Mucopolysaccharidosis 3 A (Sanfilippo A disease): deficiency of a heparin sulfamidase in skin fibroblasts and leucocytes

Cultured skin fibroblasts and peripheral leucocytes from patients with Sanfilippo A disease are strikingly deficient in sulfamidase activity (sulfamatase, EC 3.1.6.?), as measured with heparin - N³⁵SO₄. A partial sulfamidase deficiency was found in the cells of the heterozygote carriers. Since Sanfilippo A fibroblasts have normal sulfate ester hydrolase activities towards oligosaccharides prepared from ³⁵SO₄-labelled heparan sulfate by nitrous acid treatment, the basic defect in Sanfilippo A disease is considered to be the inactivity of a heparin (heparan sulfate) sulfamidase.     

Hunter's syndrome: a deficiency of L-idurono-sulfate sulfatase

[³⁵SO₄] Dermatan sulfate, isolated from normal Hurler and Hunter fibroblasts was degraded by chondroitinase A, B, C to yield mono-and disaccharides. The products were separated by ion exchange chromatography and those arising from the non-reducing terminus were characterized by paper electrophoresis. The position of sulfate substituents was established by periodate oxidation and partial acid hydrolysis. Normal dermatan sulfate terminates with GalN-SO₄ whereas IdUA-SO₄ was a prominent terminus in Hunter dermatan sulfate but not in Hurler dermatan sulfate. It is concluded that Hunter's syndrome is due to a deficiency of L-idurono-sulfate sulfatase.     

Heparan sulfate at the surface of HeLa cells

Summary HeLa cells, labeled with Na₂ ³⁵SO₄, release into the culture medium³⁵SO₄ bound to plasma membrane vesicles next to³⁵SO₄-glycoproteins and free³⁵SO₄. Plasma membrane vesicles, experimentally produced by treatment with formaldehyde, contain³⁵SO₄ and their surface can be stained with high iron diamine. Scanning of chromatograms of the trypsinate from labeled cells demonstrates radioactivity on the spot of heparan sulfate. It is concluded that HeLa cells synthesize heparan sulfate, which is incorporated at the plasma membrane and released by shedding of small vesicles.Supported by a grant from the Algemene Spaar- en Lijfrentekas Cancer Fund, Brussels, Belgium.     

Effect of stress on sulfated glycol, metabolites of brain norepinephrine.

The present study examined the effect of footshock stress on the formation of the two major metabolites of rat brain norepinephrine (NE) - the sulfate conjugates of 3-methoxy-4-hydroxyphenylglycol (MOPEG-SO₄) and 3,4-dihydroxyphenylglycol (DOPEG-SO₄). Rats receiving intraventricular injections of either ³HNE or Na₂³⁵SO₄ prior to 0.5 hour of footshock showed significant and comparable increases in both sulfated glycols labeled with ³H or ³⁵SO₄. Elevations were greatest in the hypothalumus using Na₂³⁵SO₄. In pheniprazine pretreated rats footshock did not increase the production of MOPEG-³⁵SO₄ from intraventricular labeled sulfate given alone or in combination with various doses of exogenous MOPEG. The results indicate that neuronally released brain NE is metabolized to form both MOPEG-SO₄ and DOPEG-SO₄. The increase in these metabolites results from an increased glycol production and not from a stress-induced activation of brain sulfation mechanisms.     

Lysosomal sulfate efflux following glycosaminoglycan degradation: measurements in enzyme-supplemented Maroteaux-Lamy syndrome fibroblasts and isolated lysosomes

Studies using lysosomal membrane vesicles have suggested that efflux of the sulfate that results from lysosomal glycosaminoglycan degradation is carrier-mediated. In this study, glycosaminoglycan degradation and sulfate efflux were examined using cultured skin fibroblasts and lysosomes deficient in the lysosomal enzymeN-acetylgalactosamine-4-sulfatase. Such fibroblasts store dermatan sulfate lysosomally, which could be labelled biosynthetically with Na ₂ ³⁵ SO₄. The addition of recombinantN-acetylgalactosamine-4-sulfatase to the media of³⁵S labelled fibroblasts degraded up to 82% of the stored dermatan [³⁵S] sulfate over a subsequent 96 h chase and released inorganic [³⁵S] sulfate into the medium. In the presence of 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS), sulfate was reused to a minor extent in newly synthesized proteoglycan. Isolated granules from recombinant enzyme supplemented fibroblasts degraded stored dermatan [³⁵S]sulfate to sulfate which was rapidly released into the medium at a rate that was reduced by the extra-lysosomal presence of the lysosomal sulfate transport inhibitors SITS, Na₂SO₄ and Na₂MoO₄. SITS also inhibited dermatan sulfate turnover, although it had no effect on the action of purified recombinant enzymein vitro. These data imply that sulfate clearance occurred concomitantly with dermatan sulfate turnover in the lysosome even at high substrate loading, and that lysosome-derived sulfate, while available, is reutilized minimally in synthetic pathways.Abbreviations SITS 4-acetamido-4-isothiocyanatostilbene-2,-2-disulfonic acid - GAG glycosaminoglycan - 4S N-acetylgalactosamine-4-sulfatase - r4S recombinant humanN-acetylgalactosamine-4-sulfatase - PBS phosphate buffered saline - BME basal modified Eagle's medium - FBS fetal bovine serum - GalNAc4S-GlcA-GalitolNAc4S -(N-acetyl-d-galactosamine-4-sulfate)-(1–4)--d-glucuronic acid)-(1–3)-N-acetyl-d-[1-³H]galactosaminitol-4-sulfate - DS dermatan sulfate - MPS mucopolysaccharidosis     

Distribution of inorganic sulfate between plasma, liver, and bile

The distribution of ³⁵SO₄⁼ between inorganic and other forms in plasma, liver and bile of rats was studied by use of paper electrophoresis. The overall liver sulfate space varied from 50–100% but when determined for inorganic ³⁵SO₄⁼ was constant at 34%. This is considerably higher than that expected for passive distribution and suggests SO₄⁼ is actively transported into liver cells. Overall bile/plasma ratios were always greater than 1 while the ratio for inorganic sulfate was 0.44, a value consistent with the known bile/plasma electrical potential.     

Autoradiographic detection of mucopolysaccharide accumulation in single fibroblasts

Synopsis A method is described for localizing acid mucopolysaccharides autoradiographically in cultured cells. Normal fibroblasts and fibroblasts, from patients suffering from Mucopolysaccharidosis II disease (MPS II), were cultured for six days in the presence of³⁵SO₄ and one day in unlabelled medium. The cultured cells were transferred to a plastic film dish and, after settling, they were rapidly quenched, freeze-dried, fixed in osmium tetroxide vapour and embedded in Epon. Grain counting after autoradiography in 2 m sections revealed a significant difference (P>0.001)iin³⁵SO₄ incorporation in the perinuclear cytoplasm of MPS II cells and control cells grown under the same conditions. Autoradiography was also performed after mixing MPS II cells and control fibroblasts in a ratio 11. 8 prior to freezing and the same ratio was found between labelled and unlabelled fibroblasts. These results demonstrate the feasibility of the present autordiographic technique for the detection of the acid mucopolysaccharide storage at the single cell level.     

PRODUCTS OF BIOGENIC AMINE METABOLISM IN THE LOBSTER: SULFATE CONJUGATES

Octopamine, dopamine and serotonin, the three biogenic amines found in the lobster nervous system, are each converted by lobster tissues into two principal classes of products, A and B metabolites. In this paper, evidence is presented that the B metabolites are sulfate conjugates of the amines and their A metabolites. Two double-labelled conjugates were formed from each of the three amines during incubations of lobster nerves with tritiated amine and ³⁵SO₄. When the two octopamine conjugates were hydrolyzed by mild acid, one of the conjugates was converted to a mixture of ³⁵SO₄ and [³H]-octopamine, and the other to a mixture of ³⁵SO₄, [³H]octopamine, and [³H]metabolite A. [³H]Metabolite A was also converted to octopamine by acid hydrolysis. The results indicated that one of the double-labelled conjugates was octopamine-sulfate, and the other metabolite-A-sulfate. An enzyme fraction prepared from nerve homogenates catalyzed the synthesis of double-labelled sulfate conjugates from the tritiated amines and [³⁵S]3′-phosphoadenosine-5′-phospho-sulfate. Double-labelled conjugates formed in this way contained 1 mol of sulfate per mol of amine. Indirect evidence suggested that the sulfate was in ester linkage with the ring hydroxyls of the amines. Neither monoamine oxidase, nor catechol-O-methyl transferase is found in lobster tissues; therefore, in these animals, sulfation may be a major means of inactivation of the biogenic amines following their release from nerve endings.     

The effect of chronic ethanol consumption on temperature-dependent physical properties of liver mitochondrial membranes

We have reported that the monovalent ionophore monensin causes undersulfated chondroitin sulfate biosynthesis in cultured chondrocytes. In order to clarify the mechanism of this diminished sulfation, we have measured the rate of incorporation of sulfate into chondrocytes and assayed the cellular ATP levels. We have also measured sulfatase activity, the incorporation of ³⁵SO₄ into 3′-phosphoadenosine 5′-phospho[³⁵S]sulfate and endogenous sulfotransferase activity in the cell-free extracts. We find that: (1) The incorporation of ³⁵SO₄ into the free sulfate pool in chondrocytes was not inhibited by monensin. (2) The ATP levels of monensin-treated chondrocytes were the same as control cells. (3) There was no sulfatase activity in both control and monensin-treated chondrocytes. (4) Enzymatic analyses revealed that ³⁵SO₄ incorporation into 3′-phosphoadenosine 5′-phospho[³⁵S]sulfate and subsequent sulfotransferase activity were not inhibited in the presence of monensin. At present the most tenable hypothesis to account for monensin causing undersulfated chondroitin sulfate synthesis is that the ionophore impairs the access of proteoglycans to the sulfotransferases in the luminal walls of the Golgi structures.     

Increased Retinal Synthesis of Heparan Sulfate Proteoglycan and HNK-1 Glycoproteins Following Photoreceptor Degeneration

Abstract: In an earlier analysis of the retinal biosynthesis of proteoglycan, we noted that, following photoreceptor degeneration in the rd (retinal degeneration) mouse, the remaining inner retina exhibited a marked elevation in synthesis of heparan sulfate proteoglycan (HSPG), well above the level observed in the normal (nondegenerate) retina, as well as a pronounced increase in sulfation of protein substrates. Biochemical and autoradiographic results of ³⁵S-amino acid utilization reported here confirm that the ³⁵SO₄^2? differences seen previously are accompanied by increased protein synthesis in the rd retina. An intact photoreceptor cell layer is neither a barrier to nor a sink for the amino acid precursor. Further, we have examined sulfate utilization in four other rodent strains with photoreceptor degenerations. In each of the models examined, an increase in retinal synthesis of ³⁵SO₄^2?-labeled HSPG and glycoproteins occurs following photoreceptor degeneration. We have metabolically labeled with Na₂³⁵SO₄ isolated retinal cultures from the following: (a) mice with light-induced photoreceptor degeneration; (b) rd mice; (c) transgenic mice with photoreceptor degeneration; (d) RCS rats; and (e) rats with light-induced photoreceptor degeneration. Comparisons were made with concurrent cultures of control nondegenerate retinal tissues. Protein and proteoglycan-enriched fractions were prepared from the incubation media and guanidine HCI/detergent extracts of the retinas by ion-exchange chromatography. The ³⁵SO₄^2?-proteoglycans were identified by chondroitinase ABC and nitrous acid treatments. Retinas lacking photoreceptors produced at least five times the amount of ³⁵SO₄^2?-HSPG found in control incubations. The RCS and light-damaged rats also showed increased synthesis of ³⁵SO₄^2?-chondroitin sulfate proteoglycan relative to the control, though the increase was of lesser magnitude than the HSPG effect. ³⁵SO₄^2?-protein in degenerate and light-damaged retinas always contained at least twice the radioactivity found in comparable control preparations. The bulk of the increased radiolabeling was found in N-linked oligosaccharides, including several recognized by the HNK-1 antibody. These data suggest that a sustained increase in HSPG and HNK-1 glycoprotein synthesis is a consistent response of inner retinal cells following loss of photoreceptors and is independent of the cause of photoreceptor degeneration.     

Uridine diphospho-N-acetylgalactosamine-4-sulfate sulfohydrolase activity of human arylsulfatase B and its deficiency in the Maroteaux-Lamy syndrome.

The hydrolysis of UDP-N-acetylgalactosamine-4-sulfate by human arylsulfatase B has been demonstrated with an enzyme preparation purified 200-fold from placenta. No hydrolysis was observed with arylsulfatase A. UDP-N-acetylgalactosamine-4-sulfate is the first fully characterized physiological compound shown to be a substrate for arylsulfatase B, confirming that arylsulfatase B is an N-acetylgalactosamine-4-sulfate sulfohydrolase. Cultured fibroblasts derived from patients with Maroteaux-Lamy syndrome were deficient in UDP-N-acetylgalactosamine-4-sulfate sulfohydrolase to the same extent that they were deficient in arylsulfatase B.     

The decreased synthesis of chondroitin sulfate-containing extracellular proteoglycans by SV40 transformed Balb/c 3T3 cells

Balb/c 3T3 cells synthesize 5–10 times more ³⁵SO₄^2?-labeled extracellular proteoglycan per cell than do Balb/c 3T3 cells transformed by SV40 (SV3T3). The extracellular ³⁵SO₄^2?-labeled proteoglycans of the Balb/c 3T3 and SV3T3 cells differ markedly in their acid mucopolysaccharide composition. Extracellular Balb/c 3T3 proteoglycans contain about 70–80% chondroitin sulfate, most of which is chondroitin 4-sulfate, and small amounts of heparan sulfate and/or heparin. On the other hand, extracellular SV3T3 proteoglycans contain 65–75% heparan sulfate and/or heparin and less than 15% chondroitin sulfate. Analysis of extracellular ³⁵SO₄^2?-labeled proteoglycan by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals that Balb/c 3T3 alone synthesizes a class of proteoglycans capable of migrating in a 10% separating gel. This class of proteoglycans, designated as fraction C, accounts for up to 45% of the total extracellular Balb/c 3T3 ³⁵SO₄^2?-labeled proteoglycans and contains chondroitin sulfate exclusively. It is altogether absent in the extracellular SV3T3 proteoglycans. The absence of this and other classes of chondroitin sulfate-containing proteoglycans can account for the 5–10-fold decreased synthesis of ³⁵SO₄^2?-labeled proteoglycans by SV3T3 cells when compared to Balb/c 3T3 cells.     

Sulfate restriction induces hyposecretion of the adhesion proteoglycan and cell hypomotility associated with increased 35SO42− uptake and expression of a band 3 like protein in the marine sponge,Microciona prolifera

Sulfate is an important component relating to normal proteoglycan secretion and normal motility in the marine sponge, Microciona prolifera. The following alterations were observed in sponge cells when sulfate free artificial sea water was used as the suspension medium: (1) impairment of aggregation, (2) loss of cell movements, (3) a marked reduction in the secretion of the adhesion proteoglycan (AP). Reversal of this effect occurred if sulfate depleted cells were again rotated in sulfate containing artificial sea water. Motility and reaggregation of sulfate deprived cells could be completely restored by purified AP, but only if cells were first pre-conditioned in normal sea water. Comparisons of ³⁵SO₄^2? uptake between normal and sulfate deprived cells which had been treated to reduce preformed secretions showed a marked increase in ³⁵SO₄^2? uptake and incorporation which could be greatly augmented in the presence of Ca²⁺/Mg²⁺. Excessive retention of AP in sulfate starved cells demonstrated by immunostaining suggested that AP secretion and cellular motility may be controlled by a sulfate dependent secretogogue or that undersulfated AP itself had developed a secretory defect. SDS-PAGE of Triton treated cellular extracts demonstrated a 116 kDa ³⁵SO₄^2? sulfated band which co-migrated with AP, but only in extracts derived from sulfate starved cells. Western blots prepared from such extracts incubated in the presence of a monoclonal anti-band 3 antibody demonstrated labelling of a single 97 kDa band only in material from sulfate deprived cells. The absence of this component in normal cell extracts indicated that this protein may be involved in facilitated sulfate transport. This study lends support to a heretofore unrecognized role for sulfate in cell motility and secretion.     

Sulfur dynamics in mineral horizons of two northern hardwood soils. A column study with 35S

Sulfur dynamics of two Spodosols were ascertained using soil columns constructed from homogenized mineral soil from nothern hardwood ecosystems at the Huntington Forest (HF) in the Adirondack Mountains of New York and Bear Brook Watershed in Maine (BBWM). Columns were leached for 20 weeks with a simulated throughfall solution with³⁵SO₄ ^2-. Sulfur constituents were similar to those of other Spodosols, with the organic S fractions (C-bonded S and ester sulfate) constituting over 90% of total S. HF soil columns had higher total S (14.9 mol S g^-1) than that for the BBWM soil columns (7.4 mol g^-1) primarily due to higher C-bonded S in the former.Initially, adsorbed SO₄ ^- accounted for 5 and 4% of total S for the BBWM and HF soil columns, respectively. After 20 weeks, adsorbed SO₄ ^2- decreased (81%) in BBWM and increased (33%) in HF soil columns. For both HF and BBWM soil columns, C-bonded S increased and ester sulfate decreased, but only for HF columns was there a net mineralization of organic S (5.6% of total S). The greatest decrease in ester sulfate occurred at the top of the columns.Leaching of³⁵S was less than 0.5% of the³⁵S added due to its retention in various S constituents. There was an exponential decrease in³⁵S with column depth and most of the radioisotope was found in C-bonded S (70–88 and 70–91% for BBWM and HF, respectively). The rapid turnover of adsorbed SO^2- ₄ was reflected in its high specific activity (834 and 26 kBq mol^-1 S for BBWM and HF, respectively). The lower specific activity of adsorbed SO₄ ^2- in HF was attributable to greater isotopic dilution by non-radioactive SO^2- ₄ derived from greater organic S mineralization in the HF versus the BBWM columns.Both soil columns initially had high levels of NO^- ₃ which resulted in the generation of H⁺ and net retention of SO₄ ^2- in the early phase of the experiment due to pH dependent sulfate adsorption; later NO₃ ^- decreased and SO₄ ^2- was desorbed. Leaching of NIO₃ ^- and SO₄ ^2- was correlated with losses of Mg²⁺ and Ca²⁺ of which the latter was the dominant cation.Analyses using both S mass balances and radioisotopes corroborate that for BBWM soil columns, SO^2- ₄ adsorption-desorption dominated the S biogeochemistry while in HF soil columns, organic S mineralization-immobilization processes were more important. It is suggested that similar techniques can be applied to soils in the field to ascertain the relative importances of SO₄ ^2- adsorption processes and organic S dynamics.     

Biosynthesis of lutropin in ovine pituitary slices: Incorporation of [35S]sulfate in carbohydrate units

Sulfate incorporation into carbohydrate of lutropin (LH) has been studied in sheep pituitary slices using H₂³⁵SO₄. Labeled ovine LH was purified to homogeneity by Sephadex G-100 and carboxymethyl-Sephadex chromatography from both the incubation medium and tissue extract. Autoradiography of the gel showed only two protein bands which comigrated with the α and β subunits of ovine LH in both the purified ovine LH and the immunoprecipitate obtained with LH-specific rabbit antiserum. Furthermore, [³⁵S]sulfate was also incorporated into several other proteins in addition to LH. The location of ³⁵SO₄^2? in the oligosaccharides of ovine LH was evidenced by its presence in the glycopeptides obtained by exhaustive Pronase digestion. The location and the point of attachment of sulfate in the carbohydrate unit were established by the isolation of 4-O-[³⁵S]sulfo-N-acetylhexosaminyl-glycerols and 4-O-[³⁵S]sulfo-N-acetylglucosaminitol from the Smith degradation products and by the release of ³⁵SO₄^2? by chondro-4-sulfatase. Thus, the present line of experimentation indicates the presence of sulfate on both the terminal N-acetylglucosamine and N-acetylgalactosamine in the oligosaccharide chains of the labeled ovine LH.     

A method for determining tissue sulfate

A method for the determination of the inorganic sulfate present in rat liver homogenates has been developed. In order to determine sulfate, a protein-free extract is required. The classical protein precipitation methods of preparing protein-free extracts gave 2.5–40% recovery of added ³⁵SO₄^2?. Separation of the protein by ultrafiltration gave only 29% recovery when 0.15 m KCl was the homogenizing medium. A homogenization medium containing 0.154 m NH₄OH and 20 g EDTA per liter gave 102 ± 11% recovery of added ³⁵SO₄^2? when the protein was separated by ultrafiltration.     

Sulfate flux in high sodium cat red cells

The transport of radioactive sulfate in cat red cells has been studied. The rate constant for ³⁵SO₄ inward movement under steady-state conditions is 0.24 ± 0.02/hr. This movement was found to be sensitive to osmotic changes in cell volume and to the nature of anions in the incubation medium; it increases with increasing cell volume and decreases with decreasing cell volume. The anions SCN, NO₃, and I were found to inhibit the uptake of ³⁵SO₄. Furthermore, 1-fluoro-2,4-dinitrobenzene at a concentration of 1 mM inhibits (>90%) this uptake. The inward movement of erythritol-¹⁴C shows qualitatively the same dependence on cell volume as ³⁵SO₄, but it is insensitive to the nature of the anion present in the bathing medium. It was also found that the usually observed inhibition of radioactive Na uptake by SCN in cat red cells can be reversed when cell volume is increased.     

Reduction of Sulfur Compounds in the Sediments of a Eutrophic Lake Basin

Concentrations of various sulfur compounds (SO₄²⁻, H₂S, S⁰, acid-volatile sulfide, and total sulfur) were determined in the profundal sediments and overlying water column of a shallow eutrophic lake. Low concentrations of sulfate relative to those of acid-volatile sulfide and total sulfur and a decrease in total sulfur with sediment depth implied that the contribution of dissimilatory sulfur reduction to H₂S production was relatively minor. Addition of 1.0 mM Na₂³⁵SO₄ to upper sediments in laboratory experiments resulted in the production of H₂³⁵S with no apparent lag. Kinetic experiments with ³⁵S demonstrated an apparent K_m of 0.068 mmol of SO₄²⁻ reduced per liter of sediment per day, whereas tracer experiments with ³⁵S indicated an average turnover time of the sediment sulfate pool of 1.5 h. Total sulfate reduction in a sediment depth profile to 15 cm was 15.3 mmol of sulfate reduced per m² per day, which corresponds to a mineralization of 30% of the particulate organic matter entering the sediment. Reduction of ³⁵S⁰ occurred at a slower rate. These results demonstrated that high rates of sulfate reduction occur in these sediments despite low concentrations of oxidized inorganic compounds and that this reduction can be important in the anaerobic mineralization of organic carbon.     

Kinetics of nitrate and sulfate removal using a mixed microbial culture with or without limited-oxygen fed

The biological degradation of nitrate and sulfate was investigated using a mixed microbial culture and lactate as the carbon source, with or without limited-oxygen fed. It was found that sulfate reduction was slightly inhibited by nitrate, since after nitrate depletion the sulfate reduction rate increased from 0.37 mg SO₄ ^2?/mg VSS d to 0.71 mg SO₄ ^2?/mg VSS d, and the maximum rate of sulfate reduction in the presence of nitrate corresponded to 56 % of the non-inhibited sulfate reduction rate determined after nitrate depleted. However, simultaneous but not sequential reduction of both oxy-anions was observed in this study, unlike some literature reports in which sulfate reduction starts only after depletion of nitrate, and this case might be due to the fact that lactate was always kept above the limiting conditions. At limited oxygen, the inhibited effect on sulfate reduction by nitrate was relieved, and the sulfate reduction rate seemed relatively higher than that obtained without limited-oxygen fed, whereas kept almost constant (0.86–0.89 mg SO₄ ^2?/mg VSS d) cross the six R_OS states. In contrast, nitrate reduction rates decreased substantially with the increase in the initial limited-oxygen fed, showing an inhibited effect on nitrate reduction by oxygen. Kinetic parameters determined for the mixed microbial culture showed that the maximum specific sulfate utilization rate obtained (0.098?±?0.022 mg SO₄ ^2?/(mg VSS h)) was similar to the reported typical value (0.1 mg SO₄ ^2?/(mg VSS h)), also indicating a moderate inhibited effect by nitrate.     

Central noradrenergic activity and the formation of glycol sulfate metabolites of brain norepinephrine

The present study utilized intraventricular injection of Na₂³⁵SO₄ to detect drug induced changes in the $\text{in vivo}$ formation of the two major metabolites of rat brain norepinephrine (NE) - the sulfate conjugates of 3-methoxy-4-hydroxyphenylglycol (MOPEG-SO₄) and 3,4-dihyd (DOPEG-SO₄). Assays involved the hypothalamus only. Rats pretreated with clonidine showed a reduced formation of both MOPEG-³⁵SO₄ and DOPEG-³⁵SO₄ after intraventricular Na₂³⁵SO₄ as well as reduced synthesis of ³H-NE from intraventricular ³H-tyrosine. Phenoxybenzamine (POB) produced increases in the synthesis of both ³⁵S-labeled conjugates and ³H-NE. Neither drug altered the loss of exogenous ³H-MOPEG-SO₄ but clonidine increased both the accumulation of labeled sulfate and the sulfation of exogenous MOPEG in pheniprazine treated rats. These results show that the rates of formation of the labeled glycol sulfates are sensitive indicators of changes in brain NE turnover but can also be influenced by factors involved in sulfation that are unrelated to NE turnover. Blockade of NE synthesis with alpha methyltyrosine did not affect resting or POB-elevated levels of the labeled conjugates until stores of NE were reduced by 40%. The latter findings suggest that central noradrenergic neurons can release and metabolize NE at a normal rate despite synthesis blockade so long as adequate stores of NE are available.