Debra J. Hazen-Martin

G protein-coupled receptor kinase 4 gene variants in human essential hypertension

Essential hypertension has a heritability as high as 30–50%, but its genetic cause(s) has not been determined despite intensive investigation. The renal dopaminergic system exerts a pivotal role in maintaining fluid and electrolyte balance and participates in the pathogenesis of genetic hypertension. In genetic hypertension, the ability of dopamine and D1-like agonists to increase urinary sodium excretion is impaired. A defective coupling between the D1 dopamine receptor and the G protein/effector enzyme complex in the proximal tubule of the kidney is the cause of the impaired renal dopaminergic action in genetic rodent and human essential hypertension. We now report that, in human essential hypertension, single nucleotide polymorphisms of a G protein-coupled receptor kinase, GRK4γ, increase G protein-coupled receptor kinase (GRK) activity and cause the serine phosphorylation and uncoupling of the D1 receptor from its G protein/effector enzyme complex in the renal proximal tubule and in transfected Chinese hamster ovary cells. Moreover, expressing GRK4γA142V but not the wild-type gene in transgenic mice produces hypertension and impairs the diuretic and natriuretic but not the hypotensive effects of D1-like agonist stimulation. These findings provide a mechanism for the D1 receptor coupling defect in the kidney and may explain the inability of the kidney to properly excrete sodium in genetic hypertension.

Inhibition of tumor necrosis factor-induced cell death in MCF7 by a novel inhibitor of neutral sphingomyelinase.

A high throughput screen for neutral, magnesium-dependent sphingomyelinase (SMase) was performed. One inhibitor discovered in the screen, GW4869, functioned as a noncompetitive inhibitor of the enzyme in vitro with an IC50 of 1 μm. It did not inhibit acid SMase at up to at least 150 μm. The compound was then evaluated for its ability to inhibit tumor necrosis factor (TNF)-induced activation of neutral SMase (N-SMase) in MCF7 cells. GW4869 (10 μm) partially inhibited TNF-induced sphingomyelin (SM) hydrolysis, and 20 μm of the compound was protected completely from the loss of SM. The addition of 10–20 μmGW4869 completely inhibited the initial accumulation of ceramide, whereas this effect was partially lost at later time points (24 h). These data therefore support the inhibitory action of GW4869 on N-SMase not only in vitro but also in a cellular model. The addition of GW4869 at both 10 and 20 μm did not modify cellular glutathione levels in response to TNF, suggesting that the action of GW4869 occurred downstream of the drop in glutathione, which was shown previously to occur upstream of the activation of N-SMase. Further, whereas TNF treatment also caused a 75% increase ofde novo synthesized ceramide after 20 h of incubation, GW4869, at either 10 or 20 μm, had no effect on this pathway of ceramide generation. In addition, GW4869 did not significantly impair TNF-induced NF-κB translocation to nuclei. Therefore, GW4869 does not interfere with other key TNF-mediated signaling effects. GW4869 was able, in a dose-dependent manner, to significantly protect from cell death as measured by nuclear condensation, caspase activation, PARP degradation, and trypan blue uptake. These protective effects were accompanied by significant inhibition of cytochrome c release from mitochondria and caspase 9 activation, therefore localizing N-SMase activation upstream of mitochondrial dysfunction. In conclusion, our results indicate that N-SMase activation is a necessary step for the full development of the cytotoxic program induced by TNF.

Dopamine-1 Receptor Coupling Defect in Renal Proximal Tubule Cells in Hypertension

The ability of the dopamine-1 (D1)-like receptor to stimulate adenylyl cyclase (AC) and phospholipase C (PLC), inhibit sodium transport in the renal proximal tubule (RPT), and produce natriuresis is attenuated in several rat models of hypertension. Since the inhibitory effect of D1-like receptors on RPT sodium transport is also reduced in some patients with essential hypertension, we measured D1-like receptor coupling to AC and PLC in cultures of human RPT cells from normotensive (NT) and hypertensive (HT) subjects. Basal cAMP concentrations were the same in NT (n=6) and HT (n=4). However, the D1-like receptor agonist fenoldopam increased cAMP production to a greater extent in NT (maximum response=67+/-1%) than in HT (maximum response=17+/-5%), with a potency ratio of 105. Dopamine also increased cAMP production to a greater extent in NT (32+/-3%) than in HT (14+/-3%). The fenoldopam-mediated increase in cAMP production was blocked by SCH23390 (a D1-like receptor antagonist) and by antisense D1 oligonucleotides in both HT and NT, indicating action at the D1 receptor. The stimulatory effects of forskolin and parathyroid hormone-related protein of cAMP accumulation were not statistically different in NT and HT, indicating receptor specificity and an intact G-protein/AC pathway. The fenoldopam-stimulated PLC activity was not impaired in HT, and the primary sequence and expression of the D1 receptor were the same in NT and HT. However, D1 receptor serine phosphorylation in the basal state was greater in HT than in NT and was not responsive to fenoldopam stimulation in HT. These studies demonstrate the expression of D1 receptors in human RPT cells in culture. The uncoupling of the D1 receptor in both rats (previously described) and humans (described here) suggests that this mechanism may be involved in the pathogenesis of hypertension; the uncoupling may be due to ligand-independent phosphorylation of the D1 receptor in hypertension.

Early induction of secondary injury factors causing activation of calpain and mitochondria-mediated neuronal apoptosis following spinal cord injury in rats.

To investigate a potential relationship between calpain and mitochondrial damage in spinal cord injury (SCI), a 40 gram‐centimeter force (g‐cm) injury was induced in rats by a weight‐drop method and allowed to progress for 4 hr. One‐centimeter segments of spinal cord tissue representing the adjacent rostral, lesion, and adjacent caudal areas were then removed for various analyses. Calcium green 2‐AM staining of the lesion and penumbra sections showed an increase in intracellular free calcium (Ca2+) levels following injury, compared with corresponding tissue sections from sham‐operated (control) animals. Western blot analysis showed increased calpain expression and activity in the lesion and penumbra segments following SCI. Double‐immunofluorescent labeling indicated that increased calpain expression occurred in neurons in injured segments. Western blot analysis also showed an increased Bax:Bcl‐2 ratio, indicating the induction of the mitochondria‐mediated cell death pathway in the lesion and penumbra. The morphology of mitochondria was altered in lesion and penumbra following SCI: mostly hydropic change (swelling) in the lesion, with the penumbra shrunken or normal. At 4 hr after induction of injury, a substantial amount of cytochrome c had been released into the cytoplasm, suggesting a trigger for apoptosis through caspase 3 activation. Neuronal death after 4 hr of injury was detected by a combined TUNEL and double‐immunofluoresence assay in the lesion and penumbra sections of injured cord, compared with sham controls. These results suggest that an early induction of secondary factors is involved in the pathogenesis of SCI. The increased Ca2+ levels could activate calpain and mediate mitochondrial damage leading to neuronal death in lesion and penumbra following injury. Thus, secondary injury processes mediating cell death are induced as early as 4 hr after the injury, and calpain and caspase inhibitors may provide neuroprotection.

Refractory heartburn: comparison of intercellular space diameter in documented GERD vs. functional heartburn.

OBJECTIVES:Refractory heartburn despite acid suppression may be explained by ongoing gastroesophageal reflux disease (GERD) or functional heartburn (FH), i.e., symptoms without evidence of GERD. Impedance–pH monitoring (impedance–pH) detects acid and nonacid reflux and is useful for evaluating acid-suppressed, refractory patients. Intercellular space diameter (ISD) of esophageal epithelium measured by transmission electron microscopy (TEM) is a marker of epithelial damage present in both erosive and nonerosive reflux disease. ISD has not been used to study refractory heartburn or FH. Our aim was to compare ISD in healthy controls and refractory heartburn patients with GERD and FH.METHODS:In refractory heartburn patients (heartburn more than twice/week for at least 2 months despite proton pump inhibitor (PPI) b.i.d.), erosive esophagitis and/or abnormal impedance–pH (increased acid exposure or positive symptom index) defined GERD; normal esophagogastroduodenoscopy (EGD)/impedance–pH defined FH. Asymptomatic, healthy controls had normal EGD and pH-metry. Mean ISD in each subject, determined by blinded TEM of esophageal biopsies, was the average of 100 measurements (10 measurements in each of 10 micrographs).RESULTS:In all, 11 healthy controls, 11 FH, and 15 GERD patients were studied. Mean ISD was significantly higher in GERD compared with controls (0.87 vs. 0.32 μm, P=0.003) and FH (0.87 vs. 0.42 μm, P=0.012). Mean ISD was similar in FH and controls (0.42 vs. 0.32 μm, P=0.1). The proportion of patients with abnormal ISD was significantly higher for GERD compared with FH (60 vs. 9%, P=0.014).CONCLUSIONS:ISD is increased in refractory heartburn patients with GERD but not those with FH. Our findings suggest that measurement of ISD by TEM might be a useful tool to distinguish GERD from FH in patients with refractory heartburn.

MCMV induces neointima in IFN-γR-/- mice: Intimal cell apoptosis and persistent proliferation of myofibroblasts

BackgroundCMV infections have been linked to vasculopathies like atherosclerosis and Scleroderma. CMV infects vascular endothelium with intermittent shedding of the virus and the development of latency.MethodsWe adopted a model of arteritis, developed by Presti et al. (1998), triggered by murine cytomegalovirus (MCMV) infection. Our studies focused on neointima formation. Groups of mice include: 1) immunocompetent 129S, 2) immunocompetent 129S receiving whole body irradiation and MCMV, 3) IFN-γR-/- receiving MCMV, and 4) IFN-γR-/- receiving MCMV and whole body irradiation.ResultsMice were inoculated with MCMV (5 x 104 or 1 x 105 PFUs) by i.p. injection; hearts and abdominal aortas were collected and histopathology evaluated. Infected immunocompetent animals exhibited widespread perivascular inflammation, which subsided by 8 weeks. Intimal pathology was not observed in any control group. Immunocompetent animals receiving MCMV and irradiation developed mild to moderate intimal lesions associated with medial and adventitial inflammation. IFN-γR-/- mice infected for 4 months and receiving whole body irradiation 2 months after infection developed pathology characterized by extensive adventitial and medial infiltrate and significant neointima, suggesting that infection and immunosuppression were co-requisites of neointima formation. Immunohistochemical analysis revealed myofibroblasts as a major component of neointima. The disease is characterized by up-regulation of growth factors (TGF-β1, PDGF-A and B). Apoptosis was detected in the intimal layer of affected aortas. Active proliferation of myofibroblasts and infiltrating cells was also detected.ConclusionThese results indicate that CMV infections may lead to intimal injury that results in the formation of neointima characteristic of autoimmune vasculopathies.

Identification of RPE65 in transformed kidney cells1

The protein RPE65 has an important role in retinoid processing and/or retinoid transport in the eye. Retinoids are involved in cell differentiation, embryogenesis and carcinogenesis. Since the kidney is known as an important site for retinoid metabolism, the expression of RPE65 in normal kidney and transformed kidney cells has been examined. The RPE65 mRNA was detected in transformed kidney cell lines including the human embryonic kidney cell line HEK293 and the African green monkey kidney cell lines COS‐1 and COS‐7 by reverse transcription PCR. In contrast, it was not detected in human primary kidney cells or monkey kidney tissues under the same PCR conditions. The RPE65 protein was also identified in COS‐7 and HEK293 cells by Western blot analysis using a monoclonal antibody to RPE65, but not in the primary kidney cells or kidney tissues. The RPE65 cDNA containing the full‐length encoding region was amplified from HEK293 and COS‐7 cells. DNA sequencing showed that the RPE65 cDNA from HEK293 cells is identical to the RPE65 cDNA from the human retinal pigment epithelium. The RPE65 from COS‐7 cells shares 98 and 99% sequence identity with human RPE65 at the nucleotide and amino acid levels, respectively. Moreover, the RPE65 mRNA was detected in three out of four renal tumor cultures analyzed including congenital mesoblastic nephroma and clear cell sarcoma of the kidney. These results demonstrated that transformed kidney cells express this retinoid processing protein, suggesting that these transformed cells may have an alternative retinoid metabolism not present in normal kidney cells.

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.

Cadmium nephrotoxicity in human proximal tubule cell cultures

SummaryHuman proximal tubule kidney cells grown in a serum-free tissue culture medium were exposed to concentrations of CdCl2 in a range of 0.5 to 10μg/ml. Cells were observed from 1 to 20 d upon initiation of cadmium in the culture fluid. Both confluent and subconfluent populations of cells were treated and evaluated for cytotoxicity. Both populations exhibited a concentration-dependent toxicity to ionic cadmium. For cells treated with 2.0 to 10 μg/ml Cd, the decreases in cell numbers were largely irreversible. However, cells treated with Cd in a range of 0.5 to 1.0 μg/ml exhibited a partial recovery of cell number and control morphology. In this range, recovery was more efficient in the subconfluent cultures. Fine structural alterations in Cd-treated tubule cells included condensation of nuclear chromatin, loss of microvilli structure, disorganization of lateral membrane interdigitation, as well as decreased uptake of aminoglycoside antibiotics as evidenced by decreased numbers of myeloid bodies in these cells. The results of this study imply that use of a human proximal tubule culture system has potential in discerning structural and functional effects of cadmium as well as other nephrotoxic metals and compounds on the human kidney.

Ultrastructural and immunohistochemical characterization of submandibular duct cells in culture and modification of outgrowth differentiation by manipulation of calcium ion concentration

SummaryThe recognized need for epithelial cell culture models for cystic fibrosis (CF) research has resulted in ongoing efforts to improve normal and CF submandibular duct cell culture capabilities. The duct is most likely the site of the CF defect in this and other exocrine glands. In a previous report conditions required for the successful primary explant culture of normal and CF submandibular glands were outlined; however, terminal keratinization and involution of these cultures were recognized as severe limiting factors to their utilization in CF research. This report explores the effects of calcium concentrations in the medium, growth factor supplements, and matrix components on growth and differentiation of these cultures. Results of the study further confirm the ductal origin of cells in the outgrowth and demonstrate that progressive keratinization is initiated only after cells proliferate beyond the environment of the explant fragment. Keratinization with subsequent multilayering, desmosome formation, and involution in the cell outgrowth are governed in degree by the calcium concentration of the growth medium. Upon reduction of medium calcium to 0.1 mM concentration, the cells proliferate as a monolayer and subculture through 8 to 9 passages and retain the capacity to undergo ductlike differentiation.