Intraneuronal [Ca2+] changes induced by 2-deoxy-D-glucose in rat hippocampal slices

Temporary replacement of glucose by 2-deoxyglucose (2-DG; but not sucrose) is followed by long-term potentiation of CA1 synaptic transmission (2-DG LTP), which is Ca2+-dependent and is prevented by dantrolene or N-methyl--aspartate (NMDA) antagonists. To clarify the mechanism of action of 2-DG, we monitored [Ca2+]i while replacing glucose with 2-DG or sucrose. In slices (from Wistar rats) kept submerged at 30 degreesC, pyramidal neurons were loaded with [Ca2+]-sensitive fluo-3 or Fura Red. The fluorescence was measured with a confocal microscope. Bath applications of 10 mM 2-DG (replacing glucose for 15 +/- 0.38 min, means +/- SE) led to a rapid but reversible rise in fluo-3 fluorescence (or drop of Fura Red fluorescence); the peak increase of fluo-3 fluorescence (DeltaF/F0), measured near the end of 2-DG applications, was by 245 +/- 50% (n = 32). Isosmolar sucrose (for 15-40 min) had a smaller but significant effect (DeltaF/F0 = 94 +/- 14%, n = 10). The 2-DG-induced DeltaF/F0 was greatly reduced (to 35 +/- 15%, n = 16) by,-aminophosphono-valerate (50-100 microM) and abolished by 10 microM dantrolene (-4.0 +/- 2.9%, n = 11). A substantial, although smaller effect, of 2-DG persisted in Ca2+-free 1 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N', N'-tetraacetic acid (EGTA) medium. Two adenosine antagonists, which do not prevent 2-DG LTP, were also tested; 2-DG-induced DeltaF/F0 (fluo-3) was not affected by the A1 antagonist 8-cyclopentyl-3, 7-dihydro-1,3-dipropyl-1H-purine-2,6-dione (DPCPX 50 nM; 287 +/- 38%; n = 20), but it was abolished by the A1/A2 antagonist 8-SPT; 25 +/- 29%, n = 19). These observations suggest that 2-DG releases glutamate and adenosine and that the rise in [Ca2+] may be triggered by a synergistic action of glutamate (acting via NMDA receptors) and adenosine (acting via A2b receptors) resulting in Ca2+ release from a dantrolene-sensitive store. The discrepant effects of sucrose and 8-SPT on DeltaF/F0, on the one hand, and 2-DG LTP, on the other, support other evidence that increases in postsynaptic [Ca2+]i are not essential for 2-DG LTP.     

Vasopressin modulates liver regeneration in the Brattleboro rat

Liver regeneration following partial hepatectomy is significantly impaired in rats with hereditary vasopressin deficiency (Brattleboro strain), both in rate of DNA synthesis and in return of liver DNA content to normal. Vasopressin treatment at physiological doses ameliorates the defect and thus appears to be an important modulator of liver regeneration in response to partial hepatectomy in the rat.     

Vasopressin response and terbutaline inhibition of the uterus

A study of the effects of 8-lysine vasopressin (LVP) and its long-acting analogue N-a-triglycyl-8-lysine vasopressin (TGVP) on the myometrium and vasculature of the normal human nonpregnant uterus was undertaken. The results suggest that the vasopressins decrease local endometrial blood flow both directly by effects on the uterine vascular bed, and indirectly by increasing myometrial activity. The effects in vivo can be effectively counteracted by beta2 receptor stimulation.     

Inhibition of doxorubicin-induced apoptosis in vivo by 2-deoxy-D-glucose

Previous studies have shown that DNA cleavage by mammalian topoisomerase II is ATP dependent and can be inhibited by metabolic inhibitors. Furthermore, it has been shown that metabolic inhibitors also have a cytoprotective effect in vitro against topoisomerase II-targeting antitumor drugs. However, the nature of the ATP-dependent process is not known. We have previously shown that doxorubicin induces apoptosis (programmed cell death) in the murine small intestine which can be inhibited by the protein synthesis inhibitor cycloheximide. In the present study, we have demonstrated that 2-deoxy-D-glucose reduces the incidence of doxorubicin-induced apoptosis in vivo if administered within 45 min of the doxorubicin. Maximum reduction was observed at 2 h after treatment (approximately 66%); however, significant reduction was still observable at 9 h after treatment (approximately 33%). Significant positive correlation was observed between protein synthesis inhibition and apoptosis inhibition. Other possible mechanisms of action of the inhibitor do not appear to be important in cytoprotection. The inhibitor did not reduce the uptake of doxorubicin into the intestinal epithelium; however, it caused a significant increase in retention of the drug. The kinetics of inhibition suggest that alteration of cell cycle kinetics, inhibition of formation of doxorubicin-topoisomerase II complex or induction of glucose-regulated proteins are not significant factors in cytoprotection. These studies indicate that at least in the mouse small intestinal epithelium, the ATP-dependent process in cell killing by doxorubicin may involve protein synthesis.     

Age-associated decrease in response of rat aquaporin-2 gene expression to dehydration

In biological movement systems, the level of muscular-articular links is responsible for assembling highly reliable, stable, and reproducible coordination patterns involving very many joints and muscles. Research shows that the important perceptual capabilities of this level arise from the bulk sensitivity of muscles and tendons, so-called effortful or dynamic touch, to the quantities of rotational dynamics that remain invariant (such as the inertia tensor) over variations in rotational forces and motions. The power laws characterizing this sensitivity point to underlying fractal (self-similar) processes. Other research shows that the hallmark ability of this level to produce repetitive interlimb coordinations can be addressed through a dynamics of coordination in which equations express the time-evolution of collective neuromuscular states. This research also suggests that the assembled rhythms exploit the unique blend of stability and variability characteristic of low-dimensional chaotic motion on strange attractors. In overview, research into the capabilities of the level of muscular-articular links highlights the importance of applying classical and modern (nonlinear) dynamics to understanding the assembly and perceptual control of biological movements.     

Vasopressin dilates the rat carotid artery by stimulating V1 receptors

1. The effect of the intraperitoneal administration of cholecystokinin sulphated octapeptide (CCK-8S) (10 nmol/kg i.p.) on endogenous levels of several amino acids in five areas of the rat brain was analyzed. The olfactory bulb, hypothalamus, hippocampus, cerebral frontal cortex, and corpus striatum were evaluated. In addition, the effects of CCK-8S and PD 135,158 (1 mg/kg), a selective CCK(B) antagonist, on the performance of rats submitted to a dark/light transition test were also studied. 2. Upon administration of CCK-8S, the concentration of glutamate was reduced (27%) in the olfactory bulb. The same was observed when the levels of glycine (31%) or alanine (43%) were determined. No significant effects were produced by CCK-8S on cortical and hypothalamic levels. In the hippocampus, the concentration of both glutamate (27%) and taurine (29%) were reduced, whereas the levels of GABA in the striatum (29%) were increased. 3. After a single injection of CCK-8S, the time spent by the rats in the illuminated site of the dark/light transition test box, was not changed. On the contrary, the administration of PD 135,158 increased the time spent in the lighted compartment. 4. These results show that systemic administration of CCK-8S produced regional specific changes in brain amino acids, without producing any significant behavioral modification in the rat exposed to a dark/light box. In contrast, the selective CCKB receptor antagonist, PD 135,158, induces anxiolytic-like action in an animal model of anxiety.     

Tail artery response to sound in the unanesthetized rat

Arterial pulsations have been recorded indirectly from the surface of the rat's tail. Slight heating of restrained unanesthetized rats produces vasodilatation and large amplitude pulsations that are influenced by sensory stimuli, in this case, sound. The surface-recorded pulse volume was found to be proportional to pulse pressure, indicating vasoconstriction as the cause of the decline of the pulse amplitude. A one-second noise burst elicited vasoconstriction, the duration of which was proportional to sound level and occurred as low as at hearing threshold. Under the specified conditions, reproducibility was good with no significant habituation both within session, and between sessions with a one week interval. The warming of the rats was found to be critical for the sound-elicited reactions; responses were obtained only within a narrow, individual temperature-range. The possibilities of using tail vasoconstriction for evaluation of hearing was pointed out, as well as for studies of noise effects on peripheral circulation.     

Neuropeptide Y antibody attenuates 2-deoxy-D-glucose induced feeding in rats

2-deoxy-D-glucose (2-DG) has been shown to induce increased feeding responses in animals. Recent studies suggest the possible involvement of neuropeptide Y (NPY) in 2-DG-induced feeding. The present study examined the effect of immunoneutralization of endogenous NPY on 2-DG-induced feeding. NPY antibody injected into the paraventricular nucleus of the rats significantly attenuated 2-DG-induced feeding, suggesting that hypothalamic NPY may mediate, at least partly, the effect of 2-DG on food intake.     

Alpha 2-adrenoceptors determine the response to nitric oxide inhibition in the rat glomerulus and proximal tubule

Arginine-derived nitric oxide exerts control over the processes of glomerular filtration and tubular reabsorption. The tonic influence of nitric oxide over both of these is eliminated by renal denervation. The hypothesis that the renal nerves function, in this regard, via the activation of alpha 2-adrenoceptors was tested by renal micropuncture. The physical determinants of glomerular filtration and proximal tubular reabsorption were assessed in Munich-Wistar rats before and during the administration of the nitric oxide synthase inhibitor NG-monomethyl L-arginine (L-NMMA). In one set of studies, the systemic infusion of the alpha 2-agonist B-HT 933 rendered nephron GFR, nephron plasma flow, and proximal reabsorption sensitive to reduction by L-NMMA after renal denervation. In a second set of studies, the infusion of the alpha 2 receptor antagonist, yohimbine, to rats with renal nerves intact was found to suppress the effects of L-NMMA on nephron plasma flow and proximal reabsorption. The effects of L-NMMA on nephron GFR and nephron plasma flow, afferent and efferent arteriolar resistances, and proximal reabsorption correlated with the level of underlying alpha 2-adrenergic activity. The activation of renal alpha 2-adrenoceptors increases the influence of arginine-derived nitric oxide in the glomerulus and proximal tubule.     

The response of the rat tail to hyperthermia

When the cartilage of the tail of a baby rat is exposed to temperatures between 41 degrees C and 46 degrees C either necrosis or a small degree of stunting in growth may occur. Isoeffect curves relating time and temperature for both these endpoints for normal and clamped tissue were found to be parallel, a doubling of heating time or an increase in temperature of 1 degree C having the same effect in all cases. Clamping sensitizes the tails by a factor of about three in heating time, equivalent to a temperature difference of 1.5 degrees C. Arrhenius plots show an inactivation energy of 140 kcal/mole. This is similar to that found by other workers using different endpoints, and supports the suggestion that protein denaturation is a critical target for direct heat damage.     

Antibody-mediated secondary eosinophilic response to Taenia taeniaeformis in the rat

An experimental model for neoplastic pleural effusion was made using a transplantable pleural fibrosarcoma MC-106 in ddO mice, and a local immunotherapy of neoplastic pleural effusion with oil-attached BCG cell-wall skeleton was attempted. Viable cells (3 X 10(5)) of MC-106 were injected into the right pleural cavity of the mice on day 0 with a tuberculin syringe which was joined to a two-way tap attached to a capillary manometer. All of the mice in the control group, which received intrapleurally saline solution 24 hr after the injection of tumor cells, died within 24 days and the mean survival time was 16.9 +/- 3.4 (SD) days. Macroscopically massive blooded pleural effusion in both pleural cavities and multiple tumor nodules on the surface of parietal and visceral pleura were observed. On the other hand, the mice which received intrapleurally 100 mug of oil-attached BCG cell-wall skeleton 24 hr after the injection of tumor cells survived much longer. About 50% of the mice remained alive and were killed on day 95. They revealed histologically no malignant lesion of the pleura except for residual changes of inflammatory reactions.     

Characterization of the effect of 2-deoxy-D-glucose(2-DG) on the immune system

This study was designed to characterize the effects of the metabolic stress of administration of 2-deoxy-d-glucose (2-DG, 500 mg/kg) on immune function. Male Lewis rats were exposed to one or five injections (one every 48 h) of 2-DG. Control rats received saline injections. Administration of 2-DG induced a reduction of total leukocytes in the spleen, thymus, and blood. The reduction was most prominent in animals that received five injections of 2-DG. The ratio of CD4(+)/CD8(+) in the spleen was decreased due to a significant increase of CD8(+) T-cell subpopulation. Additionally, 2-DG induced a suppression of mitogenic responsiveness and IFN-gamma production in both whole blood and spleen lymphocytes. The production of IL-1 and IL-2 was significantly reduced in the blood, but not in the spleen. Conversely, there was a significant increase in nitric oxide production in cultures of Con A-, PHA-, and LPS-stimulated splenocytes from 2-DG-injected animals compared with saline-injected controls. In blood cultures stimulated with Con A and PHA, the nitric oxide production of the group that received five injections of 2-DG was significantly higher than in the group that received one injection of 2-DG or saline. These results demonstrated that the metabolic stress 2-DG induced a downregulation of Th 1 cellular immune function in a manner similar to physical and psychological stressors. Additionally, the use of 2-DG in rats provided an important model with which to study metabolic stress.     

cAMP and Ca2+ involvement in the mitochondrial response of cultured fetal rat hepatocytes to adrenaline

The effect of adrenaline on the control of respiratory activity of mitochondria from fetal hepatocytes in primary culture was studied. In the absence of adrenaline, the respiratory control ratio (RCR) of mitochondria increased during the first 3 days of culture due to a decrease in the rate of state 4 respiration. The presence of adrenaline in the incubation medium further increased the mitochondrial RCR through a decrease in the rate of respiration in state 4 and to an increase in the respiration rate in state 3. The effect of adrenaline was mimicked by dibutyryl-cAMP, forskolin, and isobutyl methyl xanthine. All these compounds increased cAMP concentrations, suggesting that cAMP may be involved in the effect of adrenaline. The increase in intracellular free Ca2+ concentrations caused by phenylephrine, vasopressin, or thapsigargin was also accompanied by an increase in the RCR, suggesting that both phenomena are associated. Dibutyryl-cAMP also increased free Ca2+ concentrations, suggesting that the effects of cAMP may be mediated by free Ca2+ concentrations. Adrenaline, dibutyryl-cAMP, phenylephrine, vasopressin, and thapsigargin promoted adenine nucleotide accumulation in mitochondria; this may be an intermediate step in the activation of mitochondrial respiratory function. These results suggest that the stimulatory effect of adrenaline on mitochondrial maturation in cultured fetal rat hepatocytes may be exerted through a mechanism in which both cAMP and Ca2+ act as second messengers. It is concluded that the effect of adrenaline on mitochondrial maturation is exerted by both alpha- and beta-adrenergic mechanisms and is mediated by the increase in adenine nucleotide contents of mitochondria.     

Some peculiarities of the glucoregulatory response to glucose infusion in the rat

The glucoregulatory response to the i.v. infusion of different doses of glucose and glucose plus insulin was studied in anesthetized rats by using the primed constant infusion of glucose-2-3H. Infusion of glucose at the rate of 10 mg/kg/min induced a rise of about 100% in blood glucose, while the hepatic release of glucose showed only a small and transient decrease. A proportional increase of glycemia and glucose utilization (Rd) was observed without any appreciable change in the metabolic clearance rate (MCR) of glucose; a two-fold increase in plasma insulin was recorded at all times. In the group of rats receiving 20 mg/kg/min of glucose, changes in the above parameters were slightly greater; MCR showed a moderate increment in spite of the six-fold rise of plasma insulin. Finally, the influsion of large doses of insulin together with 20 mg/kg/min of glucose resulted in complete cessation of glucose release by the liver and in a remarkable increase of Rd and MCR. These results suggest a poor adaptability of the glucoregulatory system of the rat in response to glucose infusion as compared to other mammalian species.     

Vasopressin processing defects in the Brattleboro rat: implications for hereditary central diabetes insipidus in humans?

The arginine vasopressin (AVP) precursor gene of mammals contains three exons encoding the principal domains of the polyprotein precursor, including vasopressin (exon A), neurophysin (exon B), and glycopeptide (exon C). The AVP precursor (preprohormone) is processed and transported through the endoplasmic reticulum (ER), Golgi apparatus, and secretory vesicles, and finally, mature AVP is secreted from the posterior pituitary into the circulation. The exact steps of these processes during AVP translation and posttranslation events are not yet well elucidated. Defects in peptide processing are associated with several genetic disorders, including central diabetes insipidus (CDI). In the Brattleboro rat with CDI, the mRNA and protein of AVP are present in the hypothalamus, but no circulating AVP is detectable, thus suggesting a processing defect, transport defect, or both. The mutated AVP gene precursor of Brattleboro rat has a deletion of a single base, guanine, in the neurophysin coding region that leads to a frameshift resulting in the loss of the normal stop codon. It has been reported that the mutated precursor is trapped in the ER and does not reach the Golgi apparatus. Recent studies examined AVP secretion in cultured COS cells transfected with various constructs from wild-type and mutated Brattleboro AVP gene precursors. The wild-type in vitro studies demonstrated that intact neurophysin, but not the glycoprotein coding region, is necessary for normal AVP processing and secretion. Next, the results demonstrated that the guanine defect in the neurophysin coding region and the prolonged C-terminus accounted for the processing defect in the Brattleboro rat with CDI. These defects no doubt impair the folding and configuration necessary for normal processing of the AVP gene precursor in the ER. In hereditary CDI in humans, the majority of the mutations have also been shown to occur in the neurophysin coding region. However, in contrast to the recessive defect in the Brattleboro rat, in human CDI, neurotoxicity and denigration of the magnocellular neurons have been observed, and dominant inheritance occurs. Moreover, all mutations are missense, nonsense, or deletions in human CDI rather than the shift in reading frame and preserved neurons that is observed with the Brattleboro rat. Thus, the results from studies in the Brattleboro rat may only be partially applicable to hereditary CDI in humans.