Rat juxtaglomerular cells are endowed with DA-1 dopamine receptors mediating renin release

Under control conditions a primary culture containing about 80-90% of granular juxtaglomerular (JG) cells prepared from rat kidneys continuously released renin into the culture medium at a rate of 17.9 +/- 1.4 ng angiotensin I/h per mg of cell proteins per 30 min (n = 14). Dopamine (1.0 microM), the DA-1 dopamine receptor agonist fenoldopam (0.5 microM), and isoproterenol (1.0 microM) increased renin secretion markedly (130-200%). Propranolol (0.1 microM) reduced the effects of isoproterenol significantly (80%), but not those of dopamine or fenoldopam. In contrast, SCH 23390 (0.01 microM), a DA-1 dopamine receptor antagonist, inhibited markedly only the renin release evoked by the latter two agonists, whereas S-sulpiride (10 microM), a DA-2 dopamine receptor antagonist, and phentolamine (10 microM), a nonselective alpha-adrenoceptor antagonist, did not modify the effects of either dopamine or fenoldopam. In rats, pithed to eliminate reflexogenic mechanisms regulating renin release, at the end of a 15 min i.v. infusion of fenoldopam (20 micrograms/kg per min) there was a significant increase in plasma renin activity. This effect was completely prevented by SCH 23390 (0.1 mg/kg i.v.) but not significantly changed by S-sulpiride (0.3 mg/kg i.v.) or phentolamine (3.0 mg/kg i.v.) plus propranolol (0.75 mg/kg i.v.). In conclusion, these results indicate that DA-1 dopamine receptors are present in rat kidney JG cells and that pharmacological stimulation of these receptors with dopamine or fenoldopam leads to renin secretion.     

Nerve growth factor secretion by human lung epithelial A549 cells in pro- and anti-inflammatory conditions.

Nerve growth factor (NGF) has recently been presented as a possible effector of inflammation and bronchial hyperresponsiveness. However, the production of NGF in human airways as well as the regulation of its expression by inflammatory cytokines and glucocorticoids have received little attention. A549 epithelial cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% foetal bovine serum, and starved for 24 h. The effect of the pro-inflammatory cytokine interleukin-1beta (1-30 U/ml), and of the glucocorticoid dexamethasone (1 microM) on NGF secretion was studied and quantified by enzyme-linked immunosorbent assay (ELISA). In addition, NGF production within the cells was visualized by immunocytochemistry. Under basal conditions, A549 cells produced and secreted NGF (12.6+/-2.0 pg/ml). Stimulation by interleukin-1beta for 24 h induced a dose-dependent increase in NGF production (maximal at 10 U/ml with 59.6+/-3.5% increase, P<0.05). Dexamethasone (1 microM) markedly reduced the constitute NGF secretion by 44.9% (7.0+/-2.1 pg/ml, P<0.001). In addition, the interleukin-1beta-stimulated NGF secretion was inhibited to approximately the same low level (8.5+/-2.5 pg/ml, P<0.001). In conclusion, we here report that human airway A549 epithelial cells are capable of producing NGF. This production is positively regulated by the pro-inflammatory interleukin-1beta, and negatively regulated by dexamethasone.     

Renal denervation causes chronic hypotension in rats: role of beta1-adrenoceptor activity

1. Renal denervation (RDNX) chronically lowers mean arterial pressure (MAP) in normal rats but mechanisms leading to this hypotensive response remain unknown. 2. We hypothesized that this sustained decrease in arterial pressure was because of a loss of beta1-adrenoceptor mediated renin secretion. Male Sprague-Dawley rats were assigned to sham (SHAM; n = 9), unilateral (UniRDNX; n = 9), or bilateral (RDNX; n = 10) renal denervation groups and instrumented for telemetric MAP measurements, plasma renin concentration (PRC) measurements and intravenous infusion. Twenty-four h MAP, heart rate, sodium and water balances were recorded 5 days before, 3 days during and 3 days after 1-adrenoceptor blockade with atenolol. 3. The 5-day control MAP was significantly lower in RDNX (97 +/- 1 mmHg) compared to SHAM (105 +/- 2 mmHg) and UniRDNX (102 +/- 2 mmHg) rats. No significant differences in basal PRC were observed between RDNX (2.2 +/- 0.3 ngAng1/mL per h), UniRDNX (2.6 +/- 0.4 ng/Ang1/mL per h) and SHAM (2.6 +/- 0.4 ngAng1/mL per h) rats. By day 1 of atenolol, PRC was significantly lower in UniRDNX rats (1.8 +/- 0.2 ngAg1/mL per h) compared to control values, but was unchanged during atenolol infusion in the other groups. By day 3 of atenolol, MAP was significantly decreased in all groups, but the absolute levels of MAP remained statistically different between RDNX (87 +/- 1 mmHg) and SHAM (91 +/- 1 mmHg) groups. 4. We conclude that the arterial pressure lowering effect of RDNX is not solely dependent on the loss of neural control of renin release.     

Response of normal human keratinocytes to sulfur mustard (HD): cytokine release using a non-enzymatic detachment procedure

Cytokines play a major role in both acute and chronic inflammatory processes, including those produced by sulfur mustard (HD). This study describes responses of normal human epidermal keratinocyte (NHEK) cells to 2,2'-dichlorodiethyl sulfide, sulfur mustard (HD), defined by interleukin-1 beta (IL-1beta), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-alpha) release. A new method for detaching cell to cell adhesion between keratinocytes has been applied. This method permits the characterization of endogenous fluid from cellular content that could be applied for the development of therapeutic intervention. NHEK (typical average cell density 4.4 x 10(6) cells/mL) were exposed to HD (100 and 300 microM) in keratinocyte growth medium (KGM) for 24 h at 37 C in humidified air. Commercially available enzyme-linked immunosorbent assay (ELISA) kits were used to measure the cytokine release in NHEK during exposure to 100 and 300 microM of HD. Exposure to 100 microM HD increased release of cytokines. IL-1beta (exposed: 1.41 x 10(-5) pg/ cell+/-1.60 x 10(-6) pg/cell: control 7.10 x 10(-6) pg/ cell+/-1.20 x 10(-6) pg/cell), TNF-alpha (exposed: 1.06 x 10(5) pg/cell+/-7.3 x 10(-7)pg/cell; control: 4.04 x 10(-6)+/-2.80 x 10(-7) pg/cell) and IL-8 (exposed: 3.71 x 10(-5) pg/ cell+/- 3.26 x 10(-6) pg/cell; control: 2.99 x 10(-6) pg/cell+/-8.80 x 10(-7) pg/cell) were significantly enhanced when NHEK cells were detached from culture flasks by non-enzymatic procedures. Cell suspensions of NHEK released low amounts of IL-6 when exposed to 100 microM for 24 h (exposed: 1.47 x 10(-6)+/-1.60 x 10(-7) pg/cell; control: 1.28 x 10(-6)+/-8.40 x 10(-8) pg/cell). However, cell suspensions of NHEK increased levels of IL-6 after exposure to 300 microM HD (4.67 x 10(-5) pg/cell+/-3.90 x 10(-6) pg/cell; control: 3.99 x 10(-6) pg/cell+/-5.50 x 10(-7) pg/cell). The amount of IL-8 and TNF-alpha present in cell suspensions increased up to 59-fold and fourfold, respectively, above control levels when NHEK cells were exposed to 300 microM HD. Exposure of NHEK to 300 microM HD had a highly variable effect on the release of IL-1beta, where sometimes the secretion of IL-1beta increased above baseline level and other times decreased in cell suspensions. Supernatants were collected from cell culture flasks 24 h after exposure of 100 and 300 microM and significantly increased levels of IL-6 were observed. IL-6 was released in a concentration-dependent manner, 3.6-fold up to 8.4-fold, respectively, in supernatant. These pro-inflammatory mediators IL-1beta, IL-8, TNF-alpha and IL-6 may play an important role in HD injury. The present findings suggest that cytokine changes detected could be used as potential biomarkers of cutaneous vesicant injury.     

Secretion of macrophage migration inhibitory factor differs from interleukin-6 in hydrogen peroxide- and LPS-stimulated human fibroblasts

To investigate the mechanism for secretion of macrophage migration inhibitory factor (MIF) in cultured human fibroblasts, we compared it with the secretion of interleukin-6 (IL-6) after stimulation with lipopolysaccharides (LPS) and H2O2. MIF content of the medium of 2.0 x 10(6) cells/20 ml after 20 h culture of nonstimulated fibroblasts was 0.30 +/- 0.06 ng/ml, whereas LPS-stimulation (10 microg/ml) only led to a 1.5-fold increase as compared with the nonstimulated cells. In contrast, a significant increase of IL-6 was induced by LPS-stimulation (6048 +/- 488 pg/ml in LPS-stimulated cells vs. 58 +/- 36 pg/ml in control cells). On the other hand, higher concentrations of H2O2 (0.6-1.2 mM) caused an increase of MIF secretion into the culture medium irrespective of LPS-stimulation; with 1.2 mM H2O2-stimulation for 20 h, it was increased to 40-fold as compared with the nonstimulated cells. However, lower concentrations (0.1-0.4 mM) did not cause this. Interestingly, H2O2-stimulation not only failed to increase IL-6 production from fibroblasts, but also repressed induction of IL-6 by LPS-stimulation in a dose-dependent manner. Genistein, a tyrosine kinase inhibitor, and H-7, a protein kinase C inhibitor, also inhibited IL-6 secretion but not MIF secretion in both LPS- and H2O2-stimulated fibroblasts. From analysis of trypan blue exclusion, formazan formation, morphological changes, and intracellular MIF content by Western blotting, we found that MIF secretion by H2O2 seemed to be mainly due to cell death and subsequent leakage of intracellular MIF. Taken together, these results suggest that MIF secretion differs from IL-6 via LPS-mediated signaling pathways.     

Intrarenal stimulation of renin secretion by frusemide in the isolated kidney of the rat.

1. Intrarenal infusion of frusemide markedly stimulated renin secretion in the isolated perfused kidney of the rat. 2. Renin vales increased from 24+/-6 to 195+/-34 units of secretion rate (renin concentration (nmol angiotensin I/h per litre) X flow rate (ml/min)) following administration of frusemide for 8 min, compared with corresponding control values of 13 +/- 2 (P greater than 0.05) and 47 +/-18 (P less than 0.001). This stimulatory effect was also observed when urine flow was interrupted by ligation of the ureters. 3. The changes in renal perfusion pressure and perfusate flow rate were not significantly differnt from the control values. 4. These findings indicate the existence of an intrarenal site of action for frusemide on renin secretion. 5. Since frusemide did dot appear to alter perfusion pressure or flow rate, and was effective when urine flow was abolished, a dominant role for a vascular or macula densa receptor mechnaism seems unlikely. A direct effect of frusemide on the renin secreting cell is therefore suggested.     

Pharmacological manipulation of cyclo-oxygenase-2 in the inflamed hydronephrotic kidney.

1. Bradykinin (BK, 1 microgram) caused a small (2 fold at 6 h) increase in prostaglandin E2 (PGE2) in the normal rabbit kidney, perfused ex vivo. This was exaggerated (6 fold at 6 h) in the hydronephrotic kidney (HNK). The exaggerated release of PGE2 was attenuated by cycloheximide, an inhibitor of protein synthesis or by dexamethasone, a steroid known to inhibit the induction of cyclo-oxygenase (COX-2). BK (1 microgram) when injected at 6 h of perfusion increased the release of PGE2 from 90 +/- 33 pg ml-1 min-1 to 3069 +/- 946 pg ml-1 min-1. This was reduced to 200 +/- 30 pg ml-1 min-1 in kidneys infused with cycloheximide (1 microM) and to 250 +/- 40 pg ml-1 min-1 in kidneys infused with dexamethasone (n = 8). 2. When tested on human and murine recombinant COX-1 and COX-2 enzymes, DuP-697 was at least 50 fold more selective for COX-2 than for COX-1. 3. DuP-697 reduced the exaggerated release of PGE2 elicited by BK in the HNK (e.g., at 6 h of perfusion BK-evoked PGE2 release decreased from 3069 +/- 946 pg ml-1 min-1 to 187 +/- 22 pg ml-1 min-1 after perfusion with 1 microM DUP-697, n = 8). 4. Cycloheximide, dexamethasone or DuP-697 at doses used to inhibit completely the exaggerated release of PGE2 in the hydronephrotic kidney, failed to inhibit the release of PGE2 elicited by the injection of BK (1 microgram) in the normal contralateral kidney. 5. Indomethacin (1 microM), a non-selective COX-1 and COX-2 inhibitor, completely inhibited PGE2 release in the normal contralateral as well as in the hydronephrotic kidney. 6. We suggest that renal prostaglandin production in the normal kidney is driven by the activity of constitutive COX-1 while at sites of inflammation, such as the hydronephrotic kidney, there is induction of COX-2 that can be blocked selectively by anti-inflammatory glucocorticoids or selective COX-2 inhibitors.     

Effects of organochlorine pesticides on interleukin secretion from lymphocytes

Organochlorine pesticides have been used worldwide primarily as insecticides. Due to their chemical stability, they often persist in the environment long after their use has ceased. In a previous study, we found that six organochlorine compounds (alpha-chlordane, gamma-chlordane, 4,4'-DDT, heptachlor, oxychlordane, and pentachlorophenol (PCP)), at concentrations of 5 microM, were able to significantly decrease the ability of highly purified human natural killer (NK) cells to lyse tumor cells after exposures, ranging from 1 hour to 6 days. However, if T cells were present with the NK cells (T/NK cells), loss of lytic function was seen only with oxychlordane and PCP. The purpose of the current study is to begin to investigate the mechanism by which T cells may be blocking the negative effects of some organochlorine compounds on NK cell function. Here, we investigated the hypothesis that T cells could produce significant levels of NK-stimulatory interleukin(s) (ILs), and that this may account for the decreased inhibition seen with organochlorine exposures when T cells were present. Secretion of four cytokines that have a demonstrated capacity to influence NK function, and/or are secreted by T cells, was measured (IL-2, IL-4, IL-10, IL-12). We measured both the baseline levels of ILs and the effects of organochlorine compound on IL secretion in T/NK cells. The results showed that baseline levels of the NK-stimulatory IL, IL-12, were 898 +/- 264 pg/mL at 24 hours and IL-10 levels were 564 +/- 337 pg/mL. In contrast, IL-2 levels were 14 +/- 10 pg/mL, and IL-4 levels were 3 +/- 2 pg/mL at 24 hours. The two compounds that retained their capacity to decrease NK lytic function in T/NK cells, oxychlordane (5 microM) and PCP (5 and 10 microM), were able to either decrease the secretion of NK-stimulatory ILs (IL-2, IL-12 and/or IL-10) and/or increase secretion of the NK-inhibitory cytokine, IL-4, at each length of exposure tested.     

Frusemide releases renin in the rat kidney when prostacyclin synthesis is suppressed.

The effect of inhibiting prostaglandin (PG) synthesis on basal and frusemide-stimulated renin secretion was examined in the rat isolated perfused kidney. The stable PGI2 derivative, 6-keto PGF1 alpha, was measured by radioimmunoassay in urine collected from the kidney. Treatment of rats with indomethacin (3.0 mg kg-1) reduced 6-keto PGF1 alpha excretion from 121.3 +/- 39.1 (n = 9) to 15.5 +/- 6.6 (n = 9) pg min-1 (P less than 0.02) but had no effect on basal renin secretion. Renal perfusion pressure, flow rate and vascular resistance were similar in treated and control rats. Mean urine flow was lower after treatment. Infusion of frusemide (250 micrograms min-1) did not alter 6-keto PGF1 alpha excretion in control or indomethacin-treated (P greater than 0.05) rats. Although renin secretion was increased during frusemide infusion, there was no significant difference between control (1,806 +/- 384 ng angiotensin I (AI) min-1) and treated (2,310 +/- 554 ng AI min-1) rats (P greater than 0.05). Propranolol, at a dose (8 micrograms min-1) which suppressed renin secretion after isoprenaline stimulation, had no effect on the response to frusemide in indomethacin-treated rats. These results demonstrate that frusemide-stimulated renin secretion in the rat kidney does not require intact renal PGI2 synthesis and is independent of beta-adrenergic mechanisms.     

Neurohormonal and blood pressure responses to low-dose infusion of an orally active renin inhibitor, Ro 42-5892, in salt-replete men.

Renin inhibitors are an alternative means of blockade of circulating and tissue-based renin-angiotensin systems (RAS). We studied a new renin inhibitor, Ro 42-5892, by low-dose (0.1 mg/kg) intravenous (i.v.) infusion in 10 min (fast) or 6 h (slow) or placebo in a double-blind cross-over study to assess the relationship between drug concentration and response. Fasting salt-replete normotensive male volunteers (n = 9) aged 18-32 years were studied supine. There were no significant changes in blood pressure (BP) or heart rate (HR) between drug and placebo infusion. Drug concentration peaked (482 +/- 140 ng/ml) at the end of the fast infusion or showed a sustained plateau (25.9 +/- 6.1 ng/ml) with the slow infusion (mean time to peak 121 +/- 99 min). Both fast (135.2 +/- 26 ng/ml/h2) and slow (121.0 +/- 31.1 ng/ml/h2) infusions had similar area under the curve (AUC)0-24-values. Plasma renin activity (PRA) was dramatically reduced by both strategies, but AUC0-10 for PRA was significantly less for slow (1.7 +/- 0.6 ngAI/ml/h2) than fast (4.9 +/- 2.5 ngAI/ml/h2) infusions. Mean peak plasma active renin (AR) concentration was increased by both fast (102.2 +/- 65.9 pg/ml) and slow (195.2 +/- 110.5 pg/ml) infusions as compared with placebo (49.9 +/- 18.6 pg/ml). Similarly, AUC0-10 for AR was greater for slow (990.2 +/- 582.1 pg/ml/h) than fast (512.4 +/- 189.4 pg/ml/h) infusions. Plasma angiotensin-converting enzyme (ACE) activity was unaltered. Our results indicate that protracted low concentrations of Ro 42-5892 may provide more effective and long-lasting inhibition of renin.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison of the effects of verocytotoxin-1 on primary human renal cell cultures

Infection with verocytotoxin-producing Escherichia coli causes haemolytic uraemic syndrome (HUS). Verocytotoxin-1 (VT1) is cytopathic to renal microvascular endothelial cells in culture, supporting the hypothesis that the vasculopathy of HUS is caused directly by the toxic action of VT1 on cells. We provide evidence that VT1 inhibits protein synthesis in primary cultures of glomerular epithelial cells (GE), cortical tubular epithelial cells (CTE) and mesangial cells (MC). Using 100 pg/ml of VT1 we saw a decrease in protein synthesis to 14.3+/-1.9% in vero cells (a primate cell line), 1.7+/-0.3% in GE, 0.9+/-0.4% in CTE and 74.8+/-1.3% in MC at 24 h. The human renal epithelial cells are at least as sensitive as vero cells to the protein synthesis inhibitory effects of VT1 if not more so. Cell viability decreased in all cultures as measured by MTT reduction, neutral red incorporation and lactate dehydrogenase release and followed the same pattern of susceptibility as for protein synthesis inhibition. However, unlike vero cells, death occurred without DNA fragmentation. Cell sensitivity was greatest in cells which bound more VT1.     

Neurotensin potentiates the potassium-induced release of endogenous dopamine from rat striatal slices

The relationship between the renin-angiotensin system (RAS) and prostacyclin (PGI2) biosynthesis was studied in experimental diabetic rats. The group with diabetes induced by streptozotocin (STZ, 3.3 mmol/kg i.v.) showed prolonged hypertension, and plasma renin activity decreased markedly from 8.4 +/- 0.7 to 2.4 +/- 0.3 and 1.2 +/- 0.3 ng angiotensin I/ml per h at 2 and 8 weeks after STZ treatment. Plasma PGI2, determined as 6-keto-PGF1 alpha, decreased significantly at 8 weeks, with the values for the STZ-treated and control groups being 1490 +/- 99 and 2210 +/- 90 pg/ml, respectively. Significant suppression of renin release from renal cortical slices was observed at 8 weeks in the diabetic group, although no significant change was found in the renal renin content when compared with that of the controls. The release of PGI2 from the renal medullary slices of the diabetic group was suppressed at 2 and 8 weeks, with the suppression in aorta and renal cortical slices being apparent only at 8 weeks. These results indicate that suppression of the RAS may be related to PGI2 biosynthesis in diabetes mellitus.     

Effect of glucagon on renin secretion in the dog.

The effects of glucagon alone or in combination with theophylline on renin section were studied in relation to renal hemodynamic responses in anesthetized dogs. The intrarenal infusion of glucagon (0.5 microgram/kg/min) increased heart rate, renal blood flow, glomerular filtration rate and urine flow without any effect on renin secretion, but at a higher dose (1.0 microgram/kg/min) it increased renin secretion significantly. Theophylline (0.1 mg/kg/min) did not affect renal hemodynamics but caused a slight increase in renin secretion after 30--60 min infusion. The combined infusion of glucagon (0.5 microgram/kg/min) with theophylline (0.1 mg/kg/min) increased renin secretion markedly, although it produced renal hemodynamic changes similar to those induced by glucagon alone. These effects were not suppressed by d,l-propranolol (1.0 microgram/kg/min). It is suggested that the increase in renin secretion caused by the combined infusion of glucagon and theophylline resulted mainly from an increase in cyclic AMP in the juxtaglomerular cells, and not from stimulation of beta-adrenoceptors.     

Arterial and endocrine effects of a combination of an angiotensin converting enzyme inhibitor and a vasodilator in normotensive healthy volunteers.

The aim of the present study was to look for possible additive or synergistic effects of the combined oral administration of a single dose of an angiotensin converting enzyme (ACE) inhibitor, benazepril (10 mg) (B), and a long-acting vasodilator, cadralazine (5 mg) (C), on blood pressure, arterial parameters, and active plasma renin. The study was carried out in eight normotensive subjects according to a double-blind, randomized, placebo-controlled, crossover design. Blood pressure (BP), heart rate, humeral artery diameter (D), carotid-femoral pulse-wave velocity (PWV), finger-pulse ratio (FPR), and plasma active renin (PAR) were measured at baseline and every 2 h over 8 h. A significant treatment effect was observed for supine and tilted BP, FPR, PWV, and PAR. The largest decrease in supine systolic and diastolic BP was observed with the combination (10.0 +/- 6.9/7.2 +/- 3.7 mm Hg). Six hours after drug intake, the mean changes in FPR were 0.05 +/- 0.24 (P), -0.06 +/- 0.30 (C), 0.13 +/- 0.32 (B), and 0.28 +/- 0.34 (B + C), and the mean changes in PWV were 0.14 +/- 0.66 m/s (P), 0.09 +/- 0.54 m/s (C), -0.29 +/- 0.50 m/s (B), and -0.55 +/- 0.48 m/s (B + C). PAR was more markedly augmented with the combination of the two drugs (142 +/- 40 pg/ml) than with benazepril alone (90 +/- 62 pg/ml). It was concluded that a single noneffective dose of a vasodilator administered together with an ACE inhibitor in normotensives can lower blood pressure and increase arterial compliance and plasma active renin.     

Effect of prenalterol, a beta-1-adrenoceptor agonist, on renin secretion rate in the anaesthetized dog

The effects of the beta-1-adrenoceptor agonist, prenalterol, 20 microgram/kg intravenously on renin secretion rate (RSR), renal haemodynamics and sodium excretion were examined in anaesthetized dogs with innervated or denervated kidneys. In dogs with innervated kidneys, prenalterol increased RSR from 1.1 +/- 0.2 to 7.9 +/- 0.1 units X min.-1 X g-1 (P less than 0.01). Prenalterol did not affect mean arterial pressure, renal blood flow, glomerular filtration rate or sodium excretion. Heart rate was increased by 53 +/- 17 beats/min. (P less than 0.01). The increase in RSR produced by prenalterol was independent of intact renal innervation as RSR increased to the same extent in dogs with denervated kidneys. Pretreatment with the beta-1-adrenoceptor antagonist, metoprolol 0.5 mg/kg intravenously, abolished the increase in RSR produced by prenalterol. These findings suggest that prenalterol directly activates renal beta-1-adrenoceptors to increase RSR.     

Differential effects of homocysteine on porcine endothelial and vascular smooth muscle cells

High concentrations of homocysteine damage endothelial cells and lower concentrations increase vascular smooth muscle cell (VSMC) growth. This study investigated the effects of various concentrations of homocysteine on endothelial cells (VECs) and VSMCs in terms of cell survival, proliferation, and function. VECs and VSMCs from porcine thoracic aorta were studied. These cells were exposed to homocysteine in concentrations of 20 microM, 400 microM, and 1 mM every 8 h for 24 h, and its effect on cell survival, proliferation, and function were studied using methylthiazoletetrazolium assay, [3H]-thymidine incorporation test, and 6-keto-prostaglandin F1alpha enzyme-linked immunosorbent assay for VECs, and platelet-derived growth factor (PDGF) enzyme-linked immunosorbent assay for VSMCs, respectively. In VECs, 20 microM of homocysteine reduced the viable cell count to 95 +/- 31%, 400 microM reduced it to 89 +/- 35%, 1,000 microM reduced it to *58 +/- 29% (control = 100 +/- 30%, n = 18, *p < 0.05). In VSMCs, 20 microM of homocysteine slightly increased the viable cell count to 106 +/- 30%, but there was no statistical significance; 400 microM of homocysteine reduced the viable cell count to *74 +/- 29%, 1,000 microM to *50 +/- 24% (control = 100 +/- 28%, n = 18, *p < 0.05). In VECs, 20 microM of homocysteine reduced [3H]-thymidine uptake by 98 +/- 14%, 400 microM reduced it by *82 +/- 17%, 1,000 microM reduced it by *66 +/- 17% (control = 100 +/- 12, n = 6, *p < 0.05), respectively. But in VSMCs, 20 microM of homocysteine significantly increased [3H]-thymidine uptake (*131 +/- 16%), and thereafter, homocysteine decreased VSMCs [3H]-thymidine uptake, 400 microM by *24 +/- 7%, 1,000 microM by *29 +/- 10% (control = 100 +/- 16, n = 6, *p < 0.05), respectively. Homocysteine decreased VEC prostacyclin secretion in a dose-dependent manner, 20 microM by 105 +/- 0.65 pg/100 microl, 400 microM by *100 +/- 2.37 pg/100 microl, 1,000 microM by *93 +/- 2.54 pg/100 microl (control = 107 +/- 1.26 pg/100 microl, n = 6, *p = 0.007). In VSMCs, 20 microM of homocysteine slightly increased PDGF secretion by 62.2 +/- 20.7 pg/100 microl, but there was little statistical significance (p = 0.13); 400 microM of homocysteine reduced PDGF secretion by *28.9 +/- 10.7 pg/100 microl, and 1,000 microM reduced it by *21.3 +/- 4.7 pg/100 microl (control = 54.5 +/- 9.3 pg/100 microl, n = 6, *p < 0.05). High concentrations of homocysteine damaged both VECs and VSMCs with respect to cell survival, proliferation, and function. By increasing exposure to homocysteine, it was shown that physiologic high concentrations of homocysteine enhanced VSMC proliferation.     

Inhibition of hepatitis B virus polymerase-activity by various agents. Transient expression of hepatitis B virus DNA in hepatoma cells as novel system for evaluation of antiviral drugs

The effect of three putative antiviral drugs--aciclovir (acyclovir, CAS 59277-89-3), zidovudine (azidothymidine, CAS 30516-87-1) and sorangicin B (a macrocyclic lactone, CAS 100415-25-6)--on replication and gene expression of hepatitis B virus (HBV) was studied in HepG2 cells. Transfection of these cells with cloned circular HBV DNA resulted in the production and secretion of virions into the medium. When antiviral drugs were added in increasing concentrations (aciclovir at 0.5 microgram/ml to 150 micrograms/ml, zidovudine 0.1 microgram/ml to 30 micrograms/ml, sorangicin B 1 micrograms/ml to 30 micrograms/ml), the activity of the viral polymerase decreased in a dose-dependent manner. Production of viral proteins as measured by the secretion of HBsAg and HBeAg into the medium was unaffected, suggesting interference of these drugs with viral DNA/RNA synthesis. It is concluded that aciclovir, zidovudine and sorangicin B inhibit the replication of HBV. Furthermore, the cell system used in our study appears to be suitable for the rapid testing of antiviral drugs and their evaluation for possible studies in vivo.     

Beryllium-stimulated production of tumor necrosis factor-alpha by a mouse hybrid macrophage cell line

Chronic beryllium disease (CBD) results from exposure to the light-weight metal beryllium (Be). In vitro stimulation of bronchoalveolar lavage cells from CBD subjects causes the production of high levels of TNF-alpha, IFN-gamma and IL-6. We tested the hypothesis that Be-stimulation might induce the production of TNF-alpha by macrophage cell lines. We observed that H36.12j cells (12j), a mouse hybrid macrophage cell line, but not other mouse and human macrophage cell lines, produced TNF-alpha upon Be-stimulation. The response was maximal at 100 microM BeSO4 and did not occur when 12j cells were stimulated with either aluminum sulfate or cobalt sulfate. Beryllium-stimulated the production of 725+/-25 pg/ml (mean +/- SEM) TNF-alpha protein by 12j cells as measured by ELISA of culture supernatants after 24 h. As measured by RT-PCR, Be-stimulated 12j cell TNF-alpha protein production was accompanied by an increased intracellular TNF-alpha mRNA at 3 and 24 h. The addition of 10U or 100U of rMu-IFN-gamma to Be-stimulated 12j cells further increased TNF-alpha production 1.5-4 fold (1.6+/-0.1 ng/ml) respectively. Bacterial lipopolysaccharide (LPS, 1 microg/ml) stimulated production of TNF-alpha in 12j culture supernatants after 6 h (515+/-151 pg/ml). This early versus late TNF-alpha production suggests that LPS and Be both stimulate 12j cell TNF-alpha synthesis, but through different pathways. We report for the first time, the direct effects of Be stimulation on the ability of 12j cells to produce TNF-alpha. The 12j cell line, contrasted with other macrophage hybrids that do not respond to Be-stimulation, may provide a useful tool to evaluate the mechanisms by which Be stimulates macrophage cytokine production, and by which T cell derived IFN-gamma amplifies TNF-alpha production in granulomatous diseases.     

Stable expression, secretion, and characterization of active human renin in mammalian cells.

Human renin is synthesized as a 406-amino acid preprorenin protein that is processed by a signal peptidase during secretion, to release prorenin as a 386-amino acid zymogen. The 46-amino acid "pro" domain is removed by a renin-processing enzyme, to produce enzymatically active renin, by cleavage at an Arg-Leu bond. The effects of the renin-processing enzyme can be mimicked by trypsin activation, where high concentrations of trypsin are incubated with prorenin for brief periods of time, followed by excess trypsin inhibitor to minimize secondary proteolytic processing by trypsin. In order to study the role of the pro segment in the secretion, folding, and activity of human renin, we engineered a construct where the pro domain from the preprorenin cDNA was deleted. This construct was introduced into mammalian cells and its expression was assayed in transient and stable systems. In COS-1 cells transfected with the prerenin expression vector pREN3, active renin was secreted with a specific activity of 1360 micrograms of angiotensin l/min/mg, compared with trypsin-activated prorenin, which has a specific activity of 818 micrograms of angiotensin l/min/mg. The active renin secreted in this system had a significantly reduced potency for the renin inhibitor SQ 32,970. These results demonstrate that the pro segment is dispensable for the folding and secretion of renin. A permanent cell line expressing the active form of renin was obtained by co-transfection of NRP cells with pREN3 and pHyg. A colony designated B/1 was identified, subcloned, and shown to secrete active renin (110 pg of renin/10(6) cells) optimally when maintained in both G418 and hygromycin.