Cerebral norepinephrine: influence on cortical oxidative metabolism in situ.

Unilateral lesion of the locus coeruleus and the resultant norepinephrine depletion in the ipsilateral cerebrum alters the relationship between cerebral metabolic demands and local delivery of oxygen and substrates. This effect of norepinephrine depletion is demonstrated by slower recovery of the redox ratio of cytochrome a,a3 during increased metabolic demands induced by local cortical stimulation.

Putrescine as a biochemical marker of malignant brain tumors.

Putrescine, spermidine, and spermine levels were determined in normal brain and central nervous system-related tumor tissues obtained at operation from 50 patients. The biochemical data were correlated with morphological histopathological descriptions of the same tissues. There was little variation in putrescine levels in normal cerebral cortical tissue. Subcortical white matter had lower putrescine but higher spermidine content than those of the overlying cortex. Putrescine levels were elevated in all astrocytomas assayed, and the magnitude of this elevation was proportional to the malignancy of the tumor as determined by histopathological criteria. In contradistinction, putrescine content of "benign" tumors was generally equal to or lower than that of the normal cerebral cortex. Spermidine and spermine levels varied widely in the tumors assayed and did not correlate with criteria of malignancy. It is concluded that putrescine may be a good biochemical marker of malignancy in central nervous system-related tumors.

Chronic hyperglycemia increases the density of glucose transporters in human erythrocyte membranes.

We investigated the effect of chronic hyperglycemia on glucose transporters in erythrocytes of subjects with and without diabetes mellitus. We found a 22% increase in D-glucose-displaceable cytochalasin-B binding in erythrocyte membranes of diabetic subjects over those of controls (311 +/- 13 vs. 254 +/- 8 pmol/mg protein; P less than 0.001). This increased binding was due to a higher density of binding sites without a significant change in binding affinity. Cytochalasin-B binding to erythrocyte membrane correlated positively with both erythrocyte glycohemoglobin and serum glucose levels, but not with plasma C-peptide levels. The data are compatible with up-regulation of glucose transporters in the erythrocytes of subjects with chronic hyperglycemia. We suspect that this is brought about by increased synthesis and membrane incorporation of the glucose transporter during erythropoiesis.

Regional studies of blood-brain barrier transport of glucose and leucine in awake and anesthetized rats.

D-Glucose and L-leucine are transported across the blood-brain barrier (BBB) by two separate carrier-mediated facilitated diffusion mechanisms. In the awake rat there are regional differences in blood-to-brain glucose transport among the cerebral cortex, cerebellum, hippocampus, and striatum. To determine whether these are due to variations in the regional density or affinity of the glucose transporter moiety of brain capillaries or are secondary to regional tissue perfusion and capillary arrangement characteristics, we studied regional blood-to-brain transport of L-leucine in awake rats; regional blood-to-brain transport of both glucose and leucine under chloral hydrate anesthesia, a condition associated with altered regional brain blood flow (BF) and metabolism; and regional brain vascular volume, derived from the L-glucose and insulin spaces, in both awake and anesthetized rats. We found the same regional differences in blood-to-brain leucine transport in awake rats as we previously described for D-glucose transport. These regional differences in glucose and leucine transport disappear under chloral hydrate anesthesia, as regional differences in BF are abolished. However, we found regional differences in the brain vascular volumes, which are evident in wakefulness and persist during anesthesia. These results suggest that the regional differences in blood-to-brain transport are due mainly to local tissue perfusion and capillary arrangement characteristics rather than to intrinsic regional differences in the transport systems of the BBB.

Adenosine receptors and the nucleoside transporter in human brain vasculature.

Evidence suggests that adenosine modulates neuronal and cerebral vascular functions by interacting with specific receptors on brain cells and blood vessels. Adenosine and other nucleosides are also transported across the blood-brain barrier via a saturable, carrier-mediated mechanism. Using direct ligand binding methods, we studied the two adenosine receptor subtypes, A1 and A2 and the nucleoside transporter moiety in human brain microvessels, pial vessels, choroid plexus, and cerebral cortex membranes. The following specific tritiated ligands were used: cyclohexyladenosine (CHA) for A1 receptors; 5'-N-ethylcarboxamide adenosine (NECA) for A2 receptors; nitrobenzylthioinosine (NBMPR) and dipyridamole (DPY) for nucleoside transporters. We find that cerebral microvessels, pial vessels, and choroid plexus have few, if any, A1 receptors, in contradistinction to cerebral membranes, which have a 10-20-fold higher density of A1 receptor sites. Specific high-affinity NECA binding to A2 receptors in cerebral microvessels, pial vessels, and choroid plexus was saturable and was equivalent to that of cerebral cortical membranes. The Bmax and Kd of the high-affinity NECA binding to vessel preparations were approximately 1.3 pmol/mg protein and approximately 250 nM, respectively, which is similar to our previous findings in the rat and pig. NBMPR and DPY binding were also saturable and were consistent with a single class of high-affinity binding sites. The density of nucleoside transporters was approximately four-fold higher in cerebral microvessels than in cerebral cortex, pial vessels, and choroid plexus. These results suggest that human cerebral microvessels have A2, but not A1, receptors and are particularly enriched with the adenosine transporter moiety.

Bradykinin receptors of cerebral microvessels stimulate phosphoinositide turnover.

We examined by ligand binding methods whether bradykinin (BK) receptors exist in rat and pig cerebral microvessels, and in the cerebral cortex from which the microvessels were isolated. We found a high-affinity and saturable BK receptor site in both rat and pig cerebral microvessels, but not in their cerebral cortex. The maximal density of binding and the dissociation constant were 8.0 +/- 4.1 and 6.8 +/- 1.5 fmol/mg of protein and 47 +/- 24 and 150 +/- 8 pM (mean +/- SD) in cerebral microvessels of the pig and rat, respectively. The high-affinity specific binding of BK was effectively displaced by des-Arg0[Hyp3-Thi5-8,D-Phe7]BK, a specific B2 receptor antagonist, but not by des-Arg9[Leu8]BK, a specific B1 antagonist. We also demonstrated that BK increases phosphatidylinositol hydrolysis in cerebral microvessels of the rat and pig. This effect was also blocked by the B2, but not by the B1, antagonist. Increased phosphatidylinositol hydrolysis was manifested by a rapid transient increase in inositol trisphosphate and the later slow accumulation of inositol bisphosphate and inositol monophosphate. Preincubation of microvessels with phorbol ester, stable GTP analogs, pertussis toxin, or in Ca(2+)-free buffer did not influence BK activation of phosphatidylinositol hydrolysis. These results demonstrate the existence of BK receptors of the B2 subtype in brain microvessels, which may play an important role in modulation of the brain microcirculation, probably via increased phosphoinositide turnover.

Adenosine receptors of cerebral microvessels and choroid plexus.

We studied, by ligand binding methods, the two adenosine receptors, A1 and A2, in rat and pig cerebral microvessels and pig choroid plexus. Ligand binding to cerebral microvessels was compared with that to membranes of the cerebral cortex. [3H]Cyclohexyladenosine and [3H]L-phenylisopropyladenosine were the ligands used for A1-receptors, and [3H]5'-N-ethylcarboxamide adenosine ([3H]NECA) was used to assess A2-receptors. We report that cerebral microvessels and choroid plexus exhibit specific [3H]NECA binding, but have no appreciable A1-receptor ligand binding sites. Specific binding of [3H]NECA to cerebral microvessels, choroid plexus, and cerebral cortex was saturable and suggested the existence of two classes of A2-receptor sites: high-affinity (Kd approximately 250 nM) and low-affinity (Kd approximately 1-2 microM) sites. The Kd and Bmax of NECA binding to cerebral microvessels and cerebral cortex were similar within each species. Our results, indicating the existence of A2-receptors in cerebral microvessels, are consistent with results of increased adenylate cyclase activity by adenosine and some of its analogues in these micro-vessels.

Specific ouabain binding to brain microvessels and choroid plexus.

The energy-dependent transport of ions across the blood-brain barrier and the blood-cerebrospinal fluid barrier by Na+, K+-ATPase is credited with an important role in brain homeostasis. In this study, we have assessed the relative enrichment of Na+, K+-ATPase in regional brain capillary preparations and in the choroid plexus by the quantitative determination of the cardiac glycoside binding sites in these preparations using [3H]ouabain as a ligand. We find that ouabain binds specifically to brain microvessels of the rat and the pig and to the choroid plexus of the pig in a saturable manner. The maximum density of ouabain binding sites in brain microvessels of both species is about one-fourth that of the crude membranes of the cerebrum and cerebellum. The density of ouabain binding sites in the pig choroid plexus is intermediate between that of the brain and brain microvessels. We do not find regional differences in ouabain binding to membrane fractions of the cerebrum and cerebellum, nor any significant differences in ouabain binding to cerebral and cerebellar microvessels. These findings provide quantitative estimates of Na+, K+-ATPase in brain capillaries and choroid plexus.

Adrenergic and cholinergic receptors of cerebral microvessels.

The presence of alpha- and beta-adrenergic and muscarinic cholinergic receptors in cerebral microvessels of the rat and pig was assessed by ligand binding techniques. The results demonstrate the presence of specific binding to alpha 2- and beta-adrenergic receptors but no appreciable specific binding to alpha 1-adrenergic or muscarinic cholinergic receptors. beta-Adrenergic receptors of pig cerebral microvessels are similar to those of the brain and other organs in their binding characteristics to the tritiated ligand and in their stereospecificity of binding to the biologically active isomers of beta-adrenergic agonists. Further evidence derived from the differential potency of binding displacement by the various beta-adrenergic agonists and selective beta 1- and beta 2-adrenergic antagonists indicates that beta-adrenergic receptors of pig cerebral microvessels are mostly of the beta 2-subtype.

Does endogenous norepinephrine regulate potassium homeostasis and metabolism in rat cerebral cortex?

The role of endogenous cerebral norepinephrine (NE) as a modulator of transmembrane cation transport and energy metabolism was evaluated by monitoring extracellular potassium ion activity ([K+[o) in vivo and by measuring cortical Na+,K+-ATPase activity and oxygen consumption in vitro. Ipsilateral cortical NE was depleted by unilateral 6-hydroxydopamine (6-OHDA) lesions of the locus ceruleus (LC). The contralateral cortex was used for control measurements. NE depletion had no effect on resting levels of cortical [K+[o or on the rate of K+ removal from the extracellular space following direct cortical stimulation. There was also no effect of NE depletion on Na+,K+-ATPase activity in cortical homogenates nor on oxygen consumption of cortical slices over a wide range of K+ concentrations. These results indicate tht central NE depletion does not influence movements of cortical K+ either directly through an influence on Na+,K+-ATPase activity or indirectly through effects on oxidative metabolism. It is probable, therefore, that previously described effects of NE on cortical oxidative metabolism are mediated through changes in cerebral perfusion and/or modification of substrate availability in vivo.