Torben Særmark

Prevalence of hereditary haemochromatosis in late-onset type 1 diabetes mellitus: a retrospective study

BACKGROUND Although genotyping studies suggest that hereditary haemochromatosis is one of the most common genetic disorders in white people, it is still thought of as an uncommon disease. Our aim was to test the hypothesis that hereditary haemochromatosis is a disease often overlooked in patients with late-onset type 1 diabetes mellitus, a late manifestation of untreated iron overload. METHODS We did a retrospective study in which we genotyped for the C282Y and H63D mutations in the haemochromatosis gene in 716 unselected Danish patients who developed type 1 diabetes mellitus after age 30 years and 9174 controls from the general Danish population. We also screened for hereditary haemochromatosis by assessment of transferrin saturation. FINDINGS More patients with diabetes (n=9, relative frequency 1.26%, 95% CI 0.58-2.37) than controls (23, 0.25%, 0.16-0.38) were homozygous for C282Y (odds ratio 4.6, 2.0-10.1, p=0.0001). These patients had unrecognised signs of haemochromatosis. Transferrin saturation and ferritin concentrations ranged from 57% to 102% and 17 microg/L to 8125 microg/L, respectively. Frequency of compound heterozygosity (C282Y/H63D) did not differ between patients with diabetes (eight) and controls (131) (odds ratio 0.8, 95% CI 0.4-1.7). Positive and negative predictive values of transferrin saturation greater than 50%, in identification of C282Y homozygosity, were 0.26 and 1.00, respectively. A saturation of less than 50% therefore excluded C282Y homozygosity, whereas a saturation of more than 50% suggested C282Y homozygosity. INTERPRETATION Measurement of transferrin saturation followed by genetic testing could prevent liver and heart problems and improve life expectancy in patients with diabetes. Population screening before the onset of diabetes might improve the outlook of patients even further, but will be less cost effective.

Substance P in the Median Eminence and Pituitary of the Rat: Demonstration of Immunoreactive Fibers and Specific Binding Sites

The distribution of substance P was demonstrated by immunohistochemical methods, and receptor binding with 125I-8-iodinated 8-tyrosine-substance P as an analog was used to describe possible specific binding sites in the hypothalamo-hypophysial system of the rat. The immunohistochemical experiments showed fibers exhibiting substance P-immunoreactivity in the external and internal layer of the median eminence. In the external layer some of the fibers were located in apposition to the portal capillaries. In the posterior pituitary substance P-immunoreactive fibers were identified. These terminals varied in size and were located near the capillaries as well as in the neuropil. The anterior lobe contained a number of substance P-immunoreactive fibers distributed around the pituitary cells. The kinetic analysis on crude membrane fractions from anterior and posterior pituitaries revealed the presence of a specific binding site for substance P in the anterior pituitary. These data indicate that substance P may be released from the median eminence and posterior pituitary to the vascular system and reach specific receptors in the anterior pituitary. However, a direct innervation of the anterior pituitary is possible. The lack of binding sites in the posterior pituitary indicates that fibers in this organ release substance P into the blood stream.

Growth Hormone Releasing Hexapeptide (GHRP-6) Activates the Inositol (1,4,5)-Trisphosphate/Diacylglycerol Pathway in Rat Anterior Pituitary Cells

Growth hormone-releasing hexapeptide (GHRP-6) is known to stimulate secretion of growth hormone (GH) in vivo and in vitro in a variety of species. However, the cellular effects of GHRP-6 remain largely unknown. We have tested the influence of GHRP-6 on the inositol phospholipid second messenger system in cultured anterior pituitary cells. Cultured pituitary cells responded upon challenge with GHRP-6 with a dose-dependent release of GH. Moreover, incubation of GHRP-6 with pituitary cell cultures labelled with myo-[3H]inositol resulted in a dose-dependent rise in [3H]inositol phosphates. Brief stimulation of pituitary cells with GHRP-6 increased phosphorylation of MBP4-14, a specific protein kinase C substrate, when incubated with the cytosol- or plasma membrane fraction from the stimulated cells. Furthermore, introduction of MBP4-14 into the cytosol in digitonin permeabilized pituitary cells caused increased phosphorylation of this substrate. GHRP-6 induced a rise in intracellular Ca2+ in individual somatotrophs loaded with the Ca2+ indicator, Fura-2. Preincubation (3 min) with somatostatin (SRIF) diminished the Ca2+ spike elicited by GHRP-6, while no effect of SRIF was observed when added simultaneously with GHRP-6. These results indicate that GHRP-6-stimulated GH-secretion involves the diacylglycerol/inositol(1,4,5)trisphosphate pathway with a resulting rise in cytosolic Ca2+.

Calcium/sodium exchange in purified secretory vesicles from bovine neurohypophyses

Purified secretory vesicles isolated from bovine neurohypophyses take up Na+ under the same circumstances where an efflux of Ca2+ takes place, suggesting a Na+/Ca2+ exchange. Potassium cannot substitute for Na+ in this process. Also, a Ca2+/Ca2+ exchange can occur. Inhibiting the latter process by Mg2+ allowed to estimate an apparent KM of 0.7 microM free Ca2+ and a maximal uptake of 1.5 nmol X mg protein-1 X min-1 Ca2+ in exchange for Na+. The vesicles did not contain plasma membrane marker (Na+/K+ ATPase) as shown by distribution analyses on the density gradients on which they were purified. Similarly, distribution studies also showed that no other ATPase activity could be detected in the purified vesicle fraction. It is concluded that a Na+/Ca2+ exchange is operating across the secretory vesicle membrane and that it is not directly dependent on ATP hydrolysis.

Binding and internalization of a iodinated substance P analog by cultured anterior pituitary cells

Substance P binding to cultured anterior pituitary cells was characterized using Bolton-Hunter iodinated substance P as a ligand. At 0 degrees C, the interaction of the ligand and the cellular surface binding sites was found to be specific, rapid and reversible. Scatchard and Hill analysis of specific binding revealed a single class of non-interacting binding sites with a high affinity (KD = 0.48 nM) and a moderate density of binding sites (Bmax = 1187 binding sites/cell). At 37 degrees C a NaOH-soluble intracellular ligand pool was observed in addition to a surface-bound ligand pool released by a low pH buffer. Thus, substance P seems to be internalized after binding to cellular surface binding sites by means of receptor-mediated endocytosis. The internalization was rapid and could be blocked by colchicine (20 microM), an inhibitor of microtubuli assembly. Following internalization, intracellular degradation of the ligand could be demonstrated. Leupeptin (100 microM), an inhibitor of certain lysosomal enzymes could inhibit the cellular degradation of the added ligand, but had only a moderate influence on internalization. These results demonstrate that substance P after binding to a surface-localized receptor on its pituitary target cells is internalized and subsequently degraded by lysosomal enzymes.

Studies on a Ca2+-dependent nucleoside triphosphate pyrophosphohydrolase in rat liver plasma membranes.

A membrane-bound Ca2+-dependent nucleoside triphosphate pyrophosphohydrolase was solubilized in deoxycholate, separated from inorganic pyrophosphatase, and partially characterized. The Km for a variety of substrates was determined. At 10(-4) M free Ca2+ (pH 8.0) the Km values for ATP and GTP were 0.32 and 2.2 microM, respectively. With ATP as substrate, Mg2+, Sr2+, and Ba2+ could only replace Ca2+ to a limited degree. Both purine and pyrimidine nucleoside triphosphates were hydrolyzed yielding PPi and mononucleotides and similarly AMP and formed from adenosine-(beta gamma-methylene)triphosphate. UDPglucose was hydrolyzed at the pyrophosphate bond. Tripolyphosphate and phosphoribosyl-1-pyrophosphate (P-rib-PP) were not hydrolyzed. Substrate competition experiments showed that GTP inhibited pyrophosphohydrolysis of ATP competitively. However, UDP glucase and adenosine-(beta gamma-methylene)triphosphate inhibited ATP pyrophosphohydrolysis in a non-linear manner. Adenosine-(beta gamma-methylene)triphosphate inhibited pyrophosphohydrolysis of UDPglucose non-competitively, whereas UDPglucose inhibition of adenosine-(beta gamma-methylene) triphosphate pyrophosphohydr-lysis was competitive. The molecular weight of ATP pyrophosphohydrolase was estimated at 120 000 and the pI at 5.1 Pyrophosphohydrolysis of adenosine-(beta gamma-methylene)triphosphate was studied in a number of rat organs. Nearly all activity could be sedimented at 50 000 X g. Very high activities were found in liver, kidney and small intestine, whereas low activities were found in brain and blood.

Substance P and related tachykinins induce receptor-mediated hydrolysis of polyphosphoinositides in the rat anterior pituitary

In the present study characterization of phosphatidylinositol 4,5-bisphosphate-specific phospholipase C (PIP2-PLC) activity and receptor-mediated hydrolysis of PIP2 in rat anterior pituitary membranes were investigated. Incubation of the membrane fraction of anterior pituitary homogenate with [3H]inositol-labeled PIP2 in the presence of calcium increased the concentration of the water-soluble degradation product inositol trisphosphate (IP3) in a time-dependent manner. PIP2-PLC in the rat anterior pituitary had a pH optimum at 5.5 and a requirement for cations. Ca2+ and Mg2+ could activate the enzyme. Activity was maximal at a total magnesium concentration of 1 mM and at a free Ca2+ concentration of 100 microM. The addition of the detergent Triton X-100 (0.05% w/v) to the membrane fraction resulted in a 50% decrease of PIP2-PLC activity, whereas the presence of sodium deoxycholate (1 mg/ml) in the membrane fraction increased the PIP2-PLC activity by 100%. The tachykinins substance P, 8-Tyr-substance P, physalaemin, neurokinin A, eledoisin, kassinin and neurokinin B induced receptor-mediated breakdown of [3H]inositol-labeled PIP2 in the membrane fraction in a concentration-dependent manner, but with different potencies. The tachykinins displayed the following rank order of potencies: substance P greater than 8-Tyr-substance P greater than physalaemin greater than neurokinin A greater than eledoisin greater than kassinin greater than neurokinin B, which is consistent with the involvement of a NK-1 receptor. Combined treatment of anterior pituitary membranes by substance P and thyrotropin-releasing hormone (TRH) resulted in an additional increase in PIP2-PLC activity compared to stimulation with TRH alone.

Ca2+ uptake to purified secretory vesicles from bovine neurohypophyses

Purified secretory vesicles isolated from bovine neurohypophyses were found to take up Ca2+ when incubated at 30 degrees C in media containing 10(-7) to 10(-4) M free Ca2+. At 10(-4) free Ca2+ 19 nmol/mg protein were taken up within 30 min. The initial uptake at this Ca2+ concentration was about 2 nmol/mg protein per min. The uptake of Ca2+ to secretory vesicles was not affected by ATP, oligomycin, ruthenium red, trifluoperazine, Mg2+ or K+, but was inhibited by Na+ and Sr2+. From these characteristics it can be concluded that the uptake system does not utilize directly ATP (as the Ca2+-ATPases known to be present in the cell membrane and the endoplasmic reticulum) and is different from the mitochondrial Ca2+ uptake system driven by respiration and/or ATP hydrolysis. However, Ca2+-Na+ exchange may well operate: In experiments using different concentrations of Na+ we found half-maximal inhibition of Ca2+ uptake with 33.3 mM Na+. An analysis of the data in a Hill plot indicated that at least 2 Na+ would be exchanged for 1 Ca2+. Also, it was found that Ca2+ previously taken up could be released again by external Na+ but not by K+.

Cultures of Human Colonic Epithelial Cells Isolated from Endoscopical Biopsies from Patients with Inflammatory Bowel Disease. Effect of IFNγ, TNFα and IL-1β on Viability, Butyrate Oxidation and IL-8 Secretion

Cytokjne-mediated impairment of viability and metabolic function of epithelial cells has been suggested as a possible early pathogenic event in the development of inflammatory bowel disease (IBD). It is currently unknown whether pro-inflammatory cytokines have a direct effect on human nontransformed colonic epithelial cells. We investigated the effects of TNFα, IFNγ and IL-1β on viability, short chain fatty acid (butyrate) oxidation and IL-8 secretion in human colonic epithelial cell cultures in vitro obtained from macroscopically normal mucosa from IBD patients and controls. Colonic crypts were isolated from endoscopical biopsies by ultra-short (10 min) EDTA/EGTA treatment, and exposed to TNFα, IFNγ and IL-1β for 24 hours. The combination of TNFoc+IFNγ induced a significant decrease in cell viability as judged by methyltetrazoleum (MTT) metabolism which decreased to median 68 % of unexposed cultures (P<0.01). This effect was more pronounced than that observed after addition of TNFα (median 88 %) (P<0.05), but not IFNγ alone (median 78 %), whereas IL-1(3 had no significant effect. Cells from IBD patients were significantly less sensitive to TNFα + IFNγ exposure (median 74 %) compared to cells from controls (median 58 %) (P < 0.05). Butyrate oxidation, as measured by entrapment of CO2, was not inhibited in cells exposed to TNFα + IFNγ, neither from controls (median 112%) nor from IBD patients (median 108%), suggesting a relative increase of this specific metabolic function in living cells in response to immunoinflammatory stress. IL-8 levels in cell supernatants were increased by TNFα + IFNγ, supporting the role of the epithelium in signalling between luminal factors and mucosal immune cells. In conclusion, we report that TNFα and IFNγ damage and influence human colonic epithelial cell function in vitro and that such mechanisms, if operative in vivo, also may be involved in the pathogenesis of IBD

Stimulation by calmodulin of Ca2+ uptake and (Ca2+-Mg2+) ATPase activity in membrane fractions from ox neurohypophyses

Abstract Calmodulin stimulated 45 Ca 2+ uptake into a plasma membrane enriched fraction from ox neurohypophysial nerve endings and into a microsome fraction. The 45 Ca 2+ uptake and the (Ca 2+ -Mg 2+ ) ATPase activity in the plasma membrane fraction exhibited similar pCa and calmodulin sensitivities, suggesting that the enzyme activity is the biochemical expression of a high affinity Ca 2+ pump. Calmodulin thus seems to play a role in regulation of the intracellular free Ca 2+ concentration in the neurohypophysis.