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.     

Effects of protein meals on the urinary excretion of various plasma proteins in healthy subjects

To examine whether hemodynamic changes in response to acute protein loadings with different protein sources cause increases in urinary excretion of plasma proteins in healthy subjects, urinary excretions of various plasma proteins with various molecular radii and isoelectric points, namely albumin (Alb), IgG, IgG4, ceruloplasmin (CRL), and alpha2-macroglobulin (A2), were measured in healthy subjects after ingestion of a beef meal or of a tuna fish meal. Significant increases in urinary excretions of the negatively charged IgG4 and CRL and of the neutrally charged IgG were found in parallel with enhanced creatinine clearances after each protein ingestion. These renal responses returned to basal levels 9 h after the test. This finding suggests that in healthy subjects, the increase in glomerular filtration rate after acute protein loading caused selective enhancement of urinary excretions of plasma proteins with a molecular radius of approximately 55 A (the radius of IgG, IgG4, and CRL), irrespective of the charge barrier of the glomerulus. The increases in these three plasma proteins may be induced by leakage via the shunt pathway in the glomerulus, as proposed earlier (see text). In contrast, increases in urinary excretions of A2 and Alb were not found. The former finding may be explained by the possibility that A2 would not pass through this pathway, since the molecular radius of A2 (88 A) is larger than that of IgG, although the latter finding may be partially explained by preferential renal tubular reabsorption of Alb.     

Shock and acute organ dysfunction

Multiple trauma, hemorrhage, and sepsis may produce various kinds of shock, and such a host as shock could not be controlled and may easily fall into multiple organ dysfunction. Although those mechanisms on the pathogenesis of these sequential inflammatory responses have been clarified recently, the clinical outcome of such patients suffering from severe sepsis and multiple organ dysfunction is still very low. This inflammatory response against the insult shows a sequential manner; cardiovascular system failure, renal system failure, respiratory system failure, central nervous system failure, and finally, hepatic failure. However, the host response to the insult is a kind of defense against the invasion, and the clinical goal might be to stabilize hemodynamic system, metabolic system, and immunologic system. To achieve hemodynamic homeostasis, we use catecholamines and blood transfusion to improve the oxygen supply to important organs and enhance tissue repair. For metabolic homeostasis, early administration of hyperalimentation may be needed, either parenterally or enterally. Enteral feeding may also provided a route for bacterial translocation. To achieve immunologic homeostasis, prophylactic antibiotic administration and metabolic support may be required and should also protect against infection as a secondary invasion. This review explains these mechanisms in terms of the relationship between shock and organ dysfunction and the general features of clinical management.     

The occurrence of polymorphism of mannose 6-phosphate/insulin-like growth factor 2 receptor gene in laboratory and wild rats

Carcinogen-resistant inbred DRH rat strain was established from closed colony Donryu rats in the presence of 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB). Despite using 3'-Me-DAB during the stage of selection, the DRH rats developed normally and did not show any spontaneous tumor at over 1 year of age. In the present study, we examined the polymorphism in mannose 6-phosphate/insulin-like growth factor 2 receptor (M6p/Igf2r) gene and found that the DRH rat showed CCC (Proline)-type polymorphism in exon 48 and the Donryu rat had GCC (Alanine) sequence. Since the DRH rat was developed from the Donryu rat, we examined whether this polymorphism in exon 48 of M6p/Igf2r gene was due to mutation that occurred at the stage of selection in the presence of 3'-Me-DAB, using several other laboratory and wild rats. We detected the presence of polymorphism at the same site of the M6p/Igf2r gene among these rats. It is likely that the polymorphism in exon 48 of the M6p/Igf2r gene is present broadly in rats since ancient times and not due to the mutation during the course of selection unless this site is a hot spot for chemical carcinogens.     

Peripheral choriovitreal neovascularization in proliferative diabetic retinopathy: histopathologic and ultrastructural study

We describe the histopathologic and ultrastructural evidence of choriovitreal neovascularization in the peripheral fundus of a non-vitrectomized eye with proliferative diabetic retinopathy (PDR). One eye with PDR was surgically enucleated because of neovascular glaucoma and studied with light and electron microscopy. The eye had neovascular membranes at the ora serrata of the peripheral fundus. The newly formed vessels originated from the choroid, passed through Bruch's membrane and the retina, and extended into the vitreous. These vessels had either developing or mature characteristics. The endothelial cells of the developing vessels contained a bulky cytoplasm with many intracytoplasmic filaments, ribosomes and rough endoplasmic reticulum. Budding endothelial cells were frequently found in the developing vessels. The endothelial cells of the mature vessels had attenuated cytoplasm and fenestrations with diaphragms. These observations suggest that choriovitreal neovascularization in the peripheral fundus is one of the features of PDR.     

The expression of Ca2+/calmodulin-dependent protein kinase I in rat retina is regulated by light stimulation

Ca2+/calmodulin-dependent protein kinase I (CaM-kinase I) in rat retina was analyzed by immunohistochemical analysis, Western blot analysis and kinase activity assay. Western blot analysis revealed two immunoreactive bands similar to those detected in the brain. Developmental studies revealed that CaM-kinase I expression increased in accordance with postnatal development. Expression of CaM-kinase I in the retinas of rats raised in the complete darkness markedly decreased. CaM-kinase I activity assay supported these findings. Synapsin I was shown to be a possible intrinsic substrate of CaM-kinase I in rat retina. These results elucidated that CaM-kinase I is expressed in the retina and may play an important role in the retinal functions and that the expression of CaM-kinase I is regulated by light stimulation.     

Microphysiometric analysis of human alpha1a-adrenoceptor expressed in Chinese hamster ovary cells.

1. The human recombinant alpha1a-adrenoceptor (AR) has been stably expressed in Chinese hamster ovary cells. Four stable clones, aH4, aH5, aH6 and aH7, expressing 30, 370, 940 and 2900 fmol AR mg(-1) protein, respectively, have been employed to characterize this AR subtype using radioligand binding and microphysiometry to measure extracellular acidification rates. 2. Noradrenaline (NA) gave concentration-dependent responses in microphysiometry with increasing extracellular acidification rates. The potency of NA increased as the receptor density increased; pEC50 values of NA for the clones aH4, aH5, aH6 and aH7 were 6.9, 7.5, 7.8 and 8.1, respectively. This increase of potency according to receptor density indicates the presence of spare receptor for NA. Methoxamine, phenylephrine, oxymetazoline and clonidine also gave concentration-dependent responses with various intrinsic activities. 3. Antagonists shifted concentration-response curves for NA rightward in a concentration-dependent manner. Schild analysis revealed that the affinity profile of this AR subtype to antagonists in the clone aH7 had a typical pattern for the alpha1a-AR; high affinity for prazosin and WB 4101, and low affinity for BMY7378 (pA2=9.5, 9.8 and 7.3, respectively). This profile is similar in the case of the clone aH4. These affinities were in good agreement with those obtained in binding experiments. 4. These results have demonstrated that (1) classical receptor theory can be applied in microphysiometry, and (2) microphysiometry is a useful tool to investigate the pharmacological characterization of alpha1a-AR.     

The effect of nipradilol on the isoproterenol-induced depression of contractions in the cat soleus muscle

The effect of nipradilol on the isoproterelol-induced depression of contractions of the soleus muscle of the anesthetized cats was studied. Isoproterenol (0.3 microg/kg) injected intravenously decreased the tension and degree of fusion of incomplete tetanic contractions of the soleus muscle of the anesthetized cats. The effect of isoproterenol was blocked by nipradilol (> or = 3 microg/kg), desnitro-nipradilol (> or = 10 microg/kg) and propranolol (> or = 10 microg/kg), but not by nitroglycerin (10-100 microg/kg). Nipradilol (30 microg/kg) and desnitronipradilol (300 microg/kg) almost completely antagonized the depressor effects of isoproterenol. These results coupled with evidence that nipradilol does not penetrate the blood-brain barrier indicate that nipradilol exerts an anti-tremor action by blocking peripheral beta2-adrenoceptors.     

Pharmacokinetic interaction between warfarin and a uricosuric agent, bucolome: application of In vitro approaches to predicting In vivo reduction of (S)-warfarin clearance.

A uricosuric agent, bucolome, has been shown to intensify the anticoagulant effect of warfarin. The aims of the present study were to clarify its mechanism(s) and to apply in vitro approaches for predicting this potentially life-threatening in vivo interaction. An in vivo study revealed that Japanese patients given warfarin with bucolome (300 mg/day, n = 21) showed a 1.5-fold greater international normalized ratio than those given warfarin alone (n = 34) despite that the former received a 58% smaller warfarin dose than the latter. Enantioselective assays revealed that bucolome increased plasma unbound fractions of (S)- and (R)-warfarin by 2-fold (p <.01), reduced unbound oral clearances of (S)- and (R)-warfarin by 84 (p <.01) and 26% (p <.05), respectively, and inhibited the unbound formation clearance for (S)-warfarin 7-hydroxylation by 89% (p <.01). In contrast, bucolome elicited no appreciable changes in the plasma unbound (S)-warfarin concentration versus the international normalized ratio relationship. In vitro studies with recombinant human cytochrome P-450 2C9 and liver microsomes showed that bucolome was a potent mixed-type inhibitor for (S)-warfarin 7-hydroxylation, with K(i) of 8.2 and 20.2 microM, respectively. An in vitro model incorporating maximum unbound bucolome concentration in the liver estimated as a sum of hepatic artery and portal vein concentrations and in vitro K(i) made an acceptable prediction for bucolome-induced reductions in in vivo total (bound + unbound) oral clearance, unbound oral clearance, and unbound formation clearance for (S)-warfarin. In conclusion, the augmented anticoagulant effect of warfarin by bucolome due to the metabolic inhibition for pharmacologically more potent (S)-warfarin may be predictable from in vitro data.