Autoimmune polyendocrine syndrome type 1: an Italian survey on 158 patients.

BACKGROUND: Autoimmune Polyglandular Syndrome type 1 (APS-1) is a rare recessive inherited disease, caused by AutoImmune Regulator (AIRE) gene mutations and characterized by three major manifestations: chronic mucocutaneous candidiasis (CMC), chronic hypoparathyroidism (CH) and Addison's disease (AD). METHODS: Autoimmune conditions and associated autoantibodies (Abs) were analyzed in 158 Italian patients (103 females and 55 males; F/M 1.9/1) at the onset and during a follow-up of 23.7 ± 15.1 years. AIRE mutations were determined. RESULTS: The prevalence of APS-1 was 2.6 cases/million (range 0.5-17 in different regions). At the onset 93% of patients presented with one or more components of the classical triad and 7% with other components. At the end of follow-up, 86.1% had CH, 77.2% AD, 74.7% CMC, 49.5% premature menopause, 29.7% autoimmune intestinal dysfunction, 27.8% autoimmune thyroid diseases, 25.9% autoimmune gastritis/pernicious anemia, 25.3% ectodermal dystrophy, 24% alopecia, 21.5% autoimmune hepatitis, 17% vitiligo, 13.3% cholelithiasis, 5.7% connective diseases, 4.4% asplenia, 2.5% celiac disease and 13.9% cancer. Overall, 991 diseases (6.3 diseases/patient) were found. Interferon-ω Abs (IFNωAbs) were positive in 91.1% of patients. Overall mortality was 14.6%. The AIRE mutation R139X was found in 21.3% of tested alleles, R257X in 11.8%, W78R in 11.4%, C322fsX372 in 8.8%, T16M in 6.2%, R203X in 4%, and A21V in 2.9%. Less frequent mutations were present in 12.9%, very rare in 9.6% while no mutations in 11% of the cases. CONCLUSIONS: In Italy, APS-1 is a rare disorder presenting with the three major manifestations and associated with different AIRE gene mutations. IFNωAbs are markers of APS-1 and other organ-specific autoantibodies are markers of clinical, subclinical or potential autoimmune conditions.

Metabolic syndrome and diabetes mellitus in childhood cancer survivors.

BACKGROUND: The survival of children with cancer has grown considerably in recent years resulting in a marked increase of endocrine complications. increasingly recognized problems are metabolic syndrome and diabetes mellitus. DATA SOURCES: We critically analysed the most recent literature about the prevalence and molecular mechanisms of metabolic dysregulation and long-term cardio-metabolic risk in this population. RESULTS: Hypothalamic irradiation determines growth hormone deficiency and hypogonadism; moreover it is able to disrupt the appetite regulating centre leading to hyperphagia and progressive obesity. These conditions determine an insulin resistant state, contributing to the development of metabolic syndrome and diabetes mellitus. Irradiation and/or chemotherapy may lead to an insulin secretory defect through a direct damage of pancreatic beta cells. CONCLUSION: Metabolic syndrome and diabetes mellitus represent increasingly recognized long-term complications of childhood cancer treatment. The different impact of insulin resistance and secretory defects on the onset and progression of metabolic syndrome and diabetes mellitus remains unclear.

Receptor binding mode and pharmacological characterization of a potent and selective dual CXCR1/CXCR2 non-competitive allosteric inhibitor.

BACKGROUND AND PURPOSE: DF 2156A is a new dual inhibitor of IL-8 receptors CXCR1 and CXCR2 with an optimal pharmacokinetic profile. We characterized its binding mode, molecular mechanism of action and selectivity, and evaluated its therapeutic potential. EXPERIMENTAL APPROACH: The binding mode, molecular mechanism of action and selectivity were investigated using chemotaxis of L1.2 transfectants and human leucocytes, in addition to radioligand and [(35) S]-GTPγS binding approaches. The therapeutic potential of DF 2156A was evaluated in acute (liver ischaemia and reperfusion) and chronic (sponge-induced angiogenesis) experimental models of inflammation. KEY RESULTS: A network of polar interactions stabilized by a direct ionic bond between DF 2156A and Lys(99) on CXCR1 and the non-conserved residue Asp(293) on CXCR2 are the key determinants of DF 2156A binding. DF 2156A acted as a non-competitive allosteric inhibitor blocking the signal transduction leading to chemotaxis without altering the binding affinity of natural ligands. DF 2156A effectively and selectively inhibited CXCR1/CXCR2-mediated chemotaxis of L1.2 transfectants and leucocytes. In a murine model of sponge-induced angiogenesis, DF 2156A reduced leucocyte influx, TNF-α production and neovessel formation. In vitro, DF 2156A prevented proliferation, migration and capillary-like organization of HUVECs in response to human IL-8. In a rat model of liver ischaemia and reperfusion (I/R) injury, DF 2156A decreased PMN and monocyte-macrophage infiltration and associated hepatocellular injury. CONCLUSION AND IMPLICATIONS: DF 2156A is a non-competitive allosteric inhibitor of both IL-8 receptors CXCR1 and CXCR2. It prevented experimental angiogenesis and hepatic I/R injury in vivo and, therefore, has therapeutic potential for acute and chronic inflammatory diseases.

Dumping syndrome: an unusual cause of severe hyperinsulinemic hypoglycemia in neurologically impaired children with gastrostomy.

This paper describes severe hyperinsulinemic hypoglycemia during bolus enteral feeding in two neurologically impaired children. Both children were affected by dysphagia with swallowing difficulties; caloric intake was inadequate. For these reasons, percutaneous endoscopic gastrostomy had been positioned during the first months of life. In one patient due to persisting vomiting, after a few months, a gastrojejunal tube (PEG-J) was inserted. Hypoglycemia was revealed by routine blood tests, without evidence of specific symptoms. Continuous subcutaneous glucose monitoring showed wide glucose excursions, ranging from hypoglycemia to hyperglycemia. Extremely high levels of insulin were detected at the time of hypoglycemia. A diagnosis of dumping syndrome (DS) was suspected in both children. In the child with PEG, the tip of the gastrostomy catheter was found to be lying in the bulbus duodeni. Once this had been pulled back, hypoglycemic episodes disappeared. The child with PEG-J needed continuous enteral feeding to reach a normal glucose balance. DS is a relatively common complication in children with gastrostomy, but extremely irregular glucose levels, ranging from hypoglycemia to hyperglycemia, and increased insulin secretion had not been previously demonstrated. The incidence of DS is probably underestimated in children receiving enteral feeding for neurological impairment. In these patients intensive monitoring of blood glucose levels should be performed to calibrate meals. Repeated underestimated hypoglycemic episodes could worsen neurological damage and cause a deterioration in clinical conditions.

Blockade of the chemokine receptor CXCR2 ameliorates adjuvant-induced arthritis in rats.

BACKGROUND AND PURPOSE: Chemokine receptors CXCR1 and CXCR2 may mediate influx of neutrophils in models of acute and chronic inflammation. The potential benefits of oral administration of a CXCR1/2 inhibitor, DF 2162, in adjuvant-induced polyarthritis (AIA) were investigated. EXPERIMENTAL APPROACH: A model of AIA in rats was used to compare the therapeutic effects of the treatment with DF2162, anti-TNF or anti-CINC-1 antibodies on joint inflammation and local production of cytokines and chemokines. KEY RESULTS: DF2162 prevented chemotaxis of rat and human neutrophils induced by chemokines acting on CXCR1/2. DF2162 was orally bioavailable and metabolized to two major metabolites. Only metabolite 1 retained CXCR1/2 blocking activity. Treatment with DF2162 (15 mg kg(-1), twice daily) or metabolite 1, but not metabolite 2, starting on day 10 after arthritis induction diminished histological score, the increase in paw volume, neutrophil influx and local production of TNF, IL-1beta, CCL2 and CCL5. The effects of DF2162 were similar to those of anti-TNF, and more effective than those of anti-CINC-1, antibodies. DF2162 prevented disease progression even when started 13 days after arthritis induction. CONCLUSIONS AND IMPLICATIONS: DF 2162, a novel orally-active non-competitive allosteric inhibitor of CXCR1 and CXCR2, significantly ameliorates AIA in rats, an effect quantitatively and qualitatively similar to those of anti-TNF antibody treatment. These findings highlight the contribution of CXCR2 in the pathophysiology of AIA and suggest that blockade of CXCR1/2 may be a valid therapeutic target for further studies aiming at the development of new drugs for treatment of rheumatoid arthritis.

Clinical effects of early treatment with insulin glargine in patients with cystic fibrosis and impaired glucose tolerance.

Diabetes mellitus is an increasing complication of cystic fibrosis (CF), as a result of the improved life expectancy. There is clear association between diabetes and increased morbidity and mortality. Lung function and clinical status deteriorate up to 2-4 yr before the diagnosis of CF-related diabetes (CFRD). The aim of our study was to evaluate the effects, on glucose homeostasis and clinical status, of the early treatment with insulin glargine in CF patients with impaired glucose tolerance (IGT). We selected six subjects with IGT diagnosed at oral glucose tolerance test (OGTT). Median age was 18.12 yr (range 9.2-27.8). Insulin glargine was administered at the median dosage of 0.3 U/kg/day (range 0.2-0.5). After the initial adjustment of the dosage, no patient manifested hypoglycemia during treatment. Median glycosylated hemoglobin (HbA1c) did not show any significant variation during treatment: it was 5.9% at baseline (range 5.5-6.2) and 6.1% (range 5.0-6.7) at the end of follow-up (p=0.496). Median body mass index (BMI) z-score significantly increased during treatment, from -0.95 (range -3.2-+0.6) at baseline to -0.5. (range -3.0-+0.9) at the end of follow-up (p=0.026). Lung function, measured by median forced expiratory volume in the first second (FEV1%), showed a mild but significant improvement during insulin treatment. It was 72.7% at baseline (range 41.5-98.4) and 76.7% (range 42.0-106.8) at the end of follow-up (p=0.027). No significant variation was found between the number of hospitalizations for clinical exacerbation (no./patient/yr) in the last 2 yr before treatment and during follow-up. Median number at baseline was 1.95/patient/yr (range 1-3) and 2.0/patient/yr (range 1-3) at follow-up (p=0.715). Our data seem to indicate that early insulin therapy can be safe, no patient manifested hypoglycemia or other adverse effects during treatment. Insulin is an anabolic hormone implicated in both lipid and protein metabolism. The appearance of IGT out of infections can indicate an early insulin deficiency, with a potential impact on the nutritional and clinical status of the patient, even before the appearance of overt diabetes. Larger controlled trials are necessary to verify if early insulin therapy is able to reduce the deterioration of nutritional status and lung function associated with the onset of IGT.

XK-aprosencephaly and related entities.

We offer further biological characterization of the XK atelen/aprosencephaly syndrome in two infants, one with prolonged survival, the other presenting prenatally with apparent hydranencephaly and an orbital tumor (OS). Familial occurrence in the former born to presumably nonconsanguineous Lybian parents may represent parental germinal mosaicism or autosomal recessive inheritance. Both had apparently normal chromosomes; however, the Lybian infant had slightly increased induced chromosome breakage suggesting that this rare multiple congenital anomalies syndrome may involve a DNA repair defect. Virtual absence of atelen/aprosencephalic structures may lead to an arthrogryposis-like prenatal movement disorder. The orbital tumor in the Utah infant consisted of dystopic neural tissue compressing a rudimentary globe and was connected by a thin bridge of neural tissue to the small mass of disorganized brain tissue usually found in atelen/aprosencephalic infants and fetuses. No evidence of an encephaloclastic process was found in the autopsied Utah infant.

Plasma levels of adrenomedullin in patients with adrenoleukodystrophy/adrenomyeloneuropathy.

BACKGROUND/AIMS: Adrenomedullin (AM) is a recently purified hypotensive peptide and its encoding gene has been sequenced from a human pheochromocytoma. High levels of AM have been shown in Addison's disease (AD). X-linked adrenoleukodystrophy/adrenomyeloneuropathy (ALD/AMN) is a peculiar adrenal insufficiency due to an accumulation of very-long chain fatty acid in adrenal cells and it is very often associated with a devastating demyelination of the central nervous system. METHODS: We studied the AM plasma levels of 22 patients with ALD/AMN (18 with hypoadrenalism, ALDa, and 4 with normal adrenal function, ALDb) and compared them with 18 males with classical AD and 16 normal male subjects. All patients with hyposurrenalism were studied before treatment with hydrocortisone. RESULTS: Both patients with ALD/AMN and AD showed increased levels of AM and all of them showed a significant difference from the control group (p < 0.0001). The plasma renin activity was higher in all patient groups than in the control group (p <0.001 ALDa, ALDb and AD vs. control group). The aldosterone levels were higher in ALDa and ALDb groups than AD (ALDa vs. AD p < 0.01; ALDb vs. control group p < 0.05; AD vs. controls p < 0.01). ACTH plasma levels were higher in ALDa and AD than ALDb and the control group (ALDa vs. AD not significant while ALDa and AD vs. control p <0.0001). CONCLUSIONS: Our data indicate that plasma AM levels in ALDa, ALDb and AD are higher than controls. These results were previously described in untreated AD. While classical AD patients show complete adrenal insufficiency (both mineralocorticoid and glucocorticoid defects), ALD/AMN patients show a less compromised glomerular function, indicating that AM is not completely correlated with mineralocorticoid insufficiency, and that the exact mechanism responsible for the increased AM levels in ALD/AMN is still unknown.

A randomized trial of nicotinamide and vitamin E in children with recent onset type 1 diabetes (IMDIAB IX).

OBJECTIVE: Various adjuvant therapies have been introduced along with intensive insulin therapy in patients with recent onset type 1 diabetes. Nicotinamide (NA), administered at diagnosis of the disease, can have beneficial effects on the clinical remission rate, improve metabolic control and preserve or slightly increase beta-cell function, probably by reducing toxicity due to free oxygen radicals. Vitamin E, a known antioxidant, inhibits lipid peroxidation; this can lead to protection of islet beta cells from the combined effects of interleukin 1, tumor necrosis factor and gamma interferon. The aim of the present study was to investigate whether the addition of vitamin E to NA could improve metabolic control and the residual beta-cell function, as measured by C-peptide secretion, in children and adolescents with recent onset type 1 diabetes; patients were followed-up for 2 years after diagnosis. PATIENTS AND STUDY DESIGN: Recent onset type 1 diabetes patients (n=64, mean age 8.8 years) were recruited by participating centres of the IMDIAB group. Thirty-two patients were randomized to NA (25 mg/kg body weight) plus vitamin E (15 mg/kg body weight); 32 patients acted as controls and received NA only at the same dose as above. Intensive insulin therapy was applied to both treatment groups. RESULTS: There were three drop outs during the 2-year follow-up period. Overall, patients assigned to the NA+vitamin E group or the NA group did not significantly differ in terms of glycated hemoglobin (HbA1c) levels, insulin requirement or baseline C-peptide secretion. Patients diagnosed at an age of less than 9 years showed significantly reduced C-peptide levels compared with those aged over 9 years at diagnosis and at the 2-year follow-up but there were no differences between the NA and NA+vitamin E treated groups. However at 6 months, patients over 9 years of age treated with NA+vitamin E showed significantly higher C-peptide compared with the NA group (P<0.003). In both age groups and in the different treatment groups, C-peptide levels found at diagnosis were preserved 2 years later. CONCLUSIONS: The use of NA alone, or in combination with vitamin E, along with intensive insulin therapy is able to preserve baseline C-peptide secretion for up to 2 years after diagnosis. This finding is of particular interest for pre-pubertal children with type 1 diabetes and has never been reported before.

Maintenance of a normal meal-induced decrease in plasma ghrelin levels in children with Prader-Willi syndrome.

Ghrelin is a 28-amino acid peptide recently identified in the stomach as the endogenous ligand for the growth hormone secretagogue receptor (GHS-R1a). Ghrelin is a potent stimulator of GH secretion. It was recently shown that circulating ghrelin levels in humans rise shortly before and fall shortly after every meal, and that ghrelin administration increases voluntary food intake. The hypothesis that ghrelin hypersecretion might contribute to genetic obesity has never been investigated. In this context, Prader-Willi syndrome is the most common form of human syndromic obesity. As ghrelin affects appetite as well as GH secretion and both are abnormal in PWS, it has been surmised that these alterations might be due to ghrelin dysregulation. The aim of the study was to investigate whether ghrelin is suppressed by the meals differently in PWS children than in PWS adults. Overnight circulating fasting ghrelin levels and ghrelin levels 120 min after breakfast were assayed in 7 PWS children (10.2 +/- 1.7 yr), 7 subjects with morbid obesity (10.3 +/- 1.3 yr), and 5 normal controls (8.4 +/- 1.4 yr). Because of the data spread, no statistical difference was observed in fasting ghrelin levels between PWS and control children (p = NS); anyway, fasting ghrelin levels were significantly lower in obese children than in the other groups (p < 0.05 vs. control and PWS children). Ghrelin levels were slightly suppressed by the meal in control subjects (mean fasting ghrelin: 160.2 +/- 82 pg/ml; after the meal, 141.2 +/- 57 pg/ml, p = NS); the meal failed to suppress ghrelin levels in obese children (mean fasting ghrelin: 126.4 +/- 8.5 pg/ml; after the meal, 119.1 +/- 8.3 pg/ml, p = NS). Interestingly, the meal markedly suppressed ghrelin levels in PWS children (mean fasting ghrelin: 229.5 +/- 70.4 pg/ml; after the meal, 155.8 +/- 34.2 pg/ml, p < 0.01). In conclusion, since a lack of decrease in circulating ghrelin induced by the meal was previously reported in PWS adults, the finding of a meal-induced decrease in ghrelin levels in our population of young PWS would imply that the regulation of the ghrelin system involved in the orexigenic effects of the peptide is operative during childhood, although it progressively deteriorates and is absent in adulthood when hyperphagia and obesity progressively worsen.

Thioredoxin, a redox enzyme released in infection and inflammation, is a unique chemoattractant for neutrophils, monocytes, and T cells.

Thioredoxin (Trx) is a ubiquitous intracellular protein disulfide oxidoreductase with a CXXC active site that can be released by various cell types upon activation. We show here that Trx is chemotactic for monocytes, polymorphonuclear leukocytes, and T lymphocytes, both in vitro in the standard micro Boyden chamber migration assay and in vivo in the mouse air pouch model. The potency of the chemotactic action of Trx for all leukocyte populations is in the nanomolar range, comparable with that of known chemokines. However, Trx does not increase intracellular Ca2+ and its activity is not inhibited by pertussis toxin. Thus, the chemotactic action of Trx differs from that of known chemokines in that it is G protein independent. Mutation of the active site cysteines resulted in loss of chemotactic activity, suggesting that the latter is mediated by the enzyme activity of Trx. Trx also accounted for part of the chemotactic activity released by human T lymphotropic virus (HTLV)-1-infected cells, which was inhibited by incubation with anti-Trx antibody. Since Trx production is induced by oxidants, it represents a link between oxidative stress and inflammation that is of particular interest because circulating Trx levels are elevated in inflammatory diseases and HIV infection.

Interaction between interleukin-1 and ciliary neurotrophic factor in the regulation of neuroblastoma cell functions.

Human neuroblastoma cells SK-N-SH express significant numbers of IL-1R type I on their surface, as detected by saturation binding and RT-PCR, and are responsive to IL-1beta activation by producing inflammatory cytokines IL-6 and IL-8. IL-1beta can also have an indirect effect on nervous cell functions, since it is able to modulate the stimulus-induced increase of intracellular Ca++ levels, one of the first steps of the cell activation mechanism. In fact, on SK-N-SH neuroblastoma cells, IL-1beta can inhibit the Ca++ increase induced by stimulation of acetylcholine receptors with carbachol. In parallel to IL-1beta, the neurotrophic factor CNTF also shows an inhibitory effect on carbachol-stimulated Ca++ increase in CNTFRalpha-expressing SK-N-SH cells. However, when simultaneously present, the two cytokines cross-inhibit, thus allowing full cell activation in response to the cholinoceptor agonist. The inhibitory effect of CNTF on IL-1beta activities on nervous cells was confirmed in the IL-6 production assay. In fact, while CNTF could not induce IL-6 production, it could strongly inhibit cytokine production in response to IL-1beta in SK-N-SH cells. The down-modulation of IL-1 effects by CNTF could be one of the mechanisms controlling the extent of the inflammatory reaction at the nervous system level.

Activation of the Ca2+ message system by parathyroid hormone is dependent on the cell cycle.

Putative osteoblastic UMR 106-01 cells exhibit heterogeneous intracellular calcium [Ca2+]i responses to parathyroid hormone (PTH). To determine whether this phenomenon is secondary to cell cycle asynchrony, UMR 106-01 cultures were synchronized at the G1/S boundary using a sequential thymidine-aphidicolin treatment. Five hours after release from the block > 80% of the cells are in S phase, while nontreated confluent cultures are in G1. Using video image analysis of single cells loaded with fura-2, PTH (10-(7) M) induced transient increases in [Ca2+]i preferentially in cells in S phase, with 82% response frequency whereas cells in G1 phase responded poorly to PTH with only 10% response frequency. In contrast, cell response to fetal calf serum was more frequent in G1 than in S phase. Pretreatment with La3+, nifedipine or pertussis toxin reduced both the frequency and amplitude of the PTH response in S phase to values comparable to those observed in cells in G1 phase. There was no significant difference in inositol trisphosphate generated by PTH stimulation in either phase. Thus, the heterogeneous osteoblastic [Ca2+]i responsive to PTH is cell cycle-dependent with the change in the G1 to the S phase mode of response dependent on active coupling between the PTH receptor and a Ca2+ channel via a pertussis toxin-sensitive G protein.

Ligand binding to monocyte alpha 5 beta 1 integrin activates the alpha 2 beta 1 receptor via the alpha 5 subunit cytoplasmic domain and protein kinase C.

Regulation of the functional status of integrin receptors plays a critical role in inflammation and tissue remodeling, as it affects cell adherence and cytokine secretion. We have previously shown that in monocytes the binding of collagen to the alpha 2 beta 1 integrin induces the release of IL-1, an event that is potentiated by binding of fibronectin (Fn) to the alpha 5 beta 1 integrin. In this study, we have investigated the mechanisms leading to this phenomenon. Fn binding to alpha 5 beta 1 induced intracellular signals which increased the alpha 2 beta 1-dependent adhesiveness of monocytes to collagen without modifications of alpha 2 beta 1 expression. By using Abs against the intracellular region of the alpha 5 subunit of the alpha 5 beta 1 receptor, and specific inhibitors of protein kinase C (PKC), we found that the potentiation effect of Fn on monocyte IL-1 production and their adherence to collagen was dependent on an intact alpha 5 subunit cytoplasmic domain, and required PKC activation. Although the alpha 2 beta 1 could be activated by several intracellular second messengers, including protein kinase A and intracellular calcium, the potentiating effect of Fn was mediated only by PKC. These data provide an example of a novel regulatory mechanism: potentiation of beta 1 integrin-mediated events as a result of ligand binding to another integrin of the same class. They also show that the intracellular region of alpha 5 beta 1 plays a critical role in transducing signals generated by ligand binding to alpha 5 beta 1.

Norepinephrine, unlike ATP, induces all-or-none increase in cytosolic calcium in thyroid cells. The role of inositol-trisphosphate-sensitive stores and calcium channels.

The mechanism of action of norepinephrine and ATP has been analyzed in single FRTL5 cells (a normal thyroid cell line), loaded with the fluorescent Ca2+ probe Fura2. ATP increased the cytosolic Ca2+ in an apparently concentration-dependent manner with a maximal effect at 10 microM (413 +/- 26% over basal levels of 135 +/- 7 nM). In contrast, the norepinephrine-induced increase (198 +/- 5% over basal) was concentration independent in individual cells, the minimal effective concentration being 1 nM. However, the number of cells responding to norepinephrine was concentration dependent. The ATP-induced Ca2+ rise was biphasic, consisting of a rapid rise (2-4 s, 252 +/- 15%), resembling the effect of norepinephrine, followed by a slower and longer component, which reached a plateau in 0.5-2 min. The second component appeared to be related to the opening of a channel, since it required extracellular Ca2+ and was abolished by SC38249, an inhibitor of the second-messenger-operated and voltage-operated channels. Moreover, it was inhibited by 4 beta-phorbol 12-myristate 13-acetate, suggesting that protein kinase C might be involved in the modulation of this Ca2+ channel.

Analysis of protein kinase C requirement for exocytosis in permeabilized rat basophilic leukaemia RBL-2H3 cells: a GTP-binding protein(s) as a potential target for protein kinase C.

The role of protein kinase C in calcium-dependent exocytosis was investigated in permeabilized rat basophilic leukaemia cells. When protein kinase C was down-regulated by phorbol myristate acetate (1 microM for 3-6 h) or inhibited by pharmacological agents such as calphostin C (1 microM) or a protein kinase C-specific pseudo-substrate peptide inhibitor (100-200 microM), cells lost the ability to secrete in response to 10 microM free Ca2+. In contrast, a short treatment (15 min) with phorbol myristate acetate, which maximally activates protein kinase C, potentiated the effects of calcium. Biochemical analysis of protein kinase C-deprived cells indicated that loss of the Ca(2+)-induced secretory response correlated with disappearance of protein kinase C-alpha. In addition, at the concentrations effective for exocytosis, calcium caused translocation of protein kinase C-alpha to the membrane fraction and stimulated phospholipase C, suggesting that, in permeabilized cells, protein kinase C can be activated by calcium through generation of the phospholipase C metabolite diacylglycerol. The delta, epsilon and zeta Ca(2+)-independent protein kinase C isoenzymes were insensitive to phorbol myristate acetate-induced down-regulation and did not, as expected, translocate to the particulate fraction in response to calcium. Interestingly, secretory competence was restored in cells depleted of protein kinase C or in which protein kinase C itself was inhibited by non-hydrolysable GTP analogues, but not by GTP, suggesting that protein kinase C might regulate the ability of a G protein(s) directly controlling the exocytotic machinery to be activated by endogenous GTP.

Activation of the Ca2+ message system by parathyroid hormone is dependent on the cell cycle.

To verify whether the heterogeneous intracellular calcium ([Ca2+]i) responses to PTH observed in the UMR 106-01 osteogenic sarcoma cells are secondary to cell cycle asynchrony or to genotypic differences within the population, we synchronized cell monolayers at the G1/S boundary using a sequential thymidine-aphidicolin block. Video image analysis of fura-2-loaded cells revealed that PTH (10(-7) M) induced transient increases of [Ca2+]i preferentially in cells in S phase (82% response frequency, n = 63; 286 +/- 33% of baseline, n = 29), whereas cells in G1 phase responded poorly to PTH (10% response frequency, n = 51; 140 +/- 8% of baseline, n = 5). In contrast, cell exposure to 2% fetal calf serum was followed by [Ca2+]i transients in 83% (n = 42) of cells in G1 phase, but in only 25% (n = 63) of cells in S phase, with similar response amplitude. Hormonal responsiveness was heterogeneous in small clones obtained from single UMR 106-01 cells, with response frequency similar to that observed in nonsynchronized cultures. Pretreatment with either La3+, nifedipine, or pertussis toxin reduced both frequency and amplitude of PTH response in S phase to levels close to G1 phase, whereas there was no significant difference in inositol trisphosphate generated by PTH stimulation in either phase. Therefore, the heterogeneous [Ca2+]i responses of UMR 106-01 cells to hormonal stimulation is dependent on the phase of the cell cycle, rather than on genotypic heterogeneity. The switch from the G1 to the S phase mode of response is driven by active coupling between the PTH receptor and a Ca2+ channel through a pertussis toxin-sensitive G protein.

Muscarinic regulation of phospholipase A2 and iodide fluxes in FRTL-5 thyroid cells.

FRTL-5 thyroid cells express a muscarinic receptor which inhibits the phospholipase C activity in a pirenzepine-insensitive manner. We here report that the cholinergic agonist carbachol decreases in these cells the steady-state iodide content, an effect correlated with the iodination of thyroglobulin and with thyroid hormone formation. Several signal pathways may be involved in this phenomenon since carbachol in addition to inhibiting phospholipase C, increased the arachidonic acid release and modified the adenylyl cyclase activity. In FRTL-5 cells, arachidonic acid is released via the direct stimulation of phospholipase A2 by a pirenzepine-sensitive muscarinic receptor coupled to a GTP binding protein sensitive to pertussis toxin. Regarding adenylyl cyclase, carbachol potentiated the thyrotropin-induced stimulation of the enzyme, whereas it did not affect the basal levels of cAMP. In vitro binding studies revealed the presence of two muscarinic binding sites. To summarize, the analysis of signal pathways and of in vitro binding sites indicates a complex muscarinic regulation of thyroid function, which includes the modulation of iodide fluxes.

Evidence that a guanine nucleotide-binding protein linked to a muscarinic receptor inhibits directly phospholipase C.

The mechanism of phospholipase C regulation by inhibitory receptors was analyzed both in intact and in permeabilized rat thyroid cells (FRTL5). In this system, the muscarinic agonist carbachol inhibited phospholipase C, as indicated by the decrease in the basal levels of inositol 1,4,5-trisphosphate as well as by the reduced adrenergic stimulation of phosphoinositol accumulation, which was paralleled by a fall in the cytosolic Ca2+ levels. This inhibition involved an M2 muscarinic receptor because it was abolished by atropine but not by the M1 antagonist pirenzepine. Cells pretreated with pertussis toxin were not responsive to carbachol, indicating the involvement of a guanine nucleotide-binding protein in this inhibitory process. This possibility was further evaluated in permeabilized cells, where the carbachol inhibition was shown to be completely dependent on GTP. Known second messengers were not involved in this inhibitory process since Ca2+, cAMP, and activators of protein kinases were not able to mimic or prevent the carbachol effect either in intact or in permeabilized FRTL5 cells. In this system, the phospholipases C and A2 are coupled to two classes of muscarinic receptors that display a different sensitivity to pertussis toxin. The carbachol inhibitory effect occurred under conditions that prevented activation of phospholipase A2, excluding a role of the arachidonic acid metabolism in this process. Taken together these data provide the strongest support to date that an inhibitory guanine nucleotide-binding protein sensitive to pertussis toxin can directly mediate receptor-induced inhibition of phospholipase C.