Are A3 adenosine receptors expressed in the brain?

An increasing number of reports suggest a role for A3 adenosine receptors (A3ARs) in mediating adenosine action in the central nervous system. However, studies of A3AR localization in the brain have yet to be performed. To provide insights into the central sites of A3AR action, we compared patterns of A1 and A3AR mRNA and binding site expression in the brains of rats, mice and humans. We also assessed whether A3 agonists are selective for A3ARs. Whereas it was possible to detect high-level A1AR expression in many brain regions, it was not possible to detect either A3AR gene or binding site expression in the central nervous system. When we examined the affinities of the A3AR agonists CI-IAB-MECA and IAB-MECA for A1ARs, we found that these compounds bound to A1ARs with high affinity. These observations suggest that studies using A3-agonists need to consider potential effects of A1ARs activation, as A1ARs are abundantly expressed in the nervous system whereas A3AR expression in the brain cannot be directly demonstrated.

A1 adenosine receptor activation inhibits neurite process formation by Rho kinase-mediated pathways.

A1 adenosine receptors (A1ARs) are expressed in the brain during critical periods of neurogenesis and neuronal differentiation. To examine influences of A1AR activation on neuronal development we studied the effects of A1AR activation on process growth in PC12 cells expressing A1ARs and in primary cultures of cortical and hippocampal neurons. In PC12 cells, we found that A1AR activation potently inhibited nerve growth factor (NGF)-induced neurite growth and induced stress fiber formation. A1ARs action was not mediated by inhibition of p44/42 MAP kinase activity, as inhibition of MEK/MAP kinase had no effects on A1AR action. When Rho kinase activity was blocked, A1AR agonists no longer inhibited neurite growth and stress fiber formation was blocked. In neurons, A1AR activation also inhibited process growth, and A1AR action was also mediated by Rho kinase. These data show that A1AR activation inhibits neurite growth and that the inhibitory effects of A1AR are dependent on Rho kinase.

Acetaldehyde inhibits angiotensin-converting enzyme activity of bovine lung.

The role of ethanol and its primary metabolite, acetaldehyde, were investigated for their effects upon angiotensin-converting enzyme (ACE) (EC 3.4.15.1), since the enzyme plays a key role in the maintenance of blood pressure homeostasis by transforming angiotensin I into angiotensin II and degrading bradykinin. ACE was extracted from a 38,000 x g pellet of bovine lung homogenate with 0.05-M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) buffer, pH 7.0/0.4 M NaCl/10 microM ZnCl2/0.5% Triton X-100. The solubilized enzyme was preincubated with increasing concentrations of acetaldehyde (0.177-2.213 M) for 30 min at 0 degree C. Progressive inhibition of 41-84% was observed as enzyme aliquots were assayed with hippuryl-L-histidyl-L-leucine (HHL) as the substrate. The interaction of angiotensin-converting enzyme with acetaldehyde was rapid under these conditions. Ethanol appeared to to have no effect upon enzymic activity at comparable concentrations. These results suggest that acetaldehyde-mediated ACE inhibition in vivo may play a contributory role in the development of vasodilation and facial flush reaction consequent to ethanol consumption, thereby accounting for localized hypotension.

Interaction of acetaldehyde with plasma proteins of the renin-angiotensin system.

Chronic alcohol abuse may lead to hypertension by stimulating the activity of the renin angiotensin system (RAS). While there are reports on the alcohol associated increase of angiotensin II in rats and increases of plasma renin activity in rats and human alcoholics, the exact mechanisms of stimulation of the RAS activity is not clear. This study provides evidence for a biochemical interaction of acetaldehyde, the primary oxidative metabolite of ethanol, upon bilaterally nephrectomized (NEPEX) rat plasma that contains significant quantities of angiotensinogen and lacks active renin. Rat plasma served as the source of renin in this study. Preincubation of NEPEX plasma with 0.2 M acetaldehyde at 4 degrees C for 2 h resulted in a 21% increase in the angiotensin I (A I) formation by the rat plasma renin and 27% increase in the A I formation by the trypsinized rat plasma renin. When the rat plasma which contains modest quantities of endogenous angiotensinogen in addition to renin was preincubated with 0.2 M acetaldehyde at 4 degrees C for 2 h, the rate of A I formation was increased by 10%. Equivalent amounts of ethanol did not modify the rate of A I generation when added to NEPEX plasma or rat plasma. These results suggest the possibility of a biochemical interaction of acetaldehyde with the renin substrate which may enhance the activity of the RAS cascade, thereby contributing to hypertension in chronic alcoholics.

Acetaldehyde inhibits the anti-elastase activity of alpha 1-antitrypsin.

There are genetic and exogenous factors responsible for alpha 1-antitrypsin (alpha 1-AT) deficiency which may lead to cirrhosis of the liver and emphysema. The present study was initiated on a biochemical level in order to determine whether acetaldehyde, the major product of ethanol metabolism, is capable of influencing the physiological effect of alpha 1-AT upon elastase, an enzyme which is capable of inducing emphysema. The effects of acetaldehyde and ethanol upon elastase and alpha 1-AT were tested. Acetaldehyde at 0.3-M and 1.2-M concentrations inhibited the anti-elastase activity of alpha 1-AT. Acetaldehyde at 0.03-M and 0.07-M concentrations did not affect elastase activity and had a slight effect at 0.12-M levels. Equivalent amounts of ethanol were without influence upon elastase activity or alpha 1-AT function. These data provide biochemical support for the possibility that heterozygous males with lower than normal alpha 1-AT levels may be at much higher risk to develop liver disease, emphysema, and alpha 1-AT deficiency as a consequence of chronic exposure to ethanol and concomitant circulating acetaldehyde levels.

Isolation of angiotensin converting enzyme (ACE) binding protein from human serum with an ACE affinity column.

Immobilized angiotensin-converting enzyme (ACE) was utilized as an affinity ligand to isolate a naturally occurring ACE binding protein from normal human serum. The enzyme was isolated from solubilized bovine lung membrane preparations by lisinopril affinity chromatography. It had an estimated molecular weight of 180 000 and was recognized by the anti-ACE antibody for the rabbit testicular ACE in immunoblots. ACE was immobilized onto epoxy Sepharose as well as Affi-Gel 15. Immobilized ACE on Affi-Gel 15 had higher catalytic activity (0.176 U/mL) compared with the enzyme immobilized on epoxy Sepharose (0.00005 U/mL). Immobilized ACE served as the affinity ligand for the identification of the ACE binding protein in human serum with an estimated molecular weight of 14 000 as observed by SDS polyacrylamide gel electrophoresis. The identification and further characterization of ACE binding proteins in serum and tissues may facilitate the greater understanding of the endogenous regulation of this key enzyme, which is involved in blood pressure homeostasis.

Identification of a novel ankyrin isoform (AnkG190) in kidney and lung that associates with the plasma membrane and binds alpha-Na, K-ATPase.

Ankyrins are a family of adapter molecules that mediate linkages between integral membrane and cytoskeletal proteins. Such interactions are crucial to the polarized distribution of membrane proteins in transporting epithelia. We have cloned and characterized a novel 190-kDa member of this family from a rat kidney cDNA library, which we term AnkG190 based on the predicted size and homology with the larger neuronal AnkG isoform. AnkG190 displays a unique 31-residue amino terminus, a repeats domain consisting of 24 repetitive 33-residue motifs, a spectrin binding domain, and a truncated regulatory domain. Probes derived from the unique amino terminus hybridize to an 8-kilobase message exclusively in kidney and lung and specifically to the kidney outer medullary collecting ducts by in situ hybridization. Transfections of Madin-Darby canine kidney and COS-7 epithelial cell lines with a full-length AnkG190 construct result in (a) expression at the lateral plasma membrane, (b) functional assembly with the cytoskeleton, and (c) interaction with at least one membrane protein, the Na,K-ATPase. Two independent Na,K-ATPase binding domains on AnkG190 are demonstrated as follows: one within the distal 12 ankyrin repeats, and a second site within the spectrin binding domain. Thus, ankyrins may interact with integral membrane proteins in a pleiotropic manner that may involve complex tertiary structural determinants.

Observation of high and low molecular weight inhibitors of angiotensin-converting enzyme in rat lung.

Fractionation of the rat lung yielded a 54,000 g supernate, and DOC-solubilized 775 g, 3100 g and 54,000 g sediments, each of these preparations displaying an increasing angiotensin-converting enzyme activity with increasing dilution, suggesting the presence of freely reversible angiotensin-converting enzyme inhibitors. The solubilized 775 g sediment was applied to an immobilized captopril column, eluted successively with 20 mM Pi(K+), pH 7.8 buffer, buffer/0.5 M NaCl, and buffer/0.01M cysteine to obtain four major protein bands, two of which appeared with the cysteine eluant. The first two protein peaks were each pooled and subjected to ultrafiltration with 10,000 molecular weight cutoff filters. The pooled peaks, retentates and ultrafiltrates each inhibited the angiotensin-converting enzyme activity, suggesting the presence of large and small molecular weight reversible angiotensin-converting enzyme inhibitors in association with the solubilized (membranous) particulate angiotensin-converting enzyme fraction. These results expand upon earlier observations on the existence of angiotensin-converting enzyme inhibitors in mammalian serum by observing an increasing angiotensin-converting enzyme activity with increasing dilution. This activity was eluted in multiple peaks, including elution with the cysteine eluate, suggesting that the angiotensin-converting enzyme, as well as other proteins, may react covalently with the sulfhydryl functional group of the immobilized captopril in a transsulfhydration reaction cleaving the disulfide bonds in proteins. Subsequent elution with cysteine affects an additional transsulfhydration reaction, releasing the proteins from the column. It is further postulated that air oxidation of the proteins permits reformation of disulfide bonds, yielding some active angiotensin-converting enzyme. Having in mind the possibility of lipophilic angiotensin-converting enzyme inhibitors crossing the blood-brain barrier as a means of treatment of alcohol abuse, the intriguing presence of a naturally occurring angiotensin-converting enzyme inhibitors in the particulate, lipid-rich fraction of the lung cell raises the theory that inhibitors such as these might cross the blood-brain barrier to serve as downregulators of alcohol consumption.

Comparative analysis of the ontogeny of a sodium-dependent bile acid transporter in rat kidney and ileum.

An apical sodium-dependent bile acid transporter (ASBT) has recently been cloned and characterized in the rat ileum. Northern and Western blotting revealed both the ASBT mRNA and protein in rat kidney. The coding sequence of the kidney transcript was found to be identical to the previously cloned ileal ASBT. Indirect immunofluorescence studies localized the ASBT protein to the apical membrane of the renal proximal convoluted tubule. Kinetic analysis of sodium-dependent taurocholate uptake using membrane vesicles revealed a similar Michaelis-Menten constant value for taurocholate in the kidney and intestine. ASBT protein and function were present in the kidney but not the ileum from 7-day-old rats. On postnatal day 7, there was a sevenfold increase in ASBT steady-state mRNA levels in the kidney relative to the ileum, yet nuclear run-on assays revealed that the nascent transcription rates at this age were virtually the same. This suggests that the difference in the neonatal expression of the ASBT gene in the kidney and ileum may be in part due to differences in mRNA stability.

Colonic H-K-ATPase alpha- and beta-subunits express ouabain-insensitive H-K-ATPase.

Active K absorption in the rat distal colon is energized by an apical H-K-ATPase, a member of the gene family of P-type ATPases. The H-K-ATPase alpha-subunit (HKcalpha) has been cloned and characterized (together with the beta-subunit of either Na-K-ATPase or gastric H-K-ATPase) in Xenopus oocytes as ouabain-sensitive (86)Rb uptake. In contrast, HKcalpha, when expressed in Sf9 cells without a beta-subunit, yielded evidence of ouabain-insensitive H-K-ATPase. Because a beta-subunit (HKcbeta) has recently been cloned from rat colon, this present study was initiated to determine whether H-K-ATPase and its sensitivity to ouabain are expressed when these two subunits (HKcalpha and HKcbeta) are transfected into a mammalian cell expression system. Transfection of HEK-293 cells with HKcalpha and HKcbeta cDNAs resulted in the expression of HKcalpha and HKcbeta proteins and their delivery to plasma membranes. H-K-ATPase activity was identified in crude plasma membranes prepared from transfected cells and was 1) saturable as a function of increasing K concentration with a K(m) for K of 0.63 mM; 2) inhibited by orthovanadate; and 3) insensitive to both ouabain and Sch-28080. In parallel transfection studies with HKcalpha and Na-K-ATPase beta1 cDNAs and with HKcalpha cDNA alone, there was expression of ouabain-insensitive H-K-ATPase activity that was 60% and 21% of that in HKcalpha/HKcbeta cDNA transfected cells, respectively. Ouabain-insensitive (86)Rb uptake was also identified in cells transfected with HKcalpha and HKcbeta cDNAs. These studies establish that HKcalpha cDNA with HKcbeta cDNA express ouabain-insensitive H-K-ATPase similar to that identified in rat distal colon.

Partial ATP depletion induces Fas- and caspase-mediated apoptosis in MDCK cells.

Brief periods of in vitro hypoxia/ischemia induce apoptosis of cultured renal epithelial cells, but the underlying mechanisms remain unknown. We show that partial ATP depletion (approximately 10-65% of control) results in a duration-dependent induction of apoptosis in Madin-Darby canine kidney (MDCK) cells, as evidenced by internucleosomal DNA cleavage (DNA laddering and in situ nick end labeling), morphological changes (cell shrinkage), and plasma membrane alterations (externalization of phosphatidylserine). The ATP-depleted cells display a significant upregulation of Fas, Fas ligand, and the Fas-associating protein with death domain (FADD). Exogenous application of stimulatory Fas monoclonal antibodies also induces apoptosis in nonischemic MDCK cells, indicating that they retain Fas-dependent pathways of programmed cell death. Furthermore, cleavage of poly(ADP)ribose polymerase (PARP) is evident after ATP depletion, indicating activation of caspases. Indeed, the apoptotic cells display a significant increase in caspase-8 (FLICE) activity. Finally, apoptosis induced by ATP depletion is ameliorated by pretreatment with inhibitors of caspase-8 (IETD), caspase-1 (YVAD), or caspase-3 (DEVD) but is not affected by inhibitors of serine proteases (TPCK). Our results indicate that partial ATP depletion of MDCK cells results in apoptosis and that Fas- and caspase-mediated pathways may play a critical role.

Sorting of rat liver and ileal sodium-dependent bile acid transporters in polarized epithelial cells.

The rat ileal apical Na+-dependent bile acid transporter (ASBT) and the liver Na+-taurocholate cotransporting polypeptide (Ntcp) are members of a new family of anion transporters. These transport proteins share limited sequence homology and almost identical predicted secondary structures but are localized to the apical surface of ileal enterocytes and the sinusoidal surface of hepatocytes, respectively. Stably transfected Madin-Darby canine kidney (MDCK) cells appropriately localized wild-type ASBT and Ntcp apically and basolaterally as assessed by functional activity and immunocytochemical localization studies. Truncated and chimeric transporters were used to determine the functional importance of the cytoplasmic tail in bile acid transport activity and membrane localization. Two cDNAs were created encoding a truncated transporter in which the 56-amino-acid COOH-terminal tail of Ntcp was removed or substituted with an eight-amino-acid epitope FLAG. For both mutants there was some loss of fidelity in basolateral sorting in that approximately 75% of each protein was delivered to the basolateral surface compared with approximately 90% of the wild-type Ntcp protein. In contrast, deletion of the cytoplasmic tail of ASBT led to complete loss of transport activity and sorting to the apical membrane. An Ntcp chimera in which the 56-amino-acid COOH-terminal tail of Ntcp was replaced with the 40-amino-acid cytoplasmic tail of ASBT was largely redirected (82.4 +/- 3.9%) to the apical domain of stably transfected MDCK cells, based on polarity of bile acid transport activity and localization by confocal immunofluorescence microscopy. These results indicate that a predominant signal for sorting of the Ntcp protein to the basolateral domain is located in a region outside of the cytoplasmic tail. These studies have further shown that a novel apical sorting signal is localized to the cytoplasmic tail of ASBT and that it is transferable and capable of redirecting a protein normally sorted to the basolateral surface to the apical domain of MDCK cells.

Cloning and characterization of a novel peptidase from rat and human ileum.

A novel 100-kDa ileal brush border membrane protein (I100) has been purified by anionic glycocholate affinity chromatography. Polyclonal antibodies raised against this protein were utilized to clone and characterize I100 in rats. A partial length human I100 cDNA was identified by hybridization screening. In the rat, the I100 protein is a 746-amino acid glycosylated (calculated core molecular mass of 80 kDa) type II integral membrane protein found on the apical surface of ileal villus enterocytes. Its 2.6-kilobase mRNA is expressed in distal small intestine in rats and in humans. The I100 cDNA is homologous to but distinct from human prostate-specific membrane antigen and rat brain N-acetylaspartylglutamate peptidase. It is expressed on both the basolateral and apical surfaces of stably transfected Madin Darby canine kidney cells. Analysis of these stably transfected Madin Darby canine kidney cells and I100 immunoprecipitates of rat ileal brush border membrane vesicles reveals that it has dipeptidyl peptidase IV activity. Future invesitgations will need to determine the exact substrate specificity of this novel peptidase.

Ileal bile acid transport regulates bile acid pool, synthesis, and plasma cholesterol levels differently in cholesterol-fed rats and rabbits.

We investigated the effect of ileal bile acid transport on the regulation of classic and alternative bile acid synthesis in cholesterol-fed rats and rabbits. Bile acid pool sizes, fecal bile acid outputs (synthesis rates), and the activities of cholesterol 7alpha-hydroxylase (classic bile acid synthesis) and cholesterol 27-hydroxylase (alternative bile acid synthesis) were related to ileal bile acid transporter expression (ileal apical sodium-dependent bile acid transporter, ASBT). Plasma cholesterol levels rose 2.1-times in rats (98 +/- 19 mg/dl) and 31-times (986 +/- 188 mg/dl) in rabbits. The bile acid pool size remained constant (55 +/- 17 mg vs. 61 +/- 18 mg) in rats but doubled (254 +/- 46 to 533 +/- 53 mg) in rabbits. ASBT protein expression did not change in rats but rose 31% (P < 0.05) in rabbits. Fecal bile acid outputs that reflected bile acid synthesis increased 2- and 2.4-times (P < 0.05) in cholesterol-fed rats and rabbits, respectively. Cholesterol 7alpha-hydroxylase activity rose 33% (24 +/- 2.4 vs. 18 +/- 1.6 pmol/mg/min, P < 0.01) and mRNA levels increased 50% (P < 0.01) in rats but decreased 68% and 79%, respectively, in cholesterol-fed rabbits. Cholesterol 27-hydroxylase activity remained unchanged in rats but rose 62% (P < 0.05) in rabbits. Classic bile acid synthesis (cholesterol 7alpha-hydroxylase) was inhibited in rabbits because an enlarged bile acid pool developed from enhanced ileal bile acid transport. In contrast, in rats, cholesterol 7alpha-hydroxylase was stimulated but the bile acid pool did not enlarge because ASBT did not change. Therefore, although bile acid synthesis was increased via different pathways in rats and rabbits, enhanced ileal bile acid transport was critical for enlarging the bile acid pool size that exerted feedback regulation on cholesterol 7alpha-hydroxylase in rabbits.

Hepatic basolateral sodium-dependent-bile acid transporter expression in two unusual cases of hypercholanemia and in extrahepatic biliary atresia.

The recent cloning of a human sodium-dependent bile acid transporter (NTCP) permits analysis of its expression in human liver disease and investigation of potential primary defects in its expression. NTCP from normal human liver (NHL) was first characterized in detail. Northern blotting of RNA from NHL revealed a 1.8-kb NTCP transcript. Western blotting of crude NHL plasma membranes using a carboxyterminal antipeptide antibody showed that NTCP is a 39-kd polypeptide that is N-glycosylated to a final molecular weight of 56 kd. Indirect immunofluorescent analysis of NHL sections indicated that the NTCP protein is expressed on the basolateral surface of hepatocytes. We hypothesized that the clinical phenotype of a defect in NTCP might be hypercholanemia in the relative absence of liver disease. Accordingly, the coding region of the NTCP gene of two children with this phenotype was sequenced after reverse transcription/polymerase chain reaction (RT/PCR) amplification. No primary defects in the deduced NTCP amino acid sequence were found. Despite the extremely high serum bile salt levels (235 and 126 micromol/L) in these two patients, NTCP messenger RNA (mRNA) and protein expression were quantitatively normal, in contrast to the published observations in a rat model of cholestasis secondary to common bile duct ligation. Hepatic steady-state NTCP mRNA levels in a group of 23 pre- and postportoenterostomy biliary atresia patients were inversely related to total bilirubin, indicating that extrahepatic bile duct obstruction leads to down-regulation of NTCP mRNA levels, similar to that observed in rat common bile duct ligation. Therefore the lack of down-regulation in the two patients with hypercholanemia indicates that elevated serum bile salts are not sufficient to down-regulate NTCP expression, these two patients have abnormal responses to hypercholanemia, or these two patients have a defect in a gene other than NTCP that influences hepatic clearance of bile salts.