Oxygenated Krebs-Ringer Solution Research Articles

Effect of enkephalinase inhibitor on endothelin-1 induced bronchoconstriction in guinea pigs

Endothelin-1 (ET-1) is one of the most potent bronchoconstrictors in the guinea pig. The mechanism of its metabolism is still unclear. Phosphoramidon is known to be an enkephalinase inhibitor. We studied the effect of phosphoramidon on bronchoconstriction induced by ET-1. In the first in vitro study, a tracheal preparation was mounted in oxygenated Krebs-Ringer solution. The response was monitored by isometric transducer. Dose-response curves to ET-1 with or without phosphoramidon were obtained. Phosphoramidon potentiated ET-1 induced bronchoconstriction significantly. Next, the specific airway conductance (sGaw) was measured in conscious guinea pigs exposed to an aerosol of phosphoramidon or saline, followed by ET-1 aerosol inhalation. The ET-1 dose was increased by successively doubling the concentration. sGaw, after inhalation of phosphoramidon, was significantly reduced when exposed to ET-1. Phosphoramidon also potentiated ET-1 induced bronchoconstriction in vivo. Next, lung parenchymal tissues were prepared and placed in oxygenated Krebs-Ringer solution with or without phosphoramidon. ET-1 was added and incubated, and samples were injected into a high performance liquid chromatography column. Phosphoramidon inhibited an analysis of ET-1. These data suggest that enkephalinase plays a role in the break down of ET-1 in the airway of the guinea pig. Under the condition of decreased enkephalinase, ET-1 would potentiate bronchoconstriction.

Smoothelin-A Is Essential for Functional Intestinal Smooth Muscle Contractility in Mice

In patients with chronic intestinal pseudo-obstruction, intestinal motility is disturbed by either nervous or myogenic aberrations. The cause of the myogenic form is unknown, but it is likely to originate in the contractile apparatus of the smooth muscle cells. Smoothelins are actin-binding proteins that are expressed abundantly in visceral (smoothelin-A) and vascular (smoothelin-B) smooth muscle. Experimental data indicate a role for smoothelins in smooth muscle contraction. A smoothelin-deficient mouse model may help to establish the role of smoothelin-A in intestinal contraction and provide a model for myogenic chronic intestinal pseudo-obstruction. We used gene targeting to investigate the function of smoothelin-A in intestinal tissues. By deletion of exons 18, 19, and 20 from the smoothelin gene, the expression of both smoothelin isoforms was disrupted. The effects of the deficiency were evaluated by pathologic and physiologic analyses. In smoothelin-A/B knockout mice, the intestine was fragile and less flexible compared with wild-type littermates. The circular and longitudinal muscle layers of the intestine were hypertrophic. Deficiency of smoothelin-A led to irregular slow wave patterns and impaired contraction of intestinal smooth muscle, leading to hampered transport in vivo. This caused obstructions that provoked intestinal diverticulosis and occasionally intestinal rupture. Smoothelin-A is essential for functional contractility of intestinal smooth muscle. Hampered intestinal transit in smoothelin-A/B knockout mice causes obstruction, starvation, and, ultimately, premature death. The pathology of mice lacking smoothelin-A is reminiscent of that seen in patients with chronic intestinal pseudo-obstruction.

Different mechanisms of hypoxic injury on white matter and gray matter as revealed by dynamic changes in glucose metabolism in rats

Fresh rat brain slices were incubated with [ 18F]2-fluoro-2-deoxy- d-glucose ([ 18F]FDG) in oxygenated Krebs–Ringer solution at 36 °C, and the fractional rate constant (= k 3*) of [ 18F]FDG proportional to the cerebral glucose metabolic rate in white matter and gray matter was investigated with positron autoradiography. In both white matter and gray matter, the k 3* value with ≥20 min hypoxia was markedly lower than the unloaded control value, indicating irreversible hypoxic injury. Next, the neuroprotective effect against hypoxia induced by the addition of an N-methyl- d-aspartate receptor antagonist or a free radical scavenger was assessed by determining whether a decrease in the k 3* value after hypoxia loading was prevented. In gray matter, both agents exhibited a neuroprotective effect against 20 min hypoxia. In white matter, however, only the free radical scavenger was effective. These results suggest a similarity in the degree of vulnerability to hypoxia between white matter and gray matter as well as a difference in the developmental mechanism of hypoxic injury, i.e. the involvement of both glutamate and free radicals in gray matter, and the more selective involvement of free radicals in white matter.

Role of lactate in the brain energy metabolism: revealed by Bioradiography

To elucidate the role of lactate in the brain, we used a novel method, ‘Bioradiography’, in which the dynamic process could be followed in living slices by use of positron-emitter-labeled compounds and imaging plates. We studied the incorporation of 2-[ 18 F ]fluoro-2-deoxy- d-glucose ([ 18 F ]FDG) into rat brain slices incubated in oxygenated Krebs–Ringer solution. Under the glucose-free condition, [ 18 F ]FDG uptake rate in the cerebral cortex decreased with time and plateaued within 350 min but the addition of 5 mM lactate made the [ 18 F ]FDG uptake linear. When an inhibitor of the lactate transporter, 0.5 mM α-cyano-4-hydroxycinnamate (4-CIN) was applied to the glucose-free solution, the uptake rate decreased. Under the normal glucose condition, [ 18 F ]FDG uptake linearly increased for 6 h, but when 10 mM lactate was applied, the uptake rate decreased. In contrast, when 0.5 mM 4-CIN was applied to the normal glucose solution, [ 18 F ]FDG uptake rate increased. These results suggest that exogenous and endogenous lactate can substitute for glucose in the brain.

Role of acetyl- l-carnitine in the brain: revealed by Bioradiography

To elucidate the role of acetyl- l-carnitine in the brain, we used a novel method, ‘Bioradiography,’ in which the dynamic process could be followed in living slices by use of positron-emitter labeled compounds and imaging plates. We studied the incorporation of 2-[ 18F]fluoro-2-deoxy- d-glucose ([ 18F]FDG) into rat brain slices incubated in oxygenated Krebs–Ringer solution. Under the glucose-free condition, [ 18F]FDG uptake rate decreased with time and plateaued within 350 min in the cerebral cortex and cerebellum, and the addition of 1 or 5 mM acetyl- l-carnitine did not alter the [ 18F]FDG uptake rate. When a glutaminase inhibitor, 0.5 mM 6-diazo-5-oxo- l-norleucine (DON), was added under the normal glucose condition, [ 18F]FDG uptake rate decreased. Acetyl- l-carnitine (1 mM), which decreased [ 18F]FDG uptake rate, reversed this DON-induced decrease in [ 18F]FDG uptake rate in the cerebral cortex. These results suggest that acetyl- l-carnitine can be used for the production of releasable glutamate rather than as an energy source in the brain.

In vitro and in vivo contractile properties of the vastus lateralis muscle in males with COPD.

Peripheral muscle weakness is common in chronic obstructive pulmonary disease (COPD) but it is still under debate whether weakness is due to atrophy or contractile dysfunction. In vitro and in vivo contractile properties of the vastus lateralis muscle were studied in 16 patients with stable COPD (forced expiratory volume in one second 39 +/- 16% of predicted, age 67 +/- 4 yrs (mean +/- sD)) and nine sedentary control subjects. Isometric knee extensor strength was measured while mid-thigh muscle cross-sectional area (MTMCSA) was obtained using computed tomography. Muscle strips from the vastus lateralis obtained through open biopsy were rapidly suspended in an oxygenated Krebs-Ringer solution that was maintained at 35 degrees C with a pH of 7.40 to study their contractile properties. The isometric knee extensors strength/MTMCSA ratio was 0.50 +/- 0.08 versus 0.58 +/- 0.06 kg x cm(-2) for COPD and control subjects, respectively. The muscle bundle cross-sectional area (CSA) was 4.6 +/- 2.1 and 4.4 +/- 3.1 mm(-2), the length at which active tension was maximum was 15 +/- 4 and 15 +/- 3 mm, and maximal isometric peak forces normalised for CSA were 4.3 +/- 2.7 and 4.8 +/- 2.6 N x cm(-2) for COPD and control subjects, respectively. The force/frequency relationship tended to be shifted to the right in patients with COPD, meaning that a higher stimulation frequency was necessary to produce the same relative force. Patients with COPD had a lower proportion of type I fibre than controls (26 +/- 12% versus 39 +/- 11%) with reciprocal significant increase in type IIb fibre proportion (20+/-16% versus 8 +/- 4%). The proportion of type IIa fibres was similar between the two groups. These results suggest that the contractile properties of the vastus lateralis are preserved in patients with chronic obstructive pulmonary disease. Therefore, the reduction in the quadriceps strength in patients with chronic obstructive pulmonary disease cannot be explained on the basis of an alteration of the contractile apparatus.

Greater Resistance and Lower Contribution of Free Radicals to Hypoxic Neurotoxicity in Immature Rat Brain Compared to Adult Brain as Revealed by Dynamic Changes in Glucose Metabolism

Seven-day-old rat brain slices were incubated at 36°C in oxygenated Krebs-Ringer solution containing [¹⁸F]2-fluoro-2-deoxy-D-glucose ([¹⁸F]FDG), and serial two-dimensional time-resolved images of [¹⁸F]FDG uptake by the slices were obtained. The Gjedde-Patlak graphical method was applied to the image data, and the duration limit of hypoxia loading that allowed recovery of the fractional rate constant (k3*) of [¹⁸F]FDG (proportional to the cerebral glucose metabolic rate) after hypoxia loading to the unloaded control level was 50 min, and MK-801 as an N-methyl-D-aspartate antagonist had neuroprotective effects, but PBN as a free radical scavenger was ineffective. In our previous study in adult (7-week-old) rat brains [Murata et al., Exp Neurol 2000, 164:269–279], the limit of the hypoxia loading time was 20 min, and both MK-801 and PBN were effective. In the immature rat brains, the ratio of aerobic glucose metabolism to the total glucose metabolism was low compared with the adult rat brains, suggesting only a slight involvement of free radicals in hypoxic neurotoxicity. These data suggest that the higher resistance of immature brains to hypoxia compared to that of adult brains is attributable to a lower involvement of free radicals due to a lower aerobic glucose metabolic rate.

Age-Related Changes in Energy Production in Fresh Senescence-Accelerated Mouse Brain Slices as Revealed by Positron Autoradiography

To investigate age-related changes in cerebral energy production, we compared senescence-accelerated prone mice (SAMP8) as an animal model of accelerated aging and senescence-accelerated resistant mice (SAMR1) as a control. Considering that the cerebral glucose metabolic rate (CMRglc) at the time of O₂ deprivation and 2,4-dinitrophenol (DNP) loading would reflect anaerobic glycolytic capacity and mitochondrial function, respectively, we investigated dynamic changes in CMRglc before and after loading with these perturbations. Fresh brain slices were incubated with [¹⁸F]2-fluoro-2-deoxy-D-glucose ([¹⁸F]FDG) in oxygenated Krebs-Ringer solution at 36°C, and serial two-dimensional time-resolved images of [¹⁸F]FDG uptake in these slices were obtained on the imaging plates. The fractional rate constant (=k₃*) of [¹⁸F]FDG proportional to the CMRglc was evaluated by applying the Gjedde-Patlak graphical method to the image data. The k₃* value before the hypoxic perturbation in all of the brain sites analyzed was higher in SAMP8 than SAMR1 in both the 2- and 10-month-old groups. With O₂ deprivation, k₃* values were higher without site specificity in the 2-month-old SAMP8 than in 2-month-old SAMR1, whereas in 10-month-old mice, there was no significant difference between the two groups. In contrast, with DNP loading, while no significant difference was noted between 2-month-old SAMP8 and 2-month-old SAMR1, in 10-month-old mice, the SAMP8 group showed lower values in certain regions than SAMR1 mice. These results suggest that in the brain tissue of SAMP8, a marked transient enhancement of anaerobic glycolytic capacity in the 2-month-olds and a decrease in mitochondrial function in the subsequent period occur, as a result of which glucose metabolism appears to be enhanced in both the 2- and 10-month-old groups compared to SAMR1 mice.

Shape and tension distribution of the passive rat diaphragm.

We developed an in vitro preparation to investigate shape and stress distribution in the intact rat diaphragm. Our hypothesis was that the diaphragm is anisotropic with smaller compliance in transverse fiber direction than along fibers, and therefore shape change may be small. After the animals were killed (8 rats), the entire diaphragm was excised and fixed into a mold at the insertions. Oxygenated Krebs-Ringer solution was circulated under the diaphragm and perfused over its surface. A total of 20-23 small markers were sutured on the diaphragm surface. At transdiaphragmatic pressure (P(di)) of 3-15 cmH(2)O, curvature was smaller in transverse direction than along fibers. Using finite element analysis we computed membrane tension. At P(di) of 15 cmH(2)O, tension in central tendon was larger than muscle. In costal region maximum principal tension (sigma(1)) is essentially along the fibers and ranged from 6-10 g/cm. Minimum principal tension (sigma(2)) was 0. 3-4 g/cm. In central tendon, sigma(1) was 10-15 g/cm, compared with 4-10 g/cm for sigma(2). The diaphragm was considerably stiffer in transverse fiber direction than along the fibers.

Posthypoxic Reoxygenation-Induced Neurotoxicity Prevented by Free Radical Scavenger and NMDA/non-NMDA Antagonist in Tandem as Revealed by Dynamic Changes in Glucose Metabolism with Positron Autoradiography

Using a positron autoradiography technique, dynamic changes in the cerebral glucose metabolic rate (CMRglc) induced by hypoxia/reoxygenation were investigated in living brain slices. After incubating fresh rat brain slices (300 μm thick) with [18F]2-fluoro-2-deoxy-d-glucose ([18F]FDG) in oxygenated Krebs–Ringer solution at 36°C, serial two-dimensional time-resolved images of [18F]FDG uptake in the slices were obtained on imaging plates. As compared to the unloaded control values, with hypoxia-loading [18F]FDG uptake increased markedly, suggesting enhanced glycolysis. The net influx constant (K) of [18F]FDG at pre-hypoxia-loading and after reoxygenation with loading hypoxia for various periods of time was quantitatively evaluated by applying the Patlak graphical method to the image data. Regardless of the brain region, with hypoxia of ≤10-min duration, the K value returned to the preloading level, whereas with hypoxia of ≥20 min duration only partial or no recovery was seen, indicating irreversible neuronal damage. The 30-min administration of either N-methyl-d-aspartate (NMDA)/non-NMDA antagonist or a free radical scavenger at the same time as reoxygenation after 20 min hypoxia showed a neuroprotective effect inhibiting the decrease in the post-hypoxia-loading K value. In contrast, no such neuroprotective effect was evident with administration of either of these agents only during hypoxia loading, possibly indicating that immediately after reoxygenation neuronal damage was induced mediated by excitatory amino acids and free radicals in tandem. These results demonstrate that serial quantitative evaluation of CMRglc using this technique may be of use in investigating the brain tissue injury associated with hypoxia/reoxygenation as well as clarifying the underlying mechanisms and protective effect of various drugs against such injury.

Neurotoxicity after hypoxia/during ischemia due to glutamate with/without free radicals as revealed by dynamic changes in glucose metabolism

Fresh rat brain slices were incubated with [ 18F]2-fluoro-2-deoxy- d-glucose ([ 18F]FDG) in oxygenated Krebs–Ringer solution at 36°C, and serial two-dimensional time-resolved images of [ 18F]FDG uptake in the slices were obtained on imaging plates. The fractional rate constant of [ 18F]FDG (proportional to the cerebral glucose metabolic rate) from pre-loading of ischemia (O 2 and glucose deprivation)/hypoxia (O 2 deprivation) to the reperfused/reoxygenated post-loading phase was quantitatively evaluated by applying the Gjedde–Patlak graphical method to the image data. Against ischemia an N-methyl- d-aspartate antagonist and hypothermia, but not a free radical scavenger, showed a protective effect when administered during ischemia, whereas no such effect was achieved with any of the above agents when administered after reperfusion. Against hypoxia, there was no protective effect with any of the above agents when administered during hypoxia, although an effect was noted with each when administered after reoxygenation. Excitatory amino acids during ischemia loading were found to be the main factor in the neuronal damage associated with ischemia, while in hypoxia, excitatory amino acids working in tandem with free radicals immediately after reoxygenation were implicated.

Hypoxic but not ischemic neurotoxicity of free radicals revealed by dynamic changes in glucose metabolism of fresh rat brain slices on positron autoradiography.

Dynamic changes in the regional cerebral glucose metabolic rate induced by hypoxia/reoxygenation or ischemia/reperfusion were investigated with a positron autoradiography technique. Fresh rat brain slices were incubated with [18F]2-fluoro-2-deoxy-D-glucose ([18F]FDG) in oxygenated Krebs-Ringer solution at 36 degrees C, and serial two-dimensional time-resolved images of [18F]FDG uptake in the slices were obtained. In the case of loading hypoxia (oxygen deprivation)/pseudoischemia (oxygen and glucose deprivation) for various periods of time, the net influx constant (K) of [18F]FDG at preloading and after reoxygenation/pseudoreperfusion (post-loading) was quantitatively evaluated by applying the Patlak graphical method to the image data. Regardless of the brain region, with hypoxia lasting > or =20 minutes, the postloading K value was decreased compared with the unloaded control, whereas with pseudoischemia of < or =40 minutes, approximately the same level as the unloaded control was maintained. Next, the neuroprotective effect against hypoxia/pseudoischemia loading induced by the addition of a free radical scavenger or an N-methyl-D-aspartate (NMDA) antagonist was assessed by determining whether a decrease in the postloading K value was prevented. Whereas with 20-minute hypoxia, both agents exhibited a neuroprotective effect, in the case of 50-minute pseudoischemia, only the NMDA antagonist did so, with the free radical scavenger being ineffective. These results demonstrate that hypoxia causes irreversible neuronal damage within a shorter period than ischemia, with both free radicals and glutamate suggested to be involved in tandem in the neurotoxicity induced by hypoxia, whereas glutamate alone is involved in ischemic neurotoxicity.

Melatonin release from rat pineals in vitro is stimulated by both the α 2-adrenoceptor agonist medetomidine and the antagonist atipamezole

This study was done to clarify the role of α 2-adrenoceptors in the regulation of pineal melatonin synthesis. Rat pineal glands were incubated in oxygenated Krebs–Ringer solution in perifusion chambers, and perifused for 30 min with α 2-adrenoceptor ligands. The melatonin concentrations were measured from the perifusate by radioimmunoassay. Both medetomidine and atipamezole (≥10 −5 M) increased melatonin release. Yohimbine blocked the increase caused by medetomidine but not by atipamezole. The effects of medetomidine and atipamezole were also additive: the maximum response to atipamezole could be significantly increased by medetomidine. These results suggest that the two drugs stimulate the melatonin synthesis through different mechanisms: medetomidine through α 2-adrenoceptors and atipamezole possibly through nonadrenergic mechanisms. The results differ from previous in vivo experiments suggesting that α 2-adrenoceptor ligands affect melatonin synthesis both centrally and locally in the pineal gland. The local effects are most likely masked under the central regulatory systems in vivo.

Dynamic changes in glucose metabolism induced by thiamine deficiency and its replenishment as revealed by a positron autoradiography technique using rat living brain slices.

Dynamic changes in the cerebral glucose metabolic rate (CMRglc) before and after thiamine replenishment were investigated in living brain slices obtained from pyrithiamine-treated (PT) and pair-fed control rats by use of a positron autoradiography technique. Fresh rat brain slices (300 microm thick) were incubated with [18F]2-fluoro-2-deoxy-D-glucose ([18F]FDG) in oxygenated Krebs-Ringer solution at 36 degrees C, during which serial two-dimensional images of [18F]FDG uptake in the slices were constructed on the imaging plates. The net influx constant (=K) of [18F]FDG was determined by a Patlak graphical method of the image data. Prior to thiamine pyrophosphate (TPP)-loading, the K value in the neurologically symptomatic PT was higher in all brain regions except the thalamus and mammillary body than the control, suggesting compensatory enhanced glycolysis. The rapid decrease in this heightened net influx constant immediately after TPP-loading was surmised to be due to activation of pyruvate oxidation with lactate as the substrate, with this inhibiting the glycolysis. From > or = 150 min after TPP-loading, the K value continued to show low values in the thalamus and mammillary body, which are regarded as the responsible sites for Korsakoff syndrome, whereas in all other sites recovery to control values was observed. These findings suggest that using this technique the quantitative evaluation of serial local changes in CMRglc from thiamine deficiency to after its replenishment may be useful in elucidating the pathophysiology and prognosis of Wernicke's encephalopathy.

Dynamic changes in glucose metabolism of living rat brain slices induced by hypoxia and neurotoxic chemical-loading revealed by positron autoradiography.

Fresh rat brain slices were incubated with 2-deoxy-2-[18F]-fluoro-D-glucose ([18F]FDG) in oxygenated Krebs-Ringer solution at 36 degrees C, and serial two-dimensional time-resolved images of [18F]FDG uptake were obtained from these specimens on imaging plates. The fractional rate constant (= k3*) of [18F]FDG proportional to the cerebral glucose metabolic rate (CMRglc) was evaluated by applying the Gjedde-Patlak graphical method to the image data. With hypoxia loading (oxygen deprivation) or glucose metabolism inhibitors acting on oxidative phosphorylation, the k3* value increased dramatically suggesting enhanced glycolysis. After relieving hypoxia < or = 10-min, the k3* value returned to the pre-loading level. In contrast, with > or = 20-min hypoxia only partial or no recovery was observed, indicating that irreversible neuronal damage had been induced. However, after loading with tetrodotoxin (TTX), the k3* value also decreased but returned to the pre-loading level even after 70-min TTX-loading, reflecting a transient inhibition of neuronal activity. This technique provides a new means of quantifying dynamic changes in the regional CMRglc in living brain slices in response to various interventions such as hypoxia and neurotoxic chemical-loading as well as determining the viability and prognosis of brain tissues.

Triazolam-induced modulation of muscarinic acetylcholine receptor in living brain slices as revealed by a new positron-based imaging technique.

The effect of triazolam, a potent benzodiazepine (BZ) agonist, on muscarinic acetylcholinergic receptor (mAChR) binding was investigated in living brain slices by use of a novel positron-based imaging technique. Fresh rat brain slices were incubated with [11C]N-methyl-4-piperidylbenzilate ([11C]NMPB), a mAChR antagonist, in oxygenated Krebs-Ringer solution at 37 degrees C. During incubation, time-resolved imaging of [11C]NMPB binding in the slices was constructed on the storage phosphor screens. Addition of triazolam (1 microM) plus muscimol (30 microM), a GABA(A) receptor agonist, to the incubation mixture decreased the specific binding of [11C]NMPB. Ro15-1788, a BZ receptor antagonist, prevented this effect, indicating that the effect was exerted through the GABA(A)/BZ receptor complex. These results demonstrated that stimulation of the GABA(A)/BZ receptor lowers the affinity of the mAChR for its ligand, which may underlie the BZ-induced amnesia, a serious clinical side effect of BZ. No such effect in the P2-fraction instead implies that the integrity of the neuronal cells and/or their environment is prerequisite for the modulation of mAChR by GABA(A)/BZ stimulation.

Involvement of barium-sensitive K+ channels in endothelium-dependent vasodilation produced by hypercapnia in rat mesenteric vascular beds.

1. We examined the vasodilatory effect of hypercapnia in the rat isolated mesenteric vascular bed. The preparation was perfused constantly (5 ml min(-1) with oxygenated Krebs-Ringer solution, and the perfusion pressure was measured. In order to keep the extracellular pH (pHe) constant (around 7.35) against a change in CO2, adequate amounts of NaHCO3 were added to Krebs-Ringer solution. 2. In the endothelium intact preparations, an increase in CO2 from 2.5% to 10% in increments of 2.5% decreased the 10 microM phenylephrine (PE)-produced increase in the perfusion pressure in a concentration-dependent manner. Denudation of the endothelium by CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate) (5 mg l(-1), 90 s perfusion) abolished the vasodilatory effect of hypercapnia. 3. An increase in CO2 from 5% to 10% reduced the increases in the perfusion pressure produced by 10 microM PE and 400 nM U-46619 by 48% and 44%, respectively. NG-monomethyl-L-arginine (100 microM) and indomethacin (10 microM) did not affect the vasodilatory effect of hypercapnia, whereas the vasodilatory response of the preparation to hypercapnia disappeared when the preparation was contracted by 60 mM K+ instead of PE or U-46619. 4. The vasodilatory effect of hypercapnia observed in the PE- or U-46619-precontracted preparation was affected by neither tetraethylammonium (1 mM), apamin (500 microM), glibenclamide (10 microM), nor 4-aminopyridine (1.5 mM). On the other hand, pretreatment with Ba2+ at a concentration of 0.3 mM abolished the hypercapnia-produced vasodilation. 5. An increase in the concentration of K+ in Krebs-Ringer solution from 4.5 mM to 12.5 mM in increments of 2 mM reduced the PE-produced increase in the perfusion pressure in a concentration-dependent manner. Pretreatment of the preparations with not only Ba2+ (0.3 mM) but also CHAPS abolished the vasodilatory effect of K+. 6. The results suggest that an increase in CO2 produces vasodilation by an endothelium-dependent mechanism in the rat mesenteric vascular bed. The membrane hyperpolarization of the endothelial cell by an activation of the inward rectifier K+ channel seems to be the mechanism underlying the hypercapnia-produced vasodilation. Neither nitric oxide nor prostaglandins are involved in this response.

Dynamic changes in glucose metabolism by lactate loading as revealed by a positron autoradiography technique using rat living brain slices

To demonstrate the preference of lactate over glucose as an energy substrate in normal brain tissue under normoxic condition, the dynamic changes in glucose uptake by lactate loading were investigated in living rat brain slices using a positron autoradiography technique. Fresh rat brain slices were incubated with [ 18F]2-fluoro-2-deoxy- d-glucose ([ 18F]FDG) in oxygenated Krebs–Ringer solution containing 10 mM glucose at 36°C. During incubation, serial two-dimensional imaging of [ 18F]FDG uptake in the slices was constructed on the imaging plates. Lactate loading (20 mM) reversibly suppressed the [ 18F]FDG accumulation up to 80 min. Compared with the pre-loading and the unloaded control values, [ 18F]FDG uptake was suppressed to 25–45% in cerebral regions and 6–7% in cerebellum. The lactate concentration in the surrounding medium decreased after lactate loading. Hence brain tissue preferentially uses lactate over glucose under normoxic and euglycemic condition.

Receptor imaging technique with 11C-labeled receptor ligands in living brain slices: its application to time-resolved imaging and saturation analysis of benzodiazepine receptor using [ 11C]Ro15-1788

Recently we developed a novel imaging technique using positron emitter-labeled compounds as probes and a storage phosphor screen as a detector. This approach makes it possible to follow a variety of biochemical processes with spatial information in living brain slices. Further technical development is reported here in terms of time-resolved imaging and receptor characterization in a real equilibrium state. The method was validated by use of [ 11C]Ro15-1788, a benzodiazepine receptor antagonist. Fresh brain slices were incubated with [ 11C]Ro15-1788 in oxygenated Krebs-Ringer solution at 37°C, in a specially designed chamber. By placing the chamber on a storage phosphor screen, we could obtain two-dimensional images of radioactivity in the slices. Time-resolved imaging was made at 5 min intervals, revealing that it took 60 min to reach equilibrium binding. The dissociation process was observed by adding an excess amount of unlabeled Ro15-1788 to the chamber, 25 min was required for the full dissociation. In the equilibrium state, i.e. in the presence of free radio-ligand, Scatchard plot analysis was performed on the cerebral cortex (K d = 7.4 nM, B max = 146 fmol/mg tissue) and striatum (K d = 7.5 nM, B max = 107 fmol/mg tissue), suggesting the presence of a single component of binding site in these two regions. The present method, for the first time, made it possible to study a ligand-receptor interaction in living brain slices with temporal and spatial resolutions. This technique should prove useful for studies of receptor function under physiological conditions.

In vitro positron emission tomography (PET): use of positron emission tracers in functional imaging in living brain slices

Positron-emitting radionuclides have short half-lives and high radiation energies compared with radioisotopes generally used in biomedical research. We examined the possibility of applying positron emitter-labeled compounds to functional imaging in brain slices kept viable in an oxygenated buffer solution. Brain slices (300 μm thick) containing the striatum were incubated with positron emitter-labeled tracers for 30–45 min. The slices were then rinsed and placed on the bottom of a Plexiglas chamber filled with oxygenated Krebs-Ringer solution. The bottom of the chamber consisted of a thin polypropylene film to allow good penetration of β+ particles from the brain slices. The chamber was placed on a storage phosphor screen, which has a higher sensitivity and a wider dynamic range than X-ray films. After an exposure period of 15–60 min, the screen was scanned by the analyzer and radioactivity images of brain slices were obtained within 20 min. We succeeded in obtaining quantitative images of (1) [18F]fluoro-deoxyglucose uptake, (2) dopamine D2 receptor binding, (3) dopa-decarboxylase activity, and (4) release of [ 11 C]dopamine preloaded asl-[11C]DOPA in the brain slice preparation. These results demonstrate that positron emitter-labeled tracers in combination with storage phosphor screens are useful for functional imaging of living brain slices as a novel neuroscience technique.