Biomedical subjects

P K Thorne

Publications and source records attributed to P K Thorne.

7 recordsLinked to original sources

PubMed1992

Stimulation of intestinal Cl- transport by heat-stable enterotoxin: activation of cAMP-dependent protein kinase by cGMP.

Heat-stable enterotoxins activate guanylate cyclase, whereas heat-labile enterotoxins stimulate adenylate cyclase. Both classes of toxins cause secretory diarrhea at least in part by stimulating Cl- secretion in the intestine. The mechanism for regulation of Cl- secretion by guanosine 3',5'-cyclic monophosphate (cGMP) was investigated using cultured T84 intestinal cells as a model for intestinal crypt cells. Escherichia coli heat-stable enterotoxin (ST) markedly stimulated cGMP production in T84 cells. Cl- secretion across T84 cell monolayers cultured on permeable filters was stimulated by E. coli ST, cholera toxin, or 8-BrcAMP, but 8-BrcGMP was ineffective. cGMP analogues that are known to be potent and specific activators of cGMP-dependent protein kinase (cG-kinase) also had little effect on 36Cl- uptake by T84 cells cultured in plastic dishes. E. coli ST, forskolin, cholera toxin, or membrane-permeant cAMP analogues markedly increased 36Cl- uptake into T84 cells. The general protein kinase inhibitor, staurosporine, inhibited the stimulation of Cl- permeability elicited by E. coli ST, vasoactive intestinal peptide (VIP), or 8-BrcAMP. DEAE-Sephacel chromatography revealed a predominant type II isoform of cAMP-dependent protein kinase (cA-kinase) in T84 cells, whereas little or no cytosolic cG-kinase activity was found. Treatment of T84 cells with E. coli ST or VIP resulted in an increase in the cA-kinase activity ratio (-cAMP/+cAMP) if the cytosolic enzyme was assayed at reduced temperature (on ice).(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Transport↗

PubMed1990

Purification and properties of parathyroid hormone-related peptide isolated from milk.

The occurrence and properties of PTH-related peptide (PTH-RP) in milk was investigated. PTH-RP was purified to homogeneity from human and bovine milk using heat and acid to precipitate milk proteins followed by ion exchange chromatography and reverse-phase HPLC. The peak of PTH-RP from HPLC was detected using a sensitive bone cell bioassay. A single band of peptide was detected on silver-stained polyacrylamide gels, which migrated as a 20-21-kDa macromolecule. PTH-RP isolated from either human or bovine milk had similar electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The partially purified bovine PTH-RP stimulated cAMP production in UMR106-01 and OK cell lines and elicited a concentration-dependent inhibition of sodium-dependent phosphate transport in OK cells. Incubation of milk extracts with an anti-PTH antiserum did not affect their bioactivity, whereas an antihuman PTH-RP 1-34 antiserum markedly reduced the cAMP response of UMR106-01 cells to the immunoabsorbed milk extracts. A PTH antagonist, norleu PTH 3-34, blocked the stimulation of cAMP production in UMR106-01 cells treated with milk extracts. PTH-RP immunoreactivity and bioactivity occurred in milk extracts of diverse animals from both eutherian and metatherian (marsupial) species. Porcine colostrum also had immunoreactive PTH-RP, although the levels were lower than the immunoreactive PTH-RP concentrations observed in milk samples collected at 7 and 14 days of lactation. Thus, a 20-21-KDa PTH-RP is secreted into milk where it could play a role in the development of suckling, newborn animals.

Adenylyl Cyclases↗

PubMed1989

Structure-activity relationships of parathyroid hormone analogs in the opossum kidney cell line.

Structural alterations in the parathyroid hormone (PTH) molecule produce marked changes in biologic activity. We examined the relative sensitivity of PTH-stimulated cAMP formation and PTH-inhibitable Na+-dependent phosphate transport responses to bovine PTH analogs [bPTH-(1-34), bPTH-(1-84), 8,18-norleucine-34-tyrosinamide bPTH-(1-34), bPTH-(7-34)-amide, 8,18-norleucine-34-tyrosinamide bPTH-(3-34), transaminated bPTH-(1-34)] and the human PTH-related peptide of malignancy (1-34) in cultured opossum kidney cells. The rank order of potency for stimulation of cAMP formation was bPTH-(1-34) = hPTHrP-(1-34) greater than nle bPTH-(1-34) greater than bPTH-(1-84) much greater than TAbPTH-(1-34). Nle bPTH-(3-34) and bPTH-(7-34) did not affect cAMP formation in intact cells at concentrations up to 10 microM. The rank order of potency for the inhibition of phosphate transport was bPTH-(1-34) = hPTHrP-(1-34) greater than nle bPTH-(1-34) greater than bPTH-(1-84) = TAbPTH-(1-34) greater than nle bPTH-(3-34). TAbPTH-(1-34) was a full agonist and inhibited phosphate transport at concentrations that did not increase cAMP formation, but nle bPTH-(3-34) was a partial agonist in spite of its inability to stimulate cAMP formation. Bovine PTH-(7-34) had no effect on phosphate transport. This study indicates that changes in the PTH molecule produce analogs that apparently discriminate between the cAMP-stimulating activity and phosphate transport-inhibiting activities of the native hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

Amination↗

PubMed1989

Autotransplantation of avian parathyroid glands: an animal model for studying parathyroid function.

The parathyroid glands of chickens were autotransplanted and the return of parathyroid function following transplantation was determined. Parathyroidectomy (PTX) resulted in a marked hypocalcemia (5.2 +/- 0.2 mg/dl) 4 hr following PTX. Plasma calcium (PCa) had declined to 4.3 +/- 0.2 mg/dl 24 hr after PTX. Parathyroid glands were transplanted subcutaneously 24 hr after removal and 24 hr later, PCa had risen to 8.6 +/- 0.5 mg/dl. Seven days after PTX. PCa increased to 10.3 +/- 0.2 mg/dl and by 14 days was indistinguishable from control levels (10.8 +/- 0.2 mg/dl vs. 11.0 +/- 0.2 mg/dl, respectively). When chicks with transplanted glands were fed a low Ca (0.08%) diet for 2 weeks they were able to maintain plasma PCa at levels comparable to control birds. Removal of the transplanted glands resulted in marked decreases in PCa (from 9.7 +/- 0.3 to 5.6 +/- 0.8 mg/dl), in the fractional excretion of phosphate, in urine cAMP, and in renal 25OH-vitamin D3-1 alpha-hydroxylase activity. Stepwise reductions in PCa and 1 alpha-hydroxylase activity were produced in partially PTX and fully PTX chicks by removing part or all of the parathyroid tissue. These data suggest that the transplanted parathyroid tissue was the major source of circulating PTH and that it may be possible to produce different degrees of acute hypoparathyroidism by varying the amount of transplanted parathyroid tissue removed surgically. Chickens with transplanted parathyroid glands thus provide a convenient animal model in which to study parathyroid function in an avian species.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗

PubMed1989

Clonal sublines that are morphologically and functionally distinct from parental OK cells.

Three clonal subpopulations of opossum kidney (OK) cells were derived from the parental line. The distribution of apical microvilli suggested that the OK cell line was heterogeneous. The clonal OK sublines appeared homogeneous as reflected by microvilli, which were uniformly distributed on the apical surface. Parathyroid hormone (PTH), forskolin (FSK), and prostaglandin E1 (PGE1) increased adenosine 3',5'-cyclic monophosphate (cAMP) formation in OK cells and all of the clones. PTH inhibited sodium-dependent phosphate transport in parental cells and in OK/B and OK/P clones with maximal effects appearing at 4, 2, and 1 h, respectively. PTH had no effect on phosphate transport in OK/H cells. FSK inhibited phosphate transport in parental cells and OK/B and OK/P clones but was relatively ineffective in OK/H cells. PGE1 decreased phosphate transport in OK/B and OK/P cells but was ineffective in the parental line and in OK/H cells. Phorbol 12-myristate 13-acetate, a potent inhibitor of phosphate transport in the parental OK cell line, had little effect in the clonal sublines. These clonal lines have remained phenotypically stable for 10 passages and should prove useful in studying the regulation of phosphate transport by PTH as well as addressing the question of whether PTH receptor subclasses exist which couple to cAMP and/or calcium effector systems in kidney cells.

Animals↗

PubMed1988

Regulation of sodium-dependent phosphate transport by parathyroid hormone in opossum kidney cells: adenosine 3',5'-monophosphate-dependent and -independent mechanisms.

The hormonal regulation of Na+-dependent phosphate transport was studied in opossum kidney (OK) cells. PTH caused time- and concentration-dependent decreases in Na+-dependent phosphate transport, with 10 pM PTH-(1-34) producing a 19% decline in phosphate transport. The EC50 for PTH inhibition of phosphate transport was 50 pM. Kinetic analyses of phosphate transport indicated that PTH decreased the maximum velocity without affecting the Km for phosphate. PTH increased cAMP formation with an EC50 of 10 nM. 8-Bromo-cAMP and (Bu)2cAMP also inhibited phosphate transport. Forskolin increased cAMP formation and decreased phosphate transport, whereas the cyclase-inactive forskolin analog 1,9-dideoxyforskolin also inhibited phosphate transport. The PTH analog [8,18-norleucine,34-tyrosinamide]PTH-(3-34) reduced phosphate transport at concentrations from 10 nM to 30 microM, but did not increase cAMP formation at concentrations up to 10 microM. The adenylate cyclase inhibitor 2',5'-dideoxyadenosine produced concentration-dependent decreases in PTH-stimulated cAMP formation, but did not influence PTH inhibition of Na+-dependent phosphate transport. Vasoactive intestinal polypeptide and prostaglandin E1 increased cAMP formation in OK cells, but were weak inhibitors of phosphate transport. This study suggests that cAMP may not be the only transmembrane signaling mechanism involved in the regulation of Na+-dependent phosphate transport by PTH-(1-34) in OK cells.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1987

A dual mechanism for regulation of kidney phosphate transport by parathyroid hormone.

Regulation of phosphate transport by parathyroid hormone (PTH) was investigated in continuous lines of kidney cells. Phosphate transport was reduced by PTH-(1-34) at physiological concentrations (EC50 5 X 10(-11) M), whereas much higher concentrations were required to stimulate cAMP formation (EC50 1 X 10(-8) M) in opossum kidney (OK) cells. The PTH analogue [Nle]PTH-(3-34) also inhibited phosphate transport but did not enhance cAMP formation. Instead, [Nle]PTH-(3-34) was a competitive antagonist of PTH-(1-34) at cyclase-coupled receptors. PTH-(7-34) had no effect on phosphate transport or cAMP formation. Phorbol esters or mezerein were potent inhibitors of phosphate transport but did not affect cAMP synthesis. Their potencies paralleled the rank-order potency of these agents as activators of protein kinase c in other systems. Maximally effective concentrations of PTH-(1-34) and mezerein did not produce additive inhibition of phosphate transport in OK cells. Phorbol esters stimulated phosphate transport in JTC-12 cells, but PTH-(1-34) had no effect. We concluded that PTH regulates OK cell phosphate transport by interacting with two classes of receptors, and transmembrane-signaling mechanisms. Physiological levels of PTH-(1-34) may regulate phosphate transport by activation of protein kinase c, whereas higher concentrations appear to activate adenylate cyclase.

Biological Transport↗