Renal adenyl cyclase: anatomically separate sites for parathyroid hormone and vasopressin.

Adenyl cyclase from plasma membrane fractions of rat renal cortex or medulla was assayed by measuring conversion of adenosine triphosphate labeled at the alpha-phosphate with (32)P to cyclic 3',5'-adenosine monophosphate labeled with 32P. Parathyroid hormone activated the enzyme primarily in cortex; vasopressin acted primarily in medulla. These experiments support the conclusion that cyclic adenosine monophosphate mediates the action of parathyroid hormone on the kidney and show that parathyroid hormone and vasopressin stimulate adenyl cyclase at anatomically separable areas within the kidney.

Calcitonin receptors of kidney and bone.

Receptors for calcitonin, determined by activation of adenylate cyclase, were found in a distribution among zones of the kidney distinct from that of receptors for parathyroid hormone or vasopressin. Competitive binding studies showed that the receptors for calcitonin are similar in kidney and bone and that their high apparent affinity for salmon calcitonin accounts in part for the high biological potency in vivo of salmon calcitonin.

Tyrosine aminotransferase: enzyme induction independent of adenosine 3', 5'-monophosphate.

The importance of adenyl cyclase and adenosine 3',5'-monophosphate in the induction of tyrosine aminotransferase by adrenocorticosteroids has been tested in HTC cells derived from a rat hepatoma and grown in tissue culture. Adrenocorticosteroids cause a 10-to 15-fold increase in the rate of synthesis of tyrosine aminotransferase in these cells. Under various experimental conditions, with or without glucocorticoids, neither adenyl cyclase nor cyclic adenosine mono-phosphate could be detected in HTC cells. In addition, neither the cyclic nucleotide nor N(6), O(2')-dibutyryl cyclic adenosine monophosphate caused increased activity of the transaminase in HTC cells. We conclude that induction of tyrosine aminotransferase by glucocorticoids is not mediated by the adenyl cyclase-cyclic adenosine monophosphate system.

Parathyroid hormone production in vitro by human parathyroid cells transformed by Simian virus 40.

Cells froma human parathyroid adenoma were infected with simian virus 40 and maintained through 13 subcultures in monolayer tissue culture. For more than 9 months, these "transformed" cells contimed to produce parathyroid hormone which was identified by radioimmunoassay and density-gradient ultra centrifugation.

Thyrocalcitonin: ultracentrifugation in gradients of sucrose.

Analysis of thyrocalcitonin, by density-gradient ultracentrifugation at high speed, showed that its molecular weight (5000 to 6000) is considerably less than was heretofore recognized.

Beta-adrenergic receptor: stereospecific interaction of iodinated beta-blocking agent with high affinity site.

An iodine-labeled beta-adrenergic inhibitor ((125)l-hydroxybenzylpindolol) binds specifically to a site on turkey erythrocyte membranes. A series of beta-adrenergic agonists and inhibitors compete for this binding site, with apparent affinities paralleling biological effectiveness as activators or inhibitors of catecholaminestimulated adenylate cyclase. The activity of d-(+) agonists or inhibitors was 1 percent (or less) than that of the corresponding l-(-) isomers in competing for binding of the iodinated blocker as well as in affecting catecholamine-stimulated adenylate cyclase. 1-(-)-Norepinephrine was about one-tenth as active as l-(-)-isoproterenol in competing for the beta-blocking agent site. The stereospecificity of the interaction with the iodinated beta-blocking agent and the correspondence between affinity for site and biological potency of analogs suggested that this interaction is involved in function of the beta-adrenergic receptor.

Pseudohypoparathyroidism: defective excretion of 3',5'-AMP in response to parathyroid hormone.

Urinary excretion of cyclic adenosine 3',5'-monophosphate (3',5'-AMP) was tested in normal subjects and patients with pseudohypoparathyroidism, idiopathic hypoparathyroidism, surgical hypoparathyroidism, and pseudopseudohypoparathyroidism under basal conditions and after a 15 min infusion of purified parathyroid hormone. Basal excretion of the nucleotide was less than normal in the patients with hypocalcemic disorders and greater than normal in pseudopseudohypoparathyroidism. Parathyroid hormone caused a marked increase in excretion of 3',5'-AMP in all subjects except those with pseudohypoparathyroidism; nine patients with this disorder did not respond to the hormone and four showed a markedly deficient response. Radioimmunoassay showed that parathyroid hormone circulated in increased amounts in plasma from patients with pseudohypoparathyroidism and became undetectable when serum calcium was increased above 12 mg/100 ml. Suppression of parathyroid hormone secretion by induction of hypercalcemia did not alter the deficient response to exogenous hormone. The results indicate that: (a) parathyroid hormone circulates in abnormally high concentrations in pseudohypoparathyroidism and secretion of the hormone responds normally to physiological control by calcium; (b) testing urinary excretion of 3',5'-AMP in response to infusion of purified parathyroid hormone appears to be an accurate and sensitive index for establishing the diagnosis of pseudohypoparathyroidism; and (c) the metabolic defect of the disorder can be accounted for by a lack of or defective form of parathyroid hormone-sensitive adenyl cyclase in bone and kidney.

Allelic loss from chromosome 11 in parathyroid tumors.

Parathyroid tumors may occur in a sporadic fashion or, more rarely, as part of a familial syndrome (such as familial multiple endocrine neoplasia type I). The MENI gene has been mapped by linkage analysis to chromosome 11 at band q11-q13, and presumably acts as a tumor suppressor gene. In the present study, which is an extension of our previous studies, we examined 41 parathyroid tumors from patients with familial multiple endocrine neoplasia type I and 61 sporadic parathyroid tumors with markers on chromosome 11, to assess the extent of allelic loss in those tumors. Twenty-four of the MENI-associated tumors (58%) and 16 of the sporadic parathyroid tumors (26%) displayed allelic loss from chromosome 11. The region of overlap of the allelic losses in the MENI-associated tumors enables us to place the MENI gene between PGA centromerically and INT2 telomerically, a region spanning about 7.5 cM. Taken together with locus ordering by linkage analysis, this clearly localizes the MENI gene telomeric to the PGA locus. Our inability to detect allelic loss on chromosome 11 in some parathyroid tumors suggests the existence of other genes involved in the development and/or progression of this subgroup of presumably monoclonal tumors; or that localized events involving the 11q tumor suppressor gene have occurred in some parathyroid tumors whose detection is beyond the sensitivity of our analysis; or that at least some of the specimens analyzed were in fact primarily hyperplastic parathyroid tissue.

Allelic loss on chromosome 11 in hereditary and sporadic tumors related to familial multiple endocrine neoplasia type 1.

Familial multiple endocrine neoplasia type 1 (FMEN1) is an autosomal dominant disorder characterized by tumors of the parathyroid glands, pancreatic islets, and anterior pituitary. The gene for this disease maps to chromosome 11q12-11q13, and allelic loss in this region has been shown in both sporadic and FMEN1-related parathyroid tumors. FMEN1-related pancreatic islet tumors, and rarely in sporadic anterior pituitary tumors. We tested for allelic loss at 7 loci on chromosome 11 in 17 tumors outside the parathyroid. We found loss of heterozygosity in 2 of 2 FMEN1-related benign pancreatic islet tumors but in none of 8 informative sporadic islet tumors (P = 0.02) including 5 malignant gastrinomas. Of 3 islet tumors from patients who had some but not all features of FMEN1, one showed allelic loss for 5 of 5 informative restriction fragment length polymorphisms, and the other 2 retained heterozygosity for all informative markers. A bronchial carcinoid from an FMEN1 patient and 3 sporadic anterior pituitary tumors showed no allelic loss. These data provide new evidence that many sporadic pancreatic islet neoplasms, even when malignant, do not develop through homozygous inactivation of the MEN1 gene.

Effect of prostaglandin F2 alpha on human parathyroid adenomas: evidence for uncoupling of parathyroid hormone secretion and cAMP accumulation.

Human parathyroid adenomas are aberrantly regulated by extracellular calcium. We tested pertussis toxin, which ADP-ribosylates and inactivates several guanine nucleotide regulatory proteins, to test the role of these proteins in the secretory control of adenomatous parathyroid tissue. Pertussis toxin did not affect basal cAMP accumulation in 12 adenomas and enhanced parathyroid hormone (PTH) release in 6 of 10 adenomas. Prostaglandin F2 alpha (PGF2 alpha) inhibited cAMP in three of six adenomas, and pertussis toxin pretreatment did not affect this result. PTH release in 7 of 10 adenomas was inhibited by PGF2 alpha, and pertussis toxin did not significantly alter PTH release. Pertussis toxin catalyzed ADP-ribosylation of a 40-kDa protein in all adenomas tested (n = 8). We conclude that cAMP accumulation was not affected by pertussis toxin but that in 6 of 10 adenomas, the toxin enhanced PTH release. We suggest that cAMP accumulation and PTH release may be uncoupled from negative control by inhibitory ligands in adenomatous tissue or that the G-proteins involved do not couple to regulatory receptors or to effector.

Subclones of a rat parathyroid cell line (PT-r): regulation of growth and production of parathyroid hormone-related peptide (PTHRP).

Four subclones from a rat parathyroid cell line (PT-r cell) have been isolated, and morphological and functional characteristics have been examined. Subclones 1 and 2 display a polygonal shape, show growth and secretory responses to calcium (half-maximal suppressions at 1.2 and 1.7 mM, respectively), and respond to secretin with cAMP production (14.5-fold and 16.9-fold over basal) and hormone secretion (41 and 58% over basal). Subclone 4 is elongated in form and does not respond to calcium or secretin. Subclone 3 shows mixed morphology, elongated and polygonal shapes, with moderate response to calcium (half-maximal suppression at 1.7 mM) and secretin (cAMP, 3.2-fold increase and hormone secretion, 50% increase over basal). The clones were tested for content of messenger RNA (mRNA) representing parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHRP). Only PTHRP mRNA was found. The peptide released is virtually all PTHRP. PTH mRNA was not detected even with a sensitive RNA probe. The amount of mRNA for PTHRP closely paralleled the amount of PTH-like bioactivity released into the medium from each clone (144.7 +/- 12.1, 110.0 +/- 12.9, 68.0 +/- 5.6, and 39.9 +/- 2.4 pgEq of rat PTH-(-34) per 10(7) cells per 12 h in a medium with 0.7 mM ionized calcium, from subclones 1, 2, 3, and 4, respectively). Culture conditions, low-density passage (less than 1:50 split ratio) or high-density passage (greater than 1:10 split ratio), affected morphology and function of the clones 1 and 2. They became elongated and functionally dedifferentiated like subclone 4 and 3 months of high-density culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Renal receptors for calcitonin: coordinate occurrence with calcitonin-activated adenylate cyclase.

High affinity binding of 125I-labeled salmon calcitonin ([125I]SCT) and calcitonin-activated adenylate cyclase were detectable in renal plasma membranes from the rat. Addition of 5'-guanylyl-imidodiphosphate lowered the threshold for enzyme activation by peptide hormones. Renal plasma membranes from man, dog and cow contained little or no calcitonin-sensitive adenylate cyclase and showed no high affinity binding of [125I]SCT. High affinity binding sites were distributed during membrane fractionation in fractions where the specific activity of hormone-sensitive adenylate cyclase was greatest. Calcitonin binding and activation of the adenylate cyclase enzyme occurred at similar hormone concentrations. The relative potencies of calcitonin analogues were similar whether measured by competition for high affinity binding sites or by effect on adenylate cyclase. Low concentrations of Lubrol-PX, a nonionic detergent, did not affect catalytic function of the enzyme determined in the presence of sodium fluoride but caused parallel loss of high affinity [125I]SCT binding and hormonal sensitivity of the enzyme. This observation provided further evidence that interaction of calcitonin with specific receptors (identified with [125I]SCT binding) is essential for calcitonin activation of adenylate cyclase, but showed that catalytic activity of enzyme does not require functioning hormone receptors.

Inhibition of parathyroid hormone release by maitotoxin, a calcium channel activator.

Maitotoxin, a toxin derived from a marine dinoflagellate, is a potent activator of voltage-sensitive calcium channels. To further test the hypothesis that inhibition of PTH secretion by calcium is mediated via a calcium channel we studied the effect of maitotoxin on dispersed bovine parathyroid cells. Maitotoxin inhibited PTH release in a dose-dependent fashion, and inhibition was maximal at 1 ng/ml. Chelation of extracellular calcium by EGTA blocked the inhibition of PTH by maitotoxin. Maitotoxin enhanced the effects of the dihydropyridine calcium channel agonist (+)202-791 and increased the rate of radiocalcium uptake in parathyroid cells. Pertussis toxin, which ADP-ribosylates and inactivates a guanine nucleotide regulatory protein that interacts with calcium channels in the parathyroid cell, did not affect the inhibition of PTH secretion by maitotoxin. Maitotoxin, by its action on calcium channels allows entry of extracellular calcium and inhibits PTH release. Our results suggest that calcium channels are involved in the release of PTH. Inhibition of PTH release by maitotoxin is not sensitive to pertussis toxin, suggesting that maitotoxin may act distal to the site interacting with a guanine nucleotide regulatory protein, or maitotoxin could interact with other ions or second messengers to inhibit PTH release.

Preparation of viable isolated bovine parathyroid cells.

A method is described for the preparation of isolated bovine parathyroid cells. Fresh, minced bovine parathyroid tissue is incubated with 2 mg/ml collagenase and 50 mug/ml DNAse for 1 h at 37 C with periodic pipetting. Parthyroid cells are separated from contaminating fat cells and red cells by low speed centrifugation. The resulting cell preparation is indistinguishable from parathyroid cells in intact bovine glands by light and electron microscopy. Hormone release is linear for at least 3 h at both high (2.0 mM) and low (0.5 mM) calcium concentrations and is inversely proportional to divalent cation concentration between 0.3 mM and 1.0 mM calcium as been observed previously both in vivo and in vitro. As in previous studies, hormone release is also stimulated up to 2-fold by 10(6) M (-)isoproterenol, an effect blocked by 10(-6)M (-)propranolol, suggesting a beta-adrenergic effect. Such a cell preparation should be useful for studying hormone binding and effects on cyclic nudleotides and cellular transport phenomena in both normal amd abnormal tissue.

Beta-adrenergic stimulation of cyclic AMP content and parathyroid hormone release from isolated bovine parathyroid cells.

The effects of beta-adrenergic agonists and antagonists on cyclic AMP (cAMP) accumulation and parathyroid hormone (PTH) release from isolated bovine parathyroid cells have been determined. Beta-adrenergic agonists markedly stimulate cAMP production and PTH release with an order of potency (-) isoproterenol greater than (-)epinephrine greater than greater than (-) norepinephrine, suggesting a beta2-type adrenergically mediated process. Both effects are blocked by the beta-blocker propranolol with the strict stereospecificity expected for a beta-adrenergic response. Low calcium concentrations also stimulate cAMP accumulation, but the cyclic nucleotide response under these conditions is only 3% of that obtained with isoproterenol, raising the possibility that factors other than cAMP may control low calcium-mediated PTH release. The release of PTH by low calcium is also not blocked by propranolol, confirming the independence of the response to low ambient calcium from the beta-adrenergic receptor. These studies substantiate further the utility of the isolated parathyroid cell preparation for studying secretagogue-mediated alterations in cyclic nucleotides and hormone secretion. Isolated cells also also make feasible the direct identification of beta-adrenergic receptors in parathyroid cell membranes and whole cells.

Calcium-controlled secretion is effected through a guanine nucleotide regulatory protein in parathyroid cells.

Calcium regulates parathyroid secretion through mechanisms yet to be elucidated. We have investigated this phenomenon through use of pertussis toxin which catalyzes ADP-ribosylation and inactivation of a guanine nucleotide regulatory protein, possibly Ni or No. Calcium inhibition of PTH release is blocked in cells treated with pertussis toxin, and there is concomitant ADP-ribosylation of a 40-kilodalton protein. The ionophore A23187 inhibits secretion in toxin-treated as well as in control cells. We conclude that a guanine nucleotide regulatory protein is involved in calcium regulation of PTH secretion at a locus proximal to the intracellular site effecting inhibition of secretion.

Prostaglandin F2 alpha and alpha-adrenergic agonists regulate parathyroid cell function via the inhibitory guanine nucleotide regulatory protein.

Prostaglandin F2 alpha (PGF2 alpha) and alpha-adrenergic agonists inhibit cAMP production and PTH secretion in dispersed bovine parathyroid cells. We have tested the mechanism of these effects utilizing pertussis toxin which catalyzes ADP ribosylation and inactivation of the inhibitory adenylate cyclase coupling protein Ni. Dispersed bovine parathyroid cells treated with or without 0.5 micrograms/ml pertussis toxin were tested with stimulatory (epinephrine, isoproterenol) or inhibitory (PGF2 alpha) agonists for responses in cAMP accumulation (5-min incubation) or PTH (90-min incubation) release. Pertussis toxin produced an enhanced response to epinephrine (a mixed alpha-adrenergic and beta-adrenergic agonist) in cAMP production and in PTH secretion. PGF2 alpha inhibited intracellular cAMP by 40% and PTH secretion by 35%. Pertussis toxin treatment of bovine parathyroid cells reduced the PGF2 alpha inhibition. We conclude that: 1) inhibition of PTH release by PGF2 alpha and alpha-adrenergic agonists parallels inhibition of cAMP production; 2) pertussis toxin blocks the inhibitory actions of PGF2 alpha and alpha-adrenergic agents on cAMP accumulation and PTH secretion; 3) the inhibitory coupling protein Ni mediates the inhibitory effects of these agents.

Binding of radioiodinated parathyroid hormone to cloned bone cells.

We have investigated the binding of biologically active radioiodinated bovine PTH-(1-84) to cloned osteoblast-like rat osteosarcoma cells (ROS 17/2.8) and correlated binding with biological response assessed as cAMP production. The hormone was labeled with 125I on tyrosine-43 using a constant current microelectrolytic method which allows full retention of biological activity. At confluency, cells were removed from culture, and assays were carried out on intact cells in suspension. At 22 C, saturable binding reached equilibrium by 90 min. At that time, the apparent dissociation rate constant was 8 X 10(-8) min-1. At an earlier time, 10 min of incubation, the dissociation rate was twice that observed at equilibrium. Nonsaturable binding was 30-35% of the total binding. The dissociation constant derived from kinetic analysis was 41 nM and correlated with the half-maximal cAMP production at 36 nM. The results obtained suggest that cleavage amino-terminal to residue 43 is not required for binding to these bone-derived cells. Binding to receptors on the osteosarcoma cells was specific and reversible, and appeared biologically relevant, since it correlated closely with the biological response, cAMP production. The competitive inhibitor [Nle8,Nle18,Tyr34]bPTH-(3-34)amide showed the same apparent affinity in inhibiting receptor binding as in inhibiting cAMP production.

Inhibition of adenosine 3',5'-monophosphate accumulation and parathyroid hormone release by sodium nitroprusside.

Sodium nitroprusside effected a significant reduction in intracellular cAMP accumulation and parathyroid hormone release in dispersed bovine parathyroid cells. The inhibition was apparent at 3 x 10-4 M and maximal at 10-2 M nitroprusside. The effect was rapid and reversible and could be demonstrated in both the presence and absence of stimulating agonists [i.e. (-)isoproterenol, dopamine, and cholera toxin]. The inhibition was additive with that previously described for alpha-adrenergic agonists and prostaglandin F2 alpha and was not affected by phentolamine, suggesting that nitroprusside does not act through the inhibitory receptors previously described in this system. The nitroprusside effect on cAMP accumulation and parathyroid hormone release was present at virtually all concentrations of extracellular calcium tested; 2mM EGTA failed to prevent the inhibition. While extracellular calcium may play some role in this inhibition, it is not required for demonstration of the effect.

Direct determination of ligand interactions with beta-adrenergic receptors on intact turkey erythrocytes: correlation of binding with biological activity.

Previous studies on the interaction of labeled beta-adrenergic blockers with beta-adrenergic receptors have employed broken cell or membrane preparations. We have now carried out direct binding analysis on intact turkey erythrocytes employing the potent, high specific activity blocker [125I]-hydroxbenzylpindolol (HYP). [125I]HYP binds to a single class of receptor sites with a K of 5.3 X 10(10)M-1 and a binding capacity of 400-500 sites/cell. These results as well as the kinetics of association and dissociation and lack of evidence for negative cooperativity all agree well with studies reported earlier on membrane preparations from the same cells. True dissociation constants (Kd) for agonists and antagonists determined by inhibition of binding of [125I]HYP are in good agreement with results in membrane preparations. These Kd's have been compared directly with activation or inhibition constants for effects on adenylate cyclase using generation of [14C]cAMP from [14C]adenine in intact cells. The close correlation between effects on binding and adenylate cyclase activity in whole cells are similar to results obtained in membrane preparations in the presence of guanine nucleotides, suggesting the presence of an analogous regulatory substance in vivo.