) results in oxidation of glutathione, formation of malonyldialdehyde, and oxidation of hemoglobin to methemoglobin. Under these conditions, incorporation of exogenous [9,10-3H]oleic acid into phosphatidylethanolamine is enhanced while incorporation of [9,10-3H]oleic acid into phosphatidylcholine is decreased. These effects of t-butyl hydroperoxide on [9,10-3H]oleic acid incorporation are not affected by dissipating transmembrane gradients for calcium and potassium. When malonyldialdehyde production is inhibited by addition of ascorbic acid, t-butyl hydroperoxide still decreases [9,10-3H]oleic acid incorporation into phosphatidylcholine but no stimulation of [9,10-3H]oleic acid incorporation into phosphatidylethanolamine occurs. In cells pre-treated with NaNO2 to convert hemoglobin to methemoglobin, t-butyl hydroperoxide reduces [9,10-3H]oleic acid incorporation into phosphatidylcholine by erythrocytes but does not stimulate [9,10-3H]oleic acid incorporation into phosphatidylethanolamine. Under these conditions oxidation of erythrocyte glutathione and formation of malonyldialdehyde still occur. These results indicate that membrane phospholipid fatty acid turnover is altered under conditions where peroxidation of membrane phospholipid fatty acids occurs and suggest that the oxidation state of hemoglobin influences this response.

Purification and characterization of chick intestine brush border membrane. Effects of 1alpha(OH) vitamin D3 treatment.

A new technique has been developed for the isolation of membrane vesicles from the vitamin D-deficient and vitamin D-treated chick intestinal brush border membrane. The technique involves removal of nuclei from a low speed pellet by discontinuous sucrose gradient centrifugation. The resulting intact brush borders are then homogenized in 0.5 M Tris and the membrane fragments purified on a glycerol gradient. This preparation represents a 20-fold purification of the brush border marker sucrase. After 1alpha-hydroxyvitamin D3 treatment there is a significant increase in membrane phospholipid phosphorous, an alteration in the fatty acid composition of the phosphatidylcholine fraction of membrane phospholipid, and a decrease in sucrase specific activity.

Effects of inhibitors of protein and RNA synthesis on aldosterone-stimulated changes in phospholipid fatty acid metabolism in the toad urinary bladder.

The effects of an inhibitor of RNA synthesis, cordycepin, and an inhibitor of protein synthesis, cycloheximide, on aldosterone-induced changes in lipid metabolism and phospholipid fatty acid composition have been studied in the toad urinary bladder. At the concentrations employed, the inhibitors abolish the hormone-induced increases in total lipid synthesis, phospholipid fatty acid specific activities, and weight percentage of phospholipid long-chain polyunsaturated fatty acids as well as blocking the aldosterone-mediated increase in sodium transport.

Preparation of inside-out vesicles from erythrocyte membranes inactivates the pathway for oleic acid incorporation into phospholipid.

The pathway for membrane phospholipid fatty acid turnover in situ may be important in the regulation of the composition and turnover of the lipid microenvironment of membrane proteins. This pathway has been characterized further by studying the activation and incorporation of [9,10(n)-3H]oleic acid and transesterification of [1-14C]oleoyl-CoA into membrane phospholipids by isolated erythrocyte membrane ghosts and inside-out vesicles derived from these ghosts. Erythrocyte ghosts and sealed vesicles of defined orientation prepared from them have been widely employed in studies of the function of membrane proteins, particularly those which mediate the transport of ions and sugars. Preparation of inside-out vesicles from ghosts by exposure to alkaline hypotonic conditions results in elution of some membrane proteins but no loss of membrane phospholipid. Compared to ghosts, the ability of inside-out vesicles to activate and incorporate [9,10(n)-3H]oleic acid into phospholipid is diminished by over 90% and the ability of inside-out vesicles to transesterify [1-14C]oleoyl-CoA to phospholipid is diminished by over 50%. These findings indicate that exposure of erythrocyte membranes to the alkaline hypotonic conditions required for inside-out vesicle preparation results in loss or inactivation of both acyl-CoA ligase and acyl-CoA-lysophospholipid acyltransferase activities. This lability of the enzymes for in situ phospholipid fatty acid turnover should be considered in the design and interpretation of studies concerned with elucidation of the relationship between phospholipid fatty acid turnover and the regulation of membrane protein function in this membrane preparation.

Studies on the Ca2+ transport mechanism of human erythrocyte inside-out plasma membrane vesicles. V. Chlortetracycline fluorescence.

The measurement of chlortetracycline fluorescence was employed as a probe for measuring the process to calcium transport by human erythrocyte inside-out vesicles. Chlortetracycline is a divalent metal chelator which increases its fluorescence when bound to calcium in the presence of a membrane. Addition of calcium and ATP to inside out vesicles in the presence of chlortetracycline increased the chlortetracycline fluorescence as a function of time following an initial delay. Only after a threshold level of calcium had been accumulated did the fluorescence increase. The presence of both ATP and calcium were required. The addition of calmodulin increased the rate and absolute magnitude of the chlortetracycline fluorescence change. Similarly, calmodulin stimulated the rate and extent of 45Ca transport by inside-out vesicles. Moreover, the presence of saponin abolished both chlortetracycline fluorescence change and 45Ca uptake; a non-hydrolyzable ATP analog would not substitute for ATP in either 45Ca transport or chlortetracycline fluorescence experiments. Comparison between the slopes of the linear portions of chlortetracycline fluorescence change and calcium transport time courses at varied free calcium concentrations showed a consistent ratio between the slopes. This suggests that calcium transport change can be calibrated by employing chlortetracycline fluorescence. Based on this data, it is concluded that chlortetracycline fluorescence is a rapid and accurate method for monitoring calcium transport by human erythrocyte inside-out vesicles.

Hibernation activates glyoxylate cycle and gluconeogenesis in black bear brown adipose tissue.

Biochemical studies on brown adipose tissue removed from a hibernating black bear and a non-hibernating control animal demonstrate that this tissue: (1) can carry out cyanide-insensitive fatty acid oxidation, and (2) possesses catalase activity and the enzyme activities unique to the glyoxylate cycle, isocitrate lyase and malate synthase. These activities are all markedly increased in brown fat obtained from the hibernating animal. Additionally, hibernation enhances the ability of the tissue to synthesize glycogen in the presence of a fatty acid substrate. The glyoxylate cycle enzymes and the ability to convert fatty acid carbons to glucose have been generally regarded as being absent from vertebrate cells and tissues.

Serum angiotensin-converting enzyme in multiple sclerosis.

OBJECTIVE: To determine the extent and significance of serum angiotensin-converting enzyme (ACE) elevation in multiple sclerosis (MS) and the correlation between serum ACE activity and clinical and magnetic resonance imaging (MRI) indicators of disease activity. DESIGN: A retrospective cross-sectional study of 45 consecutive patients with clinically definite MS and a longitudinal study of 30 additional patients with clinically definite MS involved in a long-term study of neurologic function and MRI in MS. SETTING: Comprehensive MS center of a tertiary care university hospital. SUBJECTS: A total of 75 patients with clinically definite MS and 31 healthy controls. METHODS: Serum ACE activity was measured using a spectrophotometric assay and correlated with clinical indicators of disease activity and with total cerebral MS lesion volume measured by MRI. RESULTS: An elevated ACE activity was found in 17 (23%) of 75 patients with MS as compared with 2 (6%) of 31 healthy controls. Changes in serum ACE activity correlated with changes in total plaque volume on MRI. CONCLUSIONS: Serum ACE activity may be an indicator of disease activity in longitudinal analysis. Also, elevated ACE activity in a patient with otherwise typical MS need not raise suspicions of alternative diagnoses.

The glyoxylate cycle in rat epiphyseal cartilage: the effect of vitamin-D3 on the activity of the enzymes isocitrate lyase and malate synthase.

The effect of vitamin-D deficiency and subsequent vitamin-D replacement on the metabolism of rat epiphyseal growth plate cartilage was studied. Biochemical analyses showed the presence of the two unique glyoxylate cycle enzymes isocitrate lyase and malate synthase in cartilage. The activity of these enzymes was markedly increased after treatment with the vitamin. Additionally, rat cartilage showed the capacity to oxidize fatty acid in the presence of cyanide. This cyanide-insensitive fatty acid oxidation is characteristic of peroxisomal B-oxidation rather than mitochondrial B-oxidation. Vitamin-D treatment also increased fatty acid oxidation. Lastly, incubation of rat cartilage in the presence of a fatty acid substrate such as palmitate, resulted in a higher tissue glycogen content. Tissue glycogen was further elevated by vitamin-D. Such data indicate the presence of glyoxylate cycle enzymes in a vertebrate tissue and raise the possibility that mammalian cartilage has the capacity to convert lipid to carbohydrate.

Phenytoin hypersensitivity: a case of severe acute rhabdomyolysis.

A 22-year-old black man was hospitalized with fever, rash, myalgias, and marked periorbital and facial edema three months after beginning phenytoin therapy. His hospital course was marked by an initial serum creatine phosphokinase level of 85,000 IU/liter, which rose to a peak value of 242,000 IU/liter on the third hospital day and a striking eosinophilia (8,400/microliter). Muscle biopsy revealed only early fiber necrosis with partial dissolution of the sarcoplasm without evidence of inflammation, vasculitis, regeneration, or parasitic infection. When therapy with phenobarbital, a structural congener of phenytoin, was begun, the patient had an exacerbation of his rash and became febrile again.

Microperoxisomes in the epithelial cells of the amphibian urinary bladder: an electron microscopic demonstration of catalase and malate synthase.

All cells that comprise the epithelium of the toad urinary bladder were found to contain small ovoid to tubular membrane-bound bodies with a finely granular matrix. Such organelles were devoid of dense cores (nucleoids). These microperoxisomes reacted positively when incubated for the demonstration of catalase or malate synthase activity. In the toad liver, peroxisomes as well as microperoxisomes were seen. Histochemically, both demonstrated catalase activity; neither showed malate synthase activity. The presence of malate synthase, a glyoxylate cycle enzyme, in toad urinary bladder microperoxisomes may make these latter organelles unique among vertebrates, since malate synthase has been thought to be absent in higher animals.

Cytochemical localization of malate synthase in amphibian fat body adipocytes: possible glyoxylate cycle in a vertebrate.

The adipocytes of amphibian abdominal fat bodies contain typical microperoxisomes, as indicated by their fine structure. Electron microscopic cytochemistry showed that these organelles contain the enzymes catalase, typical for peroxisomes, and malate synthase. The latter is an enzymatic component characteristic of the glyoxylate cycle, a biochemical pathway known to exist in plant glyoxysomes (peroxisomes). This metabolic pathway makes possible the net conversion of lipid to carbohydrate. Toad adipocytes may represent yet another example of vertebrate peroxisomes which contain one of the marker enzymes (malate synthase) characteristic of the glyoxylate shunt.

Electron microscopic cytochemical localization of adenylate cyclase in amphibian urinary bladder epithelium: effects of antidiuretic hormone.

A cytochemical technique for electron microscopic localization of adenylate cyclase was used to identify this enzyme in quiescent and hormone-stimulated toad urinary bladder epithelium. In the absence of vasopressin (antidiuretic hormone), adenylate cyclase was detected along the outer surface of the basolateral plasma membranes of granular cells, mitochondria-rich cells, and basal cells, the major cell types comprising the hormone-sensitive urinary epithelium. In the presence of antidiuretic hormone, the basolateral precipitates were markedly increased. The latter was true for both tissues incubated in the presence of an osmotic gradient and those stimulated in the absence of such a gradient. A significant mucosal reaction was never seen. Such data indicate that the hormone receptors for vasopressin are located along the basolateral membranes of all epithelial cells comprising the mucosal hormone-sensitive epithelium. All cells of the epithelium also demonstrate a vasopressin-sensitive adenylate cyclase. We discuss possible mechanisms that attempt to integrate the cytochemical data into an overall scheme for the physiological action of this hormone on amphibian urinary bladder.