Dose-related influence of indomethacin on parathyroid hormone-stimulated adenosine 3',5'-monophosphate release from the perfused rat hindlimb.

We examined the effect of indomethacin (INDO) on PTH-stimulated cAMP release from the perfused rat hindlimb. Since this preparation has not been used previously to study the effects of PTH, we first determined the dose-response curve for cAMP release in response to the 1-34 fragment of synthetic bovine PTH. cAMP release peaked 3-6 min after the PTH bolus and declined gradually toward baseline, even with sustained PTH infusion. The rate of cAMP release was directly related to the PTH dose. The lowest PTH priming dose that provoked a significant increase in cAMP release was 0.6 IU. Maximal cAMP release, occurring in response to a PTH priming dose of 30 IU, was 3- to 4-fold greater than baseline. PTH caused no increase in cAMP release from or the cAMP content of isolated skeletal muscle in vitro, suggesting that cAMP released from the hindlimb in response to PTH is derived solely from bone. PTH-stimulated cAMP release was unaltered by pretreatment of the intact rat with 2 mg/kg INDO, a dose that blocks prostaglandin synthesis. In contrast, PTH-stimulated cAMP release was significantly attenuated by pretreatment with 75 mg/kg INDO. The effect was not dependent on the addition of drug to the perfusate and was not altered by thyroparathyroidectomy at the time of INDO administration. We conclude that 1) the perfused rat hindlimb can be used to examine PTH effects on bone; 2) 2 mg/kg INDO has no effect on PTH-stimulated cAMP release from the perfused rat hindlimb; and 3) INDO in high doses blunts PTH activation of adenylate cyclase.

Neurotransmitter action in osteoblasts: expression of a functional system for serotonin receptor activation and reuptake.

Neurotransmitter regulation of bone metabolism has been the subject of increasing interest and investigation. Because serotonin (5-HT) plays a role as a regulator of craniofacial morphogenesis, we investigated the expression and function of 5-HT receptors and the 5-HT transporter (5-HTT) in bone. Primary cultures of rat osteoblasts (rOB) and a variety of clonal osteoblastic cell lines, including ROS 17/2.8, UMR 106-H5, and Py1a, showed mRNA expression for 5-HTT as well as the 5-HT(1A), 5-HT(1D), 5-HT(2A), and 5-HT(2B) receptors by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Protein expression of the 5-HT(1A), 5-HT(2A), and 5-HT(2B) receptors was confirmed by immunoblot. 5-HTT binding sites were assessed in ROS 17/2.8 and UMR 106-H5 cells by binding of the stable cocaine analog [125I]RTI-55, which showed a relatively high density of nanomolar affinity binding sites. Imipramine and fluoxetine, antagonists with specificity for 5-HTT, showed the highest potency to antagonize [125I]RTI-55 binding in ROS and UMR cells. GBR-12935, a relatively selective dopamine transporter antagonist, had a much lower potency, as did desipramine, a selective norepinephrine transporter antagonist. The maximal [3H]5-HT uptake rate in ROS cells was 110 pmol/10 min per well, with a K(m) value of 1.13 micromol/L. Imipramine and fluoxetine inhibited specific [3H]5-HT uptake with IC(50) values in the nanomolar range. In normal differentiating rOB cultures, 5-HTT functional activity was observed initially at day 25, and activity increased almost eightfold by day 31. In mature rOB cultures, the estimated density of [125I]RTI-55 binding sites was 600 fmol/mg protein. Functional downregulation of transporter activity was assessed after PMA treatment, which caused a significant 40% reduction in the maximal uptake rate of [3H]5-HT, an effect that was prevented by pretreatment with staurosporine. The affinity of 5-HT for the transporter was significantly increased following PMA treatment. We assessed the functional significance of expression of the 5-HT receptors by investigating the interaction between 5-HT and parathyroid hormone (PTH) signaling. 5-HT potentiates the PTH-induced increase in AP-1 activity in UMR cells. These results demonstrate that osteoblastic cells express a functional serotonin system, with mechanisms for responding to and regulating uptake of 5-HT.

Effect of sulfonylurea therapy and plasma glucose levels on hemoglobin A1c in type II diabetes mellitus.

It is well known that hemoglobin A1c reflects plasma glucose concentrations in patients with diabetes mellitus. To examine hemoglobin A1c and plasma glucose relationships in sulfonylurea-treated patients, 25 patients with well-controlled type II diabetes (fasting plasma glucose 128 +/- 6 mg/dl, hemoglobin A1c 7.6 +/- 0.5 percent) were evaluated in a double-blind study. This study was divided into two phases (periods I and II). During period I each patient was given a diet plus a placebo and was followed every two weeks until the mean of two consecutive plasma glucose determinations was more than 50 mg/dl above the initial plasma glucose concentration obtained while the patient was taking sulfonylurea. At that point each patient was switched in a double-blind fashion to either diet plus a placebo or diet plus tolazamide. Fasting plasma glucose concentrations increased to 178 +/- 9 mg/dl (p less than 0.005) for all patients by week 2 of period I. The increase in hemoglobin A1c concentration was seen to lag behind the increasing fasting plasma glucose concentration by four to six weeks. Fasting plasma glucose and hemoglobin A1c concentrations returned to values indistinguishable from initial values in patients who were given tolazamide and who responded to it. A positive correlation was noted when the hemoglobin A1c concentration was compared with the fasting plasma glucose concentration measured four to six weeks previously.

Vitamin D insufficiency in internal medicine residents.

Medical residents may be vulnerable to low vitamin D status because of long work hours and lack of sun exposure. We conducted a prospective cohort study to measure serum 25-hydroxyvitamin D concentrations among internal medicine residents, document seasonal variation in vitamin D status, and assess risk factors for inadequate vitamin D stores. Dietary intake of calcium and vitamin D, lifestyle characteristics, and serum concentrations of 25(OH)-vitamin D and intact parathyroid hormone (iPTH) were measured in 35 resident volunteers before and after the winter season. A total of 63-69% of medical residents consumed <400 IU/day of vitamin D; 61-67% consumed <1000 mg/day of calcium. Twenty-five (74%) had lower serum 25(OH)-vitamin D concentrations and 23 (68%) had higher serum iPTH in the spring than in the fall. Nine (26%) residents had serum concentrations of 25(OH)-vitamin D of <20 ng/mL in the fall; and sixteen (47%) in the spring. Seven residents (20%) had serum concentrations of 25(OH)-vitamin D of <20 ng/mL at both time-periods; Eighteen residents (51.4%) had 25(OH)-vitamin D levels of <20 ng/mL for at least one of the time-periods. Medical residents are at risk for hypovitaminosis D, particularly during the winter months and should be aware of the need to supplement their vitamin D stores. Insufficient vitamin D status and inadequate vitamin D intake may have long-term implications for bone health in these individuals. Increased educational efforts to promote healthy dietary and lifestyle choices that allow attainment and maintenance of skeletal health are appropriate in this population.

Analysis of the relation between alopecia and resistance to 1,25-dihydroxyvitamin D.

Alopecia is a frequent feature in hereditary resistance to (1,25(OH)2D). We sought insight into this feature by analysing data from affected members of 30 kindreds. We assessed indices of mineral metabolism in one group with normal hair compared with a group with alopecia. Hereditary resistance to 1,25(OH)2D was diagnosed at an earlier age in alopecic patients (0.9 vs 3.3 years, P less than 0.05); this reflected late presentation of metabolic bone disease in some cases with normal hair and could not be attributed to early diagnosis resulting from the striking feature of alopecia. For untreated subjects, serum concentrations of calcium and 1,25(OH)2D were similar in both groups of patients. During calciferol therapy, however, the cases with alopecia showed lower serum calcium (1.9 vs 2.4 mmol/l, P less than 0.005), but higher serum 1,25(OH)2D (2900 v 340 pg/ml, P less than 0.005). Hair status did not predict the type of defect identified with cultured skin fibroblasts but did correlate with responsiveness of 25(OH)D 24-hydroxylase to 1,25(OH)2D3 in those cells. Cells from seven of eight kindreds with alopecia showed no 24-hydroxylase response to high doses of 1,25(OH)2D3 while cells from five of six kindreds with normal hair showed a 24-hydroxylase response to high doses of 1,25(OH)2D3. We conclude that in cases with hereditary resistance to 1,25(OH)2D alopecia reflects the more severe grades of this resistance based upon earlier age at time of diagnosis, lower potential for calcaemic response to calciferols, and lower potential for 24-hydroxylase response to 1,25(OH)2D3 by cultured skin fibroblasts.

Blood magnesium status of patients with parathyroid disease.

The blood magnesium status--plasma and red blood cell (RBC) concentration and mononuclear blood cell (MBC) content of magnesium and parathyroid hormone (PTH) were determined in 6 patients with chronic hypoparathyroidism and 9 patients with primary hyperparathyroidism before and after surgical cure of the disease. The magnesium content of MBCs and concentration of RBCs increased significantly (p less than 0.01) after the surgical correction of primary hyperparathyroidism. Patients with chronic hypoparathyroidism had significant increase (p less than 0.05) of magnesium in RBCs and MBCs compared with the control group. No significant difference was observed for the plasma magnesium concentration for hypoparathyroid patients compared with the control group and hyperparathyroid patients before and after surgical care. These results suggest that endogenous PTH affects the concentration of magnesium in RBCs and magnesium content in MBCs but does not alter significantly the concentration of magnesium in plasma.

Involvement of N-myristoylation in monoclonal antibody recognition sites on chimeric G protein alpha subunits.

Monoclonal antibody, LAS-2, directed against the alpha subunit of transducin (Gt alpha), inhibited Gt beta gamma-dependent, pertussis toxin-catalyzed ADP ribosylation of Gt alpha and was specific for Gt alpha. Immunoblotting studies on proteolytic fragments of Gt alpha were consistent with an amino-terminal epitope. To define the antibody recognition site, recombinant Gt alpha was synthesized in Escherichia coli cotransfected with or without yeast N-myristoyl-transferase. Amino-terminal fatty acylation of Gt alpha, verified by use of radiolabeled fatty acid, was required for immunoreactivity. LAS-2 did not react with a chimeric protein consisting of residues 1-9 of Gt alpha and the remainder Go alpha, regardless of its myristoylation. Immunoreactivity was observed when amino acids 1-17 of Gt alpha were present in a Go alpha chimera and the protein was amino-terminally myristoylated; there was no reactivity without myristoylation. It appears that the LAS-2 epitope requires both Gt alpha-specific sequence in amino acids 10-17 and a fatty acyl group in proximity to these residues. These results are consistent with the hypothesis that the myristoyl group is essential for protein structure; conceivably it "folds back" on and stabilizes the amino-terminal structure of Gt alpha as opposed to protruding from an amino-terminal alpha-helix and serving as an amino-terminal membrane anchor.

Prolonged effect of insulin on glucose uptake by rat skeletal muscle.

The fate of insulin, as it relates to its action on skeletal-muscle glucose uptake, was studied in non-cyclically perfused rat hindlimbs. Insulin (1m-i.u./ml) with and without (125)I-labelled insulin was infused intra-arterially for 5 or 6min. Net glucose uptake and the release of (125)I-labelled insulin into the venous effluent were evaluated by arteriovenous-difference measurements for an additional 24-32min. The infusion of insulin for 5min promoted glucose uptake, an effect that persisted throughout a subsequent 25min of perfusion in the absence of insulin. The addition of insulin antibody to the perfusate in the presence of insulin blocked the action of insulin on glucose uptake, but it failed to alter insulin action if the muscle tissue had been exposed to insulin before addition of antibody. When (125)I-labelled albumin was infused for 6min, venous effluent radioactivity decayed rapidly and remained HClO(4)-insoluble and there was no significant tissue retension of radioactivity. Comparable experiments in which (125)I-labelled insulin was infused for 6min revealed that the venous effluent radioactivity decayed more slowly, a significant amount of the (125)I-labelled insulin appeared as fragments (HClO(4)-soluble) and there was a significant retention of radioactivity in the tissue. Radioactivity in muscle tissue biopsies obtained 28min after infusion of (125)I-labelled insulin was associated largely with intact insulin and a peptide of mol.wt. 2400. The total radioactivity retained in the muscle at this time was 7% of the amount infused. An insulin bolus (1i.u.) failed to increase the discharge of this tissue-associated radioactivity. These results suggest that insulin and a product of insulin metabolism persists in muscle tissue long after the arterial presence of insulin ends. This tissue residence and processing of insulin may be important components of insulin's prolonged action on glucose uptake by skeletal muscle.

Perimolysis: unveiling the surreptitious vomiter.

Perimolysis is a dental condition linked to chronic regurgitation. When perimolysis is found in the patient who denies vomiting, one must suspect anorexia nervosa, a disorder with a high rate of morbidity and mortality. The dental literature has not provided guidelines for confirming the suspicion of surreptitious vomiting. The purpose of this case report is to describe our approach, using simple blood and urine studies, which establishes whether a patient who has perimolysis but denies vomiting is a surreptitious vomiter.

Mechanism of activation of cholera toxin by ADP-ribosylation factor (ARF): both low- and high-affinity interactions of ARF with guanine nucleotides promote toxin activation.

Activation of adenylyl cyclase by cholera toxin A subunit (CT-A) results from the ADP-ribosylation of the stimulatory guanine nucleotide binding protein (GS alpha). This process requires GTP and an endogenous guanine nucleotide binding protein known as ADP-ribosylation factor (ARF). One membrane (mARF) and two soluble forms (sARF I and sARF II) of ARF have been purified from bovine brain. Because the conditions reported to enhance the binding of guanine nucleotides by ARF differ from those observed to promote optimal activity, we sought to characterize the determinants influencing the functional interaction of guanine nucleotides with ARF. High-affinity GTP binding by sARF II (apparent KD of approximately 70 nM) required Mg2+, DMPC, and sodium cholate. sARF II, in DMPC/cholate, also enhanced CT-A ADP-ribosyltransferase activity (apparent EC50 for GTP of approximately 50 nM), although there was a delay before achievement of a maximal rate of sARF II stimulated toxin activity. The delay was abolished by incubation of sARF II with GTP at 30 degrees C before initiation of the assay. In contrast, a maximal rate of activation of toxin by sARF II, in 0.003% SDS, occurred without delay (apparent EC50 for GTP of approximately 5 microM). High-affinity GTP binding by sARF II was not detectable in SDS. Enhancement of CT-A ADP-ribosyltransferase activity by sARF II, therefore, can occur under conditions in which sARF II exhibits either a relatively low affinity or a relatively high affinity for GTP. The interaction of GTP with ARF under these conditions may reflect ways in which intracellular membrane and cytosolic environments modulate GTP-mediated activation of ARF.

Parathyroid mitogenic activity in plasma from patients with familial multiple endocrine neoplasia type 1.

Hyperplasia of the parathyroid glands is a central feature of familial multiple endocrine neoplasia type 1. We used cultured bovine parathyroid cells to test for mitogenic activity in plasma from patients with this disorder. Normal plasma stimulated [3H]thymidine incorporation, on the average, to the same extent as it was stimulated in a plasma-free control culture. This contrasted with the results of the tests with plasma from patients with familial multiple endocrine neoplasia type 1, in which parathyroid mitogenic activity increased 2400 percent over the control value (P less than 0.001). Plasma from these patients also stimulated the proliferation of bovine parathyroid cells in culture, whereas plasma from normal subjects inhibited it. Parathyroid mitogenic activity in plasma from the patients with familial multiple endocrine neoplasia type 1 was greater than that in plasma from patients with various other disorders, including sporadic primary hyperparathyroidism (with adenoma, hyperplasia, or cancer of the parathyroid), sporadic primary hypergastrinemia, sporadic pituitary tumor, familial hypocalciuric hypercalcemia, and multiple endocrine neoplasia type 2 (P less than 0.05). Parathyroid mitogenic activity in the plasma of patients with familial multiple endocrine neoplasia type 1 persisted for up to four years after total parathyroidectomy. The plasma also had far more mitogenic activity in cultures of parathyroid cells than did optimal concentrations of known growth factors or of any parathyroid secretagogue. This mitogenic activity had an apparent molecular weight of 50,000 to 55,000. We conclude that primary hyperparathyroidism in familial multiple endocrine neoplasia type 1 may have a humoral cause.