Neural regulation of phenylethanolamine N-methyltransferase in vivo: transcriptional and translational changes.

The hypothesis that neural regulation of rat adrenal medullary phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) occurs through transcriptional control is examined by following temporal changes in PNMT mRNA expression using paradigms of acute and chronic reserpine treatment. Concommitant changes in PNMT activity and protein were also measured to determine if reserpine induced changes depend solely on gene activation. Further, changes in adrenal corticosterone were measured to examine whether mRNA and enzyme responses might be mediated via reserpine induced changes in ACTH, and hence, corticosterone. Steady-state levels of PNMT mRNA peaked at approximately 8-fold normal by 6 h after a single reserpine injection (10 mg/kg i.p.), and then declined to control values. With continued treatment, a second, slower rise occurred after three alternate day injections (approximately 3-fold basal levels). Enzyme activity and protein rose simultaneously but were attenuated in magnitude and time course by comparison to message. With both acute and chronic treatment, activity increased 2.0-fold, peaking at 12 h after a single dose of reserpine and again after four doses of the drug. Protein, as measured by immunotitration, was elevated 1.2-and 1.4-fold, respectively. Adrenal corticosterone rose approximately 8-fold at 6 h, declined slightly at 12 h, rose again, and remained elevated thereafter. Comparison of the time courses for the various indices demonstrated that the early parallel bursts in PNMT mRNA and corticosterone are consistent with an increase in transcriptional activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Subunit characterization and primary structure of bovine adrenal medullary dopamine beta-hydroxylase.

Bovine adrenal medullary dopamine beta-hydroxylase was purified by sucrose density sedimentation, gel filtration chromatography, and Concanavalin A-Sepharose 4B affinity chromatography. Three subunits have been identified, of 71, 75, and 78 kd, present at a ratio of 1:2:1. Homogeneous subunits were isolated on denaturing polyacrylamide gels. Endoglycosidase treatment reduced each polypeptide to a 66-kd species, indicating that high and complex mannans account for the major differences in the subunits. The subunits and their 66-kd products cross-react with an anti-native dopamine beta-hydroxylase antiserum, suggesting common antigenic epitopes. Amino acid content analysis shows enrichment in glutamic acid/glutamine, aspartic acid/asparagine, glycine, and leucine, with little cysteine, tyrosine, proline, lysine, and methionine. Two to three nonidentical polypeptides have been identified from cyanogen bromide fragments. Comparison of the bovine peptide sequences to the corresponding cDNA-deduced human sequences show substantial similarity. Many of the species-specific differences in the primary structure represent conservative changes in amino acids or single base pair changes in amino acid codons.

Effects of analogs of salicylate on p-aminohippurate uptake into basal-lateral membranous vesicles.

In basal-lateral membranes of the renal cortex, thiol substituted analogs of salicylate inhibited the uptake of p-aminohippurate (PAH). The mercury-containing analogs, thimerosal and mercaptide V (formed from 2 mol of thiosalicylate and 1 mol of Hg++), were highly inhibitory. Compared with probenecid, thimerosal and mercaptide V yielded dose-response curves of steeper slope and higher maximal effect. The dose-response curves of thimerosal and mercaptide V were similar in shape, although mercaptide V was more potent. The inhibition by thimerosal, mercaptide V and mersalyl acid, an anionic sulfhydryl reagent which also inhibits uptake of PAH ( Tse et al., J. Pharmacol. Exp. Ther. 226: 19-26, 1983), was found to be competitive with Ki values of 0.39 +/- 0.05, 0.12 +/- 0.06 and 0.05 +/- 0.02 mM, respectively. The inhibitory effects of thimerosal and mercaptide V were only partially reversible. Thimerosal and mercaptide V reacted 35 and 62%, respectively, with membrane sulfhydryl groups which may explain the nonreversibility of the inhibitor. The nonreversibility is probably not due to irreversible destruction of the vesicles, as the "glucose space" of the vesicles was not affected by these two compounds. That derivatives of salicylates capable of reacting with sulfhydryl groups were more inhibitory than thiosalicylate is consistent with a hypothesis that the PAH transporter contains a sulfhydryl group(s) essential for uptake. Based on the degree of inhibition, the degree of reversibility, the degree of membrane sulfhydryl group oxidation and the computer-generated three dimensional models of thimerosal, mercaptide V and mersalyl acid, a model of the PAH transporter is proposed.

Effects of trypsin and protein modification on the renal transporter of p-aminohippurate.

Basal-lateral membranous vesicles prepared from rabbit renal cortex exhibited Mg2+-stimulated, probenecid-inhibitable transport of p-aminohippurate (PAH). This uptake could be completely eliminated by incubating the membranes with trypsin at a weight ratio of 1:700 (trypsin/membrane protein). The loss of PAH uptake activity occurred in two stages. Over the first ten minutes of the vesicles' exposure to trypsin, there was a nearly linear loss, with respect to time, of about 80% of the PAH uptake activity. The remaining 20% of activity was resistant to further trypsin digestion for the next ten minutes, but by twenty-five minutes a total inactivation of the uptake activity occurred. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of normal and trypsin-treated vesicles showed very little degradation of proteins. However, two minor polypeptides (Mr - 410,000 and 388,000) were degraded during the first ten minutes of the membranes' exposure to trypsin. After twenty minutes of exposure, two other polypeptides (Mr = 94,500 and 87,500) were degraded. Chymotrypsin and clostripain also caused a loss of PAH transport activity. However, compared to the effects of trypsin, the effects of these two proteases were less complete, slower in onset, and for clostripain, a much higher concentration of enzyme was required. Other functions or properties of the vesicles including morphological appearance, degree of vesiculation, glucose space or Na+-dependent L-glutamate transport and Na+,K+-ATPase activity were not altered by the concentration of trypsin which abolished 80% of the transport of PAH. Thus, it is possible that one or more of the degraded polypeptides detected by polyacrylamide gel electrophoresis comprises the PAH transporter.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of divalent cations and sulfhydryl reagents on the p-aminohippurate (PAH) transporter of renal basal-lateral membranes.

A specific system of transport for p-aminohippurate (PAH) is demonstrated in rabbit renal basal-lateral membrane vesicles. The PAH uptake into an intravesicular space is inhibited by probenecid in concentrations above 0.2 mM. The transport is saturable and is also temperature-dependent with an optimum between 37 and 45 degrees C. Divalent cations are able to enhance the uptake 2- to 3-fold. The stimulatory effect of the divalent cations diminishes in the following order: Mg++ = Mn++, Ba++, Ca++ and Sr++. Maximum stimulation occurs between 2.5 and 5 mM Mg++. The divalent cation stimulatory effect is not the result of changes in the size of the vesicles, in the degree of vesiculation, in the net charge of the membrane or of a transient potential difference across the membrane. Several inhibitors, more inhibitory than probenecid, were found. These are: lithium diiodosalicylate; 4-acetamido-4'-isothiocyano 2,2'-disulfonic acid stilbene; the mercurials, mersalyl acid, p-chloromercuriphenyl sulfonate and Hg++; and 5,5'-dithiobis(nitrobenzoate). Among these, mersalyl acid is the most potent inhibitor for PAH uptake. Its inhibitory effect is probably a combination of its reactivity toward sulfhydryl groups and its anionic character. The results with sulfhydryl reagents indicate that the PAH transport system contains sulfhydryl groups which are essential for the uptake activity. These sulfhydryl groups are probably buried in a hydrophobic region within the lipoprotein matrix of the basal-lateral membrane.

Basal-lateral membranes from rabbit renal cortex prepared on a large scale in a zonal rotor.

Basal-lateral membranes from the renal cortex of the rabbit were isolated by sucrose gradient centrifugation in a zonal rotor which allows for a large-scale preparation of these membranes. A heterogeneous population of membranes (P4) which contained 29% of the (Na+ + K+)-ATPase found in the homogenate of renal cortex was prepared by differential centrifugation. When pellet P4 was subjected to centrifugation in a sucrose gradient the activity of (Na+ + K+)-ATPase, a marker for basal-lateral membranes, could be separated from enzymatic markers of other organelles. The specific activity of (Na+ + K+)-ATPase was enriched 12-fold at a density of 1.141 g/cm3. Membranes (P alpha) contained in the (Na+ + K+)-ATPase-rich fractions consisted primarily of closed vesicles which exhibited probenecid inhibitable transport of rho-aminohippurate. These membranes did not exhibit Na+-dependent, phlorizin-inhibitable D-glucose transport. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of proteins from P alpha revealed at least six major protein bands with molecular weights of 91000, 81000, 73000, 65000, 47000 and 38000. A small fraction of total alkaline phosphatase found in the homogenate was found in pellet P4. Membranes containing this alkaline phosphatase activity were distributed widely over the gradient, with peak activity found at a density of 1.141 g/cm3. In contrast, when brush borders were subjected to gradient centrifugation under the same conditions as P4, alkaline phosphatase was found in a narrow distribution, with peak activity at a density of 1.158 g/cm3. The principle subcellular localization of the alkaline phosphatase found in P4 could not be determined unambiguously from the data, but the activity did not seem to be primarily associated with classical brush borders.

Electrophoretic properties of the scrapie agent in agarose gels.

The molecular properties of the scrapie agent were investigated by subjecting partially purified preparations to electrophoresis on agarose gels. When electrophoresis was performed at room temperature in the presence of sodium dodecyl sulfate (NaDodSO4), most of the recoverable agent was found at the top of the gel, consistent with previous studies indicating aggregation of the agent upon exposure to elevated temperatures. In addition, less than 5% of the agent applied to the gel was found after electrophoresis, even though the study was performed with a low concentration of NaDodSO4 (0.1%). Further studies on the inactivation of the agent by NaDodSO4 suggest that this may be, in part, a function of the NaDodSO4: protein ratio in the sample. In contrast, sodium N-lauroyl sarcosinate (Sarkosyl) did not inactivate the agent in concentrations as high as 5% (wt/vol). Virtually all of the infectivity could be recovered after electrophoresis of the agent into 0.6% agarose gels at 4 degrees C in the presence of 0.2% Sarkosyl. Digestion of the preparations with micrococcal nuclease and proteinase K prior to Sarkosyl electrophoresis caused a substantial portion of the agent to migrate ahead of DNA fragments of 1 x 10(6) daltons. The behavior of the scrapie agent in electrophoretic gels is consistent with earlier studies showing that the monomeric form of the agent has a sedimentation coefficient of less than or equal to 40 S. Thus, the smallest or monomeric form of the agent is smaller than any known animal virus.

Purification of proline-rich proteins from parotid glands of isoproterenol-treated rats.

Prolonged isoproterenol treatment of rats is known to cause hypertrophy and hyperplasia of the parotid glands. Our results show that a dramatic increase in the synthesis or accumulation in the parotid glands of a series of proteins rich in proline also occurs with isoproterenol treatment. After 10 days of treatment (5 mg of isoproterenol/day) these proline-rich proteins (PRPs) comprise more than 50% of the total soluble proteins in parotid gland homogenates. The PRPs are rapidly labeled in vivo by a single intraperitoneal injection of [3H]proline with maximum incorporation occurring at about 3. More than 90% of the [3h]proline found in parotid gland homogenates is incorporated into PRPs with less than 1% of the radioactivity in alpha-amylase. Tritium incorporated into PRPs was isolated as [3H]proline after acid hydrolysis. One acidic and six basic 3H-labeled PRPs were isolated from the 100,000 x g supernatant fraction of parotid gland homogenates by Sephadex G-100 and ion exchange chromatography. The six basic proteins accounted for about 90% of the total PRPs isolated.