The cytochemical localization of adenylate cyclase: fact or artifact?

In a study of the location of adenylate cyclase activity in rat pancreas with the method of Reik et al. (Science 168:382, 1970), as modified by Howell and Whitfield (J Histochem Cytochem 20:873, 1972) it was found that (a) unspecific staining occurs in rat pancreatic tissue fragments incubated in the Reik-Howell medium in the absence of substrate; (b) addition of adenylyl-imidodiphosphate (AMP-PNP) as substrate, either alone or together with stimulants of rat pancreas adenylate cyclase (secretin. NaF), does not result in increased precipitation; (c) cytochemical incubation of isolated rat pancreatic acinar cells and of rat liver and kidney fragments does not lead to substrate-specific precipitation. In subsequent chemical studies we have found that cyclic adenosine monophosphate (AMP) formation from [alpha32P]AMP-PNP in the presence of rat pancreatic particulate matter is very low in the Reik-Howell medium without lead ions, but is stimulated by addition of lead nitrate (4 mM). Whereas heat-treatment of the particulate matter abolishes all cyclic AMP formation in the absence of lead ions, it actually increases cyclic AMP production in the presence of 4 mM lead nitrate. This indicates that the cyclic AMP formation in the complet Reik-Howell medium occurs by a nonenzymatic mechanism. In addition, this medium shows a tendency to become turbid, particularly when calcium ions are added to the medium, suggesting a possible explanation for the apparently specific cytochemical detection observed by other authors. A revised cytochemical medium, with barium replacing lead and with a pH of 8.9 (optimal for adenylate cyclase with AMP-PNP substrate), leaves rat pancreatic adenylate cyclase activity intact and hormone sensitive, while it is still able to precipitate imidodiphosphate. However, cytochemical incubation of isolated rat pancreatic acinar cells in this revised medium in the presence of AMP-PNP and secretin does not yield an electron-dense precipitate, showing that the enzyme activity is to low to produce sufficient imidodiphosphate. These findings throw further doubt on the validity of the cytochemical detection of adenylate cyclase, reported by other investigators, notwithstanding the alleged positive results.     

Competition between ligands of glycosyltransferases and horseradish peroxidase for binding sites on intracellular and plasma membranes of HeLa cells. Application of a micro-method for the semi-quantitation of surface-bound HRP

A micro-method for the semi-quantitation of surface-bound horseradish peroxidase (HRP) was developed and was applied to study the competition between ligands of glycosyltransferases and HRP for binding sites on the surface of HeLa cells. Dried coverslip cultures of HeLa cells, fixed in methanol, were placed on 0.3 ml of the incubation medium on parafilm and were incubated for 45 min at 37 degrees C. The incubation medium contained HRP, lysozyme and Ca2+ in HEPES buffer, pH 7.2. After washing, the cells were incubated for 60 min at 37 degrees C in HEPES buffer containing 20 mM Ca2+. After this treatment, the plasma membranes showed a strong cytochemical reaction for HRP. Most of the HRP was released into buffer solution during a 5 h incubation at 37 degrees C in the absence of Ca2+, and was measured by spectrophotometry. The addition of 20 mM Ca2+ to the buffer solution prevented the release of most of the HRP from the plasma membranes thus showing that the binding of HRP required Ca2+. Ligands of glycosyltransferases were added to the incubation medium with HRP. The amount of HRP released from the cells decreased in relation to the competing potency and concentration of these ligands. The method was applied to estimate the concentration of some ligands of galactosyltransferase and sialyltransferase that caused a 50% decrease in the release of previously-bound HRP. CMP-neuraminic acid and gangliosides showed a higher competing potency to the surface binding of HRP than UDP-galactose and chitotriose. The spectrophotometric analysis was correlated (on duplicate samples) with cytochemical observations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron microscopic cytochemical localization of alpha-hydroxyacid oxidase in rat liver. Association with the crystalline core and matrix of peroxisomes

The substrate specificity and the intraperoxisomal localization of alpha-hydroxyacid oxidase in rat liver has been investigated cytochemically by the cerium technique and biochemically with a luminometric assay. Rat liver is fixed by perfusion with a low concentration (0.25%) of glutaraldehyde and vibratome sections are incubated for 60 min at 37 degrees C in a medium containing 3 mM CeCl3, 100 mM NaN3 and 5 mM of an alpha-hydroxyacid in 0.1 M of one of the following buffers: Pipes, Mops, Na-cacodylate, Tris-maleate, all adjusted to pH 7.8. Ten different alpha-hydroxyacids with a chain length between 2 and 8 carbon atoms were tested. The best results were obtained with glycolic, argininic and L-alpha-isocaproic acids. These cytochemical findings were confirmed also biochemically using purified peroxisomal fractions isolated by gradient centrifugation in metrizamide. The pattern of the intraperoxisomal localization of the enzyme was influenced markedly by the type of buffer used for the cytochemical incubation. Whereas in the Tris-maleate medium both the cores and the matrix stained with the same intensity, with all other buffers the reaction in cores was more prominent. The staining of cores was abolished by pretreating sections in Tris-maleate (pH 7.8) or alkaline pyrophosphate buffers. These observations establish the substrate specificity of alpha-hydroxyacid oxidase in rat liver and demonstrate the delicate association of this enzyme with the crystalline cores and the matrix of peroxisomes in rat liver.     

Use of strontium for bovine oocyte activation

Strontium efficiently activates mouse oocytes, however, there is limited information on its use in cattle. Thus, the objective of this study was to establish a suitable protocol for activating bovine oocyte with strontium. For pronuclear development, the absence of calcium and magnesium in the activation medium (TALP) with 10 and 50 mM strontium (34.4 and 53.1%, respectively) was superior to the complete TALP (6.5 and 19.4%, respectively). In all activation media, better results were observed with 25 and 50 mM strontium (21.9-53.1 and 19.4-53.1%, respectively). Incubation for 4 h promoted similar results in all strontium concentrations. However, strontium at 15, 20, and 25 mM for 6 and 8 h (40.7, 46.7, and 48.3%, and 29.3, 48.3, and 40.7%, respectively) were superior to control (15.5 and 10%, respectively). After in vitro maturation for 26 h, strontium (S; 20 mM in Ca2+- and Mg2+-free TALP for 6 h), ionomycin+strontium (IS), and strontium+ionomycin (SI) (60, 63.3, and 65%, respectively) were similar in pronuclear development and superior to ionomycin (I; 5 microM for 5 min; 36.7%). In treatments S and I, only 1 PN zygotes were observed. In treatment S, most of them had 1 and 2 PB (35.7 and 60.7%, respectively), and in treatment I, 0, 1, and 2 PB (14.3, 57.1, and 28.6%, respectively). Most of the zygotes in treatment IS and SI were 1 PN 2 PB (77.4 and 61.6%, respectively). The number of oocytes with clusters of cortical granules was similar in all treated groups (11-29%). Cortical granule exocytosis in treatment IS (68%) was similar to S (54%) and superior to I, SI, and control (27, 45, and 5.0%, respectively). Cleavage and blastocyst rates were similar for S, I, IS, and SI treatments (61.7-76.7, and 8.3-13.3%, respectively) and the same was observed for ICM, TE, and total cell number, and ICM/total cell ratio (22-25, 64-69, and 86-95, and 0.26-0.27). In conclusion, strontium may be efficiently applied for bovine oocyte activation at 20 mM in Ca2+- and Mg2+-free TALP medium for 6 h.     

Activation of potassium channels in erythrocytes of marine teleost Scorpaena porcus

To assess the possibility of stimulating Ca2+-activated K+ channels, marine fish erythrocytes were incubated at 20-22 degrees C in saline containing a Ca2+-ATPase inhibitor (orthovanadate), a Ca2+ ionophore (A23187), propranolol or Pb2+. Incubation of the cells for up to 2 h under control conditions or in the presence of 5 mM NH4VO3 and 1 mM Ca2+ did not affect the intracellular K+ and Na+ concentrations. About 50% cellular K+ was lost from erythrocytes incubated in the presence of 0.01 mM A23187, 1 mM EGTA and 0.4-1.0 mM Ca2+. There was a significant loss of cellular K+ after the addition of 0.05-0.2 mM propranolol to the incubation medium. The stimulatory effect of propranolol on the K+ efflux was independent of external Ca2+. Blockers of Ca2+ transport, verapamil and Co2+, caused only a small decrease in the K+ loss induced by propranolol. The treatment of erythrocytes with 1-2 microM Pb2+ led to a minor K+ loss, but at a Pb2+ concentration of 20-50 microM, about 70% cellular K+ was lost. The K+ efflux induced by propranolol or Pb2+ was completely blocked by 1 mM quinine. The induced K+ loss from the erythrocytes was accompanied by a slight increase in the intracellular Na+ concentration. These data indicate the possibility of inducing Ca2+- and Pb2+-activated potassium channels in erythrocytes of S. porcus. A distinctive feature of the cells is a high sensitivity to propranolol, which activates K+ channels in the absence of external Ca2+.     

Viability, acrosome morphology and fertilizing capacity of boar spermatozoa treated with strontium chloride

The positive effect of strontium ions (Sr2+) on sperm motility, capacitation and acrosome reaction has been demonstrated in the mouse, human, guinea pig and hamster. In the present study, we have evaluated the effect of Sr2+ on the viability and acrosome morphology of boar spermatozoa, and on the fertilization and development after the microinjection of Sr(2+)-treated spermatozoa into porcine oocytes. Before incubation, 79% of spermatozoa were classified as propidium iodide (PI)-negative (live) using the LIVE/DEAD Sperm Viability Kit. After incubation with strontium chloride (SrCl2), 39% (0 mM; no divalent cations), 25% (1.9 mM) and 24% (7.5 mM) of them were classified as PI-negative. The proportion of spermatozoa that had initiated the acrosome reaction was higher in Sr(2+)-containing medium than in Sr(2+)-free medium, when assessed by electron microscopy. There was no significant difference in percentage of spermatozoa initiating the acrosome reaction between Sr2+-treated groups (1.9 mM: 22%, 7.5 mM: 33%, p>0.05). After the microinjection of spermatozoa incubated with SrCl2, 67% (1.9 mM) and 61% (7.5 mM) of injected oocytes were successfully fertilized, and then 43% (1.9 mM) and 41% (7.5 mM) contained a fully decondensed sperm head. Sham-injected oocytes were significantly activated at a lower rate than Sr(2+)-treated groups (27%, p<0.05). Next, after microinjection of spermatozoa incubated with 1.9 mM SrCl2 (n=51), 45% of injected oocytes cleaved on day 2, and 18% developed to blastocysts on day 7 (sham-injection, n=48: 15% to cleavage and 0% to blastocyst). These results demonstrate that Sr2+ is likely to positively affect the fertilizing capacity of spermatozoa in the pig.     

Rat pancreatic adenylate cyclase. IV. Effect of hormones and other agents on cyclic AMP level and enzyme release.

1. The effects of secretin and pancreozymin-C-octapeptide and phosphodiesterase inhibitors on the concentration of adenosine 3',5'-cyclic monophosphate (cyclic AMP) and on the release of enzymes from rat pancreas have been studied. 2. In determininging cyclic AMP by means of the saturation assay of Brown et al. ((1971) Biochem. J. 121, 561-563) it is found essential to purify the pancreatic tissue extract by ion-exchange chromatography prior to the assay. 3. Injection of synthetic secretin or pancreozymin-C-octapeptide in anaesthetized rats in a secretory active dose (0.1 nmol) has no effect on the pancreatic cyclic AMP level. 4. Incubation for up to 10 min of pancreatic slices in Krebs-Ringer bicarbonate glucose medium containing 10(-2) M theophylline as phosphodiesterase inhibitor does not result in an increase of the cyclic AMP level. With 10(-2) M 1-methyl-3-isobutylxanthine as phosphodiesterase inhibitor the level is more than doubled after the first min of incubation and remains constant thereafter. 5. Addition of 3-10(-7) M secretin to slices incubated in the presence of 10(-2) M theophylline causes 84% increase of the cyclic AMP level above control, whereas the addition of 3-10(-7) M pancreozymin-C-octapeptide has no significant effect. In the presence of 10(-2) M 1-methyl-3-isobutylxanthine the latter hormone causes significant increases of up to 34% above control during 10 min of incubation. Secretin in this condition augments the cyclic AMP level by up to 296% above control during a 10 min incubation period. Addition of secretin and pancreozymin-C-octapeptide together has no greater effect than of secretin alone. 6. A broken cell fraction of rat pancreas contains adenylate cyclase activity which can be stimulated to 457 and 600% above the basal activity by 3-10(-7) M pancreozymin-C-octapeptide and secretin, respectively. Incubation of pancreatic slices with either hormone has no effect on the cyclic AMP phosphodiesterase activity in the homogenate of these slices. 7. Pancreozymin-C-octapeptide, dibutyryl cyclic AMP, 1-methyl-3-isobutylxanthine and carbamylcholine cause an elevated release of chymotrypsin from pancreatic slices incubated for 2 h in Krebs-Ringer bicarbonate medium, containing 10 mM glucose, while secretin, cyclic AMP and butyric acid have no significant effect. The release of the cytoplasmic enzyme lactate dehydrogenase is also elevated by dibutyryl cyclic AMP, 1-methyl-3-isobutylxanthine and carbamylcholine, but not significantly by pancreozymin-C-octapeptide. 8. The results support the role of cyclic AMP in the action of secretin, and do not exclude a mediating function of this nucleotide in the actions of pancreozymin in rat pancreas.     

Cytochemistry of adenylate cyclase. Quantitative analysis of the effect of cytochemical procedures on adenylate cyclase in heart tissue homogenates

The effects of different preparative and cytochemical procedures on adenylate cyclase (AC) activity in heart muscle homogenates were studied by quantitative analysis. We were mainly concerned with perfusion prefixation, using glutaraldehyde (GA) with and without DMSO, and with the influence of cytochemical incubation with lead ions as the capture reagent. Furthermore, we measured the direct effect on the AC activity of lead, cobalt, and strontium ions in prefixed heart homogenates. We also studied the influence of phosphatidylinositol and 2',5'-dideoxyadenosine. The following results were obtained: 1. Perfusion fixation using 2% GA buffered with cacodylate reduced the AC activity by about 20%. After the entire cytochemical procedure was finished, 20% of the original AC activity was still present. Stimulation by epinephrine, histamine and fluoride, which increased the activity of AC two or three times in our experiments, was only slightly reduced by the cytochemical treatments. 2. Lead ions (2 mM), added to the biochemical assay without chelating compounds, reduced the AC activity about 90%. 5 muM phosphatidylinositol stabilized the fluoride- and hormone-sensitive AC activity. 3. Co2+ also reduced the AC activity, though less than Pb2+. Sr2+ had no effect on the basic activity of the AC but had a slightly inhibitory effect on the hormone and fluoride stimulation. 4. 5% DMSO added to the fixative had no influence on the basic activity of the AC. However, this solvent definitely reduced the level of stimulation by fluoride and guanine nucleotide plus hormones. 5. A potential inhibitor of enzyme activity and of the hormone- and fluoride-sensitive AC was the adenosine derivative, 2',5'-dideoxyadenosine. This compound, at a concentration of 10(-3) M, inhibited all AC activity in the heart homogenates.     

Cytochemical localization of adenylate cyclase and of calcium ion, magnesium ion-activated ATPases in the dense tubular system of human blood platelets.

Cytochemical techniques have been employed to study the localization of adenylate cyclase and (Ca2+ + Mg2+)-stimulated ATPase activities in platelets after fixation. Biochemical analysis of adenylate cyclase demonstrated a 70% reduction in activity in homogenates from fixed cells, but the residual activity could be stimulated 10--20 times by prostaglandin E1 (1 micrometer) under the same incubation conditions as employed in the cytochemical studies (e.g. media containing 2 mM lead nitrate and 10 mM NaF). Adenylate cyclase activity employing 5'-adenylyl-imiodiphosphate (AMP-P(NH)P) as substrate was found to be associated with the dense tubular system (smooth endoplasmic reticulum) in intact fixed platelets, and was apparent only when the cells were incubated with prostaglandin E1. Less activity was found along the membranes of the surface connected open canalicular system and occasionally at the outer cell surface. Enzymatic activity was blocked by the adenylate cyclase inhibitor 9-(tetrahydro-2-furyl) adenine and was not due to AMP-P(NH)P phosphohydrolase activity. The low adenylate cyclase activity in the surface membranes may be due to enzyme inactivation as a result of fixation, since a surface membrane fraction obtained by the glycerol lysis technique from unfixed cells had an adenylate cyclase specific activity equivalent to that in the microsomal membrane fraction. (Ca2+ + Mg2+)-stimulated ATPase activity was found associated with the membranes of the surface connected open canalicular system in unfixed cells. After brief fixation (5--15 min) with glutaradehyde, strong (Ca2+ + Mg2+)ATPase activity became apparent in the dense tubular system. Longer periods of fixation inactivated enzymatic activity. Addition of Ca2+ (1.0 mM) to incubation medium with low Mg2+ (0.2 mM), or increasing Mg2+ to 4.0 mM, in both cases strongly stimulated enzyme activity. The ATPase activity in the platelet membranes was not inhibited by ouabain. It is suggested that the Ca2+-stimulated ATPase and adenylate cyclase activities in the dense tubules may possibly be involved in regulation of intracellular Ca2+ transport.     

Presence of adenylate cyclase activity in Golgi and other fractions from rat liver. II. Cytochemical localization within Golgi and ER membranes

The presence of adenylate cyclase (AC) in liver Golgi and microsomal fractions from ethanol-treated rats was tested cytochemically using 5'- adenylyl imidodiphosphate (AMP-PNP) lead phosphate method. Parallel biochemical assays showed that rat liver Golgi AC was only partially inhibited by lead: in the presence of 1 mM Pb++ 80% of the enzyme was preserved, while when 2 mM Pb++ was used 25% remained. No cAMP was formed when the AMP-PNP medium was incubated in the presence of 1 or 2 mM Pb++ but in the absence of cell fractions, indicating that at these concentrations Pb++ does not cause the nonenzymatic hydrolysis of AMP- PNP. Therefore, the reaction product observed by cytochemical localization is not due to the nonenzymatic hydrolysis of AMP-PNP by Pb++. In Golgi subfractions, lead phosphate reaction product was widely distributed among Golgi elements: it was seen in association with the majority of the very low density lipoprotein-filled secretory droplets which predominated in the two lightest Golgi fractions (GF1 and GF2) as well as within the majority of the cisternae found in the heaviest Golgi fraction (GF3). In the latter, reaction product was heaviest along the dilated peripheral rims of the cisternae. In all cases, the reaction product was localized to the outside or cytoplasmic face of the Golgi membranes. When microsomes were incubated cytochemically for AC, deposits were found on the cytoplasmic surface of smooth endoplasmic reticulum (ER) membranes, but none were observed on rough ER membranes. The results confirm the biochemical data reported previously indicating the presence of AC in Golgi and smooth microsomal fractions from rat liver and further demonstrate that the activity is indeed indigenous to Golgi elements and not due to plasma membrane contaminants. They also indicate that AC is widely distributed among Golgi and smooth ER elements. Thus, AC is not restricted in its distribution to plasma membranes as usually assumed.     

Parthenogenetic development of porcine oocytes treated by ethanol, cycloheximide, cytochalasin B and 6-dimethylaminopurine

This study was carried out to investigate the various concentrations and exposure times of ethanol, one of many intracellular calcium elevating agents, and a sequential combination of ethanol (8%), cycloheximide (CHX, 10 microg/ml), cytochalasin B (CCB, 7.5 microg/ml) and 6-dimethylaminopurine (6-DMAP, 2 mM) to improve parthenogenetic activation and development of in vitro matured porcine oocytes. Cumulus-oocyte complexes (COCs) were matured in tissue culture medium (TCM) 199 for 44 h at 38.5 degrees C, 5% CO2 in air. Cumulus-free oocytes showing first polar body were activated by concentrations of 0, 5, 6, 7, 8, 9 and 10% ethanol for 10 min and exposure times of 0, 5, 8, 10, 12 and 15 min with 8% ethanol in HEPES buffered (25 mM) NCSU-23 medium. Also, oocytes were activated with the NCSU-23 medium containing 8% ethanol for 10 min. After that, oocytes were incubated in the NCSU-23 medium supplemented with CHX, CCB, 6-DMAP, CHX + CCB, CHX + 6-DMAP, CCB + 6-DMAP and CHX + CCB + 6-DMAP for 3h, respectively. Following activation, oocytes were transferred into the NCSU-23 medium containing 0.4% BSA for further culture of 20 and 144 h at 38.5 degrees C, 5% CO2 in air. The activation rates of oocytes were higher in 6, 7 and 8% ethanol concentrations compared with 0, 5, 9 and 10% ethanol concentrations. Significantly, more oocytes (29.3-33.7%) were activated in the exposure for 8, 10, 12 and 15 min than those in the exposure for 0 and 5 min, but there was no difference due to exposure to 8% ethanol for 8-15 min. Oocytes treated by chemical agents (40.5-70.5%) after exposure to ethanol significantly improved the rate of oocyte activation compared with ethanol alone (31.2%). The percentage of cleaved oocytes was higher in the ethanol+CHX+CCB+6-DMAP treatment (66.4%) than in other treatments (24.9-57.6%). Also, the rate of blastocyst formation was higher in the ethanol+CHX+CCB+6-DMAP treatment (25.0%) than in other treatments (0.0-19.3%). In conclusion, the optimal activation treatment of ethanol exposure alone for the in vitro matured porcine oocytes was 8% ethanol for 8-15 min. Oocytes activated by 8% ethanol for 10 min and incubated in the NCSU-23 medium supplemented with CHX, CCB and 6-DMAP for 3 h were more efficient for parthenogenetic development of in vitro matured porcine oocytes.     

Properties of the guanylate cyclase-guanosine 3':5'-monophosphate system of rat renal cortex. Activation of guanylate cyclase and calcium-independent modulation of tissue guanosine 3':5'-monophosphate by sodium azide.

The effects of sodium azide on guanylate cyclase activity of homogenates of rat renal cortex and on the guanosine 3':5'-monophosphate (cGMP) content of cortical slices were examined and compared to those of carbamylcholine and NaF. In complete Krebs-Ringer bicarbonate buffer containing 10 mM theophylline, tissue cGMP content was increased 5- to 6-fold by 0.05 mM carbamylcholine or 10 mM NaN3, and 3-fold by 10 mM NaF. Increases in cGMP were maximal in response to these concentrations of the agonists and occurred within 2 min. Exclusion of Ca2+ from the incubation media reduced basal cGMP by 50% in 20 min and abolished responses to carbamylcholine and NaF, while exclusion of Mg2+ was without effect. Analogous reductions in cGMP were observed in complete buffer containing 1 mM tetracaine, an agent which blocks movement of Ca2+ across and binding to biologic membranes. By contrast, exclusion of Ca2+ or addition of tetracaine did not alter relative cGMP responses to NaN3 (6-fold increase over basal), although levels were reduced in slices exposed to these buffers for 20 min. When slices were incubated without Ca2+ or with tetracaine for only 2 min prior to addition of agonists, basal cGMP did not decline. Under these conditions, both absolute and relative increases in cGMP in response to NaN3 were comparable to those of slices incubated throughout in complete buffer, while carbamylcholine and NaF effects on cGMP were abolished. NaN3 increased guanylate cyclase activity of whole homogenates (10- to 20-fold), and of the 100,000 X g soluble (20-fold) and particulate (4-fold) fractions of cortex. Prior incubation of slices with NaN3 in the presence or absence of Ca2+ or with Ca2+ plus tetracaine also markedly enhanced enzyme activity in homogenates and subcellular fractions subsequently prepared from these slices. In the presence of 3 mM excess MnCl2, NaN3 raised the apparent Km for MnGTP of soluble guanylate cyclase from 0.11 mM to 0.20 mM, and reduced enzyme dependence on Mn2+. Thus, when Mg2+ was employed as the sole divalent cation in the enzyme reaction mixture basal and NaN3-responsive activities were 7% and 30% of those seen with optimal concentrations of Mn2+, respectively. Under a variety of assay conditions where responses to NaN3 were readily detectable, alterations in guanylate cyclase activities could not be demonstrated in response to carbamylcholine or NaF. By contrast Ca2+ increased the guanylate cyclase activity 6- to 7-fold over basal under conditions of reduced Mn2+ (0.75 mM Mn2+/1 mM GTP). This latter effect of Ca2+ was shared by Mg2+ and not blocked by tetracaine. Carbamylcholine, NaF, Ca2+, and NaN3 all failed to alter cGMP phosphodiesterase activity in cortex. Thus, while carbamylcholine and NaF enhance renal cortical cGMP accumulation through actions which are dependent upon the presence of extracellular Ca2+, NaN3 stimulates cGMP generation in this tissue through an apparently distinct Ca2+-independent mechanism.     

Gastric K+-stimulated p-nitrophenylphosphatase cytochemistry

A cytochemical study of gastric K+-stimulated p-nitrophenylphosphatase (K-NPPase) activity, corresponding to a K+-stimulated phosphoprotein phosphatase of H-K-ATPase system, has been made by a new cytochemical method. Sections of fixed guinea pig gastric mucosa in a mixture of 2% paraformaldehyde and 0.25% glutaraldehyde, were incubated with the incubation medium (1.0 M glycine-0.1 M KOH buffer, pH 9.0, 2.5 ml; 1.1 M KCl, 0.5 ml; 10 mM lead citrate dissolved in 50 mM KOH, 4 ml; levamisole, 6.0 mg; dimethyl sulfoxide, 2.0 ml; 0.1 M p-nitrophenylphosphate (Mg-salt), 1.0 ml; ouabain, 73.0 mg) for 30 min at room temperature. Under a light microscope the specific gastric K-NPPase reaction was distributed only in the parietal cells of the fundic glands. The electron microscopic cytochemistry showed that the gastric K-NPPase activity was localized on the membrane lining the apical surfaces, secretory canaliculi and tubulovesicles. On the other hand, ouabain-sensitive K-NPPase activity (Na-K-ATPase) was demonstrated to localize only in the basolateral membrane of parietal cells with Mayahara's method. These findings support the interrelationships between the apical surface membrane, secretory canalicular membrane and tubulovesicles, and the functional differentiation of the membrane between the secretory membrane and basolateral membrane.     

The sequestration of Ca2+ by mitochondria in rat heart cells

Rat heart ventricular cells, purified by Percoll density gradient centrifugation, were incubated in the presence of 1.3 mM CaCl2. After 20 min incubation, samples of the cells were lysed in medium containing 0.3 mM digitonin, ruthenium red and EGTA, and a mitochondrial fraction was isolated at intervals thereafter. Extrapolation of the mitochondrial 45Ca2+ contents to zero time enabled the endogenous 45Ca2+ to be estimated at the time of cell lysis. The lysis conditions yielded essentially complete release of lactate dehydrogenase from the cells, but caused negligible damage to the mitochondria as judged by their retention of glutamate dehydrogenase, and their ability to accumulate and retain Ca2+ in the absence of ruthenium red and EGTA. The data indicate that about 13% of total cell Ca2+ only may be mitochondrial in vivo.     

K+--induced changes of oxygen uptake by neuronal enriched and glial enriched fractions from mouse brain cortex.

Neuronal and glial enriched fractions were incubated in a medium with 10mM pyruvate, 5mM fumarate and 0.9mM 5'-AMP and the effect of increased external K+ concentrations was studied upon oxygen uptake. A concentration of 65 mM K+ had a different effect on the oxygen consumption of glial and neuronal perikarya. The rate of oxygen uptake by glia was stimulated by 52.81% whilst an insignificant decrease of 15.79% occurred in the neurones. The highest rate of oxygen uptake by incubated cells was estimated in the presence of the substrate system containing pyruvate, fumarate and 5'-AMP. The significance of components in the substrate system for a high rate of oxygen uptake by cells was also tested with 6.2 mM K+ and 65 mM K+.     

The effect of cyclic nucleotides on secretin secretion in canine duodenal mucosa in vitro

We examined the effects of cyclic nucleotides and calcium on secretin release from canine duodenal mucosal explants incubated in organ culture media. Time course studies revealed that at pH 7.4, 5 and 10 mM dibutyryl cyclic adenosine monophosphate (DBcAMP) increased secretin release progressively, reaching a peak at 2 hours. Two mM of DBcAMP at pH 7.4 did not increase secretin release but at pH 4.5, all 3 doses potentiated secretin release. DBcAMP-stimulated secretin release was not dependent on the influx of extracellular calcium. Graded doses of 3-isobutyl-1-methylxanthine (IBMX) did not stimulate secretin secretion but 1 mM IBMX with 2 mM DBcAMP increased secretin secretion significantly. Dibutyryl cyclic guanosine monophosphate, cholera toxin and 5'-guanylyl-imidodiphosphate (GPP(NH)p) did not stimulate basal secretion release. The release of secretin from our explants incubated at pH 7.4 was not due to specific leakage because all of our viability studies revealed that our explants were functionally intact at the end of 2 hours. Our observations suggest that cyclic nucleotides may participate in the intracellular regulation of secretin secretion.     

Electron microscopic cytochemical localization of alpha-hydroxyacid oxidase in rat kidney cortex. Heterogeneous staining of peroxisomes

The substrate specificity of alpha-hydroxyacid oxidase in the rat kidney has been investigated cytochemically by the cerium technique and biochemically with a luminometric assay applied to isolated renal peroxisomes. Rat kidneys were fixed by perfusion via the abdominal aorta with a low concentration (0.25%) of glutaraldehyde. Vibratome sections were incubated for 60 min at 37 degrees C in a medium containing 3 mM CeCl3, 100 mM NaN3 and 5 mM of an alpha-hydroxyacid in 0.1 M Pipes or 0.1 M Tris-maleate buffer both adjusted to pH 7.8. Ten aliphatic alpha-hydroxyacids with chain lengths between 2 and 8 carbon atoms and two aromatic substrates were tested. The alpha-hydroxyacid oxidase in the kidney exhibited a markedly different substrate specificity than the corresponding enzyme in the liver. Thus glycolate gave a negative reaction while two aromatic substrates, mandelic acid and phenyllactic acid, stained prominently. With aliphatic substrates a stronger reaction was obtained in Pipes than in the Tris-maleate buffered incubation media. The best reaction in the kidney was obtained with hydroxybutyric acid. These cytochemical findings were confirmed by the luminometric determination of the oxidase activity in isolated purified peroxisome fractions. By electron microscopy the electron dense reaction product of cerium perhydroxide was found in the matrix of peroxisomes in the proximal tubules. The intensity of reaction varied markedly in neighbouring epithelial cells but also in different peroxisomes within the same cell. Thus heavily stained particles were seen next to lightly reacted ones. These observations establish the substrate specificity of alpha-hydroxyacid oxidase in the rat kidney and demonstrate the marked heterogeneity in the staining of renal peroxisomes for this enzyme.     

Renaissance of cytochemical localization of membrane ATPases in the myocardium

ATPases of cardiac cells are known to be among the most important enzymes to maintain the fluxes of vital cations by hydrolysis of the terminal high-energy phosphate of ATP. Biochemically the activities of Ca²⁺-pump ATPase, Ca²⁺/Mg²⁺-ecto ATPase, Na⁺,K⁺-ATPase and Mg²⁺-ATPase are determined in homogenates and isolated membranes as well as in myofibrillar and mitochondrial fractions of various purities. Such techniques permit estimation of enzyme activitiesin vitro under optimal conditions without precise enzyme topography. On the other hand, cytochemical methods demonstrate enzyme activityin situ, but not under optimal conditions. Until recently several cytochemical methods have been employed for each enzyme in order to protect its specific activity and precise localization but the results are difficult to interpret. To obtain more consistent data from biochemical and cytochemical point of view, we modified cytochemical methods in which unified conditions for each ATPase were used. The fixative solution (1% paraformaldehyde –0.2% glutaraldehyde in 0.1 M Tris Base buffer, pH 7.4), the same cationic concentrations of basic components in the incubation medium (0.1 M Tris Base, 2mM Pb(NO₂)₃, 5 mM MgSO₄, 5 mM ATP) and selective stimulators or inhibitors were employed. The results reveal improved localization of Ca²⁺-pump ATPase, Na⁺–K⁺ ATPase and Ca²⁺/Mg²⁺-ecto ATPase in the cardiac membrane.     

Photodynamic action of endogenously synthesized porphyrins from aminolevulinic acid,using a new model for assaying the effectiveness of tumoral cell killing

• 1.1. The effectiveness of the photodynamic action of porphyrins, was studied by means of the tissue explant culture technique. A murine tumor tissue explant was incubated in a medium containing 0.6 mM of ALA for periods of 1 and 2 hr; total porphyrins synthesized under these conditions were of the same level as those found in our previous in vivo experiments. The explants were then irradiated for 30 min with He-Ne laser of 3.5 mW output power placed at a distance of 10 cm. Controls of non-irradiated tumor tissue slices incubated with and without ALA were performed. Immediately after irradiation, innocula of exactly 1 mm³ of the irradiated and non-irradiated tissue were subcutaneously injected under the right and left flanks of the same animal, respectively. The growth of the tumor was measured 15, 20 and 25 days after implantation.
• 2.2. Results obtained showed that the explants that were incubated for 1 hr with ALA and irradiated, reaching a concentration of 2.8 μg porphyrins/g tissue, produced a reduction of 50–70% of tumor size as compared with the non-irradiated controls incubated with ALA. Explants incubated for 2 hr, reaching a concentration of 4.6 μg porphyrins/g tissue, produced from 60% to complete lack of tumor growth. The effectiveness index (EI) of photoirradiation was calculated on the basis of the tumor growth in irradiated and non-irradiated tumors. EI was nearly 100% showing almost complete tumor cell destruction for tumor irradiated for 2 hr with 0.6 mM ALA.
• 3.3. As indicators of cell injury and subsequent death and necrosis, LDH activity in the incubation medium and intracellular potassium content were measured. Results indicated that as a consequence of irradiation of porphyrin loaded tumor explants, significant release of LDH to the medium and loss of intracellular potassium occurred. These findings show great to complete tumor destruction by combination of porphyrins endogenously formed from ALA and low irradiance with laser.

     

Dimethylnitramine metabolism in vitro by NZR rat liver slices

The net metabolism of dimethylnitramine (DMNO) was studied in NZR rat liver slices in tissue culture medium (Dulbecco's MEM). In rats, mice and fish, liver is the principal target organ for DMNO carcinogenesis. Destruction of DMNO in vitro with oxygenated medium was linear with amount of tissue (0.3-3.0 g liver), and with substrate concentration (0.14-4.44 mM). Substrate destruction (initially 0.2 mM DMNO) was linear for 60 min (average rate 0.9 +/- 0.1 microgram DMNO/g liver/min) and then slowed to become linear again at about half the initial rate from 90 min to longer than 5 h. In anoxic (N2) conditions DMNO metabolism slowed or stopped completely after 70 min. Metabolism of dimethylnitrosamine (DMN) was studied in the same preparation. DMN destruction rates were initially about 50% higher than DMNO, but were equal at longer incubation times. Simultaneous metabolism of DMNO and DMN by the same tissue slices showed DMNO rates unaltered in the presence of equimolar DMN (0.24 mM), but DMN rates were 20-40% depressed. No evidence was found for the oxidation of DMN to form DMNO, or for reduction of DMNO to DMN.