Effect of aldosterone on incorporation of amino acids into renal medullary proteins

Summary Studies on the effects of pretreatment with aldosterone on the incorporation of³H leucine or³H methionine into proteins in renal slices were carried out in Joklik-modified minimal essential medium. Administration of aldosterone (2 g/100 g body wt) to adrenalectomized rats increased³H leucine incorporation into trichloroacetic acid insoluble fractions of crude homogenates of cortical slices by 15.5±0.4% and of medullary slices by 53.5±1.3%. No increase in isotope incorporation was observed in slices of renal papilla or spleen prepared from the same rats. Aldosterone had no effect on the³H-leucine content of the trichloroacetic acid-soluble fractions of all three renal zones and the spleen. The dose of aldosterone that elicited a half-maximal increase in³H-methionine incorporation into proteins of renal medullary slices (0.45 g of aldosterone/100 g body wt) was indistinguishable from that needed to elicit a halfmaximal increase in the urinary K⁺/Na⁺ ratio (0.35 g of aldosterone/100 g body wt). Dexamethasone, a potent glucocorticoid, at a dose of 0.8 g/100 g body wt did not augment³H-leucine incorporation into renal medullary proteins but was effective at 8 g/100 g body wt. Spirolactone (SC-26304), a potent anti-mineralocorticoid, abolished the effect of aldosterone on amino acid incorporation into medullary proteins when administered at a 100-fold higher dosage [i.e., 80 gvs. 0.8 g (per 100 g body wt)]. These results imply that the action of aldosterone on amino acid incorporation is mediated by the mineralocorticoid rather than the glucocorticoid pathway, presumably the mineralocorticoid receptors. Moreover, pretreatment of the rats with actinomycin D (70–80 g/100 g body wt) erased the effect of aldosterone (0.8 g/100 g body wt) on amino acid incorporation into medullary proteins.In paired experiments with³H and³⁵S methionine, aldosterone (0.8 g/100 g body wt) increased methionine incorporation into trichloroacetic acid precipitable proteins of subcellular fractions of the renal medulla. The effect of aldosterone on incorporation of methionine into medullary cytosol proteins was analyzed further by polyacrylamide gel electrophoresis at pH 8.3 in tris-glycine buffer. The gel profiles indicate that aldosterone significantly increased methionine incorporation into at least one protein (independent of the isotope) with a molecular weight of 31,000. This increase was inhibited by either pretreatment of the rat with actinomycin D (70–80 g/100 g body wt or SC-26304 (80 g/100 g body wt). Dexamethasone (0.8 g/100 g body wt) did not increase incorporation of methionine into the medullary cytosol proteins resolved by polyacrylamide gel electrophoresis.     

Effect of 2-fluoropalmitate,cerulenin and tunicamycin on the palmitoylation and intracellular translocation of myelin proteolipid protein

We have investigated the effect of documented protein palmitoylation inhibitors on the fatty acylation and intracellular transport of myelin proteolipid protein (PLP). To this end, brain slices from 20-day-old rats were incubated with either [³H]palmitate or [³H]leucine in the presence or absence of various concentrations of 2-fluoropalmitate (FP), cerulenin (CER), or tunicamycin (TM). FP ( 10 M) decreased the cellular uptake of [³H]palmitate and consequently reduced the labeling of palmitoyl-CoA, glycerolipids and PLP. CER ( 1 mM) reduced the palmitoylation of PLP with a concomitant decline in protein thiols. Consistent with being a fatty acyl-CoA analogue, TM ( 200 M) diminished the palmitoylation of PLP and lipids while increasing the amount of [³H]palmitoyl-CoA. Although both CER and TM decreased protein palmitoylation, only the latter affected the appearance of newly synthesized PLP into myelin. Because TM, but not CER, also reduced the formation of lipids, it is concluded that palmitoylation is not required for intracellular transport. Finally, comparison of the effect of TM in brain slices and in a cell-free system suggests that palmitoylation of PLP in whole cells may be an enzymatic process.     

Axonal transport of phospholipids in rabbit optic pathway

The uptake of different labeled precursors, their incorporation into lipids, and transport along the rabbit optic pathway [ipsilateral retina and optic nerve (ON), and contralateral optic tract (OT), lateral geniculate body (LGB), and superior colliculus (SC)] were investigated. Albino rabbits were used. The following radioactive precursors, either combined or separately, dissolved in 50 l of saline containing 15% BSA, were injected into vitreous body: [2-³H]glycerol (50 Ci), [1-¹⁴C]palmitate (15 Ci), and [1-¹⁴C]linoleate (7.5 Ci). Animals were killed at different time intervals from 1 hr up to 24 days. The radioactivity of total lipids and of different phospholipid classes from total tissue was measured. One hour after the administration of precursors, the radioactivity into the retina was high and the incorporation of [³H]glycerol and [¹⁴C]palmitate increased until 12 hr and 24 hr, respectively. The incorporation of [¹⁴C]linoleate reached a maximum on the second day. The phospholipids of LGB and SC were intensively labeled after 4–8 hr, and their radioactivity increased up to the 10th day after injection, independent of the precursor employed. The results obtained indicate that the labeled hydrophilic and hydrophobic precursors used were actively incorporated into the retina. The phospholipids were later transported at a rapid rate along the optic pathway.A preliminary report of this study has been presented at the Satellite ISN Meeting, Istanbul, September 8–10, 1979.     

Change in substrate metabolism in isolated mouse hearts following ischemia-reperfusion

Several genetic and transgenic mouse models are currently being used for studying the regulation of myocardial contractility under normal conditions and in disease states. Little information has been provided, however, about myocardial energy metabolism in mouse hearts. We measured glycolysis, glucose oxidation and palmitate oxidation (using ³H-glucose, ¹⁴C-glucose and ³H-palmitate) in isolated working mouse hearts during normoxic conditions (control group) and following a 15 min global no-flow ischemic period (reperfusion group). Fifty min following reperfusion (10 min Langendorff perfusion + 40 min working heart perfusion) aortic flow, coronary flow, cardiac output, peak systolic pressure and heart rate were 44 ± 4, 88 ± 4, 57 ± 4, 94 ± 2 and 81 ± 4% of pre-ischemic values. Rates of glycolysis and glucose oxidation in the reperfusion group (13.6 ± 0.8 and 2.8 ± 0.2 mol/min/g dry wt) were not different from the control group (12.3 ± 0.6 and 2.5 ± 0.2 mol/min/g dry wt). Palmitate oxidation, however, was markedly elevated in the reperfusion group as compared to the control group (576 ± 37 vs. 357 ± 21 nmol/min/g dry wt, p < 0.05). This change in myocardial substrate utilization was accompanied by a marked fall in cardiac efficiency measured as cardiac output/oxidative ATP production (136 ± 10 vs. 54 ± 5 ml/mol ATP, p < 0.05, control and reperfusion group, respectively). We conclude that ischemia-reperfusion in isolated working mouse hearts is associated with a shift in myocardial substrate utilization in favour of fatty acids, in line with previous observations in rat.     

Mercury budget of an upland-peatland watershed

Inputs, outputs, and pool sizes oftotal mercury (Hg) were measured in a forested 10 hawatershed consisting of a 7 ha hardwood-dominatedupland surrounding a 3 ha conifer-dominatedpeatland. Hydrologic inputs via throughfall andstemflow, 13±0.4 g m^–2 yr^–1over the entire watershed, were about doubleprecipitation inputs in the open and weresignificantly higher in the peatland than in theupland (19.6 vs. 9.8 g m^–2 yr^–1). Inputs of Hg via litterfall were 12.3±0.7g m^–2 yr^–1, not different in thepeatland and upland (11.7 vs. 12.5 g m^–2yr^–1). Hydrologic outputs via streamflow were2.8±0.3 g m^–2 yr^–1 and thecontribution from the peatland was higher despiteits smaller area. The sum of Hg inputs were lessthan that in the overstory trees, 33±3 gm^–2 above-ground, and much less than eitherthat in the upland soil, 5250±520 gm^–2, or in the peat, 3900±100 gm^–2 in the upper 50 cm. The annual flux of Hgmeasured in streamflow and the calculated annualaccumulation in the peatland are consistent withvalues reported by others. A sink for Hg of about20 g m^–2 yr^–1 apparently exists inthe upland, and could be due to either or bothstorage in the soil or volatilization.     

The contribution of size-fractionated plankton to biomass and primary production of phytoplankton in saline–alkaline ponds

Primary productivity, biomass and chlorophyll-a of size fractionated phytoplankton (<0.22 m, <3 m, <8 m, <10 m, <40 m, <64 m, <112 m and <200 m) were estimated in 6 ponds and 5 experimental enclosures. The results showed that the planktonic algae less than 10 m are important in the biomass and production of phytoplankton in saline–alkaline ponds. The production of size fractionated phytoplankton corresponding to <112 m, <10 m and <3 m in saline–alkaline ponds were 10.5 ± 6.6 , 8.6 ± 5.4 and 0.33 ± 0.1 mgC l^–1 d^–1, respectively. Mean community respiration rate was 1.80 ± 0.73, 1.69 ± 0.90 and 1.38 ± 1.12 mgC l^–1 d^–1, respectively. The average production of phytoplankton corresponding to micro- (10–112 m), nano- (3–10 m) and pico- (<3 m) were 1.61, 8.30 and 0.33 mgC l^–1 d^–1, respectively. The ratio of those to the total phytoplankton production was 15%, 79% and 3%, respectively. The mean respiration rate of the different size groups was 0.11, 0.31 and 1.38 mgC l^–1 d^–1; the ratio of those to total respiration of phytoplankton was 6%, 17% and 77%, respectively. The production of size-fractionated phytoplankton corresponding to <200 m, <10 m and <3 m in enclosures was 2.19 ± 1.63, 2.08 ± 1.75 and 0.22 ± 0.08 mgC l^–1 d^-1, respectively. Mean community respiration rates were 1.25 ± 1.55, 1.17 ± 1.42 and 0.47 ± 0.32 mgC l^–1 d^–1, respectively. The average production of phytoplankton corresponding to micro- (10–200 m), nano- (3–10 m) and pico- (<3 m) plankton was 0.11, 1.86 and 0.22 mgC l^–1 d^–1, respectively. The ratio of those to the total production of phytoplankton was 5%, 85% and 10%, respectively. The mean respiration rate of different size groups were 0.08, 0.72 and 0.46 mgC l^–1 d^–1, the ratio of those to total respiration of phytoplankton was 6%, 57% and 37%, respectively. The concentrations of chlorophyll-a of the phytoplankton in the corresponding size of micro- (10–112 m), nano- (3–10 m) and pico- (<3 m) plankton in the experimental ponds were 19.3, 98.2 and 11. 9 g l^–1, respectively. The ratio of those to the total chlorophyll-a was 15%, 76% and 9%, respectively. The concentrations of chlorophyll-a of phytoplankton micro- (10–200 m), nano- (3–10 m) and pico- (<3 m) plankton in enclosures were 1.7, 34.3 and 3.0 g l^–1, respectively. The ratio of those to the total chlorophyll-a was 4%, 88% and 8%, respectively.     

Localization of sodium absorption and chloride secretion in an intestinal epithelium

Summary Hen coprodeum absorbs sodium electrogenically and, when stimulated by theophylline, secretes chloride. In this study the vibrating microprobe technique was used to localize the transport of these ions to intestinal villi/folds and crypts. With the isolated, stretched epithelium, controlled by light microscopy and scanning electron microscopy, in open circuit, currents were inward, 40±7 A/cm², 50 m vertically above villi, and outward, 36±7 A/cm² above crypts. The currents decayed exponentially to near zero at 300 m with the same length constant. A physical model simulating the observed loci of current sources and sinks predicts potential profiles consistent with our data. Extrapolation of the currents gives a surface potential of 45 V, negative on villi and positive above crypts. Short circuiting increased villus current to 86±27 A/cm² at 50 m, and amiloride treatment reduced it to –8 A/cm²; in both cases crypt currents were abolished. The inward currents are compatible with sodium absorption. Induction of chloride secretion after amiloride treatment, resulted in current circuits similar to those induced by sodium absorption, with villus currents of 23±7 A/cm². This is in accord with chloride secretion at the villi. Quantitative estimates of crypt number (860/cm²) and opening diameter (15 m), in conjunction with isotopic measurements of active and electrical potential-driven ion fluxes demonstrate, however, that only 4% of the potential-driven co-ion transport occurs through the crypts. This indicates that nearly all chloride secretion comes from the sodium-absorbing villar area. Were the chloride secretion to occur solely from the crypts, the current should have been in the opposite direction and 10,000-fold larger.     

Dry to wet weight biomass conversion constant for Tetrahymena elliotti (Ciliophora,Protozoa)

Summary The wet and dry weights of both axenic and monoxenic cultures of the ciliate Tetrahymena were determined directly. These estimates are dependent upon the method of volume determination. Assuming a prolate spheroidal shape for the ciliate, we calculate a mean wet weight of 0.4157±0.0713 pg m^-3 and a mean dry weight of 0.2793±0.0652 pg m^-3. Using electronic cell sizing, our estimates are 0.7869±0.1659 pg m^-3 and 0.5239±0.1101 pg m^-3, respectively. Independent of the method of volume determination, we estimate a mean biomass conversion ratio (dry weight/wet weight) of 0.59±0.08.     

Biosorption and solubilization of copper oxychloride fungicide by Aspergillus niger and the influence of calcium

The biosorption of copper oxychloride fungicide particulates(1 m diameter), at concentrations ranging from 25 to 500 ppm active ingredient (ai), by pelleted mycelium of Aspergillus niger grown on Czapek Dox medium was evaluated. The concentration of the fungicide adsorbed to the mycelium, remaining suspended or solubilized in the medium, was determined by analysis of its copper content (CuF)using atomic absorption spectrophotometry (AAS). 2-day-old pellets exhibited highbiosorption efficiency ranging from 97 ± 1.0 to 88 ± 1.2% of the initially added fungicide concentrations, respectively, within 10 min. However, underthe same conditions, amounts of the removed fungicide by 6-day-old mycelial pellets were significantly lower and ranged from 0.5 ± 0.03 to 0.15 ± 0.01%. Scanning electron microscopy studies of 2-day-old pellets supplemented with thefungicide revealed predominant aggregations of clumps and dense particulates on the hyphal tips. The adsorbed CuF of 125 ppm ai fungicide subsequently decreased from 7.5 ± 0.5 to 2.1 ± 0.1 mol Cu (mg dry wt)^-1 after 12 h incubation. Simultaneously, the soluble portion of CuF remaining in the medium increased from 0.9 ± 0.6 to4.9 ± 0.2 mol Cu ml^-1. The presence of 50 mM CaCl₂ resulted in a decrease of the adsorbed CuF to 3.5 ± 0.5 mol Cu (mg dry wt)^-1 and solubilizedcopper in the medium increased to 5.9 ± 0.8 mol Cu ml^-1. Additionally, the cellular copper contents attained after 2 h were 0.08 ± 0.01 and 0.16 ± 0.007 mol Cu (mg dry wt)^-1 in absence and presence of calcium, respectively. The addition of calcium to glucose-starved pellets greatly increased the medium [H⁺] which was conclusively discussed in relation to Ca²⁺/H⁺ exchangecapacity of the fungal cells. These results are of potential environmental,biotechnological and agricultural importance.     

Three-dimensional and quantitative observation of the hypertrophy of rat corticotrophs following adrenalectomy

Summary The three-dimensional form of corticotrophs in the anterior pitiutary gland of rats was studied by reconstruction from serial semi-thin sections both in control rats and rats one week after adrenalectomy. The corticotrophs have large depressions and cup-shaped cavities on their surface, and these features became more conspicuous after adrenalectomy. The hypertrophy of corticotrophs in adrenalectomized rats was quantified by measuring the area and perimeter of all serially sectioned profiles. The volume of the whole cell increased from 1129±114 m³ to 2902±201 m³ (P<0.01) after adrenalectomy, while the surface area of the cells increased from 690±45 m² to 1431±116 m² (P<0.01). The volume of the nucleus increased from 87±11 m³ to 172±14 m³ (P<0.05). Though the complexity of the form of corticotrophs seems to be increased after adrenalectomy, the ratios (adrenalectomized/control) of cell volume and surface area were 2.57 and 2.07, respectively; this indicates that the increase of the cell volume was greater than that of the surface area.     

Mercury contamination in Lake Xolotlán (Managua)

Total mercury was measured in different compartments of Lake Xolotlán's (Managua) ecosystemviz., sediments, water, fish and men. Sediments from 18 localities at 5 depths inside the sediment (5, 10, 15, 20 and 25 cm) contained an average concentration of 0.62 g Hg.g^–1±0.46 at the surface, with extreme values of 0.16 and 1.8 g.g^–1. The highest concentration was observed at 25 cm depth in front of the chlor-alkaly factory (ELPESA). This maximum is associated with the period of highest production of this factory. The highest mercury concentrations in water were also measured close to the discharge of ELPESA,viz. 787 g.Hg^–1 in January and 506 g.g^–1 in April. The mean mercury concentrations measured in the muscles of the most consumed fish were 0.63 g.g^–1±0.22 (extreme values 0.22 and 1.45) inCichlasoma managuense, and 0.07 g.g^–1±0.14 (extreme values 0.004 and 0.63) inC. citrinellum. The concentration in the liver was 0.79 g.g^–1±1.29 inC. managuense and 0.62 g.g^–1±0.44 inC. citrinellum. Human hairs (n=98) of fishermen and their families contained 5.03 g.g^–1±6.2 (extreme values 0.02 and 38.22). The mean concentration measured in men was 6.22 g.g^–1±6.34 (n=58), and in women 3.39 g.g^–1±5.7 (n=40). The average mercury concentration of hairs of workers of ELPESA was 91.24 g.g^–1±156.9 (extreme values 0.46 and 724.53; n=32). We conclude that total mercury levels in the various ecosystem compartments are very high and mercury contamination in the lake may be considered as dangerous for human health.     

Simultaneous use of 14C and 3H to determine autotrophic production and bacterial protein production in periphyton

A method of simultaneously quantifying photoautotrophic (algae and cyanobacteria) and bacterial production in periphyton communities by ¹⁴C-bicarbonate and ³H-leucine incorporation was investigated and applied to communities subjected to specific intensities of photosynthetically active radiation (400–700 nm). Maximum photosynthetic output (2.23 ± 0.29 (SE) g C cm^-2 h^-1) and bacterial production (0.07 ± 0.006 g C cm^-2 h^-1) occurred at the highest photon flux density (400 mol m^-2 s^-1). Over a photon flux density range of 20–400 mol m^-2 s^-1, bacterial and autotroph productivity were significantly and positively correlated (r = 0.89). Furthermore, application of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea, a photosystem 11 inhibitor, to periphyton films reduced bacterial production by 46%, but it had no such effect on bacteria-only cultures. Therefore, the magnitude of bacterial production in periphyton was coupled to the photosynthesis/metabolism of algae and/or cyanobacteria.     

Transfer of axonally transported phospholipids into myelin isolated from the rabbit optic pathway

The contribution of the axonal transport to the biosynthesis of myelin phospholipids was investigated in the rabbit optic pathway. A double labeling technique was used. The same animals were injected with one isotope intravitreally and the other intraventricularly. This procedure allows double labeling of the optic nerves, optic tracts, lateral geniculate bodies (LGB), and superior colliculus (SC). The precursors simultaneously injected were: [1-¹⁴C]palmitate (15 Ci intravitreally in both eyes or 50 Ci intraventricularly) and [2-³H]glycerol (50 Ci intravitreally in both eyes or 100 Ci intraventricularly). Twenty four hours and 10 days after the injections, myelin was purified from pooled optic nerves and optic tracts as well as from pooled LGBs or SCs. The phospholipids were extracted and then separated by thin-layer chromatography; the specific radioactivity of the various classes of phospholipids was determined. Using both administration routes of¹⁴C-or³H-precursors, the distribution of label and specific radioactivity of myelin phospholipids in the retina and in all other optic structures were very similar. Phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine + phosphoinositol were preferentially labeled with both precursors. These results suggest that, in the rabbit optic pathway the phospholipids synthesized in the retinal ganglion cells and transported along the axons, could undergo transaxonal transfer into myelin.     

Intracellular distribution of biotin-14COOH in rat liver

Biotin clearance, its distribution in liver and liver fractions after intravenous administration of 5 Ci/100 g body weight (21.55 g) of biotin-¹⁴COOH in normal and biotin-deficient rats are reported. In the biotin deficient animal there is a more rapid disappearance of the labeled biotin from the blood stream. Biotin-¹⁴COOH incorporation in the liver of the deficient rat is more rapid and larger than the incorporation in normal rat liver. Almost all the biotin recovered from liver homogenate is found in the mitochondria and in the pH 5.2 cytosol fraction; whereas in the microsomes only a very small amount is present. The intracellular distribution of biotin is in agreement with its known metabolic roles.     

Effects of Pb2+, Ni2+, Hg2+ and Se4+ on cultured cells. Analysis of uptake, toxicity and influence on radiosensitivity

Effects of Pb²⁺, Ni²⁺, Hg²⁺ and Se⁴⁺ on cultured human glioma U-343MG cells were investigated considering uptake, toxicity and, in combination with radiation, clonogenic cell survival. The cells were exposed to 0-100 m of the metals for a week before the evaluation. The tests showed a tendency to toxicity with 10 m nickel although not significant (P > 0.05). Selenium, lead and mercury exerted a significant toxicity (P < 0.05) at 2.5 m, 10 m and 1 m, respectively. To challenge the clonogenic cell survival capacity, the cells were irradiated with⁶⁰Co photons after being exposed to the highest nontoxic concentration of the different metals. The clonogenic cell survival tests, after irradiation, showed no significant change if the cells were exposed to 5 m nickel, 0.5 m selenium or 5 m lead compared with those not exposed. Mercury, 0.1 m, gave a relative reduction in survival compared with only irradiated cells of 58 ± 17%. Thus, only mercury affected the radiation-induced damage and/or repair. When exposed to the highest nontoxic concentrations of the different metals, the cultures did not display a significant uptake ratio (metal concentration ratio of exposed cells to control cells) of nickel (3.1 ± 3.3), only a small uptake ratio of selenium (4.0 ± 0.4), while there was a large uptake ratio of both lead (2.6 ± 1.7) x 10² and mercury (1.5 ± 0.2) x 10¹. The results indicated that nickel was neither especially toxic nor influenced the clonogenic cell survival after irradiation. Mercury was more toxic and also influenced the radiation sensitivity. Lead was taken up strongly but did not influence the radiation sensitivity. Selenium accumulated but gave no detectable effect on the radiation sensitivity.     

Endogenous phosphorylation of rat brain synaptosomal plasma membranes in vitro: Some methodological aspects

The time course of endogenous phosphorylation in vitro of total or separted synaptic plasma membrane proteins (SPM) has been correlated with that of hydrolysis of the phosphate donor (ATP) in the incubation medium. The ATP/SPM ratio in the medium was varied. In a low-ratio medium (7.5 M ATP; 2.2 g SPM/l) a complete hydrolysis of ATP occurred almost instantaneously as was measured by the release of free phosphate in and the disappearance of ATP from the medium. As a consequence, only a very short peak of phosphorylation, followed by dephosphorylation was observed. However, when higher ATP/SPM ratios were used (200 M ATP; 0.4 g SPM/l and 500 M ATP; 0.4 g SPM/l), the incorporation of phosphate into SPM proteins was linear for 20 sec, and the maximum level of phosphate incorporation was increased. Similar results were obtained after separation of³²P-labeled phosphoproteins by slab gel electrophoresis. However, analysis of the autoradiographs obtained fromone SPM preparation under different ATP/SPM ratios revealed dependence of phosphorylation of individual protein bands on the conditions used.     

Ultrastructural immunocytochemical localization of l-glutamate decarboxylase and GABA in rat pancreatic zymogen granules

Summary The ultrastructural immunohistochemical localization of gamma aminobutyric acid (GABA) and its regulating enzymes, l-glutamate decarboxylase (GAD) and gamma aminobutyrate--ketoglutarate transaminase, was determined utilizing an immunogold post-embedding protocol in pancreatic exocrine tissue. Within the acinar cell, GABA and its biosynthetic enzyme, GAD, were localized in zymogen granules. Quantitative analysis of the GABA immunoreactivity in the acinar cell revealed 1.7±0.5 gold particles/m² over the cytoplasm, 36.6±14.1 gold particles/ m² over the zymogen granules, and 2.9±2.1 gold particles /m² over the mitochondria. Quantitative analysis of the distribution of colloidal gold particles, representing glutamate decarboxylase immunoreactivity in the acinar cells, revealed 38.4±2.5 gold particles/m² over the zymogen granules, 4.7±1.1 gold particles/m² over the mitochondria and 6.3±0.5 gold particles/m² over the remainder of the cytoplasm. Substitution of normal sheep serum for the sheep anti-glutamate decarboxylase serum revealed a significant (p< 0.001) decrease of the colloidal gold particle distribution over the zymogen granules and cytoplasmic compartments of the acini. Gamma aminobutyrate --ketoglutarate transaminase, the catabolic enzyme for GABA, was not detected in the mitochondria, zymogen granules, and cytoplasm of the acinar cell, suggesting that GABA is not catabolized within the acinar cell. Preabsorption and substitution controls resulted in an absence of labeling. These results suggest that GABA may act extracellularly and/or have a role within the zymogen granule in the exocrine pancreas.     

Properties of the sarcoplasmic reticulum Ca2+-pump in coronary artery skinned smooth muscle

Pig coronary artery cultured smooth muscle cells were skinned using saponin. In the presence of an ATP-regenerating system and oxalate, the skinned cells showed an ATP-dependent azide insensitive Ca²⁺-uptake which increased linearly with time for >1 h. The Ca²⁺-uptake occurred with K_m values of 0.20±0.03 M for Ca²⁺ and 400±34 M for MgATP^2–. Thapsigargin and cyclopiazonic acid inhibited this uptake with IC₅₀ values of 0.13±0.02 and 0.56±0.04 M, respectively. These properties of SR Ca²⁺-pump are similar to those reported for membrane fractions isolated from fresh smooth muscle of coronary artery and other arteries. However, optimum pH of the uptake in the skinned cells (6.2) was lower than that reported previously using isolated membranes (6.4–6.8).Abbreviations SR sarcoplasmic reticulum - ER endoplasmic reticulum - PM plasma membrane - CPA cyclopiazonic acid - DTT dithiothreitol     

Retinoylation Reaction of Proteins in Leydig (TM-3) Cells

The covalent incorporation of [³H]all-trans-retinoic acid into proteins has been studied in Leydig (TM-3) cells. The maximum retinoylation activity of Leydig cells proteins was 570± 27 fmoles/8×10⁴ cells at 37C. About 95% of [³H]retinoic acid was trichloroacetic acid-soluble after proteinase-K digestion or after hydrolysis with hydroxylamine. Thus, retinoic acid is most probably linked to proteins as a thiol ester. The retinoylation process was inhibited by 13-cis-retinoic acid and 9-cis-retinoic acid with IC₅₀ values of 0.6 and 1.2 M respectively. Dibutyryl-cAMP and forskolin increased the retinoylation activity by 75 and 81% at 500 and 25 M respectively. Also hCG increased the retinoylation binding activity of 110% at 250 ng/mL. After cycloheximide treatment of the Leydig cells the binding activity of [³H]RA was about the same that in the control, suggesting that the bond occurs on proteins in pre-existing cells. Retinoylation was not inhibited by high concentrations of palmitic or myristic acids (500 M); on the contrary, there was an increase of the binding activity of about 60 and 50% respectively.This paper is dedicated to the memory of Prof. J. A. Olson.     

Locations and central projections of neurons associated with the retrocerebral neuroendocrine complex of the cockroach Periplaneta americana (L.)

Summary Retrograde diffusion and precipitation of Co²⁺ reveals in the ipsilateral pars lateralis (PL) and contralateral pars intercerebralis (PI) of the brain neurons that enter the corpus cardiacum (CC), and, possibly, the corpus allatum (CA) on each side. The PL group consists of 29.6±8.4 somata that fill. Of these, 5.6±0.6 exceed 25 m in diameter, 14.3±2.7 range from 15–25 m, and 9.6±7.6 are smaller than 15 m. After CoCl₂ was applied to the right CC-CA of two males, 239 and 265 somata in the left PI stained. Except for 16 ranging from 30–45 m and chiefly located anteriorly, a majority of these somata measured 10–25 m.The only somata revealed by staining whole brains with the performic acid-resorcin fuchsin method are neurosecretory cells 10–20 m in diameter located within the PI. In starved adult males there are 92.4±8.1 on the right, and 93.2±6.9 on the left. The largest somata in the PL group contain numerous granules that stain with paraldehyde fuchsin. These somata also fill with Co²⁺, and belong to neurosecretory cells that extend into the CC-CA.The cerebral distribution of branches from the PL group, and the relationship of these to the corpora pedunculata, central body, and arborizations from the PI decussation are described.