Ashwini Mokashi

Comparative distribution of neuropeptide Y Y1 and Y5 receptors in the rat brain by using immunohistochemistry

Neuropeptide Y (NPY) Y1 and Y5 receptor subtypes mediate many of NPYs diverse actions in the central nervous system. The present studies use polyclonal antibodies directed against the Y1 and Y5 receptors to map and compare the relative distribution of these NPY receptor subtypes within the rat brain. Antibody specificity was assessed by using Western analysis, preadsorption of the antibody with peptide, and preimmune serum controls. Immunostaining for the Y1 and Y5 receptor subtypes was present throughout the rostral‐caudal aspect of the brain with many regions expressing both subtypes: cerebral cortex, hippocampus, hypothalamus, thalamus, amygdala, and brainstem. Further studies using double‐label immunocytochemistry indicate that Y1R immunoreactivity (‐ir) and Y5R‐ir are colocalized in the cerebral cortex and caudate putamen. Y1 receptor ir was evident in the central amygdala, whereas both Y1‐ and Y5‐immunoreactive cells and fibers were present in the basolateral amygdala. Corresponding with the physiology of NPY in the hypothalamus, both Y1R‐ and Y5R‐ir was present within the paraventricular (PVN), supraoptic, arcuate nuclei, and lateral hypothalamus. In the PVN, Y5R‐ir and Y1R‐ir were detected in cells and fibers of the parvo‐ and magnocellular divisions. Intense immunostaining for these receptors was observed within the locus coeruleus, A1–5 and C1–3 nuclei, subnuclei of the trigeminal nerve and nucleus tractus solitarius. These data provide a detailed and comparative mapping of Y1 and Y5 receptor subtypes within cell bodies and nerve fibers in the brain which, together with physiological and electrophysiological studies, provide a better understanding of NPY neural circuitries. J. Comp. Neurol. 464:285–311, 2003.

GLUT-1 GLUCOSE TRANSPORTERS IN THE BLOOD-BRAIN BARRIER: DIFFERENTIAL PHOSPHORYLATION

Glucose is the primary metabolic fuel for the mammalian brain, and a continuous supply is required to maintain normal CNS function. The transport of glucose across the blood–brain barrier (BBB) into the brain is mediated by the facilitative glucose transporter GLUT‐1. Prior studies (Simpson et al. [2001] J Biol Chem 276:12725–12729) had revealed that the conformations of the GLUT‐1 transporter were different in luminal (blood facing) and abluminal (brain facing) membranes of bovine cerebral endothelial cells, based on differential antibody recognition. This study has extended these observations and, by using a combination of 2D‐PAGE/Western blotting and immunogold electron microscopy, determined that these different conformations are exhibited in vivo and arise from differential phosphorylation of GLUT‐1 and not from alternative splicing or altered O‐ or N‐linked glycosylation.

An active transport system in the blood-brain barrier may reduce levodopa availability.

Levodopa, the primary drug used to treat patients with Parkinsons disease, is transported into the brain by the facilitative amino acid transporter (L1). We present here an unanticipated discovery: levodopa may be pumped out of the brain by a Na(+)-dependent transport system that couples the naturally occurring Na(+) gradient existing between the brains extracellular fluid and the cytoplasm of capillary endothelial cells. The activity of this system reduces the net availability of levodopa.

Glutamate permeability at the blood-brain barrier in insulinopenic and insulin-resistant rats

The influence of diabetes on brain glutamate (GLU) uptake was studied in insulinopenic (streptozotocin [STZ]) and insulin-resistant (diet-induced obesity [DIO]) rat models of diabetes. In the STZ study, adult male Sprague-Dawley rats were treated with STZ (65 mg/kg intravenously) or vehicle and studied 3 weeks later. The STZ rats had elevated plasma levels of glucose, ketone bodies, and branched-chain amino acids; brain uptake of GLU was very low in both STZ and control rats, examined under conditions of normal and greatly elevated (by intravenous infusion) plasma GLU concentrations. In the DIO study, rats ingested a palatable, high-energy diet for 2 weeks and were then divided into weight tertiles: rats in the heaviest tertile were designated DIO; rats in the lightest tertile, diet-resistant (DR); and rats in the intermediate tertile, controls. The DIO and DR rats continued to consume the high-energy diet for 4 more weeks, whereas the control rats were switched to standard rat chow. All rats were studied at 6 weeks (subgroups were examined under conditions of normal or elevated plasma GLU concentrations). The DIO rats ate more food and were heavier than the DR or control rats and had higher plasma leptin levels and insulin-glucose ratios. In all diet groups, the blood-brain barrier showed very low GLU penetration and was unaffected by plasma GLU concentration. Brain GLU uptake also did not differ among the diet groups. Together, the results indicate that the blood-brain barrier remains intact to the penetration of GLU in 2 models of diabetes under the conditions examined.

Improved membrane protein solubilization and clean-up for optimum two-dimensional electrophoresis utilizing GLUT-1 as a classic integral membrane protein

Two-dimensional (2-D) electrophoresis remains a primary resolving tool for proteomic analyses. The final number of proteins resolved by 2-D electrophoresis depends on their respective solubility, size, charge, and isoelectric point. While water-soluble cytosolic proteins have often been well represented in 2-D maps, the same is not true with membrane proteins. Highly hydrophobic in nature, membrane proteins are poorly resolved in 2-D gels due to problems associated primarily with sample preparation. This is of especial concern in neuroscience studies where many proteins of interest are membrane bound. In the current work, we present a substantially improved sample preparation protocol for membrane proteins utilizing the GLUT-1 glucose transporter from brain microvessels as an example of a typical membrane protein. GLUT-1 (SLC2A1; solute carrier family 2 (facilitated glucose transporter), member 1) is a 55kD glycoprotein that contains 12 membrane-spanning alpha helices that impart the protein its characteristic hydrophobicity. GLUT-1 based on its amino acid sequence has a theoretical isoelectric point (pI) of 8.94. Using a combination of the non-ionic detergents, n-dodecyl-beta-maltoside (DDM) and amido sulphobetaine-14 (ASB-14) for sample solubilization, and a modification of the Bio-Rad 2-D clean-up protocol involving trichloroacetic acid (TCA)/acetone, we obtained near complete solubilization of GLUT-1 and greater than 90% recovery of this membrane protein in 1-D and 2-D Western blots. The total number of proteins resolved also increased dramatically in Deep Purple total protein stains using our improved protocol.

Pyroglutamate stimulates Na+‐dependent glutamate transport across the blood–brain barrier

Regulation of Na+‐dependent glutamate transport was studied in isolated luminal and abluminal plasma membranes derived from the bovine blood–brain barrier. Abluminal membranes have Na+‐dependent glutamate transporters while luminal membranes have facilitative transporters. This organization allows glutamate to be actively removed from brain. γ‐Glutamyl transpeptidase, the first enzyme of the γ‐glutamyl cycle (GGC), is on the luminal membrane. Pyroglutamate (oxoproline), an intracellular product of GGC, stimulated Na+‐dependent transport of glutamate by 46%, whereas facilitative glutamate uptake in luminal membranes was inhibited. This relationship between GGC and glutamate transporters may be part of a regulatory mechanism that accelerates glutamate removal from brain.

Na+-dependent transport of taurine is found only on the abluminal membrane of the blood–brain barrier

Luminal and abluminal plasma membranes were isolated from bovine brain microvessels and used to identify and characterize Na(+)-dependent and facilitative taurine transport. The calculated transmembrane potential was -59 mV at time 0; external Na(+) (or choline under putative zero-trans conditions) was 126 mM (T=25 °C). The apparent affinity constants of the taurine transporters were determined over a range of taurine concentrations from 0.24 μM to 11.4 μM. Abluminal membranes had both Na(+)-dependent taurine transport as well as facilitative transport while luminal membranes only had facilitative transport. The apparent K(m) for facilitative and Na(+)-dependent taurine transport were 0.06±0.02 μM and 0.7±0.1 μM, respectively. The Na(+)-dependent transport of taurine was voltage dependent over the range of voltages studied (-25 to -101 mV). The transport was over 5 times greater at -101 mV compared to when V(m) was -25 mV. The sensitivity to external osmolality of Na(+)-dependent transport was studied over a range of osmolalities (229 to 398 mOsm/kg H(2)O) using mannitol as the osmotic agent to adjust the osmolality. For these experiments the concentration of Na(+) was maintained constant at 50mM, and the calculated transmembrane potential was -59 mV. The Na(+)-dependent transport system was sensitive to osmolality with the greatest rate observed at 229 mOsm/kg H(2)O.