Pseudohypoparathyroidism type Ia from maternal but not paternal transmission of a Gsalpha gene mutation.

While loss-of-function mutations in Gsalpha are invariably associated with the short stature and brachydactyly of Albright hereditary osteodystrophy (AHO), the association with hormone resistance (to parathyroid hormone and thyrotropin) typical of pseudohypoparathyroidism type Ia (PHP-Ia) is much more variable. Observational studies and DNA polymorphism analysis suggest that maternal transmission of the Gsalpha mutation may be required for full expression of clinical hormone resistance. To test this hypothesis, we studied transmission of a frameshift mutation in Gsalpha through three generations of a pedigree affected by AHO and PHP-Ia. While all family members carrying this loss-of-function mutation in one Gsalpha allele had AHO, neither the presence of the mutation nor the degree of reduction of erythrocyte Gsalpha bioactivity allowed prediction of phenotype (AHO alone versus AHO and PHP-Ia). Paternal transmission of the mutation (from the patriarch of the first generation to three members of the second generation) was not associated with concurrent PHP-Ia, but maternal transmission (from two women in the second generation to four children in the third generation) was invariably associated with PHP-Ia. No expansion of an upstream short CCG nucleotide repeat region was detected, nor was there evidence of uniparental disomy by polymorphism analysis. This report, the first to document the effects across three generations of both paternal and maternal transmission of a specific Gsalpha mutation, strongly supports the hypothesis that a maternal factor determines full expression of Gsalpha dysfunction as PHP-Ia.

Desensitization of vascular tissue to parathyroid hormone and parathyroid hormone-related protein.

Although PTH and PTH-related protein (PTHrP) are vasodilators, prolonged exposure to elevated levels of PTH is often associated with hypertension. We investigated the effects of prolonged incubation with PTH or PTHrP on arterial segments and cultured vascular smooth muscle cells (VSMC). PTH or PTHrP transiently relaxed precontracted arterial segments within 10 min. Additional PTH or PTHrP added after 40-min exposure to these peptides had little effect on vascular tone, whereas forskolin, isoproterenol, isobutylmethyl-xanthine, or acetylcholine were still potent. In fura 2-loaded VSMC, 5-min incubation with PTH or PTHrP attenuated angiotensin II (Ang II)-induced calcium mobilization, an effect that was reduced by preincubation of VSMC with PTH for 1.5 h. Similarly, 1.5-h preincubation with PTH or PTHrP decreased the cAMP response to these peptides but not to forskolin or NaF. Ang II potentiated the cAMP response to PTH and PTHrP but was also subject to desensitization. Nle8, 18Tyr34 bovine PTH(3-34) amide did not desensitize vascular tissue to PTH or PTHrP. Our results suggest that homologous desensitization to PTH or PTHrP in vascular tissue requires receptor stimulation, occurs proximal to G stimulatory protein, and impairs attenuation of calcium mobilization by PTH or PTHrP. This may be a mechanism by which vasodilator effects of these peptides are decreased with prolonged elevation of PTH levels.

Parathyroid hormone analogues inhibit calcium mobilization in cultured vascular cells.

Parathyroid hormone and parathyroid hormone-related protein lower blood pressure and relax contracted arteries. Parathyroid hormone also attenuates angiotensin II-induced vasoconstriction. To determine the cellular mechanism or mechanisms by which parathyroid hormone analogues antagonize pressor effects, we examined the effect of these peptides on angiotensin II-induced calcium mobilization in fura 2-AM-loaded cultured rat vascular smooth muscle cells. Either 100 nmol/L parathyroid hormone or parathyroid hormone-related protein significantly reduced the amount of calcium mobilized by 100 nmol/L angiotensin II. The attenuating effect of these peptides was mimicked by 10 mmol/L forskolin and 10 mmol/L isobutylmethylxanthine and was not dependent on the presence of extracellular calcium. This effect of the parathyroid hormone analogues was reduced when cells were pretreated with 100 mmol/L 2',5'-dideoxyadenosine, an adenylate cyclase inhibitor. Combined inhibition of cyclic nucleotide-dependent protein kinases eliminated the inhibitory effect of parathyroid hormone, whereas protein kinase C inhibition had no effect. Parathyroid hormone analogues decreased the amount of calcium released by inositol 1,4,5-trisphosphate in digitonin-permeabilized vascular smooth muscle cells. This effect was inhibited by treatment with 2',5'-dideoxyadenosine. These results suggest that these peptides attenuate inositol 1,4,5-trisphosphate-sensitive calcium mobilized by angiotensin II via an adenylate cyclase-dependent mechanism. This may be a mechanism by which acute administration of parathyroid hormone or parathyroid hormone-related peptide antagonizes vasoconstriction.

Parathyroid localization, three-dimensional modeling, and percutaneous ablation techniques.

When available, state of the art noninvasive localization studies should be utilized routinely in previously unexplored patients for localizing parathyroid pathology, even when exceptional surgical experience exists. These studies can both minimize the 3 to 20% incidence of missed pathology and promote an approach of limited neck exploration with consequent lowering of morbidity, complications, and costs. Choice of imaging modalities for localizing these small masses is largely dependent on the level of state of the art of available equipment, the interest and experience of the performing physicians, and the attention to technical detail for each of the modalities at an individual institution. In choosing a single test, CT, and most recently cine CT with three-dimensional modeling, is favored because of higher probability of providing the kinds of information most useful to the surgeon. This includes precise anatomic localization and identification of locations likely to be missed by the surgeon (such as mediastinum, deep neck) and the capability for predicting multiple gland disease, for detecting smaller lesions, and for lower incidence of false-positive results. Ultrasound is attractive because of the low cost and noninvasiveness, and it is particularly sensitive in the thyroid region and upper neck. In difficult cases, CT, cine CT, and ultrasound may be augmented by needle aspiration of fluid for PTH assay. Thallium-technetium scanning and MRI are useful alternatives. In the previously explored patient and in patients with difficult diagnostic problems (such as ectopic adenoma, parathyroid carcinoma), the use of multiple noninvasive studies is strongly recommended, preferably CT (particularly, cine CT with three-dimensional imaging) and isotope scanning or MRI. The concurrence of two or more of these studies has a relatively high predictive value (82 to 88%) for localization. However, highly selective venous catheterization and selective magnification arteriography remain the most accurate modalities in these patients (91 to 95% sensitivity with few false-positive results) and may be combined with interventional radiologic techniques for tumor ablation in selected patients without compromising subsequent surgical alternatives. Stereotactic ablation techniques are in development.

Decreased vascular reactivity to norepinephrine in Fischer rats with neoplasia-induced hypercalcemia.

The effect of a hypercalcemia-producing Leydig cell tumor on vascular reactivity in Fischer rats was studied. Seven to eight days after tumor implantation, there was no difference between tumor (T) and control (C) animals in serum calcium, serum phosphate, plasma catecholamine levels, mean arterial pressure (MAP), or blood pressure responses to norepinephrine (NE) infusion. At day 12-13 of tumor growth, the serum calcium in the tumor-bearing rats was significantly higher (12.2 +/- 0.8 vs. 9.7 +/- 0.3 mg%, P less than .01) and their serum phosphate significantly lower (4.5 +/- 0.3 vs. 5.7 +/- 0.4 mg%, P less than .01) than controls. Plasma epinephrine (E) (497 +/- 154 vs. 62 +/- 13 pg/ml, P less than .05), and norepinephrine (NE) (686 +/- 85 vs. 329 +/- 75 pg/ml, P less than .01) were markedly elevated in the tumor rats. MAP and the blood pressure responses to graded NE infusions were significantly lower in tumor animals at Day 12-13, whereas there was no change in sensitivity to angiotensin II (AII) infusions. In vitro contractile responses of tail artery segments to transmural nerve stimulation (TNS) in animals with tumors were lower than in controls but there were no differences in sensitivity to exogenous NE in vitro. These results suggest that the tumor stimulates production of a circulating factor which desensitizes NE receptors and that this tumor also decreases neurovascular function by an undefined mechanism.

A systematic approach to parathyroid surgery.

In the last 8 years 33 patients with hyperparathyroidism have been surgically treated by the authors. Thirty patients had primary, 2 secondary and 1 tertiary hyperparathyroidism. In the 30 patients with the primary disorder, 26 had a single adenoma and 28 of these patients had normal calcium levels postoperatively. Two patients were hypocalcaemic following surgery and required calcium supplements for 3 months and 9 months respectively. It is recommended that when a single adenoma is found its removal will render the patient normocalcaemic. When all 4 glands are hyperplastic the surgery should be subtotal (3 1/2 glands) parathyroidectomy except in the case of secondary hyperparathyroidism when total parathyroidectomy with autotransplantation and cryopreservation of the remainder should be performed.

Responses to glucagon infusion in pseudohypoparathyroidism.

Single or graded doses of glucagon (Eli Lilly) were given to patients with pseudohypoparathyroidism (PsHP) type I to examine the possible presence of hormone resistance. The doses of glucagon ranged from 0.25-15 micrograms/kg. The following individuals were studied: 13 normal subjects, 5 patients with low erythrocyte N-protein activity (PsHP type Ia), and 7 patients with normal erythrocyte N-protein activity (PsHP type Ib). Two additional patients with treated primary hypothyroidism who were relatives of a patient with PsHP type Ib were also studied. The patients with PsHP type Ia had blunted plasma cAMP responses to all glucagon doses. In contrast, the patients with PsHP type Ib had normal cAMP responses to glucagon infusion. However, the 2 relatives of the patient with PsHP type Ib had clearly decreased cAMP responses to glucagon infusion; both had normal renal responses to PTH and were clinically and biochemically euthyroid at the time of study. Glucose responses to glucagon were normal in both PsHP groups; the glucose response per unit cAMP response was slightly, but not significantly, enhanced in PsHP type Ia patients. Glucagon resistance appears to be a common finding in patients with PsHP type Ia, but not in those with PsHP type Ib. However, the observation of reduced glucagon responsivity in association with familial hypothyroidism in a kindred with PsHP type Ib suggests the possibility that this disorder may also cause disturbances in several hormone systems.

Sodium transport in red blood cells from dialyzed uremic patients.

Studies on red blood cell (RBC) sodium (Na) transport in chronic renal failure have described abnormalities in the ouabain-sensitive Na, K pump. We now report Na transport in RBC using cation flux methodology, measuring both the ouabain-sensitive Na, K pump and the ouabain-insensitive Na, K cotransport (CoT) and Na, lithium (Li) countertransport (CTT) in 28 subjects on hemodialysis, eight subjects on chronic ambulatory peritoneal dialysis (CAPD) and 29 control subjects. Intracellular cation content and passive permeability of Na were also examined. Mean Na efflux through the ouabain-sensitive Na, K pump was not reduced in dialysis patients when compared to normal subjects, whether measured in fresh cells (1.41 +/- 0.05 vs. 1.30 +/- 0.03 mmole/liter RBC/hr; P less than 0.05) or in Na-loaded cells (7.10 +/- 0.24 vs. 6.90 +/- 0.22; NS). There was, however, a marked and uniform suppression of the CoT pathway in Na-loaded cells from dialysis patients versus controls (0.14 +/- 0.02 vs. 0.41 +/- 0.05 mmole/liter RBC/hr; P less than 0.001). Mean CTT activity, as measured by Li efflux, was not different between dialysis and normal subjects. Uremic and normal RBC had similar intracellular Na or K content as well as passive permeability for either ion. This indicates that intracellular cationic homeostasis is maintained, perhaps secondary to balanced changes in cationic flux activity through these transport pathways.

Hypercalcemia in rats bearing growth hormone- and prolactin-secreting transplantable pituitary tumors.

We evaluated the effects of chronic massive elevations of serum GH and PRL on calcium metabolism in rats bearing the MStT/W15 and 7315a transplantable pituitary tumors. MStT/W15 tumor rats manifest elevated serum GH and PRL levels, hypercalcemia, hypercalciuria, and elevated serum levels of PTH and 1,25-dihydroxyvitamin D. The hypercalcemia was not reversed by dexamethasone or propranolol treatment, but was ameliorated by starvation. Parathyroidectomy produced hypocalcemia in the MStT/W15 tumor rats, confirming the parathyroid dependence of the hypercalcemia. The 7315a tumor produced a milder degree of hypercalcemia, along with elevated serum levels of PRL, ACTH, and corticosterone; serum GH was normal. In high concentrations, PRL and/or GH may stimulate the secretion of PTH as well as enhance dietary calcium absorption, in part through the mediation of 1,25-dihydroxyvitamin D.

Normal free fatty acid response to isoproterenol in pseudohypoparathyroidism.

The lipolytic response to isoproterenol infusion was examined in seven normal subjects and six patients with pseudohypoparathyroidism type I [two had deficiency of the hormone receptor-cyclase coupling protein (N-protein) and four did not]. Despite blunted plasma cAMP responses to isoproterenol in both subgroups of pseudohypoparathyroidism patients, the serum FFA responses were normal. We conclude that changes in plasma cAMP do not reflect the adequacy of the lipolytic response to isoproterenol.

Blunted plasma cyclic adenosine monophosphate response to isoproterenol in pseudohypoparathyroidism.

Plasma cAMP, serum glucose, and cardiovascular responses to isoproterenol infusion were examined in seven normal subjects and seven patients with pseudohypoparathyroidism (PHP), type I (three with deficiency of the hormone receptor-cyclase coupling protein (N-protein) and four who were N-protein replete). Although plasma cAMP responses were blunted in both subgroups of PHP patients, the cardiovascular and glucose responses to isoproterenol were normal. We conclude that the blunted plasma cAMP response to isoproterenol in PHP does not depend on deficient N-protein coupling of adenylate cyclase to the beta-adrenergic receptor and that the cardiovascular and glucose responses to isoproterenol are not reflected in changes in plasma cAMP.

Preliminary trials with 24,25-dihydroxyvitamin D3 in dialysis osteomalacia.

Fifteen patients with dialysis osteomalacia were treated with 24,25-dihydroxyvitamin D3 in dosages up to 10 micrograms per day for two to 24 months. All had previously had no improvement during treatment with calcitriol but had been remarkably susceptible to hypercalcemia. When 24,25-dihydroxyvitamin D3 was given with either calcitriol or dihydrotachysterol, serum calcium levels were significantly lower than during treatment with calcitriol or dihydrotachysterol alone. Eight of nine patients who received combined therapy with 24,25-dihydroxyvitamin D3 and calcitriol for longer than two months had clinical improvement; six patients underwent repeated bone biopsy and showed evidence of improved bone mineralization. Patients who received 24,25-dihydroxyvitamin D3 alone did not improve clinically. Since 24,25-dihydroxyvitamin D3 appears to improve calcium homeostasis and bone mineralization in some patients with severe dialysis osteomalacia when administered with 1-hydroxylated vitamin D metabolites, further controlled studies are warranted.

Platelets of pseudohypoparathyroid patients: evidence that distinct receptor-cyclase coupling proteins mediate stimulation and inhibition of adenylate cyclase.

We studied platelets of patients with the genetic disorder pseudohypoparathyroidism (PHP) to test whether the nucleotide-binding proteins mediating stimulation of adenylate cyclase (termed N(s)) are identical to those mediating inhibition of cyclase (termed N(i)). Functional responses to hormones that work through stimulation of adenylate cyclase are blunted in PHP patients. The erythrocytes of many of these patients (PHP-Ia) have previously been shown to have decreased N(s) activity whereas those of other PHP patients (PHP-Ib) have normal N(s) activity. We find that this decreased N(s) activity (measured by the ability to restore adenylate cyclase activity to membranes prepared from S49 cyc(-) cells) also occurs in the platelets of PHP-Ia but not of PHP-Ib patients. Platelets from both groups of patients accumulate less cAMP in response to prostacyclin than do platelets from control subjects. In contrast to the decreased N(s) function in patients with PHP-Ia, we find that N(i) function in platelets is similar in these patients and control subjects in several types of experiments: (i) epinephrine-mediated inhibition of prostacyclin-stimulated cAMP production in intact platelets; (ii) the affinity of platelet alpha(2)-adrenergic receptors for epinephrine, as determined by competition for [(3)H]yohimbine binding; (iii) the decrease in receptor affinity for epinephrine produced by Na(+) and GTP; and (iv) the concentration dependence of GTP for decreasing the affinity of these receptors for epinephrine. Because N(i) is expressed normally in platelets from patients that are genetically deficient in N(s), we conclude that N(s) and N(i) are likely to be distinct gene products.

Deficient activity of receptor-cyclase coupling protein is transformed lymphoblasts of patients with pseudohypoparathyroidism, type I.

Erythrocytes of many patients with pseudohypoparathyroidism, type 1 (PHP-I) exhibit quantitatively reduced activity of the N protein, the guanine nucleotide-binding regulatory component of adenylate cyclase. We have designated this group of patients PHP-Ia to distinguish them from PHP-Ib patients, in whom erythrocyte N activity is quantitatively normal. In virus-transformed lymphoblasts of three normal, three PHP-Ia, and two PHP-Ib subjects, we compared N and adenylate cyclase activities, as well as cAMP accumulation and susceptibility to radiolabeling in the presence of [32P]NAD and cholera toxin. In comparison to normal lymphoblasts, N activities were reduced by approximately 50% in lymphoblasts of the PHP-Ia patients, but were not reduced in lymphoblasts from the PHP-Ib patients. Toxin-catalyzed radiolabeling of the 42,000 molecular weight subunit of the N protein was also reduced in lymphoblasts of the PHP-Ia patients. These results are consistent with the hypothesis that N deficiency is generalized in tissues of PHP-Ia patients. Deficient lymphoblast N activity in PHP-Ia was not associated with decreases in adenylate cyclase activity or cAMP accumulation, probably because these activities involve many potential regulable cellular components in addition to the N protein.

Hypercalcemia in association with a Leydig cell tumor in the rat: a model for tumor-induced hypercalcemia in man.

The etiology of tumor-induced hypercalcemia was investigated in a transplantable Leydig cell tumor of the Fischer rat. In this model, serum calcium rose from a baseline of 10.4 +/0 0.3 mg/dl to 12.5 + 0.4 mg/dl at day 10 and 16.4 +/- 1.3 mg/dl (p less than 0.001) at day 13 post transplant. Urinary calcium also increased from 1.52 +/- 0.17 mg/d to 3.52 + 0.72 mg/d (Day 12, p less than 0.01). Serum phosphate decreased from a baseline of 7.5 +/- 0.3 mg/dl to 5.5 +/- 0.6 mg/dl at day 13 (p less than 0.05). At day 13 serum immunoreactive parathyroid hormone levels fell 76% from baseline (p less than 0.01). Calcitonin increased from 59 +/- 2 pg/ml to 88 +/- 9 pg/ml (p less than 0.02). The plasma prostaglandin E metabolite, 13,14-dihydro-15-keto-PGE2 increased from 407 +/- 103 pg/ml to 647 +/-62 pg/ml (p less than 0.05) and the active Vit D compound 1,25(OH)2D increased from 94.8 +/- 5.2 pg/ml to 162.3 +/- 11.8 pg/ml (p less than 0.01). Urinary cyclic AMP did not decrease in parallel with the parathyroid hormone level and, in fact, increased from 146 +/- 3 nmol/d to 172 +/- 27 nmol/d (NS). Administration of the cyclooxygenase inhibitor indomethacin (20 mg/Kg/d) or hydrocortisone (50 mg/Kg/d) did not prevent the development of hypercalcemia. This model is similar to many patients with humoral hypercalcemia of malignancy who demonstrate suppression of parathyroid hormone with elevated urinary cyclic AMP excretion and may prove useful in the understanding of the responsible mechanisms.

Bone aluminum and histomorphometric features of renal osteodystrophy.

To evaluate the relationship between aluminum and the characteristics of bone disease in uremia, bone aluminum content and quantitative histomorphometric analysis of bone were evaluated in bone biopsies from 59 uremic patients undergoing maintenance hemodialysis. Biopsies were classified as showing 1) pure osteomalacia (OM) in 23 cases, 2) osteitis fibrosa (OF) in 13, 3) mixed in 7, and 4) mild lesions in 16. There were no significant differences in levels of serum calcium or alkaline phosphatase between the groups, but serum phosphorus levels were slightly higher in those with OF. Serum immunoreactive parathyroid hormone levels were greater in the patients with OF and mixed lesions than in patients with OM or mild lesions (P less than 0.01). Bone aluminum exceeded normal in all groups (P less than 0.01), with values of 175 +/- 18 mg/kg dry wt in OM patients, 46 +/- 7 of OF patients, 81 +/- 29 in mixed subjects, and 67 +/- 7 in patients with mild lesions. Bone aluminum was significantly higher in the OM patients than in any other group (P less than 0.01); also, bone aluminum correlated with the quantitative measure of unmineralized osteoid in OM (r = 0.67; P less than 0.001); no correlations existed for the other groups. There were inverse correlations between bone aluminum and the serum immunoreactive parathyroid hormone (r = -0.35; P less than 0.01) and resorbing surface on biopsy (r = -0.44; P less than 0.001). Bone aluminum correlated with the duration of hemodialysis in patients with OF with mixed and mild lesions (r = 0.49); no relation was seen in OM patients, and bone aluminum was higher for the duration of dialysis, suggesting that aluminum may accumulate more rapidly in OM subjects. These findings are consistent with but do not prove the hypothesis that aluminum plays a pathogenic role in dialysis osteomalacia; the mechanism by which aluminum accumulates remains unknown.

Fibroblast defect in pseudohypoparathyroidism, type I: reduced activity of receptor-cyclase coupling protein.

Erythrocytes of many patients with pseudohypoparathyroidism, type I (PHP-I), exhibit reduced activity of the N protein, a guanine nucleotide-binding regulatory component of hormone-sensitive adenylate cyclase. We compared N and adenylate cyclase activities and the accumulation of cAMP in fibroblasts propagated from skin biopsies of six normal subjects and seven PHP-I patients. N activities were reduced by approximately 40% in fibroblasts as well as erythrocytes of five PHP-I patients. N activities in fibroblasts from two PHP-I patients with normal erythrocyte N activities were within the normal range. These results are consistent with the hypothesis that N deficiency is generalized in tissues of most PHP-I patients and is the primary defect responsible for their resistance to metabolic effects of hormones that work by stimulating adenylate cyclase. Fibroblast N deficiency was not associated with decreases in hormone-stimulated adenylate cyclase or cAMP accumulation in fibroblasts, probably because these activities involve many potentially regulable cellular components in addition to the N protein.

Vitamin-D-resistant osteomalacia in hemodialysis patients lacking secondary hyperparathyroidism.

We describe a sporadic, vitamin-D-resistant osteomalacic syndrome in 19 patients undergoing hemodialysis. The syndrome was found in less than 1.5% of patients from referring dialysis centers. All 19 patients had multiple fractures, severe myopathy, and many developed spontaneous hypercalcemia. Severe osteomalacia without evidence of secondary hyperparathyroidism distinguished this syndrome from other forms of renal osteodystrophy. Bone aluminum, measured in six patients, was greatly elevated. Therapy with calcitriol (1 alpha, 25-dihydroxycholecalciferol) lad to clinical improvement in seven patients with reduced pain and myopathy, decreased serum alkaline phosphatase, or both, but no improvement in bone histology. Patients who did not respond clinically to calcitriol developed marked hypercalcemia. The cause of this severe osteomalacia, which occurs despite normal or slightly elevated levels of serum calcium and phosphorus and fails to mineralize with calcitriol, is unclear.

Pseudohypoparathyroidism: inheritance of deficient receptor-cyclase coupling activity.

Pseudohypoparathyroidism, type I (PHP-I) is an inherited disorder of primary resistance to multiple hormones that work by stimulating adenylate cyclase. In an attempt to clarify the mode of inheritance of PHP-I, we measured the activity of the N protein, a receptor-cyclase coupling component, in erythrocyte membranes. Erythrocyte N-protein activity was reduced by approximately 50% in erythrocytes of 15 PHP-I patients and was normal in 19 of their clinically normal first degree relatives. Reduced N-protein activity and the PHP-I phenotype in these families exhibited both dominant and recessive patterns of inheritance. This suggests that at least two distinct genetic loci are involved in inheritance of N-protein deficiency. In two additional families, dominant inheritance of the PHP-I phenotype was associated with normal activities of erythrocyte N protein. Thus, it appears that mutation of at least one additional genetic locus, not involving the N protein, can produce PHP-I.