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The hippocampal slice preparation has contributed greatly to analysis of the basic neurophysiology of brain neurons. In addition, because traumatic dissociative procedures are not used, the in vitro slice is particularly well suited for studies of electrophysiological properties of hippocampal neurons in young and aged rodent brain. Using the slice, we have previously observed an aging-dependent enhancement of voltage-activated Ca2+ influx using a combination of intracellular sharp electrode current-clamp and voltage-clamp techniques. The Ca2+-dependent afterhyperpolarization as well as the Ca2+ action potential were significantly larger in aged rat neurons. Using the sharp electrode switch clamp method, similar effects were found for high voltage-activated whole-cell Ca2+ currents. In order to study the mechanistic bases of these aging phenomena at the single-channel level, we have recently focused on recording cell-attached patches from neurons in the partially dissociated hippocampal slice (‘zipper’ slice). This technique, developed by Gray et al. in 1990, subjects slices to a mild enzymatic treatment resulting in the exposure of individual neurons for patch-clamp procedures. Using this technique, we are currently recording single Ca2+ channel activity in hippocampal slices from 4- to 29-month-old rats.

Macromolecular imaging probes (or sensors) of enzymatic activity have a unique place in the armamentarium of modern optical imaging techniques. Such probes were initially developed by attaching optically "silent" fluorophores via enzyme-sensitive linkers to large copolymers of biocompatible poly(ethylene glycol) and poly(amino acids). In diseased tissue, where the concentration of enzymes is high, the fluorophores are freed from the macromolecular carrier and regain their initial ability to fluoresce, thus allowing in vivo optical localization of the diseased tissue. This chapter describes the design and application of these probes and their alternatives in various areas of experimental medicine and gives an overview of currently available techniques that allow imaging of animals using visible and near-infrared light.

This paper reviews evidence that brain aging and Alzheimer's disease (AD) are somehow closely related and that the hippocampus (CA1) is highly vulnerable to cell loss under both conditions. In addition, two current lines of evidence on the mechanisms of hippocampal cell loss with aging are considered, including studies of neuronal calcium dysregulation and studies of cumulative glucocorticoid (GC) neurotoxicity. Moreover, recent electrophysiological studies have shown that excess glucocorticoid activation of hippocampal neurons increases the influx of calcium through voltage-activated calcium channels. Second messenger systems may mediate the steroid modulation of calcium channels. Therefore, it is hypothesized that excess glucocorticoid activation and neuronal calcium dysregulation may be two phases of a single process that increases the susceptibility of neurons to neurodegeneration during aging and Alzheimer's disease.

Ischemia/reperfusion injury is regarded as the main cause of failure in revascularization of limbs and transfer of free flaps in the so called nonreflow phenomenon. This type of damage is caused by the production of free radicals, above all, of neutrophils that release great quantities of extracellular superoxide through the action of a membrane enzyme. In our study we used 40 white rabbits. Rabbit rectus femoris muscle is perfused by a single artery and vein and is therefore a valuable model for study of ischemia-induced reperfusion injury of skeletal muscle. The objective of this study was to individualize a valid method of protection for the muscle from damage by ischemia-induced reperfusion injury. We have tested the effectiveness of WEB2170, a PAF antagonist, of hyperbaric oxygen therapy one (HBO), and of combined employment of WEB2170 and HBO. The results show that both PAF and HBO play important protective roles against damage from ischemia/reperfusion injury, and that the combined employment of both therapies has a synergistic effect. We propose therefore a new therapeutic protocol for the prevention of damage resulting from ischemia/reperfusion injury with the simultaneous employment of this PAF and HBO.

The distance between the beta-subunits of Na+/K+-ATPase isolated from pig dark red kidney medulla was determined by Förster energy transfer. First, oligosaccharides of the beta-subunit were shown to be labelled with three fluorophores: Lucifer yellow (LY), Lissamine rhodamine B sulfonyl hydrazine (LRSH) and Cascade blue (CB). Further, LY and LRSH were used as the donor and the acceptor, respectively, for Förster energy transfer studies to determine the localization of the beta-subunit in the native enzyme which is known to be formed as a tetramer (alphabeta)2. It was found that the beta-subunits in the functional enzyme complex in the membrane are not localized next to each other but are spatially separated. The distance between fluorophores covalently attached to the beta-subunits was found to be 5.1 nm. This conclusion was confirmed by measurements with another donor-acceptor pair CB-LY. The results also support the idea of a direct interaction of the beta-subunit with the extracellular part of the alpha-subunit. These interactions were modified in the presence of millimolar concentrations of magnesium ions. This indicates a crucial role of magnesium in extracellular interactions between the alpha and beta subunits.

A membrane fraction enriched with plasma membranes was isolated from rat ileal brush-border cells before and after five-day starvation of the animals. Cholesterol/phospholipid ratio of the standard cell membranes decreased highly significantly (0.42 to 0.18), as did the microviscosity of the membranes determined by polarization of fluorescence (0.187 to 0.142). Concomitantly, the specific activity of Na,K-ATPase in the basolateral membranes significantly increased (59 to 83 mumol ATP hydrolyzed per mg protein per min).

The imbalance of cation transport is considered to play an important role in the development of hypertension, and this also applies to hypertension during pregnancy. Magnesium (Mg) is one of the factors that regulate cation transport across the cell membrane. We therefore studied the effect of a magnesium-deficient diet on the activity of erythrocyte Na/K-ATPase and Mg-ATPase from six pregnant rabbits and compared the results to those obtained from six controls on a normal diet. None of the rabbits on the deficient diet developed hypertension or intrauterine growth retardation; nevertheless the activity of both enzymes was significantly reduced compared to the group on the normal diet. Since the reduced activity of these enzymes can determine sodium or calcium retention in the cell, Mg deficiency could be the basis of the onset of some forms of hypertension in pregnancy.

1. Mitochondria isolated from porcine adrenal cortex under State 3 conditions oxidized succinate with a rate of 47 +/- 4.48 na oxygen/min/mg/protein and with ADP:O ratio 0.98 +/- 0.09. In the presence of 15 microM deoxycorticosterone the rate of succinate oxidation was 36.8 +/- 3.08 na oxygen/min/mg/protein. 2. Under the same conditions the rate of glutamate oxidation was 22.8 +/- 2.21 and 16.8 +/- 0.65 na oxygen/min/mg/protein, respectively. ADP:O ratio was 1.45 +/- 0.14. 3. Introduction of trace amounts of malate into the mitochondria oxidizing glutamate only slightly increased the rate of O2 uptake. 4. The glutamate dehydrogenase activity in these mitochondria was 12.5 +/- 0.69 nmol/min/mg.

The innervation of guinea-pig and rat cardiac tissue consists of thin noradrenergic axons running parallel to the myocardial fibres and producing well-developed fluorescent networks which are denser in guinea-pig tissue. Transverse sections of arterial vessels exhibit a marked polar-like disposition of adrenergic nerves, which mainly appear to be concentrated in two opposite areas. Tissue sections from daunomycin-treated animals fluoresced a striking orange-red. This drug-specific fluorescence emanated from all parts of the ventricular tissue, with particular intensity in the nuclei. Neither the density nor the shape of the adrenergic nerves were affected by treatment with the drug. Despite penetration and storage of the antibiotic into the ventricular tissue, cardiotoxicity was not found in the present study.

We have studied the effects of anesthetics on synaptic membranes obtained from pig brain by using stearic acid spin labels. The anesthetics used (butanol, halothane, ketamine) affect the rotational mobility of 16-doxylstearate and the order parameter of 5-doxylstearate. The changes in mobility of 16doxylstearate show a stronger fluidization in the membrane core than in vesicles of lipids extracted therefrom. This effect may be operationally described as a disruption of lipid-protein interactions involving hydrophobic proteins. In fact no disordering is induced on the surface of synaptic membranes as shown by the order of Soioxylstearate, indicating a highly immobilized state of the lipids on the membrane surface. The results are discussed in view of our working hypothesis concerning the role of lipids in modulating protein conformation.

We have studied the effect of general anesthetics on the kinetic properties of the mitochondrial Mg2+-dependent ATPase. The enzyme is inhibited by anesthetics (alcohols, halotane, pentrane, ketamine) at concentrations of the order of those found to affect lipid-protein interactions. The inhibition appears usually uncompetitive with respect to the substrate, ATP, with a decrease of both Vmax and KM, indicating a possible stabilization of the enzyme-substrate complex. Arrhenius plots of ATPase activity show a striking increase in activation energy below 17-20 degrees C. Anesthetics affect the temperature dependence by increasing the activation energy above the break or abolishing the break whatsoever. An exception is diethyl ether, that induces a decrease in activation energy and a shift of the break to lower temperatures. Anesthetics make the ATPase insensitive to energy transfer inhibitor, oligomycin and dicyclohexyl carbodiimide. At low anesthetic concentration the oligomycin inhibition curve is changed from sigmoidal to hyperbolic, showing a loss of cooperativity in the inhibition.

We have investigated the effect of n-butanol on the spectroscopic properties of two types of probes, that are usually employed to detect membrane fluidity, in mitochondrial phospholipid vesicles and mitochondrial membranes. Although both a spin label (16-doxylstearate) and the fluorescent probe perylene detect a higher immobilization of the membrane in comparison with phospholipid vesicles, the effect of butanol in the case of the membrane is strikingly different with the two techniques employed, showing increased fluidity in the case of the spin label and decreased fluidity with the fluorescent probe.

We have tested the working hypothesis that anesthetics, by labilizing lipid-protein interactions, induce conformational changes in membrane proteins involved in the transmission of neural impulses. In the first communication of this series we report that general anesthetics induce changes in the fluorescence of the probes ANS and NPN in model membranes, lipid vesicles and mitochondria. The changes observed concern the quantumyield but not the position of the emission maximum. Such changes may be interpreted as due to fluidization of the membrane core (NPN), accompanied by variable effects in the membrane surface(ANS).

An investigation was undertaken of the capacity of thepirubicin (THP), a new anthracycline derivative with reduced cardiotoxicity, to be fixed in the myocardial fibre, as compared with that of adriamycin hydrochloride (ADR), using a method based on the histofluorescence of tissues treated in vivo with anthracyclines. Histological examination of heart sections from rats perfused with either THP or ADR showed similar results. The major difference was the lower quantitative localization of THP in the cytoplasm, although this quantitative difference was not so pronounced in the nuclei. This suggests that cardiac lesions may be due not to the basic action of anthracyclines on DNA but to collateral effects not yet identified.

2-(p-Nitrophenyl)-4-isopropylmorphine (V), an analog of 1-(p-nitrophenyl)-2-isopropylaminoethanol (INPEA, I) in which the OCHCHN chain of I is locked in a morpholine ring, loses the beta-receptor blocking activity of I on various isolated preparations. The same ineffectiveness is observed in the O-methyl (II), N-methyl (III), and N,O-dimethyl analog (IV) of I. However, some other properties which are present in I, such as inhibitory effect on acetylcholine or on 5-HT, intrinsic alpha-sympathomimetic activity, and potentiation of catecholamines, are maintained; this demonstrates a complete dissociation of these effects from beta-receptor blockade. The interactions with the alpha-adrenoceptors and with the uptake mechanism are discussed on the basis of the structure-activity relationship between I and its analogs II-V.

The responses of the isolated rabbit and human renal pelvis to drugs and to electrical stimulation have been investigated. Regular spontaneous changes in tension occur in the circular smooth muscle, which in the rabbit are synchronous with electrical waves. The responses to electrical stimulation seem to be due to the release of catecholamines from adrenergic nerves. Such nerves have been observed by means of a fluorescence technique. The cholinergic system appears functionally unimportant. The adrenergic responses in human and rabbit seem to be mediated exclusively by α-adrenoceptors.

Anesthesia by means of ketamine induces an increase of fluidity in the hydrophobic area of rat brain synaptic membranes, as proved by the spin label 16-doxyl stearate, in contrast with no change induced in the order parameter of 5-doxylstearate that probes the membrane surface. These effects are qualitatively similar to those observed by ketamine addition in vitro. On the contrary, acetylcholinesterase from synaptic membranes, although inhibited in vitro by ketamine, is activated in ketamine-anesthetised rats suggested that metabolic changes are at the basis of the effects observed in vivo.

In this communication we report the effects of general anesthetics on the mobility and order of spin labeled stearic acid derivatives in synaptic membranes and in bilayers formed from the lipids extracted therefrom. The anesthetics studied abolish the immobilization induced by synaptic membrane proteins on the membrane lipids : this effect, observed particularly in the bilayer core, is interpreted as a labilization of lipid-protein interactions induced by anesthetics.

We have investigated the effect of general anesthetics (the normal alcohol series up to pentanol, halothane, pentrane, diethyl ether and ketamine) on lipid fluidity of phospholipid vesicles and mitochondrial membranes and on the kinetics of mitochondrial ATPase, a lipid-requiring enzyme. Lipid fluidity has been studied by using spin labels (5- and 16-doxyl stearic acids) and the fluorescent probe N -phenyl naphthylamine (NPN). The spin labels show that all anesthetics tested have a slight fluidizing effect on lipid vesicles but induce a very strong increase in mobility in mitochondrial membranes. Similar effects have been found by analysing the decrease in fluorescence of NPN induced by anesthetics: the decrease is due to decreased quantum yield as shown by double reciprocal plots of probe fluorescence against membrane concentration. The fluorescence decrease is interpreted as an increase in fluidity of the bilayer and not as an increase in polarity of the probe environment, since there is no significant shift in the fluorescence spectra induced by anesthetics. The effects are stronger in mitochondrial membranes than in lipid vesicles, showing a labilization of lipid-protein interactions induced by anesthetics. The mitochondrial ATPase is inhibited by anesthetics at concentrations of the order of those found to affect lipid-protein interactions. The inhibition appears usually uncompetitive with respect to ATP, with a decrease of both V max and K M indicating a possible stabilization of the enzyme-substrate complex. Arrhenius plots of ATPase activity show a striking increase in activation energy below 17–20°; anesthetics affect the temperature dependence in a variable way depending on the specific anesthetic used; most increase the activation energy above the break or abolish the break completely. Anesthetics make the ATPase insensitive to the energy transfer inhibitor oligomycin. At low anesthetics concentration the oligomycin inhibition curve is changed from sigmoidal to hyperbolic, showing a loss of cooperativity in the inhibition.

alpha-Naphthyl-isothiocyanate (ANIT) is a well known toxic substance which induces characteristic hepatic lesions. Its distribution in some organs and body fluids was investigated by using spectrophotometry, gas-chromatography and thin-layer chromatography. The results indicate that ANIT concentrates in the liver, kidneys and blood but not in the brain, urine and bile. Another five ANIT-like substances have also been examined, but no trace of them has been found in these organs and body fluids. The authors suppose that ANIT is metabolized to an unknown metabolite which is responsible for the toxic action of ANIT. None of the ANIT-like substances examined by us can be indentified with this metabolite.

The fluidity of the liver plasma membrane during the first cell cycle of hepatectomized rats has been investigated by means of stearic acid spin labels and of the fluorescence polarization of perylene. The order parameters of 5-doxylstearate, that probes the membrane surface, have been found lowered in the hepatectomized rats after 15 hours from operation, in comparison with anesthetised and sham-operated controls at the same time; on the contrary, the fluidity in the membrane core, as probed by 16-doxylstearate, is increased in all groups. The fluorescence polarization of perylene is also lower in the hepatectomized rats. The results suggest that liver regeneration is accompanied by a fluidity increase of the plasma membrane.

This study was undertaken to investigate aclacinomycin distribution in the rat, using a method based on the histofluorescence of tissues treated in vivo with anthracyclines. The target organs were the kidney, lung and pancreas; a fainter fluorescence was also detected in the heart compared with adriamycin due to a quantitatively different fixing of the two anthracyclines. Our findings revealed a preferential uptake into the cell nucleus in all tissues examined except the adrenal gland medulla where a slight fluorescence appeared only in the cytoplasm.

The pattern of noradrenergic innervation of guinea-pig hepatic tissue was examined by using the fluorescence method of Falk et al. (1962). The distribution of monoaminergic nerve fibres in untreated animals was clearly evident at the level of the portal vein, the hepatic artery and the bile duct. The hepatic artery was the most densely innervated. Mast cells and Kupffer cells had no connection with the adrenergic fibres. In daunomycin-pretreated animals, the hepatic cells appeared to be deep red in colour owing to the presence of the antibiotic. Some noradrenergic nervous fibres, never previously described, were unexpectedly distinguishable in the liver parenchyma against the background red colour of daunomycin-pretreated tissue. They appeared to be contiguous to the hepatocytes and were of different shapes: comma-like, V-like, or like a string of pearls. The nature and the functional role of these elements is briefly discussed.

Investigations were carried out into morphological differences between the uptake of adriamycin and its 13-OH metabolite adriamycinol into rat liver and heart. In the liver parenchyma, adriamycinol is mainly stored in the central area of the hepatic lobule, while adriamycin shows a more uniform distribution. In cardiac tissue, adriamycin accumulates within chromatin masses, whereas adriamycinol scarcely enters myocardial cells. The cardiotoxicity of both anthracyclines might be related to interactions with cytomembrane binding sites.