Decrease In Light Scatter Research Articles

Compensatory role of the NBCn1 sodium/bicarbonate cotransporter on Ca2+-induced mitochondrial swelling in hypertrophic hearts.

NBC Na+/HCO3- cotransporter (NBCn1) and NHE1 Na+/H+ exchanger have been associated with cardiac disorders and recently located in coronary endothelial cells (CEC) and cardiomyocytes mitochondria, respectively. Mitochondrial NHE1 blockade delays permeability transition pore (MPTP) opening and reduces superoxide levels, two critical events exacerbated in cells of diseased hearts. Conversely, activation of NBCn1 prevented apoptosis in CEC subjected to ischemic stress. We characterized the role of the NHE1 and NBCn1 transporters in heart mitochondria from hypertrophic (SHR) and control (Wistar) rats. Expression of NHE1 was analyzed in left ventricular mitochondrial lysates (LVML), by immunoblots. NHE1 expression increased by ~40% in SHR compared to control (P<0.05, n=4). To examine NHE1-mediated Na+/H+ exchange activity in cardiac hypertrophy, mitochondria were loaded with BCECF-AM dye and the maximal rate of pHm change measured after the addition of 50mM NaCl. SHR mitochondria had greater changes in pHm compared to Wistar, 0.10±0.01 vs. 0.06±0.01, respectively (P<0.05, n=5). In addition, mitochondrial suspensions from SHR and control myocardium were exposed to 200μM CaCl2 to induce MPTP opening (light-scattering decrease, LSD) and swelling. Surprisingly, SHR rats showed smaller LSD and a reduction in mitochondrial swelling, 67±10% (n=15), compared to control, 100±8% (n=13). NBC inhibition with S0859 (1μM) significantly increased swelling in both control 139±10% (n=8) and SHR 115±10% (n=4). Finally, NBCn1 Na+/HCO3- cotransporter increased by twofold its expression in SHR LVML, compared to normal (P<0.05, n=5). We conclude that increased NBCn1 activity may play a compensatory role in hypertrophic hearts, protecting mitochondria from Ca2+-induced MPTP opening and swelling.

GABA UPTAKE BY GAT1 MODULATES LONG-TERM OPTICAL CHANGES FOLLOWING ELECTRICAL STIMULATION OF THE PITUITARY GLAND NEUROINTERMEDIATE LOBE

Intrinsic optical changes that follow infundibular stalk stimulation of the neurointermediate lobe of the mouse pituitary gland exhibit three different phases that reflect three distinct physiological events. The first (E-wave) is the rapid light-scattering increase that is associated with a nerve terminal volume increase (mechanical spike), and that accompanies excitation of the neurohypophysial terminals by the invading action potential; the second (S-wave) is the slower light-scattering decrease that is tightly correlated with the secretion of the peptide hormones oxytocin and arginine vasopressin, and the third is the long-duration response (R-wave) that reflects cell volume changes in the pars intermedia. We have studied the E-wave and the S-wave in earlier publications. The R-wave, considered here, is sensitive to chloride replacement as well as to blockade of chloride channels. By blocking GABAAreceptors (which are ligand-gated chloride channels) with pharmacological agents, and by applying GABA directly into the bathing solution, or evoking its release from GABAergic inputs, we have demonstrated that this long-duration optical response is sensitive to chloride movements and reflects GABA-induced changes in the intrinsic optical properties of the pars intermedia. The full time-course of this optical response takes minutes and, therefore, has to embody some other process (or processes) related to the restoration of resting physiological chloride concentrations, following the opening and closing of GABAA-receptor channels. Here we demonstrate that the shape of the R-wave, the long-lasting light-scattering signal, is indeed affected by the activity of GAT1, one of the sodium- and chloride-dependent GABA transporters.

Anoxia induces matrix shrinkage accompanied by an increase in light scattering in isolated brain mitochondria

It is important to monitor mitochondrial conditions, and light scattering (LS) measurements have been applied to the detection of morphological changes in mitochondria in vivo. Little is known about the morphological and LS responses of brain mitochondria to oxygen withdrawal, a critical factor in cell death. We have therefore investigated the morphological and LS responses of isolated brain mitochondria to anoxia. Anoxia induced an increase in LS, reflecting mitochondrial matrix shrinkage. This response was reversible, but was reduced by adding digitonin, which disrupted the outer membrane selectively. This suggested that integrity of the outer membrane was necessary for the matrix response. We further examined the effects of Mg 2+ and ATP on the responses because both exist in cells and modulate the changes in matrix volume. Although Mg 2+ and ATP reduced the rates of increase and decrease in LS, respectively, the magnitudes of the increases in LS caused by anoxia stayed at over 80% of the control level (no Mg 2+) in the presence of Mg 2+ and ATP. This suggested that the increase in LS occurred in cells containing Mg 2+ and ATP during anoxia. In contrast, that caused by inhibitors of the electron transport chain was reduced to below 30% of the control level in the presence of Mg 2+. The present in vitro study provides a basis for interpretation of LS signals from mitochondria in brain research during oxygen withdrawal.

Light scattering in rat neocortical slices differs during spreading depression and ischemia

Spreading depression (SD) and ischemia are different pathophysiological events but have similar characteristics. This study investigated whether similarity exists in the light scattering (LS) properties during SD and ischemia in rat neocortical slices. SD was induced by injection of K + while ischemia was simulated by removing oxygen and glucose. LS was simultaneously recorded with changes in extracellular direct current (DC) potential and extracellular space (ECS) volume. LS was measured using a photon counting fiber optic system and the ECS volume change was determined by measuring the ECS concentration of tetramethylammonium (TMA +). Slices maintained in normal artificial cerebrospinal fluid (ACSF) showed a consistent LS increase during SD, but exhibited two different LS behaviors during 6 min of ischemia. In eight slices, LS decreased and remained so until the end of the ischemic challenge. In another 10 slices, LS diminished initially but, after 2 min, suddenly reversed sign, accompanied by a rapid negative shift in extracellular DC potential. When 50 or 91% of Cl − in the ACSF was replaced by membrane-permeable propionate, LS retained its increase during SD, but always showed the sudden LS reversal during ischemia. In contrast, when Cl − was substituted with membrane-impermeant methylsulfate, the SD-induced LS increase was replaced by an LS decrease, and the sudden LS reversal during ischemia was absent. While the LS signal showed different characteristics during SD and ischemia, the DC potential always presented negative shifts and the ECS volume always exhibited similar decreases. These results suggest that the polarity of the LS signal is determined by the competition of at least two factors: cell swelling and anion influx.

Two different mechanisms underlie reversible, intrinsic optical signals in rat hippocampal slices.

Intrinsic optical signals (IOSs) induced by synaptic stimulation and moderate hypotonic swelling in brain tissue slices consist of reduced light scattering and are usually attributed to cell swelling. During spreading depression (SD), however, light-scattering increases even though SD has been shown to cause strong cell swelling. To understand this phenomenon, we recorded extracellular voltage, light transmission (LT), which is inversely related to light scattering, and interstitial volume (ISV) simultaneously from the same site (stratum radiatum of CA1) in both interface and submerged hippocampal slices. As expected, moderate lowering of bath osmolarity caused concentration-dependent shrinkage of ISV and increase in LT, while increased osmolarity induced opposite changes in both variables. During severe hypotonia, however, after an initial increase of LT, the direction of the IOS reversed to a progressive decrease in spite of continuing ISV shrinkage. SD caused by hypotonia, by microinjection of high-K(+) solution, or by hypoxia, was associated with a pronounced LT decrease, during which ISV shrinkage indicated maximal cell swelling. If most of the extracellular Cl(-) was substituted by the impermeant anion methylsulfate and also in strongly hypertonic medium, the SD-related decrease in LT was suppressed and replaced by a monotonic increase. Nevertheless, the degree of ISV shrinkage was similar in low and in normal Cl(-) conditions. The optical signals and ISV changes were qualitatively identical in interface and submerged slices. We conclude that there are at least two mechanisms that underlie reversible optical responses in hippocampal slices. The first mechanism underlies light-scattering decrease (hence enhancing LT) when ISV shrinks (cell swelling) under synaptic stimulation and mild hypotonia. Similarly, as result of this mechanism, expansion of ISV (cell shrinkage) during mild hypertonia leads to an increased light scattering (and decreased LT). Thus optical signals associated with this first mechanism show expected cell-volume changes and are linked to either cell swelling or shrinkage. A different mechanism causes the light-scattering increase (leading to a LT decrease) during severe hypotonia and various forms of SD but with a severely decreased ISV. This second mechanism may be due to organelle swelling or dendritic beading but not to cell-volume increase. These two mechanisms can summate, indicating that they are independent in origin. Suppression of the SD-related light-scattering increase by lowering [Cl(-)](o) or severe hypertonia unmasks the underlying swelling-related scattering decrease. The simultaneous IOS and ISV measurements clearly distinguish these two mechanisms of optical signal generation.

Small and Large Unilamellar Vesicle Membranes as Model System for Bile Acid Diffusion in Hepatocytes

Uptake of bile acids into the liver cell occurs via active transport or passive diffusion. In a model system, passive diffusion was studied in liposomes using pyranine fluorescence. Rate constants for the diffusion of diverse more polar or more apolar bile acids were examined. Hydrophobic lithocholic acid (LCA) revealed a maximal rate constant of 0.057 s−1; with the polar ursodeoxycholic acid (UDCA), the value was 0.019 s−1. UDCA (3 mol%) effectively decreased the rate constant of 0.1 mM chenodeoxycholic acid (CDCA), whereas cholesterol reached a similar decrease only between 5 and 10 mol%. At higher concentrations of CDCA (above 1 mM) or LCA (0.3–0.4 mM), breaking up of liposomal structure was confirmed by light-scattering decrease and increase of carboxyfluorescein fluorescence. Changes in lipid composition of phosphatidylcholine (PC)- small unilamellar vesicles (SUVs) or large unilamellar vesicles (LUVs) also caused decreasing rate constants. For a cardiolipin (CL):PC ratio of 1:20 the CDCA (0.1 mM) rate constant was 71% lower (0.015 s−1) and for a sphingomyelin (SM):PC ratio of 2:1 the rate constant was 50% lower (0.026 s−1). Changes in membrane fluidity were detected using membrane anisotropy measurements with the 1,6-diphenyl-1,3,5-hexatriene (DPH) method. Membrane fluidity was reduced with cholesterol- but not with CL- or SM-containing SUVs (ratio: cholesterol, CL, SM:PC of 1:5). This model system is currently used for the analysis of more complex lipid vesicles resembling the plasma/hepatocyte membrane, which is either stabilized or destabilized by appropriate conditions. The results should become clinically relevant.

Rapid Changes in Light-Scattering in the Prism ofTorpedoElectric Organ Slice Associated with the Production of Postsynaptic Potentials

Using a bifurcated lightguide placed on the surface of a prism of the electric organ slice ofTorpedo,the generation of postsynaptic potentials by direct stimulation was found to be associated with readily detectable, disphasic optical changes. The changes consisted of a decrease in light-scattering during the rising phase of the electric potential, followed by a prolonged increase in scattering. The effects of varying the calcium-ion concentration in the medium strongly support the view that replacement of transmitter molecules for bound Ca-ions in the receptor proteins is at the base of these changes. The effects of synaptically active reagents on these optical signals were demonstrated.

Solubilization of unilamellar phospholipid bilayers by nonionic surfactants

The mechanisms governing the solubilization of neutral or electrically charged unilamellar liposomes by a series of octylphenol polyethoxylated surfactants (average of ethylene oxide units between 8.5 and 20.0) were investigated. Solubilization was detected as a decrease in light-scattering of liposome suspensions. To this end, in accordance with the nomenclature adopted by Lichtenberg, three parameters were considered as corresponding to the effective surfactant/lipid molar ratios (Re) at which the surfactant (a) saturated the liposomesRe sat; (b) resulted in a 50% solubilization of vesiclesRe 50% and (c) led to a total solubilization of liposomesRe sol. These parameters, corresponded to theRe at which light scattering starts to decrease, reaches 50% of the original value and shows no further decrease. It is noteworthy that theRe sat parameter decreases as the EO contents or the surfactant critical micellar concentration (CMC) increases. However, theRe 50% and theRe sol parameters show the lowest values for the surfactant with 12.5 EO units in its molecular structure regardless of the electrical charge of the lipid bilayers. As a consequence, these last parameters are not linearly correlated with the CMC of these surfactants. The CMC values of the surfactant/lipid systems at 0.5 mM lipid concentration corresponded in all cases to the surfactant concentration at which liposomes were saturated by surfactants (S sat).

GTP-induced fusion of isolated pancreatic microsomal vesicles is increased by acidification of the vesicle lumen

Using the ‘fusogen’ polycthyleneglycol (PEG), Dawson et al. [1] have concluded that both guanosine triphosphate (GTP)-mduced calcium efflux and the enhancement of IP 3-promoted calcium release from rat liver microsomal vesicles could be attributed to a GTP-dependent vesicle fusion. We have studied GTP-induced fusion of microsomal vesicles from rat exocrine pancreas using light scatter and fluorescence dequenching methods. In the presence of PEG (3%), GTP (10 μM) induced a decrease in light scatter and an increase in fluorescence in the fluorescence dequenching assay (GTP-effect) indicating fusion of the vesicles. Guanosine 5'- O-(3-thiotriphosphate) (10 μM) had no effect on its own and inhibited the GTP-induced signals. Preincubation of the vesicles with adenosine triphosphate (ATP) (4 mM) increased the GTP-effect by 80%. whereas bafilomycin B 1, a specific inhibitor of vacuolar type H +-ATPases, and the protonophore CCCP (10 μM) inhibited only the ATP-dependent part of the GTP-effect. Inhibitors of the vacuolar type H + -ATPase, which are also SH-alkylating reagents such as N-ethylmaleimid (100 μM) and the tyrosine-,cysteinc- and lysine-reactive reagent 7-chloro-4-nitrobenz-2-exa-1,3-diazole (10 μM) [2,3j. abolished the GTP-effect in the absence or presence of ATP. We conclude that GTP induces fusion of pancreatic microsomes which is increased by an H + gradient established by a vacuolar type H +-ATPase.

Detergent solubilization of phospholipid vesicle. Effect of electric charge

In order to explore the effect of electric charge on detergent solubilization of phospholipid bilayers, the interaction of nine electrically charged surfactants with neutral or electrically charged liposomes has been examined. The detergents belonged to the alkyl pyridinium, alkyl trimethylammonium or alkyl sulphate families. Large unilamellar liposomes formed by egg phosphatidylcholine plus or minus stearylamine or dicetyl phosphate were used. Solubilization was assessed as a decrease in light-scattering of the liposome suspensions. The results suggest that electrostatic forces do not play a significant role in the formation of mixed micelles and that hydrophobic interactions are by far the main forces involved in solubilization. In addition, from the study of thirty different liposome-surfactant systems, we have derived a series of empirical rules that may be useful in predicting the behaviour of untested surfactants: (i) the detergent concentration producing the onset of solubilization (Don) decreases as the alkyl chain length increases; the decrease follows a semi-logarithmic pattern in the case of alkyl pyridinium compounds; (ii) for surfactants with critical micellar concentrations (cmc) less than 6 x 10(-3) M, Don. is independent of the nature of the detergent and the bilayer composition; for detergents having cmc greater than 6 x 10(-3) M, Don. increases linearly with the cmc; and (iii) Don. varies linearly with the surfactant concentration that produces maximum solubilization.

Partial inhibition by cyclosporin A of the swelling of liver mitochondria in vivo and in vitro induced by sub-micromolar [Ca2+], but not by butyrate. Evidence for two distinct swelling mechanisms

1. The effects of cyclosporin A on the increase in matrix PPi and consequent swelling of energized liver mitochondria incubated with 1 mM-butyrate, 30 microM-bongkrekic acid or 0.1-35 microM-Ca2+ [Halestrap (1989) Biochim. Biophys. Acta 973, 355-382] were studied. 2. Cyclosporin (1 microM) had no significant effect on the swelling induced by butyrate, bongkrekic acid or Ca2+ at concentrations of less than 0.3 microM. 3. At higher [Ca2+] (greater than 0.3 microM), swelling became progressively inhibited by cyclosporin, although the increase in matrix PPi was slightly greater in the presence than in the absence of cyclosporin. 4. Titration with cyclosporin indicated that there are 128 pmol of relevant cyclosporin-binding sites per mg of mitochondrial protein, with a Ki of about 5 nM. 5. The decrease in light-scattering by hepatocytes induced by butyrate [Davidson & Halestrap (1988) Biochem. J. 254, 379-384] was unaffected by cyclosporin, whereas that induced by vasopressin was inhibited by 20-30% without a significant change in cellular PPi content. 6. It is suggested that there are two mechanisms for the increase in mitochondrial volume induced by Ca2+: a PPi-mediated mechanism that is insensitive to cyclosporin and an additional Ca2(+)-mediated effect that is inhibited by cyclosporin. The nature of these pathways and their inter-relationship is discussed in the following paper [Halestrap & Davidson (1990) Biochem. J. 268, 153-160].

Membrane ionic currents in Rhodobacter capsulatus

1. The cytoplasmic membrane ionic current of cells of Rhodobacter capsulatus, washed to lower the endogenous K+ concentration, had a non-linear dependence on the membrane potential measured during photosynthetic illumination. Treatment of the cells with venturicidin, an inhibitor of the H(+)-ATP synthase, increased the membrane potential and decreased the membrane ionic current at values of membrane potential below a threshold. 2. The addition of K+ or Rb+, but not of Na+, led to an increase in the membrane ionic current and a decrease in the membrane potential in either the presence or absence of venturicidin. Approximately 0.4 mM K+ or 2.0 mM Rb+ led to a half-maximal response. At saturating concentrations of K+ and Rb+, the membrane ionic currents were similar. The membrane ionic currents due to K+ and Rb+ were not additive. The K(+)-dependent and Rb(+)-dependent ionic currents had a non-linear relationship with membrane potential: the alkali cations only increased the ionic current when the membrane potential lay above a threshold value. The presence of 1 mM Cs+ did not lead to an increase in the membrane ionic current but it had the effect of inhibiting the membrane ionic current due to either K+ or Rb+. 3. Photosynthetic illumination in the presence of either K+ or Rb+, and weak acids such as acetate, led to a decrease in light-scattering by the cells. This was attributed to the uptake of potassium or rubidium acetate and a corresponding increase in osmotic strength in the cytoplasm. 4. The addition of NH4+ also led to an increase in membrane ionic current and to a decrease in membrane potential (half-maximal at 2.0 mM NH4+). The relationship between the NH4(+)-dependent ionic currents and the membrane potential was similar to that for K+. The NH4(+)-dependent and K(+)-dependent ionic current were not additive. However, illumination in the presence of NH4+ and acetate did not lead to significant light-scattering changes. The NH4(+)-dependent membrane ionic current was inhibited by 1 mM Cs+ but not by 50 microM methylamine. 5. It is proposed that the K(+)-dependent membrane ionic current is catalysed by a low-affinity K(+)-transport system such as that described in Rb. capsulatus [Jasper, P. (1978) J. Bacteriol. 133, 1314-1322]. The possibility is considered that, as well as Rb+, this transport system can also operate with NH4+. However, in our experimental conditions NH4+ uptake is followed by NH3 efflux.(ABSTRACT TRUNCATED AT 400 WORDS)

Inorganic pyrophosphate is located primarily in the mitochondria of the hepatocyte and increases in parallel with the decrease in light-scattering induced by gluconeogenic hormones, butyrate and ionophore A23187.

1. The effects of a variety of hormones on the PPi content and light-scattering of isolated rat liver cells was studied. 2. The basal PPi content was about 130 pmol/mg of cell protein, and increased after hormone addition, in parallel with a decrease in light-scattering which we have observed previously [Quinlan, Thomas, Armston & Halestrap (1983) Biochem. J. 214, 395-404]. 3. The mean increases in PPi content with the agonists shown (as pmol/mg of protein) were: 0.1 microM-glucagon, 25; 20 microM-phenylephrine, 30; 25 nM-vasopressin, 127; glucagon + phenylephrine, 115; glucagon + vasopressin, 382; 100 microM-ADP, 50; 15 microM-A23187, 72; 1 mM-butyrate, 80. 4. In the absence of extracellular Ca2+, vasopressin had little effect on either the PPi content or the light-scattering of hepatocytes. 5. The magnitude of the increase in PPi content correlated with that of the decrease in light-scattering irrespective of the stimulating agent, provided that the PPi did not exceed 300 pmol/mg of protein. Above this value little additional change in light-scattering was observed. 6. Subcellular fractionation showed that over 90% of the cellular PPi was intramitochondrial in both control and stimulated cells. 7. The data support the conclusions of previous experiments using isolated liver mitochondria [Davidson & Halestrap (1987) Biochem. J. 246, 715-723] that hormones increase the mitochondrial matrix volume through a Ca2+-induced rise in matrix [PPi]. 8. It is further proposed that this increase in mitochondrial [PPi] allows entry of ADP into the mitochondria in exchange for PPi and is therefore responsible for the increase in total mitochondrial adenine nucleotides observed after hormone treatment.

Fluorometer based multi-parameter analysis of phagocytic cell activation.

Simultaneous measurements of the calcium rise, membrane potential change, and 90 degrees light scatter (shape change) responses exhibited by neutrophils upon activation, can be obtained with identical result as that obtained when independently performing each measurement. The putative intracellular mediator diacylglycerol depolarizes membrane potential and causes a decrease in light scatter. Leukotriene B4 causes a rise in calcium and a decrease in light scatter. The chemotactic peptide, N-formylmethionyl-leucyl-phenylalanine, causes a depolarization of membrane potential, a calcium rise, and a decrease in light scatter. The fura 2 measurements of intracellular free calcium indicate that the calcium concentration of unstimulated cells is much lower than previously thought based on quin 2 studies.

The regulation of the oxidation of fatty acids and other substrates in rat heart mitochondria by changes in the matrix volume induced by osmotic strength, valinomycin and Ca2+.

1. The rate of ADP-stimulated respiration with various substrates and the matrix volume of rat heart mitochondria were measured over a range of osmolarities of the medium. 2. The rate of oxidation of palmitoylcarnitine (in the presence of malate) was stimulated 7-fold by increasing the matrix volume from 0.6 to 1.0 microliter/mg of protein. Oxidation of octanoate showed a similar sensitivity to the matrix volume, whereas oxidation of other substrates showed little sensitivity until the volume fell below 0.7 microliter/mg of protein. 3. The matrix volume of heart mitochondria incubated under physiological conditions was about 0.8 microliter/mg of protein. 4. Low concentrations of valinomycin added to mitochondria incubated under such physiological conditions could activate the rate of ADP-stimulated palmitoylcarnitine oxidation by at least 100%. 5. Decreasing the matrix volume increased the reduction of the electron-transferring flavoprotein (ETF), suggesting an effect on electron flow between ETF and ubiquinone, as has been observed for liver mitochondria [Halestrap & Dunlop (1986) Biochem. J. 239, 559-565]. 6. A rapid decrease in light-scattering by heart mitochondria incubated in State 4 was induced by addition of Ca2+, reaching 50% of the maximal effect after about 30 s at 30 degrees C and with K0.5 for Ca2+ of 0.3 microM. This was not associated with a change in matrix volume, and is discussed in terms of a conformational change whose identity remains to be determined. 7. However, incubation of heart mitochondria at 37 degrees C in the presence of 0.65 microM-Ca2+ for 4 min did increase the matrix volume significantly, by 0.181 +/- 0.029 microliter/mg of protein (n = 7, P less than 0.001), similar to the Ca2+-induced changes observed with liver mitochondria [Halestrap, Quinlan, Whipps & Armston (1986) Biochem. J. 236, 779-787]. 8. The possible significance of these results in the co-ordinate regulation of fatty acid oxidation and the citric acid cycle in the heart responding to increased work load or hormonal stimulation is discussed.

Caffeine blocks activation of cyclic AMP synthesis in Dictyostelium discoideum

Cyclic AMP (cAMP) appears to play multiple roles in the development of the social ameba Dictyostelium discoideum, serving as the chemoattractant mediating aggregation, and perhaps also regulating gene transcription in both early and late stages of differentiation. Progress in understanding the mechanism of activation of the adenylate cyclase in D. discoideum has been frustrated by the inability to obtain its activation in vitro. Also, the lack of defined cAMP-defective mutants has prevented a causal relationship from being established between cAMP levels and gene expression. As an alternative approach to studying the role of cAMP in D. discoideum development, we have sought a compound which inhibits cAMP synthesis in a reasonably specific manner. Here we identify caffeine as a compound which rapidly and reversibly inhibits cAMP-dependent activation of the adenylate cyclase without affecting either cell viability or intracellular levels of ATP or GTP. Using this drug, we show that cAMP synthesis is not required for the cAMP-stimulated decrease in lightscattering, the increase in cyclic GMP synthesis, or for chemotaxis toward cAMP. Studies of the mechanism of action of caffeine show that the drug does not act by inhibiting a cAMP phosphodiesterase, by inhibiting binding of cAMP to its receptor, by itself binding to a physiological adenosine receptor, or by directly inhibiting the adenylate cyclase. Instead, caffeine blocks the cAMP-dependent activation of the adenylate cyclase. Since similar effects are obtained with the cation ionophore A23187, it is possible that caffeine exerts its effect by altering intracellular calcium distribution.

Release of acid and changes in light-scattering properties following fertilization of Urechis caupo eggs

The release of a fertilization acid, monitored by measuring the pH of egg suspensions, begins within 10 sec of insemination of Urechis caupo eggs. This is 4 min before the vitelline layer begins to elevate and is apparently unrelated to that process. The eggs of two molluscs, Mytilus californianus and Acmaea incessa, do not form a fertilization acid. The acid of Urechis eggs is not accompanied by release of “fertilization” carbohydrate, sulfate, or a nonvolatile weak acid into the seawater. The light-scattering properties of Urechis eggs change during the first 10 min after insemination. A decrease in light scattering begins by 10 sec and is complete by 1 min (Phase I). This is followed by a further decrease (3–6 min, Phase II) and an increase (6–10 min, Phase III). In striking contrast to an overtly similar situation in sea urchin eggs (fertilization acid and coincident light-scattering decrease), the release of acid and the initial light-scattering change are not the result of cortical granule discharge, and the acid, at least, is not related to the changes in shape or surface area which the eggs undergo. The processes underlying these rapid events are not yet known.

The Proportion of Volatile Aerosols on the Island of Hawaii

Abstract Nephelometry, in conjunction with a tube furnace and an Aitken nuclei counter, has been applied to the investigation of the volatile component of the aerosol budget at Cape Kumukahi, Hilo. and Mauna Loa Observatory, Hawaii. It was found that heating of the incoming air sample resulted in a decrease in light scattering above 100C due to the loss of organics and other easily volatilized compounds, and a drastic increase in Aitken nuclei counts at temperatures above 150C in the presence of ammonium sulfate. In the marine aerosol, a decrease in light mattering at about 45C was observed which is probably due to the loss of moisture during the phase transition from droplet to crystal. A second decrease near 120C is probably caused by the volatilization of organics from the droplet aerosol. In heating to 150C, the amount of light-scattering decrease was found to depend on the air mass. On occasions when volcanic effluent was apparently present, an increase in Aitken nuclei was noted in the heated air. T...

III. Interaction between synthetic adenosine triphosphate analogues and actomyosin systems

The following compounds were synthesized as analogues of ATP:6-morpho 5′-triphosphate (III) and 2,6-dimethlymercapto-9-β- d-ribofuranosylpurine 5′-triphosphate (V). The interactions of these analogues with actomyosin system were investigated, together with those of 3′-deoxythymidine 5′-triphosphate (I), thymidine 5′-triphosphate (II)and 2′,3′- O-isopropylidene adenosine 5′-triphosphate (IV). The degrees of decrease in light-scattering of myosin B on addition of these analogues were similar to that induced by ATP, except in the case of Compound I. The rates of hydrolysis of analogues by myosin B in 0.6 M KCl and 7 mM Ca 72+ were in the decreasing order of ATP > IV ≈ II > V > I ≈ III, while the order of hydrolysis in 0.075 M KC1 and 2mM Mg 2+ was II > ATP > IV > I > III > V. Compounds II and IV, as well as ATP, induced contraction of myofibrils, while Compounds I, II and V did not. It was concluded that hydrogen bondings at the 6-N or O of the base and the 3′-O of ribose with myosin are necessary for the rapid hydrolysis fo an ATP analogue and for contraction of myofibrils by the analogue.

Permeability of Escherichia coli to organic compounds and inorganic salts measured by light-scattering

Resting cells of Escherichia coli strain B are impermeable to the chloride salts of sodium, potassium, ammonium, magnesium, and calcium and are readily plasmolysed in their presence at concentrations of 300 mM. Plasmolysis was monitored by the increase in light scattered by the suspension. Glycerol and sodium formate rapidly equilibrate across the cell membrane, are respired, and do not affect the light-scattering of the suspension. l-Glutamate, l-aspartate, proline, α-ketoglutarate, succinate and hexoses, are accumulated energetically by cells grown with glucose, are respired, and cause a deplasmolysis of the cells with a resultant decrease in light-scattering. The d-isomers of glutamate and aspartate, however, are neither accumulated nor respired. 2,4-Dinitrophenol and 4-hydroxybenzalmalononitrile inhibit the accumulation of substrates but stimulate respiration. Rapid recording techniques were employed to simultaneously record the changes in O 2 consumption and light-scattering in the bacterial suspensions. This approach provided a sensitive method for investigating the penetration of substances into the cells, and the metabolism of the cell; the relationship between these two parameters is discussed.