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J T Potts

Publications and source records attributed to J T Potts.

At least 199 records · Page 11Linked to original sources
PubMed1977

Effect of ethane-1-hydroxy-1,1-diphosphonate (EHDP) on the ultrastructure of parathyroid glands and plasma immunoreactive parathyroid hormone in pregnant cows fed a low calcium diet.

The long term (70 days) effects of administering ethane-1-hydroxy-1,1-diphosphonate (EHDP) (4 mg. per kg. per day) on parathyroid function was investigated in pregnant cows fed a low calcium diet. Serum calcium and phosphorus were significantly lower at parturition and postpartum in EHDP-treated cows compared to pregnant control cows fed the low calcium diet. Plasma immunoreactive parathyroid hormone levels were similar prepartum, at parturition, and postpartum in cows administered EHDP and control cows. Immediately available calcium reserves were greater preparation in control cows than in cows receiving EHDP as indicated by a more rapid rate of return of serum calcium toward normal levels following ethylenediaminetetraacetic acid (EDTA)-induced hypocalcemia approximately 10 days prepartum. EHDP-treated cows responded to the hypocalcemic challenge with similar changes in plasma immunoreactive parathyroid hormone levels as in control cows; however, urinary hydroxyproline excretion increased at certain intervals only in control cows. Ultrastructurally, chief cells in parathyroid glands of both groups of cows were in an active stage of the secretory cycle with well developed organelles concerned with hormonela synthesis. Chief cells in cows administered EHDP were degranulated and contained fewer secretory granules in response to the hypocalcemia than those in control cows. Chief cells in EHDP-treated cows often had prominent perinuclear accumulations of microfilaments, scattered vacuolated mitochondria, and lysosomal bodies in the cytoplasm. Thyroid C-cells were densely granulated and thyroid calcitonin content was similar in both groups of cows. The principal defect in calcium homeostasis of EHDP-treated cows appeared to be an impairment both in bone calcium mobilization and bone matrix catabolism in response to the secretion of parathyroid hormone. In vitro uptake of 45Ca by duodenal mucosa and urinary excretion of cyclic adenosine monophosphate were similar in both groups of cows. The ability of the parathyroid glands to synthesize and secrete parathyroid hormone in response to hypocalcemia induced either by EDTA or associated with parturition was not impaired by the administration of EHDP.

Animals↗
PubMed1976

Conversion of proparathyroid hormone to parathyroid hormone: studies in vitro with trypsin.

The conversion of proparathyroid hormone to parathyroid hormone (PTH) was studied in vitro employing pancreatic trypsin as a prototype converting enzyme. Digestion of intact radiolabeled bovine prohormone with trypsin (0.1%) (w/w) resulted in release of a peptide comigrating with intact hormone marker in systems resolving both on the basis of charge (urea polyacrylamide gels, pH 4.4) and size (sodium dodecyl sulfate-urea polyacrylamide gels, pH 7.2). Tryptic digestions of a synthetic analogue of bovine prohormone, ProPTH-(-6 + 34), consisting of the prohormone hexapeptide covalently bonded to the NIH2 terminus of the active fragment of the hormone, released in high yield the hexapeptide and the intact active hormone fragment before any other smaller fragments. Analyses of digestions were by: (i) thin-layer chromatography and amino acid analysis of digestion products; (ii) comparison of the biological activity of the prophormone substrate and the products of digestion; and (iii) peptide end-group analysis by the Edman method during progressive tryptic hydrolysis over 22 h. The latter experiments demonstrated cleavage of more than 75% of the hexapeptide-hormone peptide bond before cleavage of other trypsin-sensitive sites within the molecule. It is concluded that the specificity of cleavage at the hexapeptide-hormone bond in the process of intracellular hydrolysis of proparathyroid hormone resides primarily in the sequence and/or conformation of the precursor molecule; inasmuch as conversion of prohormone to hormone can be efficiently accomplished by pancreatic trypsin in vitro, there is, therefore, no need to postulate the existence of an intracellular converting enzyme within the parathyroid cell that possesses unique hydrolytic specificity.

Adenylyl Cyclases↗
PubMed1976

Pre-proparathyroid hormone. Evidence for an early biosynthetic precursor of proparathyroid hormone.

Pre-proparathyroid hormone, discovered previously by the translation of messenger RNA from parathyroid glands in heterologous cell-free systems, is a polypeptide of 115 amino acids consisting of 25 amino acids added to the NH2 terminus of proparathyroid hormone (90 amino acids), the immediate biosynthetic precursor of parathyroid hormone (84 amino acids). We now have detected pre-proparathyroid hormone formation within intact parathyroid cells, thus providing direct evidence that it is a biosynthetic precursor of proparathyroid hormone. A radiolabeled protein synthesized by slices of bovine parathyroid glands during 1 to 10 min of incubation with [35S] methionine was identified that co-migrated on both urea-acetate and urea-SDS acrylamide gels with 3H-labeled pre-proparathyroid hormone prepared by mRNA translation in a cell-free system derived from wheat germ. The amount of radiolabeled protein reached a maximum 3 min after exposure of the tissue to[35S] methionine and decreased during a 3-min chase incubation with unlabeled methionine. It bound to antisera to both proparathyroid hormone and parathyroid hormone and contained 35S-labeled tryptic peptides that migrated identically with peptides of pre-proparathyroid hormone prepared from the wheat germ system. Radiolabeled proparathyroid hormone was identified in the tissue after 1 min of incubation with [35S] methionine. These findings indicate that pre-proparathyroid hormone is an early biosynthetic precursor of proparathyroid hormone in parathyroid tissue and that this precursor is converted to proparathyroid hormone within 1 min after completion of its synthesis on the rough endoplasmic reticulum.

Animals↗
PubMed1976

Pre-proparathyroid hormone: analysis of radioactive tryptic peptides and amino acid sequence.

The amino acid sequence of bovine pre-proparathyroid hormone has been partially determined by analysis of the polypeptide labeled selectively with radioactive amino acids. Analysis of tryptic peptides containing methionine or lysine indicated that parathyroid hormone, proparathyroid hormone, and pre-proparathyroid hormone had several common peptides. Two lysine-containing peptides present in proparathyroid hormone but not in parathyroid hormone were also present in pre-proparathyroid hormone. In addition, pre-proparathyroid hormone contained several additional lysine- and methionine-containing peptides not present in parathyroid hormone or proparathyroid hormone. Analysis by repetitive Edman degradation of the polypeptide labeled with lysine, methionine, and other amino acids indicated that pre-proparathyroid hormone contained 25 additional amino acids at the amino terminus of proparathyroid hormone; the identities of 17 of the 25 amino acids have been established. An unusual feature found was the presence of methionyl-methionyl at the amino terminus and the presence of 5 methionines within the first 14 amino acids.

Amino Acid Sequence↗
PubMed1976

Pre-proparathyroid hormone: fidelity of the translation of parathyroid messenger RNA by extracts of wheat germ.

Pre-proparathyroid hormone is the major protein synthesized in wheat-germ extracts in response to addition of an 8-15S fraction of parathyroid RNA. The accuracy of the translation of the mRNA from parathyroid tissue was examined by analysis of the carboxyl-terminal tryptic peptide and the amino-terminal amino acid of the protein, by analysis of the size distribution of the mRNA, and by translation of the mRNA in a second cell-free extract. When 8-15S RNA was fractionated on a sucrose gradient containing formamide, RNA that supported the synthesis of pre-proparathyroid hormone was present in a single symmetrical peak, suggesting that it was homogeneous. Analyses by paper chromatography and electrophoresis of the proline-containing tryptic peptides of pre-proparathyroid hormone indicate that they are identical with the corresponding proline-containing peptides of parathyroid hormone. Because the COOH-terminal tryptic peptide of parathyroid hormone contains proline, the data indicate that the COOH termini of pre-proparathyroid hormone and parathyroid hormone are identical. Methionine from initiator [35S]Met-tRNAfMet was rapidly incorporated into pre-proparathyroid hormone by the wheat-germ extract, and a single-step Edman degradation selectively removed almost all of the initiator [35S]methionine present in pre-proparathyroid hormone. Translation of the 8-15S RNA in a cell-free extract from Krebs-II ascites cells resulted in a protein that comigrated with pre-proparathyroid hormone on sodium dodecyl sulfate-acrylamide gel electrophoresis. These data support the conclusion that the wheat-germ system accurately translates the mRNA for parathyroid hormone, and they strengthen the contention that pre-proparathyroid hormone is the initial biosynthetic product.

Animals↗
PubMed1976

Chemical and biological properties of synthetic, sulfur-free analogues of parathyroid hormone.

Several analogues of the biologically active fragment of bovine parathyroid hormone (bPTH), based on the sequence of the NH2-terminal 34 amino acids, were prepared by solid phase synthesis and bioassayed in the in vitro adenylyl cyclase assay to provide further information concerning structure-activity relations in parathyroid hormone. In two analogues both methionines of the natural hormone were replaced with the sulfur-free and closely isosteric amino acid norleucine (Nle). The synthetic analogue [Nle-8, Nle-18]bPTH-(1-34) was highly active in the in vitro rat adenylyl cyclase bioassay, thus demonstrating that neither of the methionines, found in the native sequence, is indispensable for biological activity. Tyrosine was substituted for phenylalanine at position 34 in the synthesis of two other hormone analogues, [Try-34]bPTH-(1-34) and [Nle-8,Nle-18,Tyr-34]bPTH-(1-34). Both derivatives were exposed to conventional iodination procedures involving use of the oxidant chloramine T. Although iodination of [Try-34]bPTH-(1-34) resulted in virtually complete loss of biological activity, [Nle-8,Nle-18,Tyr-34]-bPTH-(1-34), which lacks methionine, could be exposed to oxidants and labeled efficiently with iodine with retention of nearly complete biological activity. These findings confirm that the loss of biological activity after oxidation of bPTH, as previously observed with the native hormone, is indeed attributable to the oxidation lability of methionine rather than to any other modifications. This sulfur-free, radioiodinated, biologically active analogue of parathyroid hormone may prove useful in studies of interaction of the hormone with the membrane receptors of target tissues and in studies of the metabolism of parathyroid hormone.

Adenylyl Cyclases↗
PubMed1976

Metabolism of amino- and carboxyl-sequence immunoreactive parathyroid hormone in the bovine: evidence for peripheral cleavage of hormone.

Radioimmunoassays that detect specifically peptide sequences within either the biologically active amino region (N-assay) or inactive carboxyl region (C-assay) of parathyroid hormone (PTH) were used to evaluate the metabolism of PTH during and after infusion and injection of homogeneous (containing less than 0.1% hormonal fragments) intact bovine PTH (bPTH) into calves. During continuous infusions of hormone, when constant blood levels of immunoreactive PTH were reached, a dissociation between the concentrations of amino versus carboxyl immunoreactivity was observed; concentrations of hormone measured by the C-assay rose to a level of approximately three times higher than that measured by the N-assay. Analysis by gel filtration of immunoreactive PTH in plasma samples from calves after injection of hormone showed the rapid disappearance of intact hormone (N- and C-assays) and the appearance of a large fragment detected by the C-assay but not by the N-assay. The hormonal fragment lacked antigenic determinants within the amino peptide sequence required for biologic activity. No additional fragments of PTH were detected by gel filtration using the N- and C-assays. No detectable conversion of intact PTH to hormonal fragments occurred during incubation in vitro in bovine serum. The results are consistent with the concept that PTH is metabolized after entry into the circulation at peripheral sites located outside the vascular space, resulting in the rapid disappearance from blood of intact hormone and the appearance of a biologically inactive hormonal fragment(s). These studies done in calves agree with earlier studies done in dogs and man and point to the existence in mammals of common pathways for the peripheral metabolism of PTH.

Amino Acid Sequence↗
PubMed1976

Dietary calcium, calcium kinetics and plasma parathyroid hormone concentration in cows.

Kinetic analysis of radiocalcium data was combined with radioimmunoassays of plasma parathyroid hormone in nonpregnant nonlactating cows 4.5 to 7.8 years of age whose calcium intake was varied from 0.05% to 1.4% of the diet. Calcium intake had no significant effect on plasma concentration of calcium or parathyroid hormone. It had a slight but significant effect on the plasma phosphorus concentration. As calcium intake increased, calcium absorption increased but calcium removal from bone decreased so that total calcium transport into the exchangeable pool remained fairly constant. A decline in calcium clearance with age was accompanied by a fall in plasma parathyroid hormone concentration, and a decrease in calcium transport into the pool. These observations are consistent with feedback controls between plasma calcium, parathyroid hormone and calcium transport operating to maintain plasma calcium homeostasis as calcium clearance declined with advancing age.

Age Factors↗
PubMed1976

Chemistry and physiology of parathyroid hormone.

Many recent studies, only a few of which are reviewed here (see Habener & Potts, 1976, for a detailed summary) have led to considerable clarification concerning critical features of the chemistry, biosynthesis, secretion and mode of action of parathyroid hormone. Certain of the confusion recently experienced in measurements of parathyroid hormone in blood is clearly explained by the heterogeneity arising from various forms of parathyroid hormone arising during biosynthesis and metabolism. Ultimately, as critical features of biosynthesis and metabolism of PTH as well as chemical definition of various molecular forms are achieved, considerable refinement in immunoassays should result. These advances in turn should provide better insights into the role of parathyroid dysfunction in various disorders of calcium and skeletal metabolism. From the physiological viewpoint, a clearer appreciation of the homeostatic role of parathyroid hormone in calcium metabolism is emerging as the essential features of biosynthesis, release, metabolism and action of the hormone are uncovered.

Amino Acid Sequence↗
PubMed1976

Metabolism and biological activity of parathyroid hormone in renal cortical membranes.

Recent studies from several laboratories have documented the presence of fragments of parathyroid hormone in blood or peripheral tissues or in both. Inasmuch as amino-terminal fragments are known to be biologically active, it has been suggested that fragments, rather than the intact polypeptide of 84 amino acids, might be the active molecular species in tissue fluids. Accordingly, the metabolism of native bovine parathyroid hormone, bPTH-(1-84), was studied in purified renal cortical membranes from several species and correlated with hormonal stimulation of adenylyl cyclase in these membranes in vitro. Analysis of whole incubation mixtures or membrane-bound hormone by gel electrophoresis and gel chromatography after incubation of [3H]bPTH-(1-84) or 125-I-labeled bPTH-(1-84) or unlabeled biologically active bPTH-(1-84) with purified canine renal cortical membranes revealed no evidence of proteolysis, and yet the uncleaved hormone readily stimulated adenylyl cyclase. Kinetic studies of hormone-stimulated adenylyl cyclase activity revealed no difference in rate of onset of activity between bPTH-(1-84) And the active synthetic amino-terminal tetratriacontapeptide bPTH-(1-34), and hence there was no evidence of precursor-product relationship between the native hormone and an active amino-terminal fragment. The results suggest, insofar as the activity detected in these membranes reflects the biological response of the hormone in vivo, that the native hormone is indeed biologically active at the receptor level directly without the requirement for cleavage into active fragments.

Adenylyl Cyclases↗
PubMed1976

Parathyroid hormone secretion in vivo. Demonstration of a calcium-independent nonsuppressible component of secretion.

The response of normal bovine parathyroid glands to hypercalcemia was assessed in vivo by radioimmunoassay of immunoreactive parathyroid hormone concentrations in parathyroid effluent blood obtained by surgical cannulation of both anesthetized and nonanesthetized calves. Hypercalcemia was induced for periods of 0.3-35 h by intravenous infusion of a solution of calcium chloride. Assessment of immunoreactivity in effluent and peripheral blood included measurements of selected samples by use of a radioimmunoassay specific for a site residing in the biologically active portion of the hormone molecule. In all instances, the concentration of immunoreactive parathyroid hormone in hypercalcemic venous effluent from a superior parathyroid gland exceeded that of the peripheral blood. Failure of hypercalcemia to suppress completely secretion by normal parathyroids indicates that a portion of parathyroid hormone secretion occurs independent of blood calcium concentration. Consequently, continued parathyroid hormone secretion despite hypercalcemia can no longer be regarded as a unique feature of parathyroid neoplasia.

Amino Acid Sequence↗
PubMed1976

Relative effectiveness of magnesium and calcium on the secretion and biosynthesis of parathyroid hormone in vitro.

The effects of varying extracellular concentrations of calcium and magnesium were compared on the release of 3H-labeled and immunoreactive parathyroid hormone (PTH) by bovine parathyroid slices incubated for 4 h in vitro in media containing [3H]leucine. The quantity of [3H]PTH or immunoreactive PTH released was determined by polyacrylamide gel electrophoresis and by radioimmunoassay, respectively. Biosynthesis of PTH in response to extracellular concentrations of magnesium and calcium was evaluated by radioimmunoassay of amounts of proparathyroid hormone (PROPTH) in extracts of the parathyroid tissues and by electrophoretic analyses of [3H]ProPTH synthesized during a 35 min pulse-labeling of parathyroid slices. Rates of hormone release were found to vary in response to changes in concentrations of either calcium or magnesium. The effects, however, of these two cations on hormone release, although additive, were not equipotent. On a molar basis, approximately 2.5 times as much magnesium as calcium was required to produce equivalent secretory responses. Effects of the cations on hormone biosynthesis wer small; magnesium had no effect and calcium altered synthesis by approximately 15%. Neither cation affected conversion of ProPTH to PTH. The results agree with those reported previously from studies of the effects on hormone secretion of calcium and magnesium in vivo and serve to emphasize that, within normal physiological concentrations of these two cations, calcium is the predominant regulator of PTH secretion and synthesis.

Animals↗
PubMed1976

Immunological comparisons of two synthetic human parathyroid hormone-(1-34) peptides.

The immunological properties of human parathyroid hormone-)1-34) synthesized in accord with the sequence reported by Brewer et al. and the different sequence found by Niall et al. were compared with those of highly purified native human hormone-)1-84). Analyses were performed by radioimmunoassay using 7 anti-bovine parathyroid hormone antisera and bovine parathyroid hormone-)1-34) as tracer. Whereas human parathyroid hormone-(1-34) synthesized in accord with the sequence of Niall et al. was immunologically indistinguishable from native human hormone in all 7 assay systems, striking differences were seen between human parathyroid hormone-(1-34) synthesized in accord with the sequence of Brewer et al. and the native hormone. In none of the 7 assay systems did human parathyroid hormone-(1-34) synthesized in accord with the sequence of Brewer et al. give the same displacement slopes that the reference preparation gave. The observation that immunologic probes easily discriminate between the two different human parathyroid hormone-(1-34) peptides suggests that similar immunologic approaches will be of value in exploring several important issues, particularly those relating to the sequence and conformational properties of human parathyroid hormone and its synthetic peptides and to the question of the existence of isohormonal forms.

Animals↗
PubMed1976

Metabolism of radioiodinated bovine parathyroid hormone in the rat.

Metabolism of bovine 125I-labeled parathyroid hormone was studied in the rat by gel filtration and by sequence analysis of the iodinated fragments. Analysis of the kinetics of hormone metabolism shows that iodinated intact hormone has a multiexponential disappearance curve with a rapid (3 min) initial and a slower (48 min) second component. Iodinated fragments, which rapidly increase during the first 12 min after injection of the intact hormone, subsequently disappear from the circulation with a t1/2 of no greater than 48 min. Plasma samples collected at various time-intervals after intravenous injection of bovine 125I-labeled parathyroid hormone were gel filtered on Bio-gel P-100. Four radioactive peaks were seen. The first and second peaks eluted, respectively, at the void volume of the column and at the position of intact hormone. The third peak consisted of iodinated fragments, and the last peak eluted at the salt volume of the column. Sequence analysis of the iodinated fragments in the third peak showed that it was heterogeneous, containing several different, but closely related, polypeptides. Before 48 min after injection, the most-abundant fragment is one whose amino-terminal amino acid is residue 34. The amino-terminal residue of the next most-common fragment is the amino acid at position 37. No fragments representing cleavages closer to the amino-terminus than residue 34 were seen. The results of these studies are virtually identical with those previously obtained in the dog. The similarities found in the sites of hormone proteolysis and in the kinetics of hormone metabolism in the rat and dog, coupled with the less direct evidence indicating that similar cleavages are also present in man and bovine, are consistent with the view that proteolysis of parathyroid hormone is peripheral tissues is specific, at least in mammalian species, and may be a critical step in controlling the availability of biologically active hormone.

Animals↗
PubMed1976

Effects of bone in vitro of bovine parathyroid hormone and synthetic fragments representing residues 1-34, 2-34 and 3-34.

The biological activities of bovine parathyroid hormone (BPTH) and fragments comprising portions of its amino-terminal sequence have been compared in three different assay systems using embryonic rat bone in vitro. Whereas the 3-34 fragment was without significant activity the 1-34 fragment caused all the actions characteristic of BPTH 1-84, extending to bone previous evidence that the amino-terminal residues are sufficient for expression of the biological effects of intact parathyroid hormone. However, the relative potencies of the fragment and the intact hormone were different in the various systems. BPTH 1-34 showed relatively low osteolytic activity and induced anabolic effects in both osteoblasts and cartilage cells of cultivated embryonic mouse radii which were not evoked by the intact hormone. Further work is required to determine the mechanisms responsible for these interesting alterations in relative potency of fragment and native hormone.

Animals↗
PubMed1976

Role of 5'-guanylylimidodiphosphate in the activation of adenylyl cyclase by parathyroid hormone.

We have studied the effects of guanylylimidodiphosphate (Gpp(NH)p), an analogue of GTP, on the stimulation of renal cortical adenylyl cyclase by bovine parathyroid hormone (bPTH, or bPTH-(1-84)). Incubation of canine renal membranes with bPTH-(3-34), a specific antagonist of parathyroid hormone, in either the presence or absence of Gpp(NH)p, prevented subsequently added bPTH-(1-84) from stimulating adenylyl cyclase. The addition of the antagonist to a cyclase system previously activated by both bPTH-(1-84) and Gpp(NH)p, however, produced no inhibition of enzyme activity. Removal of bPTH by washing the membranes virtually abolished activity, but washing after addition of bPTH plus Gpp(NH)p did not prevent continued accumulation of cAMP. The persistence of the activity of the enzyme brought about by the addition of Gpp(NH)p plus bPTH, despite washing or addition of specific inhibitor of bPTH action, indicates that the activity of the hormone-specific adenylyl cyclase in membrane suspensions is independent of cintinuous occupancy of the peptide-hormone receptor by bPTH in the presence of the guanyl-nucleotide analogue.

Adenylyl Cyclases↗