Potentiation of angiotensin II action by corticosteroids in vascular tissue.

OBJECTIVES: Studies were performed to determine if corticosteroids act directly on the vasculature to potentiate the vasoconstrictor action of angiotensin II and to determine whether corticosteroids upregulate angiotensin II receptors by receptor redistribution or by synthesis of new receptors. METHODS: Aortic rings from normal Sprague-Dawley rats were incubated ex vivo with corticosteroids in aerated Krebs-Henseleit buffer to avoid secondary systemic effects prior to stimulated contraction. In cultured vascular smooth muscle cells, these experimental techniques were used: colchicine (blocker of microtubule assembly), chloroquine (inhibitor of endosomal pH gradients), measuring surface-bound 125I-Ang II internalization rate, immunoblotting of angiotensin AT1 receptor protein, and incorporation of [35S]methionine into AT1 receptor protein. RESULTS: Contractions to 100 nM angiotensin II in rings incubated with 1 microM aldosterone or dexamethasone for 10 min ex vivo were not different from contractions in control rings. However, angiotensin II-stimulated (but not KCl-stimulated) contractions were enhanced by almost 100% if ex vivo incubation with aldosterone (or corticosterone) lasted for 24 h. Endothelium-dependent relaxation was not significantly reduced by aldosterone pre-incubation. Incubation of cultured vascular smooth muscle cells with a number of corticosteroids for > 8 h resulted in concentration-dependent upregulation of angiotensin II receptor binding and was reversible upon removal of the corticosteroid. Aldosterone did not affect the rate of internalization of surface-bound angiotensin II. In addition, concomitant incubation of colchicine or chloroquine with aldosterone did not hamper angiotensin II receptor upregulation. Incubation of cells with various concentrations of aldosterone for 24 h resulted in concentration-dependent increases in total cell angiotensin II receptor protein content and increases in [35S]methionine incorporation into immunoprecipitated AT1 receptor protein. CONCLUSIONS: At least a portion of the enhancement of angiotensin II action by corticosteroids is via direct interaction of corticosteroids with the vasculature. Corticosteroids appear to upregulate angiotensin II receptors by synthesis of new receptor protein rather than by alterations in receptor trafficking.

Carbenoxolone damages endothelium and enhances vasoconstrictor action in aortic rings.

Carbenoxolone causes hypertension indirectly by inhibition of 11beta-hydroxysteroid dehydrogenase and consequent elevation of intracellular glucocorticoid levels and enhancement of vasoconstrictor action. We performed the present study to determine whether carbenoxolone also enhances vascular tone directly by mechanisms independent of glucocorticoids and other systemic influences. Exposure of rat aortic rings to 10 to 100 micromol/L carbenoxolone in aerated Krebs-Henseleit buffer for 24 hours resulted in concentration-dependent increases in angiotensin II (Ang II) (100 nmol/L)-stimulated contractions and significant shifting of the phenylephrine cumulative contraction curve to the left but not increases in KCI (120 mmol/L)-stimulated contractions. Maximal enhancement of Ang II contraction was 39 percent. In contrast, brief (15-minute) exposure to 100 micromol/L carbenoxolone did not alter Ang II contractions. Mechanical denudation of the endothelium obviated enhancement of Ang II contractions by carbenoxolone, suggesting interaction of carbenoxolone with the endothelium. Endothelium-dependent relaxation of precontracted rings to acetylcholine or ATP was reduced by more than 90 percent by 24-hour pretreatment with 100 micromol/L carbenoxolone but not with 100 micromol/L deoxycorticosterone acetate (a mineralocorticoid) or 100 mu mol/L glycyrrhizic acid (a natural 11beta-hydroxysteroid dehydrogenase inhibitor). Vascular smooth muscle relaxation with sodium nitroprusside was not inhibited by carbenoxolone. Incubation of cultured endothelial cells with 100 mu mol/L carbenoxolone for 24 hours did not inhibit nitric oxide synthase activity, as measured by conversion of [3H]L-arginine to [3H]L-citrulline. Electron micrography demonstrated that endothelial cell ultrastructure but not vascular smooth muscle cell ultrastructure was abnormal after incubation of rings for 24 hours with 100 micromol/L carbenoxolone. These studies suggest that carbenoxolone concentrations higher than 10 micromol/L enhance vasoconstrictor action via selective toxicity to the endothelium and elimination of endothelium-dependent relaxation.

Corticosterone metabolism and effects on angiotensin II receptors in vascular smooth muscle.

It has been postulated that mineralocorticoids can bind to corticosteroid receptors in the kidney, because glucocorticoids are metabolized to inactive compounds. The present study was performed to delineate glucocorticoid metabolism by rat vascular tissue and to determine the activity of these metabolites. Vascular segments converted 25% to 30% of corticosterone (compound B), the major glucocorticoid in the rat, to 11-dehydrocorticosterone (compound A) but not to aldosterone or 6 beta-hydroxycorticosterone. In cultured vascular smooth muscle cells, 10% of compound B was converted to compound A, whereas > 60% of compound A was converted to compound B. The 11 beta-hydroxysteroid dehydrogenase inhibitor carbenoxolone (1 mumol/L) completely blocked conversion in both directions. Whereas 6 beta-hydroxycorticosterone did not upregulate angiotensin II receptor binding (a marker for corticosteroid action in vascular smooth muscle), compound A caused concentration-dependent upregulation. Compound A was almost (75%) as effective and as potent as compound B in upregulating angiotensin II binding. Upregulation elicited by exposure to compound A persisted in the presence of 1 mumol/L carbenoxolone, which completely prevented the conversion of compound A to compound B. Compound A, even in the presence of carbenoxolone, effected other glucocorticoid actions by inhibiting cell growth and potentiating angiotensin II-stimulated inositol phosphate formation. In summary, compound B and compound A are interconverted in vascular tissue, and the latter displays significant glucocorticoid action. The concentration excess of compound B in the circulation and the activity of its metabolite compound A will make it difficult for mineralocorticoids to gain access to corticosteroid receptors in the vasculature.

Potentiation of angiotensin II-stimulated vascular contraction by lithium.

Previous studies have suggested that lithium prolongs or enhances vascular contractions stimulated by alpha-adrenergic agents. The present study was performed to determine whether a similar phenomenon occurs with angiotensin II (ANG II)-stimulated contractions and whether this phenomenon results from interactions with the phosphoinositide signaling system. Contractions of rat aortic rings with 100 nM ANG II were 38% greater in the presence of 20 mM LiCl than in its absence (0.47 +/- 0.07 vs. 0.34 +/- 0.05 g tension/mg dry tissue wt, P < 0.01). The effects of lithium on inositol phosphate responses, diacylglycerol responses, and intracellular calcium concentration on single or repeated stimulations with ANG II were then examined in vascular smooth muscle cells cultured from rat aorta. Cells exposed twice to 100 nM ANG II contained 50% lower inositol trisphosphate levels (InsP3) and 10% lower diacylglycerol levels than cells exposed to ANG II only once. LiCl or lithium acetate abolished these desensitizations in a concentration-dependent manner. Similarly, InsP3 and diacylglycerol responses to a single exposure of ANG II were heightened by lithium (by 75 and 25%, respectively), and the duration of the responses was prolonged by lithium (5- and 2-fold, respectively). In contrast, ANG II-stimulated calcium transients were not enhanced or prolonged by lithium, nor was desensitization of ANG II-stimulated cytosolic calcium mobilization upon serial exposures abolished by lithium. When ring contraction studies were repeated in the presence of the protein kinase C inhibitor staurosporine (150 nM), lithium no longer potentiated ANG II contractions [0.38 +/- 0.03 (control) vs. 0.35 +/- 0.06 g tension/mg dry tissue wt (lithium)].(ABSTRACT TRUNCATED AT 250 WORDS)

Electron probe microanalysis of silicon and the role of the macrophage in proximal (capsule) and distant sites in augmentation mammaplasty patients.

Electron probe x-ray microanalysis was used to locate silicon (Si) within macrophages from 12 women who had previously undergone polymer prosthesis augmentation or reconstruction. Silicon was identified within macrophages and extracellularly in all fibrous breast capsules. In four women with arthritic joint pain and one woman with sclerodermatous skin lesions, silicon also was identified within macrophages residing in joint synovium and skin, respectively. It is suggested that the silicon-laden macrophages observed in the remote lesions may have accumulated silicon from other macrophages that had previously resided in the connective-tissue capsule around the silicone breast implants.

Cryo-jet preservation of calcium in the rat spinal cord.

In order to determine the distribution of diffusible ions, especially calcium, in rat spinal cord axons before and after trauma, sham, 6 hours post 6 gm/cm and 20 gm/cm trauma cords were cryo-jet frozen in situ and cryo-sections were subjected to electron probe X-ray microanalysis. The results confirm part of the Ca hypothesis, i.e., some spinal cord axons accumulate intracellular Ca after traumatic injury. In addition, 6 hours after trauma, Ca is only accumulated by axons that have also lost homeostasis. Thus, it is likely that observations of the continual rise of Ca in the traumatized spinal cord are due to an increase in the number of axons that have lost homeostasis rather than an overall increase in Ca within surviving cells.

Endoscopic treatment of reflux: migration of Teflon to the lungs and brain.

We set out to determine experimentally whether particles of polytetrafluoroethylene migrate to the lungs and brain when relatively small volumes of Teflon paste are injected into the bladder in the manner used to correct reflux. Numerous particles of polytetrafluoroethylene were recovered from these organs within 2 weeks of injection. Those in the brain measured up to 15 microns in diameter, indicating that the pulmonary bed is an inefficient filter of particles gaining access to the venous circulation. Although clinically no adverse neurological effects have hitherto been reported, this study suggests that following the 'sting' procedure, some particles may lodge in the brain where they can block the cerebral microcirculation. We believe these findings represent a contraindication to the use of Teflon paste in children.

An improved cryo-jet freezing method.

A new cryo-jet freezing apparatus is described that is easy to use and gives good results using a propane-butene mixture (3:1). Our use of the freezer in the study of mouse spinal cord explant cultures is discussed. At the tissue surface, the quality of tissue preservation from freezing, followed by freeze substitution, rivals that of conventional electron microscopic methods. Certain intracellular structures are better visualized using our methods. There is no evidence of the tissue being distorted by the cryogen jet when the freezer is operated correctly. A new freeze substitution device is also discussed.

Subcellular electrolyte shifts during in vitro myocardial ischemia and reperfusion.

Isolated perfused rabbit right ventricular wall was studied with electron probe microanalysis (EPMA) under three conditions: 1) control (37 degrees C, 1.2 Hz), 2) 60 min global ischemia, and 3) ischemia plus 5 min of reperfusion. After 60 min of ischemia, only one cell population was evident; the variance of intracellular electrolyte concentrations was the same as in controls. When compared with controls, there was no change in Ca concentration within any region of the cell, but mitochondria were swollen with K-rich fluid. Two cell populations were evident after 5 min of reperfusion. The severely injured cells were markedly swollen, exhibited hypercontraction bands, and had electrolyte profiles similar to extracellular fluid. The moderately injured cells were normal in appearance, still retained electrolyte gradients, but had elevated Na and Cl concentrations in all compartments. Cell Ca did not increase in the moderately injured cells, but the region of the cell containing the sarcoplasmic reticulum (SR) lost 90% of its Ca. Accompanying this loss were large increases in myofibrillar and mitochondrial Ca concentration. It appears that release of SR Ca, loss of SR Ca-accumulating capacity, and increased intracellular Na are the principal electrolyte shifts in functional cells during early reperfusion.

Cellular compartmentation in ischemic myocardium: indirect analysis by electron probe.

Electron probe microanalysis (EPMA) was carried out directly on myocardial cells and on the myofibrils and the mitochondria within them. A third subcellular compartment, which contains sarcoplasmic reticulum (SR), was measured indirectly. The percent of the total cell calcium content that resides within this "hidden" compartment was calculated from cell data minus weighted myofibril and mitochondria data. This approach was applied to control, ischemic, and reperfused myocardium, and other elements were also quantified. We found that the calcium content of this third compartment is little changed during global ischemia but is markedly depleted after 5 min reperfusion. We conclude that these changes are ascribable to changes in SR function.

Passage and Survival of Chlamydospores of Phytophthora cinnamomi Rands, the Causal Agent of Forest Dieback Disease, Through the Gastrointestinal Tracts of Termites and Wild Birds.

Chlamydospores of Phytophthora cinnamomi Rands have been shown to survive in the intestinal tracts of termites (Nasutitermes exitiosus) and two species of forest birds indigenous to West Australian jarrah forests. Viable chlamydospores were recovered from bird feces within the normal rate of passage time for food through the gut. The above factors would allow these creatures to function as vectors for the spores.