Direct measurement of ferrous ion transport across membranes using a sensitive fluorometric assay.

The fluorophore, Phen Green SK (PGSK), was assessed for its suitability to be used in an assay for ferrous ion transport into membrane vesicles. The long wavelengths of excitation and emission (506 and 520 nm, respectively) enable PGSK fluorescence to be detected in membranes, such as the chloroplast inner envelope, that contain high levels of carotenoids which absorb light at lower wavelengths. At low concentrations of Fe2+, less than 3 microM, the interaction between PGSK and Fe2+ appears to result in both static and dynamic quenching of the PGSK fluorescence. The characteristics of this quenching were used to develop a calibration curve to determine the concentration of free Fe2+ at these low concentrations. Pronounced quenching of PGSK fluorescence entrapped within chloroplast inner envelope membrane vesicles was observed when Fe2+ was added. The extent of quenching of PGSK fluorescence trapped inside asolectin vesicles on Fe2+ addition was much less. The kinetics of the quenching of PGSK fluorescence by Fe2+ in vesicles was quite different from that for PGSK and Fe2+ in solution. Using the calibration curve developed for interaction of PGSK and low Fe2+ concentrations the initial rates of iron transport could be determined for the chloroplast inner envelope membranes.

Resonance energy transfer between tryptophan 57 in the epsilon subunit and pyrene maleimide labeled gamma subunit of the chloroplast ATP synthase.

The intrinsic fluorescence of the catalytic portion of the chloroplast ATP synthase (CF1) is quenched when cysteine 322, the penultimate amino acid of the gamma subunit, is specifically labeled with pyrene maleimide (PM). The epsilon subunit of CF1 contains the only two residues of tryptophan, which dominate the intrinsic fluorescence of unlabeled CF1. CF1 deficient in the epsilon subunit (CF1-epsilon) was reconstituted with mutant epsilon subunits in which phenylalanine replaced tryptophan at position 15 (epsilonW15F) and position 57 (epsilonW15/57F). CF1(epsilonW15F) containing a single tryptophan, epsilonW57, was labeled with PM at gammaC322. Resonance energy transfer (RET) from epsilonW57 to PM on gammaC322 occurred with an efficiency of energy transfer of 20%. RET was also observed from epsilonW57 to PM attached to the disulfide thiols of the gamma subunit (gammaC199,205) with an efficiency of approximately 45%. The R(o) (the distance at which the efficiency of energy transfer is 50%) for the epsilonW57 and PM donor/acceptor pair is 30 A, indicating that both gammaC322 and gammaC199,205 must be within 40 A of epsilonW57. These RET measurements show that both gammaC322 and gammaC199,205 are located near the base of the alpha/beta hexamer. This places the C-terminus of CF1 gamma much closer to epsilon than hypothesized based on homology to crystal structures of mitochondrial F1. These new RET measurements also allow the alignment of the predicted epsilon subunit structure. The orientation is similar to that predicted from cross-linking and mutational studies for the epsilon subunit of Escherichia coli F1.

Fluctuations in brain glucose concentration during behavioral testing: dissociations between brain areas and between brain and blood.

Traditional beliefs about two aspects of glucose regulation in the brain have been challenged by recent findings. First, the absolute level of glucose in the brain's extracellular fluid appears to be lower than previously thought. Second, the level of glucose in brain extracellular fluid is less stable than previously believed. In vivo brain microdialysis was used, according to the method of zero net flux, to determine the basal concentration of glucose in the extracellular fluid of the striatum in awake, freely moving rats for comparison with recent hippocampal measurements. In addition, extracellular glucose levels in both the hippocampus and the striatum were measured before, during, and after behavioral testing in a hippocampus-dependent spontaneous alternation task. In the striatum, the resting extracellular glucose level was 0.71 mM, approximately 70% of the concentration measured previously in the hippocampus. Consistent with past findings, the hippocampal extracellular glucose level decreased by up to 30 +/- 4% during testing; no decrease, and in fact a small increase (9 +/- 3%), was seen in the striatum. Blood glucose measurements obtained during the same testing procedure and following administration of systemic glucose at a dose known to enhance memory in this task revealed a dissociation in glucose level fluctuations between the blood and both striatal and hippocampal extracellular fluid. These findings suggest, first, that glucose is compartmentalized within the brain and, second, that one mechanism by which administration of glucose enhances memory performance is via provision of increased glucose supply from the blood specifically to those brain areas involved in mediating that performance.

Hormonal responses to exercise in chronic fatigue syndrome.

Chronic fatigue syndrome (CFS) is a debilitating disease characterized by severe, unexplained fatigue and postexertional exacerbation of symptoms. We examined basal endocrine function in a group of CFS patients and a carefully matched group of sedentary controls. The subjects then completed a graded, maximal exercise test on a treadmill, and additional blood samples were drawn 4 min and a day after the end of exercise. There were no differences in basal hormone levels before exercise. Plasma adrenocorticotropin, epinephrine, prolactin and thyrotropin responses 4 min after exercise were lower in the CFS group, but the growth hormone response may have been exaggerated, and the plasma norepinephrine response was similar to that in controls. The next day, there were no differences in hormone levels between the groups, which suggests that long-term changes in endocrine function are unlikely to be a cause of the prolonged fatigue that occurs in CFS patients after a bout of exertion.

Simultaneous measurement of deltapH and electron transport in chloroplast thylakoids by 9-aminoacridine fluorescence.

Electron transport and the electrochemical proton gradient across the thylakoid membrane are two fundamental parameters of photosynthesis. A combination of the electron acceptor, ferricyanide and the DeltapH indicator, 9-aminoacridine, was used to measure simultaneously electron transport rates and DeltapH solely by changes in the fluorescence of 9-aminoacridine. This method yields values for the rate of electron transport that are comparable with those obtained by established methods. Using this method a relationship between the rate of electron transport and DeltapH at various uncoupler concentrations or light intensities was obtained. In addition, the method was used to study the effect of reducing the disulfide bridge in the gamma-subunit of the chloroplast ATP synthase on the relation of electron transport to DeltapH. When the ATP synthase is reduced and alkylated, the threshold DeltapH at which the ATP synthase becomes leaky to protons is lower compared with the oxidized enzyme. Proton flow through the enzyme at a lower DeltapH may be a key step in initiation of ATP synthesis in the reduced enzyme and may be the way by which reduction of the disulfide bridge in the gamma-subunit enables high rates of ATP synthesis at low DeltapH values.

Influences of energization and nucleotide binding on the reaction of Lucifer Yellow vinyl sulfone with the alpha subunits of the chloroplast ATP synthase.

The catalytic portion of the chloroplast ATP synthase (CF(1)) consists of five different polypeptides in the stoichiometry alpha(3)beta(3)gammadeltaepsilon and is structurally asymmetric. Asymmetry is readily apparent in the properties of the six nucleotide binding sites and the single-copy, smaller subunits. Asymmetry is also detected in the alpha subunits by the rapid and covalent binding of Lucifer Yellow vinyl sulfone (LY) to one of the three chemically identical alpha subunits. The binding of LY to a single alpha subunit has allowed the investigation of whether asymmetry in the alpha subunits is a permanent feature of CF(1). The development of an electrochemical proton gradient across illuminated thylakoid membranes and the preincubation of CF(1) in solution with Mg(2+)-ATP were found to alter the LY distribution such that multiple alpha subunits were labeled with LY. Illumination of thylakoid membranes doubled the extent of LY labeling, and fluorescence resonance energy transfer measurements indicated that LY was bound to more than one alpha subunit. Since the change in LY distribution was inhibited by proton ionophores (uncouplers), alteration of alpha conformation by illumination is a result of the generation of a proton gradient. Preincubation of CF(1) in solution with Mg(2+)-ATP had no effect on the extent of LY labeling but resulted in multiple alpha subunits binding LY as determined by fluorescence resonance energy transfer measurements. Adenine nucleotides at substrate level concentrations inhibit the reaction of LY with the alpha subunits. No increase in LY labeling was observed when thylakoids were illuminated under conditions in which CF(1) was catalytically active.

THE CHLOROPLAST ATP SYNTHASE: A Rotary Enzyme?

The chloroplast adenosine triphosphate (ATP) synthase is located in the thylakoid membrane and synthesizes ATP from adenosine diphosphate and inorganic phosphate at the expense of the electrochemical proton gradient formed by light-dependent electron flow. The structure, activities, and mechanism of the chloroplast ATP synthase are discussed. Emphasis is given to the inherent structural asymmetry of the ATP synthase and to the implication of this asymmetry to the mechanism of ATP synthesis and hydrolysis. A critical evaluation of the evidence in support of and against the notion that one part of the enzyme rotates with respect to other parts during catalytic turnover is presented. It is concluded that although rotation can occur, whether it is required for activity of the ATP synthase has not been established unequivocally.

Regulation of proton flow and ATP synthesis in chloroplasts.

The chloroplast ATP synthase is strictly regulated so that it is very active in the light (rates of ATP synthesis can be higher than 5 micromol/min/mg protein), but virtually inactive in the dark. The subunits of the catalytic portion of the ATP synthase involved in activation, as well as the effects of nucleotides are discussed. The relation of activation to proton flux through the ATP synthase and to changes in the structure of enzyme induced by the proton electrochemical gradient are also presented. It is concluded that the gamma and epsilon subunits of CF(1) play key roles in both regulation of activity and proton translocation.

Enhanced release of norepinephrine in rat hippocampus during spontaneous alternation tests.

Recent evidence suggests that release of acetylcholine (ACh) in the hippocampus is associated with performance on a spontaneous alternation task and with enhancement of that performance by systemic and central injections of glucose. The present study extended these findings by examining norepinephrine (NE) release in the hippocampus using in vivo microdialysis while rats were tested for spontaneous alternation performance with and without prior injections (ip) of glucose. Microdialysis samples were collected every 12 min and assayed for NE content by HPLC-ECD. Like ACh, NE release in hippocampus increased during spontaneous alternation testing. As in past experiments, administration of glucose (250 mg/kg) significantly enhanced alternation scores. However, glucose did not influence NE release either during behavioral testing or at rest. These findings contrast with prior evidence showing that glucose augments testing-related increases in ACh release. The findings suggest that norepinephrine is released within the hippocampus while rats are engaged in alternation performance. However, increased release of norepinephrine apparently does not contribute to the enhancement of alternation scores produced by glucose.

Altered NGF regulation may link a genetic predisposition for hypertension with hyperactive voiding.

PURPOSE: Hyperactive voiding and elevated smooth muscle NGF output are traits of the spontaneously hypertensive rat (SHR). Elevated target-derived NGF is associated with hypertension and hyperactive voiding in SHRs. In the present study, we tested for possible genetic links between hypertension, hyperactive voiding and augmented bladder smooth muscle cell (BSMC) NGF secretion. MATERIALS AND METHODS: We crossed SHRs with WKYs to produce a gene segregating F2 population. We measured F2 mean arterial blood pressure (BP) and six-hour voiding frequency. BSMCs were cultured from 'Low BP F2s' (95+/-2) and 'High BP F2s' (141+/-3 mm. Hg) and conditioned medium tested for NGF with a two-site ELISA. The NGF regulators isoproterenol, platelet-derived growth factor (PDGF) and phorbol-12-myristate-13-acetate were tested in F2 BSMC cultures. RESULTS: A positive correlation (r = 0.75) between blood pressure and voiding frequency existed in this F2 population. As BP rose voiding frequency increased and volume per void decreased such that there were no significant changes in total urine voided (Low BP F2s: 1.0+/-0.5; High BP F2s: 6.2+/-0.5 voids/6 hours). Low BP F2s (2.0+/-0.2) secreted NGF at a higher basal rate than High BP F2s (0.7+/-0.1 fg NGF/hr/100 cells). However, High BP F2s (1,620 and 3,850) were oversensitive to isoproterenol and PDGF-induced increases in NGF output, compared with Low BP F2s (219 and 1,282% control, respectively). CONCLUSIONS: Elevated tissue NGF due to a hypersensitivity to NGF regulating stimuli, rather than alterations in basal NGF, may genetically link hypertension and hyperactive voiding.

Attenuation of morphine-induced behavioral changes in rodents by D- and L-glucose.

Administration of d-glucose enhances learning and memory in several tasks and also attenuates memory impairments and other behavioral effects of several drugs, including morphine. The present experiment compared the effects of peripherally administered d-glucose with those of l-glucose, a stereoisomer of d-glucose that is not metabolized and does not readily cross the blood-brain barrier. Like d-glucose, though at somewhat different doses, peripherally administered l-glucose attenuated morphine-induced deficits in spontaneous alternation performance in rats and mice and attenuated morphine-induced hyperactivity in mice. l-Glucose did not raise circulating levels of plasma d-glucose, suggesting that the effects of l-glucose are not secondary to increased availability of d-glucose. Using direct injections of d- and l-glucose and morphine into the medial septum of rats, the findings indicate that d-glucose but not l-glucose attenuated morphine-induced deficits in spontaneous alternation performance; indeed, intraseptal injections of l-glucose alone impaired spontaneous alternation performance. These findings suggest that peripheral l-glucose antagonizes morphine-induced behavioral effects by a peripheral signaling mechanism, one distinct from the mechanisms that mediate at least some of the effects of d-glucose on brain function.

Influence of divalent cations on nucleotide exchange and ATPase activity of chloroplast coupling factor 1.

The ATPase activity of the catalytic part of ATP synthases is inhibited by free Mg2+, even though MgATP is the substrate. Here we show that the inhibition of the MgATPase activity of chloroplast coupling factor 1 deficient in its epsilon subunit (CF1-epsilon) by Mg2+ is complex. The hydrolysis of MgATP by CF1-epsilon that contains tightly bound ADP, but no bound Mg2+, is initially rapid and decreases within about 1 min to a steady-state rate. The bound MgADP content of CF1-epsilon was varied. The initial fast phase of MgATP hydrolysis is eliminated when the molar ratio of MgADP to CF1-epsilon approaches 2. Loosely bound Mg2+ also affects the initial kinetics of the enzyme that contains bound MgADP. At molar ratios of bound MgADP to enzyme in excess of 1, the initial ATPase activity was low and reached the steady state after about 30 s. Free Mg2+ in the assay mix also inhibited steady-state ATP hydrolysis by all forms of the enzyme. The results are consistent with a model in which two Mg2+ bind cooperatively, probably to the dissociable nucleotide-binding sites on CF1-epsilon. Thus, four different nucleotide-binding sites may be involved in the inhibition of the MgATPase activity of CF1-epsilon. Three of these sites are potentially catalytic, and the fourth may be regulatory. The exchange of bound trinitrophenyl-ADP induced by the addition of MgATP or CaATP was found to be fast enough for the site to be involved in catalysis.

Arterial nerve growth factor (NGF) mRNA, protein, and vascular smooth muscle cell NGF secretion in hypertensive and hyperactive rats.

Elevated levels of nerve growth factor (NGF) protein and NGF mRNA have been reported in the vessels of spontaneously hypertensive rats (SHR: hypertensive, hyperactive) compared to Wistar-Kyoto (WKY) rats. Elevated NGF may be involved in the development of hypertension in SHRs. We examined vascular NGF mRNA and protein content and the regulation of NGF secretion by vascular smooth muscle cells (VSMCs) from two inbred strains (WKHT: hypertensive; WKHA: hyperactive) derived from SHRs and WKYs. Our goal was to determine if receptor-mediated defects in NGF regulation play a role in increased secretion of VSMC NGF from hypertensive animals. Tissue NGF mRNA content was determined by competitive, quantitative RT-PCR. Tissue NGF and NGF content in cultured VSMC-conditioned medium was quantified using a two-site ELISA. Tail artery NGF mRNA was elevated in WKHTs compared to WKHAs. Tissue NGF protein was elevated in WKHT aorta, mesenteric, and tail artery compared to WKHAs. Pharmacologically induced increases in NGF output were blocked with inhibition of transcription or protein synthesis. Basal NGF secretion by WKHT VSMCs was significantly higher than WKHAs. The observed increases in VSMC NGF output in SHRs over WKYs in response to beta-adrenergic agents are not preserved in the WKHT:WKHA comparison. Protein kinase C-dependent increases in SHR VSMC NGF appear in both WKHTs and WKHAs. In contrast, elevated NGF levels due to disturbances in alpha-adrenergic, peptidergic, and purinergic control of NGF output are features common to both genetic models of hypertension (SHR and WKHT). These results suggest that the defect in smooth muscle NGF metabolism observed in SHRs cosegregates with a hypertensive rather than a hyperactive phenotype. Moreover, altered receptor-mediated regulation (alpha-adrenergic, peptidergic, and purinergic) of VSMC NGF production may contribute to elevated vascular tissue NGF, suggesting a mechanism leading to the high levels of NGF associated with hypertension in SHRs and WKHTs.

Effect of prenatal stress on plasma corticosterone and catecholamines in response to footshock in rats.

The effect of prenatal stress was investigated on the sympathoadrenal response to novelty and footshock by measuring the time course of the changes in circulating corticosterone (COR) catecholamines and their metabolites. Pregnant rats were subjected to noise and light stress, three times weekly on an unpredictable basis throughout gestation. When the male offspring of stressed rats (PS) and those of unstressed mothers (C) were 4.5-5 months of age, they were prepared with indwelling catheters in the tail artery 24 h before the experiment. Resting levels of plasma COR, noradrenaline (NA), adrenaline (AD), dihydroxyphenylglycol (DHPG), dihydroxyphenylacetic acid (DOPAC), and dihydroxyphenylalanine (DOPA) were measured. Further blood samples were taken within 3 min of their transfer to the shock box, 1-2, 5, 15, and 45 min after footshock. Plasma COR was significantly higher in PS than in C rats at rest, but those of adrenaline, NA, and their metabolites did not differ in the two groups. Transfer of the rats to the shock box increased plasma COR, NA, adrenaline, and dihydroxyphenylglycol in both groups, and dihydroxyphenylalanine and dihydroxyphenylacetic acid only in PS rats. All the catechols increased further 2-3 min after footshock, except dihydroxyphenylalanine in PS rats. Plasma NA and dihydroxyphenylglycol levels were significantly higher in PS than in C rats immediately after footshock, indicating a greater activation of the sympathetic nervous system in PS rats. The findings demonstrate for the first time that prenatal stress can induce long term changes in the sensitivity of the sympathoadrenal system to stress.

Altered regulation of bladder nerve growth factor and neurally mediated hyperactive voiding.

Elevated bladder smooth muscle cell (BSMC) nerve growth factor (NGF) secretion and related neuroplasticity are associated with hyperactive voiding in spontaneously hypertensive rats (SHRs: hypertensive, behaviorally hyperactive), compared with control Wistar-Kyotos (WKYs). We used two inbred strains (WKHT: hypertensive; WKHA: hyperactive) to further investigate this phenomenon. WKHA BSMCs secreted higher basal levels of NGF than WKHT BSMCs. Antagonists did inhibit NGF output in WKHA but not WKHT cultures. Thus augmented basal secretion of NGF cosegregates with a hyperactive phenotype, whereas a lack of regulatory inhibition of NGF output cosegregates with a hypertensive phenotype. Bladder norepinephrine content paralleled NGF content, with WKHTs > SHRs > WKHAs > WKYs, providing evidence that a lack of inhibition is the greatest contributor to elevated bladder NGF and noradrenergic innervation. Protein kinase C (PKC) agonists affected NGF production differentially depending on strain, suggesting that altered PKC signaling may contribute to strain differences in NGF secretion. Finally, 6-h voiding frequency differed between the strains, with SHRs > WKHTs = WKHAs > WKYs. Thus aspects of both the hypertensive and hyperactive phenotypes may be associated with elevated SHR bladder NGF and hyperactive voiding.

Gender differences in sympathoadrenal activity in rats at rest and in response to footshock stress.

A comparison was made of the dynamics of sympathoadrenal activity in 11 age-matched male and female rats, under basal conditions and after exposure to footshock. Rats were prepared with indwelling catheters in the tail artery 24 h before the experiment. Measurements were made of plasma corticosterone (COR), norepinephrine (NE), epinephrine (EPI), dihydroxyphenylalanine (DOPA), dihydroxyphenylglycol (DHPG) and dihydroxyphenylacetic acid (DOPAC) under resting conditions, after transfer to the shock box (novelty) and at various times after footshock. Under basal conditions, males have significantly higher blood pressure and plasma DHPG/NE ratios but lower plasma levels of COR, NE and DOPAC than females. Three min after exposure to the shock chamber (novelty stress) there were significant increases in COR, EPI, NE and DHPG in both sexes, while DOPA increased only in females and DOPAC remained unchanged in both sexes. Footshock produced a further increase in EPI, NE and DOPAC within 2 min, which lasted about 15 min. There were significant sex differences in the extent and duration of the response of COR, EPI and DHPG. The data show that the female sympathoadrenal system is more reactive than that of the male to the stresses of a novel environment and footshock. The smaller DHPG/NE ratios in females at rest and after stress suggest that neuronal uptake of NE is lower in females than in males. The finding that stress produces larger increments of plasma DOPA and DOPAC in female rats indicates that tyrosine hydroxylase in the sympathetic nerve terminals and adrenal medulla may also be higher than in males.

Neurally mediated hyperactive voiding in spontaneously hypertensive rats.

The development of hypertension in spontaneously hypertensive rats (SHR) and hyperactive voiding in rats with urethral obstruction are characterized by abnormal smooth muscle growth, increased tissue levels of nerve growth factor (NGF) and altered patterns of innervation. The present study was undertaken to determine if bladder smooth muscle from SHRs contains and secretes elevated levels of NGF, and if so, whether the augmented NGF contributes to changes in bladder innervation and function without tissue hypertrophy. Voiding behavior was monitored using specially designed metabolic cages. NGF levels in tissue homogenates and conditioned cell culture media were measured by ELISA. NGF mRNA in cultured bladder smooth muscle cells (BSMCs) was quantified using reverse transcriptase PCR. Noradrenergic innervation was assessed by staining with glyoxylic acid and assaying norepinephrine (NE) content in bladders with high performance liquid chromatography. SHRs voided more frequently than WKY rats. NGF content was higher in bladders from adult SHRs when compared to Wistar-Kyoto normotensive rats (WKYs). No significant difference in NGF mRNA content was observed between SHR and WKY BSMCs. However, SHR BSMCs secreted NGF at a higher rate and amount per unit mRNA than did WKY BSMCs. SHR bladders contained more NE and were more densely stained for catecholaminergic fibers than bladders from WKY rats. The results support the hypothesis that elevated NGF secretion by bladder smooth muscle is associated with hyperinnervation of bladder and hyperactive voiding in SHRs. Thus, the SHR strain may represent a genetic model to study changes in bladder function resulting from altered patterns of innervation.

Activity-dependent regulation of dopamine content in the olfactory bulbs of naris-occluded rats.

Several lines of evidence strongly suggest that reduced olfactory nerve activity results in decreased bulb dopamine content. In the present study, high performance liquid chromatography with electrochemical detection was used to assess catecholamine levels in bulbs from postnatal day 60 rats that had undergone either unilateral naris cautery or a sham surgery on day 30. Thirty days of odor deprivation dramatically reduced dopamine and dihydroxyphenylacetic acid levels in functionally-deprived bulbs (ipsilateral to occluded nares) as compared to contralateral controls, while norepinephrine and dihydroxyphenylglycol levels were unchanged. The loss of dopamine was more severe in medial as compared to lateral aspects of experimental bulbs, while the loss of dihydroxyphenylacetic acid was similar on the two sides. To test directly the hypothesis that afferent activity regulates dopamine and dihydroxyphenylacetic acid content, 1 h of high frequency tetanic nerve stimulation was provided to the rostral-medial olfactory nerve layer in deprived olfactory bulbs, and catecholamine levels were assessed from 6 to 192 h later. Partial and temporary recovery of dopamine was observed in medial aspects of the bulb when rats were examined 96 h later, while consistent recovery of dihydroxyphenylacetic acid content was not apparent. These data corroborate evidence that olfactory nerve activity is a potent regulator of bulb dopamine and indicate that continued afferent input is necessary to maintain dopamine levels.