Ulrich Hopfer

Role of hypoxia-induced Bax translocation and cytochrome c release in reoxygenation injury.

We investigated mechanisms of cell death during hypoxia/reoxygenation of cultured kidney cells. During glucose-free hypoxia, cell ATP levels declined steeply resulting in the translocation of Bax from cytosol to mitochondria. Concurrently, there was cytochrome c release and caspase activation. Cells that leaked cytochrome c underwent apoptosis after reoxygenation. ATP depletion induced by a mitochondrial uncoupler resulted in similar alterations even in the presence of oxygen. Moreover, inclusion of glucose during hypoxia prevented protein translocations and reoxygenation injury by maintaining intracellular ATP. Thus, ATP depletion, rather than hypoxia per se, was the cause of protein translocations. Overexpression of Bcl-2 prevented cytochrome c release and reoxygenation injury without ameliorating ATP depletion or Bax translocation. On the other hand, caspase inhibitors did not prevent protein translocations, but inhibited apoptosis during reoxygenation. Nevertheless, they could not confer long-term viability, since mitochondria had been damaged. Omission of glucose during reoxygenation resulted in continued failure of ATP production, and cell death with necrotic morphology. In contrast, cells expressing Bcl-2 had functional mitochondria and remained viable during reoxygenation even without glucose. Therefore, Bax translocation during hypoxia is a molecular trigger for cell death during reoxygenation. If ATP is available during reoxygenation, apoptosis develops; otherwise, death occurs by necrosis. By preserving mitochondrial integrity, BCL-2 prevents both forms of cell death and ensures cell viability.

Perturbation of D1 Dopamine and AT1 Receptor Interaction in Spontaneously Hypertensive Rats

Abstract—The dopaminergic and renin-angiotensin systems interact to regulate blood pressure. Because this interaction may be perturbed in genetic hypertension, we studied D1 dopamine and AT1 angiotensin receptors in immortalized renal proximal tubule (RPT) and A10 aortic vascular smooth muscle cells. In normotensive Wistar-Kyoto (WKY) rats, the D1-like agonist fenoldopam increased D1 receptors but decreased AT1 receptors. These effects were blocked by the D1-like antagonist SCH 23390 (10−7 mol/L per 24 hours). In spontaneously hypertensive rat (SHR) RPT cells, fenoldopam also decreased AT1 receptors but no longer stimulated D1 receptor expression. Basal levels of AT1/D1 receptor coimmunoprecipitation were greater in WKY RPT cells (29±2 density units, DU) than in SHR RPT cells (21±2 DU, n=7 per group, P <0.05). The coimmunoprecipitation of D1 and AT1 receptors was increased by fenoldopam (10−7 mol/L per 24 hours) in WKY RPT cells but decreased in SHR RPT cells. The effects of fenoldopam in RPT cells from WKY rats were similar in aortic vascular smooth muscle cells from normotensive BD IX rats, that is, fenoldopam decreased AT1 receptors and increased D1 receptors. Our studies show differential regulation of the expression of D1 and AT1 receptors in RPT cells from WKY and SHR. This regulation and D1/AT1 receptor interaction are different in RPT cells of WKY and SHR. An altered interaction of D1 and AT1 receptors may play a role in the impaired sodium excretion and enhanced vasoconstriction in hypertension.

Dopamine1 Receptor, Gsα, and Na+-H+ Exchanger Interactions in the Kidney in Hypertension

The ability of dopamine1 (D1) receptors to inhibit luminal Na+-H+ exchanger (NHE) activity in renal proximal tubules and induce a natriuresis is impaired in spontaneously hypertensive rats (SHR). However, it is not clear whether the defect is at the level of the D1 receptor, Gs&agr;, or effector proteins. The coupling of the D1 receptor to Gs&agr; and NHE3 was studied in renal brush border membranes (BBM), devoid of cytoplasmic second messengers. D1 receptor, Gs&agr;, and NHE3 expressions were similar in SHR and their normotensive controls, Wistar-Kyoto rats (WKY). Guanosine-5′-O-(3-thiotriphosphate) (GTP&ggr;S) decreased NHE activity and increased NHE3 linked with Gs&agr; similarly in WKY and SHR, indicating normal Gs&agr; and NHE3 regulation in SHR. However, D1 agonists increased NHE3 linked with Gs&agr; in WKY but not in SHR, and the inhibitory effects of D1 agonists on NHE activity were less in SHR than in WKY. Moreover, GTP&ggr;S enhanced the inhibitory effect of D1 agonist on NHE activity in WKY but not in SHR, suggesting an uncoupling of the D1 receptor from Gs&agr;/NHE3 in SHR. Similar results were obtained with the use of immortalized renal proximal tubule cells from WKY and SHR. We conclude that the defective D1 receptor function in renal proximal tubules in SHR is proximal to Gs&agr;/effectors and presumably at the receptor level. The mechanism(s) responsible for the uncoupling of the D1 receptor from G proteins remains to be determined. Because the primary structure of the D1 receptor is not different between normotensive and hypertensive rats, differences in D1 receptor posttranslational modification are possible.

Activation of D3 Dopamine Receptor Decreases Angiotensin II Type 1 Receptor Expression in Rat Renal Proximal Tubule Cells

The dopaminergic and renin angiotensin systems interact to regulate blood pressure. Disruption of the D3 dopamine receptor gene in mice produces renin-dependent hypertension. In rats, D2-like receptors reduce angiotensin II binding sites in renal proximal tubules (RPTs). Because the major D2-like receptor in RPTs is the D3 receptor, we examined whether D3 receptors regulate angiotensin II type 1 (AT1) receptors in rat RPT cells. The effect of D3 receptors on AT1 receptors was studied in vitro and in vivo. The D3 receptor agonist PD128907 decreased AT1 receptor protein and mRNA in WKY RPT cells and increased it in SHR cells. PD128907 increased D3 receptors in WKY cells but had no effect in SHR cells. D3/AT1 receptors colocalized in RPT cells; D3 receptor stimulation decreased the percent amount of D3 receptors that coimmunoprecipitated with AT1 receptors to a greater extent in WKY than in SHR cells. However, D3 receptor stimulation did not change the percent amount of AT1 receptors that coimmunoprecipitated with D3 receptors in WKY cells and markedly decreased the coimmunoprecipitation in SHR cells. The D3 receptor also regulated the AT1 receptor in vivo because AT1 receptor expression was increased in kidneys of D3 receptor–null mice compared with wild type littermates. D3 receptors may regulate AT1 receptor function by direct interaction with and regulation of AT1 receptor expression. One mechanism of hypertension may be related to increased renal expression of AT1 receptors due decreased D3 receptor regulation.

Interaction of Angiotensin II Type 1 and D5 Dopamine Receptors in Renal Proximal Tubule Cells

Angiotensin II type 1 (AT1) receptor and D1 and D3 dopamine receptors directly interact in renal proximal tubule (RPT) cells from normotensive Wistar-Kyoto rats (WKY). There is indirect evidence for a D5 and AT1 receptor interaction in WKY and spontaneously hypertensive rats (SHR). Therefore, we sought direct evidence of an interaction between AT1 and D5 receptors in RPT cells. D5 and AT1 receptors colocalized in WKY cells. Angiotensin II decreased D5 receptors in WKY cells in a time- and concentration-dependent manner (EC50=2.7×10−9 M; t1/2=4.9 hours), effects that were blocked by an AT1 receptor antagonist (losartan). In SHR, angiotensin II (10−8 M/24 hours) also decreased D5 receptors (0.96±0.08 versus 0.72±0.08; n=12) and to the same degree as in WKY cells (1.44±0.07 versus 0.92±0.08). However, basal D5 receptors were decreased in SHR RPT cells (SHR 0.96±0.08; WKY 1.44±0.07; n=12 per strain; P<0.05) and renal brush border membranes of SHR compared with WKY (SHR 0.54±0.16 versus WKY 1.46±0.10; n=5 per strain; P<0.05). Angiotensin II decreased AT1 receptor expression in WKY (1.00±0.04 versus 0.72±0.08; n=8; P<0.05) but increased it in SHR (0.96±0.04 versus 1.32±0.08; n=8; P<0.05). AT1 and D5 receptors also interacted in vivo; renal D5 receptor protein was higher in mice lacking the AT1A receptor (AT1A−/−; 1.61±0.31; n=6) than in wild-type littermates used as controls (AT1A+/+; 0.81±0.08; n=6; P<0.05), and renal cortical AT1 receptor protein was higher in D5 receptor null mice than in wild-type littermates (1.18±0.08 versus 0.84±0.07; n=4; P<0.05). We conclude that D5 and AT1 receptors interact with each other. Altered interactions between AT1 and dopamine receptors may play a role in the pathogenesis of hypertension.

Angiotensin II Regulation of AT1 and D3 Dopamine Receptors in Renal Proximal Tubule Cells of SHR

Abstract—Dopamine and angiotensin II negatively interact to regulate sodium excretion and blood pressure. D3 dopamine receptors downregulate angiotensin type 1 (AT1) receptors in renal proximal tubule cells from normotensive Wistar-Kyoto rats. We determined whether AT1 receptors regulate D3 receptors and whether the regulation is different in cultured renal proximal tubule cells from normotensive and spontaneously hypertensive rats. Angiotensin II (10−8M/24 hours) decreased D3 receptors in both normotensive (control, 36±3; angiotensin II, 24±3 U) and hypertensive (control, 30±3; angiotensin II, 11±3 U; n=9 per group) rats; effects that were blocked by the AT1 receptor antagonist, losartan (10−8M/24 hours). However, the reduction in D3 expression was greater in hypertensive (60±10%) than in normotensive rats (32±9%). In normotensive rats, angiotensin II (10−8M/24hr) also decreased AT1 receptors. In contrast, in cells from hypertensive rats, angiotensin II increased AT1 receptors. AT1 and D3 receptors co-immunoprecipitated in renal proximal tubule cells from both strains. Angiotensin II decreased D3/AT1 receptor co-immunoprecipitation similarly in both rat strains, but basal D3/AT1 co-immunoprecipitation was 6 times higher in normotensive than in hypertensive rats. Therefore, AT1 and D3 receptor interaction is qualitatively and quantitatively different between normotensive and hypertensive rats; angiotensin II decreases AT1 expression in normotensive but increases it in hypertensive rats. In addition, angiotensin II decreases D3 expression to a greater extent in hypertensive than in normotensive rats. Aberrant interactions between D3 and AT1 receptors may play a role in the pathogenesis of hypertension.

Anion transport in brush border membranes isolated from rat small intestine

Abstract Addition of sodium salts to isolated membrane vesicles induced a rapid release of protons from the vesicles, followed by a slower reuptake. As proton reuptake under the experimental conditions is dependent on simultaneous anion translocation, the rates of anion transport could be calculated from the changes in medium pH. Anion transport was measured in the absence and presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, a proton carrier. It is concluded that the brush border membrane is relatively impermeable to cyclamate and that it contains conductance pathways for thiocyanate and nitrate as well as a chloride transport system catalyzing an electroneutral chloride-cation co-transport (chloride-hydroxyl exchange). For acetate, the protonated form rather than the anion appears to be the permeant species.

Renal Protein Phosphatase 2A Activity and Spontaneous Hypertension in Rats

The impaired renal paracrine function of dopamine in spontaneously hypertensive rats (SHR) is caused by hyperphosphorylation and desensitization of the renal D1 dopamine receptor. Protein phosphatase 2A (PP2A) is critical in the regulation of G-protein–coupled receptor function. To determine whether PP2A expression and activity in the kidney are differentially regulated in genetic hypertension, we examined the effects of a D1-like agonist, fenoldopam, in renal cortical tubules and immortalized renal proximal tubule cells from normotensive Wistar-Kyoto rats (WKY) and SHR. In cortical tubules and immortalized proximal tubule cells, PP2A expression and activities were greater in cytosol than in membrane fractions in both WKY and SHR. Although PP2A expressions were similar in WKY and SHR, basal PP2A activity was greater in immortalized proximal tubule cells of SHR than WKY. In immortalized proximal tubule cells of WKY, fenoldopam increased membrane PP2A activity and expression of the regulatory subunit PP2A-B56&agr;, effects that were blocked by the D1-like antagonist SCH23390. Fenoldopam had no effect on cytosolic PP2A activity but decreased PP2A-B56&agr; expression. In contrast, in immortalized proximal tubule cells of SHR, fenoldopam decreased PP2A activity in both membranes and cytosol but predominantly in the membrane fraction, without affecting PP2A-B56&agr; expression; this effect was blocked by the D1-like antagonist SCH23390. We conclude that renal PP2A activity and expression are differentially regulated in WKY and SHR by D1-like receptors. A failure of D1-like agonists to increase PP2A activity in proximal tubule membranes may be a cause of the increased phosphorylation of the D1 receptor in the SHR.

ATP-stimulated electrolyte and mucin secretion in the human intestinal goblet cell line HT29-C1.16E

The response of confluent monolayers of HT29-Cl.16E cells to stimulation by extracellular ATP and ATP analogues was investigated in terms of mucin and electrolyte secretion. Mucin secretion was measured as release of glucosamine-labeled macromolecules trapped at the stacking/running gel interface of polyacrylamide gels and electrolyte secretion as shortcircuit current (Isc). Luminal ATP stimulated a transient increase in the release of mucins and of Isc corresponding to a secretory Cl− current. Both secretions peaked at 3 to 5 min after addition of ATP. Maximal ATP-stimulated mucin secretion over 15 min was up to 18-fold above control with an apparent ED50 of approximately 40 μm. Maximal peak Isc after stimulation with ATP was approximately 35 μA/cm2 with an apparent ED50 of about 0.4 mm. ATP-dependent Isc was at least in part due to Cl− secretion since removal of Cl− from the medium reduced the peak Isc by 40% and the Isc integrated over 40 min by 80%. The secretory responses were not associated with cell damage as assessed by failure of ethidium bromide to enter into the cells, absence of release of lactate dehydrogenase, maintenance of monolayer conductance, viability, and responses to repeated applications of ATP. The order of efficacy of nucleotide agonists was similar for both processes with ATP>ADP>AMP≥adenosine. Luminal ATP was much more effective than basolateral addition of this compound. These results suggest involvement of a luminal P2-type receptor which can initiate signaling pathways for granule fusion and mucin release as well as for activation of Cl− channels. P2-receptor-stimulated mucin and Isc release was strongly inhibited by a 30 min preincubation with the classical K+ channel blockers quinine (1 mm), quinidine (1 mm), and Ba2+ (3 mm). Experiments with amphotericin B to measure separately the conductance changes of either luminal or basolateral plasma membrane revealed that quinidine did not directly block the ATP-induced basolateral K+ or the luminal anion channels. The quinidine inhibition after preincubation is therefore most easily explained by interference with granule fusion and location of anion channels in granule membranes. Luminal P2 receptors may play a role in intestinal defense mechanisms with both fluid and mucin secretion aiding in the removal of noxious agents from the mucosal surface.

Purification of brush border membrane by thiocyanate treatment.

Rat small intestinal brush border membranes are purified from brush borders by homogenization in relatively high concentrations of thiocyanate salts (0.56 M LiSCN, 0.41 M NaSCN, or 0.52 M KSCN), removal of this salt, and differential centrifugation to separate cytoskeletal material from membranes. The marker enzyme, sucrase, is enriched 98-fold in the final membranes over the starting homogenate of intestinal scrapings at a yield of about 20%. The isolated membranes are capable of secondary active sodium-dependent glucose transport as demonstrated by sodium gradient-supported overshooting glucose uptake.