Medizin - Open Access LMU - Teil 10/22

HYPERGLYCAEMIA impairs recovery from transient cerebral ischaemia: the importance of tissue acidification for this phenomenon has not been clarified in detail. We investigated this issue in a less complex in vitro preparation of isolated rat dorsal spinal roots exposed for 30 min to hyperglycaemic hypoxia. Peak height of compound action potentials recovered minimally in 5 mM bicarbonate. However, recovery was greatly improved by addition of the weak base trimethylamine during re-oxygenation. Addition of the weak acid propionate had no such effect. Cytoplasmic alkalinization improved recovery in a brief time window only: application of trimethylamine after 15 min of re-oxygenation was without beneficial effect. These data emphasize the importance of cytoplasmic acidification for neurophysiological recovery from hyper-glycaemic hypoxia during the initial period of re-oxygenation.

1. Electrotonic responses to 150 ms current pulses were recorded from isolated rat dorsal roots incubated for at least 3 h with either normal (5 mM) or high (25 mM) D-glucose solutions, and with either normal (25 mM) or low (5 mM) bicarbonate concentrations. 2. On replacement of O2 by N2 for 50 min, all the roots depolarized, but the changes in electrotonus differed systematically. With normal glucose, the depolarization was accompanied by an increase in input conductance. In contrast, for the hyperglycaemic roots the depolarization was slower and accompanied by a fall in input conductance which was exacerbated in low bicarbonate concentrations. 3. The changes induced by hyperglycaemic hypoxia in low bicarbonate could be mimicked by exposure of the roots either to 100% CO2 or to a combination of 3 mM tetraethylammonium chloride and 3 mM 4-aminopyridine, to block both fast and slow potassium channels. 4. These results indicate that the primary mechanism of hypoxic depolarization of these sensory axons is altered by hyperglycaemia. In normoglycaemia, the changes in electrotonus are consistent with an increase in axonal potassium conductance. The block of potassium channels seen in hyperglycaemic hypoxia is attributed to intra-axonal acidification by anaerobic glycolysis and may contribute to the pathogenesis of diabetic neuropathy.

We have compared the expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) genes in various human tissues using a quantitative polymerase chain reaction technique. Tissues of three human subjects, obtained at autopsy, were analyzed. BNP transcripts could be detected in the central nervous system, lung, thyroid, adrenal, kidney, spleen, small intestine, ovary, uterus, and striated muscle. ANP transcripts could also be demonstrated in various human extracardiac tissues including several endocrine organs. In all peripheral tissues, the level of both natriuretic peptide transcripts was approximately 1-2 orders of magnitude lower than in cardiac ventricular tissues. This distribution is in marked contrast to the much lower level of ANP and BNP transcripts present in extracardiac rat tissues (generally less than 1/1000 of ventricles). These data suggest differential expression of the two natriuretic peptide genes in cardiac and extracardiac tissues in man. Furthermore, the presence of local synthesis of ANP and BNP in various peripheral organs suggests paracrine and/or autocrine function of these natriuretic peptides.

Since the 64kDa-protein glutamic acid decarboxylase (GAD) is one of the major autoantigens in T-cell mediated Type 1 diabetes, its relevance as a T-cell antigen needs to be clarified. After isolation of splenic T-cells from non-obese diabetic (NOD) mice, a useful model for human Type 1 diabetes, we found that these T-cells proliferate spontaneously when incubated with human GAD65, but only marginally after incubation with GAD67, both recombinated in the baculovirus system. No effect was observed with non-diabetic NOD mice or with T-cells from H-2 identical NON-NOD-H-2g7 control mice. It has been published previously that NOD mice develop autoantibodies against a 64kDa protein detected with mouse beta cells. In immunoprecipitation experiments with sera from the same NOD mice and 33S-methionine-labelled GAD, no autoantibody binding could be detected. We conclude firstly that GAD65 is an important T-cell antigen which is relevant early in the development of Type 1 diabetes and secondly that there is an antigenic epitope in the human GAD65 molecule recognized by NOD T-cells, but not by NOD autoantibodies precipitating conformational epitopes. Our results therefore provide further evidence that GAD65 is a T-cell antigen in NOD mice, being possibly also involved in very early processes leading to the development of human Type 1 diabetes.

By using an immunoprecipitation assay, we analysed reactivity of autoantibodies to human recombinant GAD65 and GAD67 in sera from patients with autoimmune polyendocrine syndrome Type II (APS II) with and without Type 1 (insulin-dependent) diabetes mellitus (IDDM) compared to patients with organ-specific autoimmunity. Overall antibodies to GAD65 were correlated with IDDM in all study groups, whereas GAD67 antibodies were associated with IDDM when APS II coexists. Antibodies to GAD65 and GAD67 were detected in 13 (44.8%) and 7 (24.1%) out of 29 APS II patients with IDDM, but in only 4 (13.8%) and 2 (6.9%) out of 29 APS II patients without IDDM, respectively (p < 0.05). In short-standing IDDM (< 1 year), antibodies to GAD67 were significantly more frequent in patients with APS II (5 of 9 [55.6%] subjects) compared to matched diabetic patients without coexisting polyendocrinopathy (1 of 18 [5.6%] subjects) (p < 0.02). The levels of GAD65 (142 ± 90 AU) and GAD67 antibodies (178 ± 95 AU) were significantly higher in patients with polyglandular disease than in patients with isolated IDDM (91 ± 85 AU and 93 ± 57 AU) (p < 0.02). Interestingly, all 11 GAD67 antibody positive subjects also had GAD65 antibodies (p < 0.0001), and in 10 of 11 anti-GAD67 positive sera the GAD67 antibodies could be blocked by either GAD67 or GAD65, suggesting the presence of cross-reactive autoantibodies. No correlation was observed between GAD antibodies and age, sex or any particular associated autoimmune disease, besides IDDM. GAD antibodies were present in only 1 of 6 (16.7%) patients with APS Type I, in 1 of 26 (3.9%) patients with autoimmune thyroid disease but in none of the patients with Addison's disease (n = 16), pernicious anaemia (n = 7) or normal controls (n = 50). Our data suggest distinct antibody specificities reactive to GAD isoforms in APS II and IDDM, which might reflect different mechanisms of autoimmune response in IDDM with coexisting autoimmune polyendocrine autoimmunity.