Burhan Mahmood

Ontogeny of P-Glycoprotein in Mouse Intestine, Liver, and Kidney

Background P-glycoprotein (Pgp) is an ATP-dependent, integral plasma-membrane efflux pump that is constitutively expressed on (i) adult apical brush-border epithelial cells of the intestine, (ii) the bile canalicular face of hepatocytes, and (iii) the brush border epithelium of renal proximal tubules. This Pgp tissue distribution and localization affects the absorption, distribution, metabolism, and excretion of Pgp substrates. Little is known regarding the ontogeny of Pgp expression in these tissues. Methods Postnatal expression of Pgp on brush border membranes of small intestine, liver, and kidney as a function of maturity from birth through adulthood was determined using Western immunoblotting and immunohistochemical techniques. Tissue was isolated from FVB mice at four different ages: day of life 0 (D0), day of life 7 (D7), day of life 21 (D21), and adult (Ad). The relative expression of Pgp protein on Western immunoblots was assessed by scanning densitometry and indexed as a percentage (mean±SEM) of the adult levels. Results On Western immunoblots, Pgp expression was limited at birth (19±6% of Ad) and increased significantly with maturation in intestine (ANOVA, P <0.005). In contrast, hepatic (113±12% of Ad) and renal (96±15% of Ad) Pgp expression were at adult levels at birth. The tissue-specific developmental pattern of Pgp expression was confirmed by immunohistochemistry. Conclusions We conclude that Pgp is expressed in a tissue-specific and developmentally regulated fashion and speculate that developmental modulation of intestine-Pgp expression may affect the oral bioavailability of Pgp substrates.

P-Glycoprotein and Bilirubin Disposition

P-glycoprotein (Pgp), an ATP-dependent plasma membrane efflux pump, is expressed in abundance on the luminal aspect of brain capillary endothelial cells and astrocytes of the blood–brain barrier where it limits the passage of a variety of lipophilic substrates into the central nervous system. This review summarizes current evidence characterizing (1) unconjugated bilirubin as a potential substrate for Pgp and (2) the ontogeny of Pgp expression at the blood–brain barrier and apical brush border epithelium of the gastrointestinal tract, findings that may provide insights regarding the disposition of bilirubin in immature subjects.

Unconjugated bilirubin efflux by bovine brain microvascular endothelial cells in vitro.

Objectives: The passage of unconjugated bilirubin (UCB) across the blood-brain barrier into the central nervous system is a crucial first step in the development of kernicterus. The objective of the current study was to characterize the passage of UCB across primary bovine brain microvascular endothelial cell (BBMVEC) monolayers in vitro. Design: Experimental study. Setting: Research institute. Subjects: BBMVECs. Interventions: Tritiated UCB (3H-UCB) transport at 60, 80, 100, 200, 300, and 400 nM concentrations was tested in both the apical to basolateral (A→ B) and basolateral to apical (B→A) directions in BBMVEC monolayers in vitro with or without preincubation with pharmacologic active transport inhibitors cyclosporine A, indomethacin, or MK571. Measurements and Main Results: The rate of 3H-UCB transport in the B→A direction was 6.2- to 7.3-fold higher than in the A→B direction, suggesting active efflux of UCB. Cyclosporine A (5 &mgr;M), a model inhibitor of P-glycoprotein, enhanced A→B while decreasing B→A UCB transport, resulting in an overall decrease in BBMVEC UCB efflux of between 46% and 54%. Indomethacin (10 &mgr;M) and MK-571 (50 &mgr;M), respectively a substrate and potent inhibitor of multidrug resistance-associated protein-1, had no effect. Conclusions: We conclude that 1) UCB is transported by BBMVEC monolayers in vitro in a net B→A direction (i.e., active efflux); and 2) cyclosporine A partially inhibits such transport. We speculate that the blood-brain barrier limits the passage and central nervous system retention of UCB by active transport and that this may be accounted in part by P-glycoprotein.

Current trends in neonatal ECMO

Extracorporeal membrane oxygenation (ECMO) is a life-saving therapy for patients with respiratory and cardiac failure refractory to maximal medical management. The extracorporeal life support organization registry is the largest available resource for describing the population and outcomes of patients treated with this therapy. The use of ECMO for neonatal patients is decreasing in proportion to the total annual ECMO runs most likely due to advancements in medical management. Although the overall survival for neonatal ECMO has decreased, this is likely a reflection of the increasingly complex neonatal patients treated with this therapy. Although many patient and mechanical complications are decreasing over time, there remains a high percentage of morbidities and risks associated with ECMO. Continued refinements in management strategies are important to improving overall patient outcomes.

Bilirubin Efflux By Brain Capillary Endothelial Cell Monolayers in Vitro: Role of P-Glycoprotein.

Background: The passage of bilirubin across the blood-brain barrier into the CNS is central to the development of kernicterus. Indirect in vivo evidence suggests that P-glycoprotein (Pgp), a multidrug transporter expressed on brain capillary endothelial cells, may limit the influx and CNS retention of unconjugated bilirubin (Pediatr Res 44:763–766, 1998). This phenomenon has not been studied in brain capillary endothelial cell monolayers in vitro.Objective: To test the hypothesis that Pgp mediates bilirubin transport across brain capillary endothelial cell monolayers in vitro.Design/Methods: Bovine brain capillary endothelial cells (Cell Systems Corp.) were grown in confluent monolayers at a density of 5 × 104 cells/insert in 1 cm2 Transwell dishes. [3H]-bilirubin (100nM) transport (pnol/cm2/min) was tested in both the apical to basolateral [A→B] and basolateral to apical [B→A] directions in the presence and absence of a Pgp inhibitor (cyclosporin A[5uM]). Involvement of a Pgp mediated efflux mechanism is suggested by a B→A/A→B ratio of greater than 1.5 (Pharm Res 16:1206, 1999). Brain capillary endothelial cell Pgp expression was confirmed by Western immunoblotting techniques.Results: Brain capillary endothelial cells express Pgp as seen on Western immunoblots. Bilirubin transport in the B→A direction (0.67±0.05 pmol/cm2/min) was 6.4 fold higher than the rate for A→B direction (0.11±0.02) suggesting active efflux of bilirubin across brain capillary endothelial cell monlolayers. B→A bilirubin transport decreased (0.60±0.07) and A→B bilirubin transport was enhanced (0.17±0.03) in the presence of the Pgp inhibition, with an overall decrease in bilirubin efflux of 27% suggesting that bilirubin transport by brain capillary endothelial cells is mediated in part by Pgp.Conclusions: We conclude that i) bilirubin is transported by brain capillary endothelial cell monolayers in vitro in a net B→A direction (i.e. activie efflux); ii) Pgp plays an active role in this barrier function, and iii) unconjugated bilirubin is a substrate for Pgp. We speculate that i) brain capillary endothelial Pgp limits the CNS passage and retention of unconjugated bilirubin and ii) that low brain capillary endothelial cell Pgp expression, as reported in premature neonates, may enhance brain bilirubin levels during hyperbilirubinemia.Disclosure: Supported by NINDS (038993), the 25 Club of Magee-Womens Hospital and the Mario Lemieux Centers for Patient Care and Research.

Mid-Aortic Syndrome in a Preterm Infant: A Rare Cause of Hypertension

An extremely premature male of 27 0/7 weeks gestation was born via spontaneous vaginal delivery. The postnatal course was significant for systemic hypertension with systolic blood pressures ranging from 80–90mmHg (greater than 95% percentile of normal for this gestational age) (1). The infant had a significant patent ductus arteriosus (PDA), which required surgical ligation. The hypertension continued despite surgical closure of the PDA and medical management with hydralazine, propranolol, isradipine and diuretics. Diagnostic imaging of the abdominal vessels demonstrated narrowing of the suprarenal abdominal aorta, however renal arteries and veins were reported as patent. The infant subsequently developed multiorgan failure and disseminated intravascular coagulation, resulting in bilateral grade IV intraventricular hemorrhages (IVH) and family elected to redirect care to comfort measures given the grim prognosis. n nPost-mortem examination demonstrated coarctation of the abdominal aorta at the level of the renal arteries with fibrointimal thickening of the suprarenal aorta extending into both renal arteries (Figure), consistent with a diagnosis of mid-aortic syndrome (MAS) (2). Additionally, the renal arteries were both severely hypoplastic with numerous collateral vessels, resulting in congested kidneys and acute tubular necrosis. Cardiac evaluation demonstrated biventricular hypertrophy, likely secondary to systemic hypertension. n n n nFigure n nA, Gross image in situ at autopsy showing the aortic narrowing as a ridge (arrow) above the opening of the renal artery. B, Histologic image showing fibrointimal plaque in the aorta (thin arrow) and near-total occlusion at origin of renal artery with ... n n n nThis case provides documentation of the youngest premature infant with MAS. Although a rare condition, a diagnosis of MAS should be considered in infants with refractory systemic hypertension. Four additional reported cases of MAS in neonates (3–6) have described a mortality of 100%, secondary to IVH or cardiac dysfunction/failure (4, 5). n nImproved outcomes for premature infants and advances in imaging technology have given us the ability to diagnose MAS in smaller infants. However, recent improvements in diagnosis have not been accompanied by advancements in treatment modalities. Current treatment focuses on medical management, allowing for infant growth until surgical intervention is feasible (7, 8).

Small Intestinal P-Glycoprotein mRNA Levels and Protein Expression Increase with Postnatal Maturation in Mice

Small Intestinal P-Glycoprotein mRNA Levels and Protein Expression Increase with Postnatal Maturation in Mice