Focal ischemia of the rat brain,with special reference to the influence of plasma glucose concentration

Summary Focal cerebral ischemia was induced by occlusion of the right middle cerebral artery in hypoglycemic, normoglycemic, as well as in acute and chronic diabetic rats. The brain damage was studied after 4 days. The volume of infarction was decreased in hypoglycemia (29±19 mm³ (mean±SD) versus 58±35 mm³,P<0.0046), unaltered in acute diabetes (61±45 mm³), and increased in chronic diabetes (91±22 mm³,P<0.0463). The cortex adjacent to the infarct showed selective neuronal injury affecting the cortical layers 2 and 3. The damage was enhanced by hypoglycemia and prevented in most of the diabetic animals. The findings indicate that different mechanisms cause infarction and selective neuronal injury outside infarcts, but that both are influenced by the plasma glucose concentration.     

Release of 3H-5-Hydroxytryptamine from Isolated Rabbit Aorta

Abstract: The main aim of the present investigation was to study systematically the passive and stimulation-evoked release of ³H-5-hydroxytryptamine (³H-5-HT) from rabbit isolated aorta. This was accomplished by preloading rings of aorta with ³H-5-HT (10^–6M) and then monitoring by fractional collection the basal ³H-outflow and stimulation-evoked ³H-overflow. The basal ³H-outflow from aorta preloaded with 10^–6M of either ³H-5-HT or (-)-³H-noradrenaline (³H-NA) leveled off about 100 min. after the onset of wash-out and remained almost constant thereafter (100–240 min.). The basal ³H-outflow from tissue preloaded with ³H-5-HT was almost 3-fold higher (70–240 min.) than that seen after preloading with ³H-NA. Cocaine (3x10^–5M) did not alter the basal ³H-outflow (15–240 min.) from tissue preloaded with ³H-5-HT, while pargyline (5X10^–4M) decreased it by about 66% (100–240 min.). Electrical-field stimulation (S₁S₇, 200 mA, 600 pulses, 0.5 msec, 3 Hz) were applied to the tissue. The initial stimulation-evoked ₃H-overflow from aorta preloaded with ³H-5-HT was higher than the subsequent ones (S₁-S₇: 100, 35, 35, 35, 35, 37, and 40%). Similar results to these were obtained with tissues preloaded with ³H-NA. The stimulation (S₁-S₇; 200 mA; 600 pulses, 0.5 msec, 3 Hz)-evoked ³H-overflow increased in an apparent linear manner with the amount of current used (50–200 mA). This was also the case for number of pulses (100–900) in the stimulus. The stimulation-evoked ³H-overflow depended in part on the stimulation frequency: unchanged at 2–4 Hz; small increase at 8 Hz; and a 15-fold increase at 16 Hz relative to 2 Hz. Tetrodotoxin (10^–6M) decreased the stimulation-evoked ³H-overflow from aorta preloaded with ³H-5-HT (S₂-S₆) by about 60%, while S₁ was not affected. The inhibitory effect of tetrodotoxin was fully reversed by washing the aorta with drug-free salt solution. Omission of Ca²⁺ from the salt solution reduced the stimulation ³H-overflow by 47–57% (S₂-S₆) while S₁ was unaffected. 6-Hydroxydopamine markedly increased the stimulation-evoked ³H-overflow from ³H-5-HT preloaded rings (180–323% of control). Pargyline (5x10^–4M), cocaine (3x10^–5M) and removal of endothelium did not alter the stimulation-evoked ³H-overflow evoked by stimulation (S₁-S₆) of aorta preloaded with ³H-5-HT. It is concluded that ³H-5-HT can be released by electrical-field stimulation as a “false transmitter'’from rabbit isolated aorta. Most of the release is probably of neuronal origin. However, some of the stimulation-evoked ³H-overflow is derived from extraneuronal sites.     

Presynaptic action of adrenaline on adrenoceptors modulating stimulation-evoked 3H-noradrenaline release from rabbit isolated aorta

Summary The purpose of this investigation was to study the effect of adrenaline on presynaptic adrenoceptors by recording the release of ³H-noradrenaline evoked by electrical-field stimulation. Adrenaline (10^–10–³ × 10^–9 mol/l) had no effect on the ³H-overflow evoked by stimulation of aorta preloaded with ³H-noradrenaline. At 10^–8 and 3 × 10^–8 mol/l, the ³H-overflow was decreased by up to 47%. The maximum decrease was more marked in the presence of either cocaine (3 × 10^–5 mol/l) plus corticosterone (4 × 10^–5 mol/l), cocaine (3.3 × 10^–6 mol/l) plus normetanephrine (4 × 10^–5 mol/l), or desipramine (10^–6 mol/l) plus normetanephrine (10^–5 mol/l). The relationship between adrenaline-induced decrease and stimulation-frequency was dependent on the experimental design: either the decrease was the same at all frequencies (1–16 Hz) or it was more marked, the lower the frequency (1 > 3 > 8 Hz). Phentolamine and rauwolscine (both 10^–6 mol/l) antagonized the inhibitory effect of adrenaline (10 ^{– 8}–10^–6 mol/l). Phenoxybenzamine (10^–6 mol/l), prevented the inhibitory effect. No enhancing effect of adrenaline (10^–9–10^–6 mol/l) was observed in the presence of these three -adrenoceptor antagonists. Our results suggest that adrenaline activates inhibitory ₂-adrenoceptors, but not facilitatory -adrenoceptors on postganglionic sympathetic nerve terminals in rabbit aorta. Send offprint requests to J. Abrahamsen at the above address     

The 'novel' 'uncoupling' proteins UCP2 and UCP3: what do they really do? Pros and cons for suggested functions

The scientifically novel, but evolutionarily ancient, so-called uncoupling proteins 2 and 3 (UCP2, UCP3) are structurally similar to the archetypical uncoupling protein UCP1. A series of suggestions have been forwarded for their physiological function. We discuss systematically here the pros and cons for these suggestions. We conclude that the novel UCPs do not seem to be physiologically relevant uncoupling proteins; the uncoupling property was apparently a late introduction into the subfamily through the evolution of UCP1. Physiological functions ascribed to UCP2 and UCP3 based on their purported uncoupling property may have to be revised (i.e. any type of thermogenesis, including protection against obesity, protection against the formation of reactive oxygen species and thermogenic involvement in the fever response). The presence of a mixed genetic background in most published studies of UCP2 or UCP3 gene-ablated mice also means that data concerning marked differences in diabetes propensity, infection sensitivity and production of reactive oxygen species may require confirmation in backcrossed mice. The increased expression of UCP2 and UCP3 under conditions of increased fatty acid metabolism implies an as yet undefined role in lipid metabolism. Thus, the novel UCPs should probably be considered as mitochondrial carriers, and the challenge now is to identify the transported molecule.     

Cell density in the border zone around old small human brain infarcts

Nine brain autopsy cases of small old cerebral infarcts were selected for neuropathological studies. Eight of the patients had cortical infarcts, in two cases with extension into the striate body. In one case the infarct involved the striate body only. The density of neurons and glial cells was measured in the coronal and the horizontal planes at various distances from the margin of the infarct. Corresponding counting points in the contralateral hemisphere served as control. On light microscopy, the infarcted cortex was irregularly shaped, but on serial sections the bulging parts appeared to be cut off from the infarcted tissue ("pseudo-infarct islands"). The zone of transition from infarcted to normal brain tissue was less than a few mm wide. In one patient, tomographic measurements of the cerebral blood flow (CBF) and a CT scan could be compared with the neuropathological findings. In this patient, CBF in the surroundings of the infarct was decreased despite a normal neuronal density. The study supports the traditional view held by pathologists that a sharp transition exists between infarcted and normal brain tissue and suggests that the hypoperfusion zone surrounding the region of complete infarction may be due to mechanisms other than selective loss of neurons.     

Lack of presynaptic modulation by isoprenaline of 3H-noradrenaline release from rabbit isolated ear artery

Summary The aim of the present investigation was to examine whether or not presynaptic facilitatory -adrenoceptors are detectable on the postganglionic nerves in the rabbit isolated ear artery. Strips of rabbit central ear artery were incubated with ³H-noradrenaline (10^–7 mol/l; 30 min or 10^–6 mol/l; 60 min). Subsequently, they were washed repeatedly with physiological salt solution. The strips were subjected to electrical-field stimulation (S₁–S₈) and the resultant ³H-overflow was determined.When the ear artery was stimulated with 150 pulses (0.5 ms; 3 Hz; 225 mA), isoprenaline (10^–9–10^–6 mol/l) either alone or in the presence of either rauwolscine (10^–6 mol/l) or phentolamine (10^–6 mol/l) did not alter the stimulation-evoked ³H-overflow. This was also the case in the presence of rauwolscine (10^–6 mol/l) plus either the selective phosphodiesterase inhibitor ICI 63 197 (3 × 10^–5 mol/l) or forskolin (10^–6 mol/l). When the ear artery was stimulated with 300 pulses (1 ms; 5 Hz; 225 mA), isoprenaline had no effect on the stimulation-evoked ³H-overflow. This was also the case when phentolamine (10^–6 mol/l) was present. Propranolol (10^–7–10^–5 mol/l) did not alter the stimulation-evoked ³H-overflow. In some experiments, the stimulation current was reduced to 175 mA in order to obtain similar reference release (S₃) values despite the presence of rauwolscine (150 pulses; 0.5 ms; 3 Hz). Even then, isoprenaline (10^–9–10^–6 mol/l) did not change stimulation-evoked ³H-overflow. The results suggest that postganglionic sympathetic nerves in rabbit central ear artery do not possess presynaptic facilitatory -adrenoceptors. Send offprint requests to J. Abrahamsen at the above address     

HE4 as a predictor of adjuvant chemotherapy resistance and survival in patients with epithelial ovarian cancer

The aim of this study was to investigate the value of serum human epididymis protein 4 (HE4) and HE4 tissue protein expression to predict tumor resistance to adjuvant chemotherapy, progression‐free survival (PFS), and overall survival in patients with epithelial ovarian cancer (EOC). Consecutive inclusion of 198 patients diagnosed with EOC was conducted. Blood samples were collected prior to surgery and tissue samples during surgery. Patient data were registered prospectively in the Danish Gynecologic Cancer Database. The association between serum HE4 and HE4 tissue protein expression, resistance to adjuvant chemotherapy, PFS, and overall survival were analyzed in univariate analyses and in multivariate analyses adjusted for age, performance score, surgical outcome, stage, grade, and histological subtype. Serum HE4 levels predicted chemotherapy resistance, PFS, and overall survival correlated significantly (p < 0.001) in the univariate analyses; but after adjustment in a multivariate model, serum HE4 was insignificant, except in a subgroup analysis of postmenopausal women, where serum HE4 significantly predicted resistance to chemotherapy and progression‐free survival. HE4 tissue protein expression predicted PFS (p = 0.022) and overall survival (p = 0.047) in the univariate analysis, while HE4 tissue protein expression failed to predict these outcomes in the adjusted multivariate analyses. Serum HE4 or HE4 tissue protein expression are not independent factors of chemotherapy resistance or survival in patients with EOC, but serum HE4 might predict chemotherapy resistance and PFS in postmenopausal women.     

Connexins regulate calcium signaling by controlling ATP release

Forced expression of gap junction proteins, connexins, enables gap junction-deficient cell lines to propagate intercellular calcium waves. Here, we show that ATP secretion from the poorly coupled cell lines, C6 glioma, HeLa, and U373 glioblastoma, is potentiated 5- to 15-fold by connexin expression. ATP release required purinergic receptor-activated intracellular Ca²⁺ mobilization and was inhibited by Cl⁻ channel blockers. Calcium wave propagation also was reduced by purinergic receptor antagonists and by Cl⁻ channel blockers but insensitive to gap junction inhibitors. These observations suggest that cell-to-cell signaling associated with connexin expression results from enhanced ATP release and not, as previously believed, from an increase in intercellular coupling.     

Target-mediated clearance and bio-distribution of a monoclonal antibody against the Kunitz–type protease inhibitor 2 domain of Tissue Factor Pathway Inhibitor

Introduction

A humanised monoclonal antibody, concizumab, that binds with high affinity to the Kunitz-type protease inhibitor (KPI) 2 domain of human tissue factor pathway inhibitor (TFPI) is in clinical development. It promotes coagulation by neutralising the inhibitory function of TFPI and may provide a subcutaneous prophylaxis option for patients with haemophilia. We aimed to study biodistribution and pharmacokinetics (PK) of concizumab.

Materials and Methods

Blockage of cellular TFPI by concizumab was measured by tissue factor/Factor VIIa-mediated Factor X activation on human EA.hy926 cells. Biodistribution of concizumab was analysed in rabbits by immunohistology, and the PK was measured in rabbits and rats.

Results and Conclusions

Concizumab bound to cell surface TFPI on EA.hy926 cells and neutralised TFPI inhibition of Factor X activation. The antibody cross-reacted with rabbit TFPI, but not with rat TFPI, allowing for comparative PK studies. PK data in rats described a log-linear profile typical for a non-binding antibody, whereas PK data in rabbits revealed a non-linear, dose-dependent profile, consistent with a target-mediated clearance mechanism. Immunohistology in rabbits during target-saturation showed localisation of the antibody on the endothelium of the microvasculature in several organs. We observed a marked co-localisation with endogenous rabbit TFPI, but a negligible sub-endothelial build-up. Concizumab binds and neutralises the inhibitory effect of cell surface-bound TFPI. The PK profile observed in rabbits is consistent with a TFPI-mediated drug disposition. Double immunofluorescence shows co-localisation of the antibody with TFPI on the endothelium of the microvasculature and points to this TFPI as a putative target involved in the clearance mechanism.     

Alpha1- and beta1-adrenoceptor signaling fully compensates for beta3-adrenoceptor deficiency in brown adipocyte norepinephrine-stimulated glucose uptake

To assess the relative roles and potential contribution of adrenergic receptor subtypes other than the beta3-adrenergic receptor in norepinephrine-mediated glucose uptake in brown adipocytes, we have here analyzed adrenergic activation of glucose uptake in primary cultures of brown adipocytes from wild-type and beta3-adrenergic receptor knockout (KO) mice. In control cells in addition to high levels of beta3-adrenergic receptor mRNA, there were relatively low alpha1A-, alpha1D-, and moderate beta1-adrenergic receptor mRNA levels with no apparent expression of other adrenergic receptors. The levels of alpha1A-, alpha1D-, and beta1-adrenergic receptor mRNA were not changed in the beta3-KO brown adipocytes, indicating that the beta3-adrenergic receptor ablation does not influence adrenergic gene expression in brown adipocytes in culture. As expected, the beta3-adrenergic receptor agonists BRL-37344 and CL-316 243 did not induce 2-deoxy-d-glucose uptake in beta3-KO brown adipocytes. Surprisingly, the endogenous adrenergic neurotransmitter norepinephrine induced the same concentration-dependent 2-deoxy-D-glucose uptake in wild-type and beta3-KO brown adipocytes. This study demonstrates that beta1-adrenergic receptors, and to a smaller degree alpha1-adrenergic receptors, functionally compensate for the lack of beta3-adrenergic receptors in glucose uptake. Beta1-adrenergic receptors activate glucose uptake through a cAMP/protein kinase A/phosphatidylinositol 3-kinase pathway, stimulating conventional and novel protein kinase Cs. The alpha1-adrenergic receptor component (that is not evident in wild-type cells) stimulates glucose uptake through a phosphatidylinositol 3-kinase and protein kinase C pathway in the beta3-KO cells.