Albumin uptake in OK cells exposed to rotenone: a model for studying the effects of mitochondrial dysfunction on endocytosis in the proximal tubule?

BACKGROUND: The renal proximal tubule (PT) is clinically vulnerable to mitochondrial dysfunction; sub-lethal injury can lead to the Fanconi syndrome, with elevated urinary excretion of low-molecular-weight proteins. As the mechanism that couples mitochondrial dysfunction to impaired PT low-molecular weight protein uptake is unknown, we investigated the effect of respiratory chain (RC) inhibitors on endocytosis of FITC-albumin in PT-derived OK cells. METHODS: Uptake of FITC-albumin was quantified using confocal microscopy. Cytosolic ATP levels were measured in real time using both luciferin/luciferase assays and measurements of free [Mg(2+)]. Reactive oxygen species production was measured using mitosox. RESULTS: RC blockade produced only a small decrease in cytosolic ATP levels and had minimal effect on FITC-albumin uptake. Inhibition of glycolysis caused a much bigger decrease in both cytosolic ATP levels and FITC-albumin endocytosis. Rotenone led to higher rates of reactive oxygen species production than other RC inhibitors. Rotenone also caused widespread structural damage on electron microscopy, which was mimicked by colchicine and prevented by taxol; consistent with inhibition of microtubule polymerisation as the underlying mechanism. CONCLUSIONS: Endocytosis of FITC-albumin is ATP-dependent in OK cells, but the cells are very glycolytic and therefore represent a poor metabolic model of the PT. Rotenone has toxic extra-mitochondrial structural effects.

Diabetes mellitus-related morphoquantitative changes in the celiac ganglion neurons of the dog.

Diabetes mellitus is the most common endocrine disturbance of domestic carnivores and can cause autonomic neurological disorders, although these are still poorly understood in veterinary medicine. There is little information available on the quantitative adaptation mechanisms of the sympathetic ganglia during diabetes mellitus in domestic mammals. By combining morphometric methods and NADPH-diaphorase staining (as a possible marker for nitric oxide producing neurons), type I diabetes mellitus-related morphoquantitative changes were investigated in the celiac ganglion neurons in dogs. Twelve left celiac ganglia from adult female German shepherd dogs were examined: six ganglia were from non-diabetic and six from diabetic subjects. Consistent hypertrophy of the ganglia was noted in diabetic animals with increase of 55% in length, 53% in width, and 61.5% in thickness. The ordinary microstructure of the ganglia was modified leading to an uneven distribution of the ganglionic units and a more evident distribution of axon fascicles. In contrast to non-diabetic dogs, there was a lack of NADPH-diaphorase perikarial labelling in the celiac ganglion neurons of diabetic animals. The morphometric study showed that both the neuronal and nuclear sizes were significantly larger in diabetic dogs (1.3 and 1.39 times, respectively). The profile density and area fraction of NADPH-diaphorase-reactive celiac ganglion neurons were significantly larger (1.35 and 1.48 times, respectively) in non-diabetic dogs compared to NADPH-diaphorase-non-reactive celiac ganglion neurons in diabetic dogs. Although this study suggests that diabetic neuropathy is associated with neuronal hypertrophy, controversy remains over the possibility of ongoing neuronal loss and the functional interrelationship between them. It is unclear whether neuronal hypertrophy could be a compensation mechanism for a putative neuronal loss during the diabetes mellitus.

Chlorimipramine: a novel anticancer agent with a mitochondrial target.

Mitochondria have been suggested to be a potential intracellular target for cancer chemotherapy. In this report, we demonstrate the ability of the tricyclic antidepressant chlorimipramine to kill human glioma cells in vitro by a molecular mechanism resulting in an increase in caspase 3 activity following inhibition of glioma oxygen consumption. Studies with isolated rat mitochondria showed that chlorimipramine specifically inhibited mitochondrial complex III activity, which causes decreased mitochondrial membrane potential as well as mitochondrial swelling and vacuolation. The use of chlorimipramine in human as an effective, non-toxic cancer therapeutic having a strong selectivity between cancer cells and normal cells on the basis of their mitochondrial function is discussed.

Human saphenous vein and coronary bypass surgery: ultrastructural aspects of conventional and "no-touch" vein graft preparations.

Coronary artery bypass graft surgery (CABG) is routinely used to restore blood flow to diseased cardiac muscle due to coronary artery disease. The patency of conventional grafts decreases with time, which is due to thrombosis and formation of neointima. A primary cause of graft failure is the mechanical damage inflicted to the graft during harvesting, including removal of surrounding tissue accompanied by high pressure saline distension to overcome vasospasm (both causing considerable mechanical trauma). The aim of this study was to compare the ultrastructural features of human saphenous vein (SV) grafts harvested conventionally and grafts prepared using an atraumatic 'no-touch' harvesting technique introduced by Souza (1996). The results of this study showed a better preservation of the lumenal endothelium and medial vascular smooth muscle (SM) in 'no-touch' versus conventional grafts. A 'fast' (within 30 min) response of SM cells to conventional harvesting was noted where features of both SM cell division and apoptosis were observed. It is concluded that the 'preserved' nature of the 'no-touch' aortocoronary SV grafts renders them less susceptible to thrombotic and atherosclerotic factors than grafts harvested conventionally. These features are suggested to contribute to the improved early patency rate described using the no-touch technique of SV harvesting.

Basilar artery of the capybara (Hydrochaeris hydrochaeris): an ultrastructural study.

The present study investigated the ultrastructural features of the basilar artery of the largest rodent species, the capybara. The study suggests that the general ultrastructural morphological organization of the basilar artery of the capybara is similar to that of small rodents. However, there are some exceptions. The basilar artery of the capybara contains a subpopulation of 'granular' vascular smooth muscle cells resembling monocytes and/or macrophages. The possibility cannot be excluded that the presence of these cells reflects the remodelling processes of the artery due to animal maturation and the regression of the internal carotid artery. To clarify this issue, more systemic studies are required involving capybaras of various ages.

P2X4 and P2X6 receptors associate with VE-cadherin in human endothelial cells.

We investigated the expression of P2X4 and P2X6 receptors on human umbilical vein endothelial cells (HUVECs) and found that both P2X receptor subtypes on plasma membranes are largely restricted to areas of cell-cell contact. Co-labelling experiments at the confocal and electron microscopy levels revealed that P2X4 and P2X6 receptors are strongly co-localised with the cell adhesion molecule VE-cadherin. The P2X4 and P2X6 receptors on plasma membranes at cellular junctions are rapidly (within 5 min) internalised specifically after decreasing extracellular [Ca2+]. Disruption of microfilaments, microtubules and integrin-mediated adhesion or stimulation of P2 receptors with ATP did not alter P2X4 and P2X6 receptor expression on HUVEC plasma membranes. Membraneous P2X4 and P2X6 receptors resisted extraction with Triton-X 100, whereas cytoplasmic P2X receptors were Triton-X 100 soluble. P2X4 receptors, but not P2X6 receptors, could be co-immunoprecipitated with VE-cadherin and vice versa. We conclude that P2X4 and P2X6 receptors are associated with VE-cadherin at HUVEC adherens junctions.

Perivascular nerves and vascular endothelium: recent advances.

Within the last few years, advances have been made regarding perivascular nerves and the endothelium of the vascular system, both potentially important in the understanding of the mechanisms of local control of blood flow. Endothelin-1 (ET-1) has been identified in rat cerebrovascular nerves, neuropeptide Y (NPY) has been demonstrated in umbilical endothelium, the arginine-vasopressin (VP) system has been discovered in the heart (including coronary endothelium), and P2X receptors have been observed in vascular endothelial cells. After a brief introduction to vascular biology, this review will focus on the above-mentioned new data.

Ultrastructural identification of P2Y2 receptor mRNA in the rat thymus.

In situ hybridisation to identify P2Y(2) receptor mRNA was performed for the first time at the ultrastructural level on the thymus of adult male rats. These studies revealed transcripts for P2Y(2) receptors in cortical T cells and endothelial cells of thymic blood vessels. These transcripts are likely to be linked with the production of functional P2Y(2) receptors in these cells. In the T cells, transcripts for the P2Y(2 )receptor were localised in the cytoplasm as well as on the smooth endoplasmic reticulum and cell membrane. Dividing T cells also expressed P2Y(2 )receptor mRNA, mostly in the cytoplasm around chromosomal material. Endothelial cells displaying labels for P2Y(2 )receptor transcripts were of cortical arteries/arterioles and capillaries and of postcapillary venules in the corticomedullary junction. P2Y(2) mRNA transcripts were localised in the cytoplasm of endothelial cells, although they did not appear to be specifically associated with subcellular organelles or structures. In postcapillary venules, T cells displaying labelling for the P2Y(2) receptor were seen migrating across the P2Y(2) receptor mRNA-positive endothelium. Our findings are discussed in terms of the relationship between thymic immune cells and the endothelium. This includes the issue of immune cell trafficking into the circulation, and the ATP-related regulatory role and involvement of P2Y(2) receptors in the rat thymus.

Immunoreactivity to P2X(6) receptors in the rat hypothalamo-neurohypophysial system: an ultrastructural study with extravidin and colloidal gold-silver labelling.

The distribution of the purine receptor P2X(6) subtype was studied in the rat hypothalamo-neurohypophysial system at the electron microscope level. Receptors were visualised with ExtrAvidin peroxidase conjugate and immunogold-silver pre-embedding immunocytochemistry using a polyclonal antibody against an intracellular domain of the receptor. Application of ExtrAvidin labelling revealed P2X(6) receptors in subpopulations of: (i) neurosecretory cell bodies, neurosecretory and non-neurosecretory axons and dendrites of neurones in the paraventricular and supraoptic nuclei; and (ii) pituicytes and neurosecretory axons of the neurohypophysis. Some of the neurosecretory granules observed in the supraoptic and paraventricular nuclei neurone cell bodies, dendrites and axons as well as those in neurohypophysial axons were also positive for the P2X(6) receptors. In the paraventricular nucleus, some axons and dendrites of non-neurosecretory neurones positive for P2X(6) receptors formed synapses between themselves. Using the immunogold-silver method, the electron-dense particles labelling P2X(6) receptors were found in neurosecretory cell bodies of the supraoptic and paraventricular nuclei, in relation to the cytoplasm, endoplasmic reticulum, Golgi complex and neurosecretory granules. The particles indicative of P2X(6) receptors were also located in neurosecretory and non-neurosecretory axons including axonal buttons making synapses with P2X(6)-negative dendrites. In the neurohypophysis, the electron-dense particles were localised in a subpopulation of pituicytes and neurosecretory axons. In neurohypophysial axons, particles were at times seen over the membrane of some neurosecretory granules (immunogold label) or microvesicles (immunoperoxidase label). We speculate that the P2X(6) receptors at the neurohypophysial level may be implicated not only in hormone release from the axon terminals, but also in membrane recycling of the granular vesicles and microvesicles.

Ultrastructural localisation of ATP-gated P2X2 receptor immunoreactivity in vascular endothelial cells in rat brain.

In this report, we show for the first time that P2X2 receptors--ATP-gated cation channels--can be demonstrated in endothelial cells of small cerebral vessels of rat. Immunoreactivity to P2X2 receptors was visualised at the ultrastructural level with electron-immunocytochemistry (ExtrAvidin-horseradish peroxidase technique) using a polyclonal antibody against the fragment of an intracellular domain of the receptor. The possibilities that these receptors may regulate the formation of gap and/or tight junctions between adjacent endothelial cells influencing the blood-brain barrier, or modulate the contractility of capillary endothelial cells are discussed.

Ultrastructural localization of arginine vasopressin in coronary vessels of newborn rat.

We report on the ultrastructural distribution of arginine-vasopressin (AVP) in the heart of newborn rats using pre-embedding peroxidase-antiperoxidase immunocytochemistry with a polyclonal AVP antibody for electron microscopy. Positive labelling for AVP was localized in endothelial cells of main coronary arteries and cardiac vessels of smaller diameter (microvessels). Examination of the right coronary artery showed that approximately 58% of the endothelial cells were positive for AVP. Immunoreactivity to AVP in the cytoplasm of arterial endothelium predominated in association with the membranes of granular endoplasmic reticulum and in subplasmalemmal areas. The endothelium of small vessels exhibits less endoplasmic reticulum, but still shows AVP immunoprecipitate in the cytoplasm. It is suggested that endothelial AVP may contribute to vasomotor control of the coronary circulation in the early stages of postnatal development. AVP antibody also labelled some fibroblast/fibroblast-like cells associated with the coronary arteries and microvessels; thus, these cells as well as the endothelium appear to be a source of AVP in the newborn rat heart. The functional significance of these findings is discussed.

Neural endothelin in hypertension: increased expression in ganglia and nerves to cerebral arteries of the spontaneously hypertensive rat.

Endothelin has previously been localised in perivascular nerves of the rat basilar artery. Considering its potent vasoconstrictor and mitogenic properties on vascular smooth muscle, the potential role of a neural source of this peptide in hypertension has been investigated. The trigeminal, superior cervical and sphenopalatine ganglia of Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) at 16 weeks of age have been examined for immunolocalisation of endothelin at the light and electron microscope level. At the light microscope level, neurones immunopositive for endothelin were detected in these ganglia of the SHR but were not seen in ganglia from WKY rats. This difference was particularly marked in the trigeminal ganglia where endothelin-positive neurones colocalised with substance P immunoreactivity. Using in situ hybridisation techniques, endothelin-1 mRNA was localised to the cytoplasm of neurones in the ganglia and was more prominent in the SHR. At the electron microscope level, endothelin-immunoreactivity was localised at the peripheral perikarya of some neuronal cell bodies of the trigeminal, superior cervical and sphenopalatine ganglia of WKY rats but was more prominent with heavy labelling throughout the cytoplasm of neurones in the SHR. Notably, in the trigeminal ganglia of the SHR only, some endothelin-immunopositive nerve fibres appeared to be damaged and contained vacuoles with granular material. Ultrastructural examination of the basilar artery revealed an increased number of endothelin-positive axons in the SHR, but these axons usually showed selective damage. In summary, in the SHR, there was a marked increase in endothelin particularly in sensory neurones projecting to the basilar artery which also appear to be undergoing degenerative changes. An increased neural source of endothelin in the SHR may contribute to the development of hypertension or may be a consequence of selective degenerative change.

Induction of proliferation and apoptotic cell death via P2Y and P2X receptors, respectively, in rat glomerular mesangial cells.

BACKGROUND: Cell surface receptors for adenosine 5'-triphosphate (ATP; P2 receptors) have been subdivided into two families: ligand-gated ion channels (P2X1-7) and G-protein-coupled (P2Y1-8) receptors. We investigated the potential role of P2 receptors on rat glomerular mesangial cells. METHODS: To investigate cell proliferation, DNA synthesis was assayed by measuring [3H]thymidine incorporation into DNA. For detecting apoptosis, morphological features, DNA fragmentation, and exposure of phosphatidylserine on the outside surface of the cell membrane were investigated. Expression of mRNA and distribution of receptors were detected by reverse transcription-polymerase chain reaction and immunohistochemistry, respectively. RESULTS: ATP triggered a dose-dependent increase in DNA synthesis. This response was also induced by uridine triphosphate (UTP), an agonist equipotent with ATP at P2Y2 and P2Y4 receptors; both P2Y2 and P2Y4 mRNA are expressed in glomerular mesangial cells and isolated glomeruli. In contrast, the P2X7 receptor agonist 2'-83'-O-(4-benzoyl benzoyl) ATP (BzATP) caused a decrease in cell number. BzATP produced DNA cleavage and exposure of phosphatidylserine on the outside of the cell membrane. P2X7 receptors were distributed heterogeneously in unstimulated cells. The expression of P2X7 mRNA was maintained at a low level, but was induced by tumor necrosis factor-alpha. CONCLUSIONS: Stimulation of glomerular mesangial cells via P2Y2 and/or P2Y4 and via P2X7 receptors can induce proliferation and apoptotic cell death, respectively. The balance between proliferation and apoptosis will depend on the relative stimulation and expression of these P2 receptor subtypes, and could play an important role in normal and abnormal glomerular function.

Endothelin immunoreactivity and mRNA expression in sensory and sympathetic neurones following selective denervation.

The localization of endothelin (ET) in perivascular nerve varicosities supports pharmacological evidence that ET is a neurotransmitter in the autonomic nervous system. To examine the potential source of ET previously localized in cerebrovascular nerves, ganglia which send projections to these vessels were immunolabelled for ET and examined at the ultrastructural level. The trigeminal (TG) and superior cervical ganglia (SCG) were examined in control rats and following either sensory denervation or sympathectomy. In control TG, ET immunolabelling was detected throughout the cytoplasm of a subpopulation of neurones whereas in the SCG only the occasional ET-positive neurone was seen. Following sensory denervation with capsaicin, very few ET-immunoreactive nerve cell bodies or nerve fibres were detected in the TG compared with control ganglia, suggesting that ET is predominantly localized in primary afferent neurones, although some remaining myelinated nerve fibres stained positively. ET labelling of neurones in the SCG was unaffected by sensory denervation. Following selective damage to sympathetic nerves with 6-hydroxydopamine, there was a marked increase in intensity of ET-labelling of nerve fibres in the TG, probably due to increased availability of nerve growth factor for sensory nerves. There was no effect on ET immunoreactivity in the nerve cell bodies and nerve fibres within the SCG. However, in situ hybridization techniques demonstrated that 6-hydroxydopamine sympathectomy resulted in a marked increase in ET-1 mRNA expression in the SCG neurones. In conclusion, sensory nerves projecting from the TG are a more likely source of ET-positive perivascular nerves in cerebral arteries than sympathetic nerves from the SCG. Damaged sympathetic neurones markedly increase ET mRNA expression. In view of the neuroprotective properties of ET, this may represent a compensatory mechanism to promote repair.

Electron-immunocytochemical studies of perivascular nerves of mesenteric and renal arteries of golden hamsters during and after arousal from hibernation.

Electron immunocytochemistry was used to examine perivascular nerves of hamster mesenteric and renal arteries during hibernation and 2 h after arousal from hibernation. Vessels from cold-exposed but nonhibernating, and normothermic control hamsters were also examined. During hibernation the percentage of axon profiles in mesenteric and renal arteries that were immunopositive for markers of sympathetic nerves, tyrosine hydroxylase (TH) and neuropeptide Y (NPY), were increased 2-3 fold compared with normothermic and cold control animals. This increase was reduced markedly only 2 h after arousal from hibernation. The small percentage of nitric oxide synthase-1-positive axon profiles found in mesenteric (but not renal) arteries was also increased during hibernation and returned towards control values after arousal. In contrast, the percentage of perivascular axons immunostaining for vasoactive intestinal polypeptide (VIP), a marker for parasympathetic nerves, was reduced in mesenteric arteries during hibernation. There was no labelling of perivascular nerves for substance P in either mesenteric or renal arteries. It is suggested that the increase in percentage of TH- and NPY-immunostained perivascular nerves may account for the increased vasoconstriction associated with high vascular resistance that is known to occur during hibernation. The reduction in the percentage of axons positive for VIP in hibernating animals would contribute to this mechanism since this neuropeptide is a vasodilator.

Ultrastructural localization of nitric oxide synthase and endothelin in the renal and mesenteric arteries of the golden hamster: differences during and after arousal from hibernation.

This is a study of the electron-immunocytochemical localization of nitric oxide synthase (type III) and endothelin in renal and mesenteric artery endothelial cells of normal (active) and hibernating hamsters, as well as hamsters exposed to the cold but not hibernating, and hamsters aroused for 2h following hibernation. In the renal artery of hibernating hamsters and cold-exposed hamsters, a subpopulation of nitric oxide synthase-positive endothelial cells displayed immunoprecipitate predominantly in the vicinity of the Golgi complex indicating intracellular translocation from the cytoplasm to the Golgi complex. In hibernating animals, the percentages of both nitric oxide synthase-positive and endothelin-positive endothelial cells were notably lower than those observed either in active, cold-exposed or aroused animals. These changes may reflect a reduced endothelial contribution to the maintenance of vascular tone in these vessels during hibernation and an upregulation of expression of nitric oxide synthase and endothelin in the endothelium early on during arousal from hibernation.

Nitric oxide and endothelin-1 in coronary and pulmonary circulation.

Since the discovery of the vasorelaxant properties of nitric oxide and the vasoconstrictor effect of endothelin-1, there have been many studies of the distribution and functional significance of these agents in various vascular beds. In the coronary and pulmonary circulation nitric oxide and endothelin-1 actions have been largely investigated in terms of an imbalance between the opposing effects of these vasoactive agents leading to pathophysiological conditions. This article review functional and immunocytochemical studies with emphasis on the ultrastructural localization of nitric oxide synthase and endothelin-1 in the coronary and pulmonary vascular beds. Localization of nitric oxide synthase (type III or I or II) has been shown in endothelial cells, smooth muscle, and perivascular nerves of the coronary and pulmonary vascular beds and in the neurons, nerve fibers, and the small granule-containing cells within cardiac ganglia. Endothelin-1 was mainly localized in subpopulations of coronary and pulmonary endothelial cells. These immunocytochemical studies provide information about the sources of nitric oxide and endothelin-1 that contribute to the vasomotor control of cardiac and pulmonary circulation under normal and pathophysiological conditions.

Ultrastructural localisation of ATP-gated P2X2 receptor immunoreactivity in the rat hypothalamo-neurohypophysial system.

The distribution of the P2X(2) subtype of the purine receptor associated with the extracellular signalling activities of ATP was studied in the rat hypothalamo-neurohypophysial system at the electron microscope level. Receptors were labelled with ExtrAvidin-horseradish peroxidase preembedding immunocytochemistry using a polyclonal antibody against a fragment of an intracellular domain of the receptor. Immunoreactivity to P2X(2) receptors was localised in: (i) paraventricular and supraoptic nuclei-in subpopulations of endocrine neurones, neurosecretory and non-neurosecretory axons and dendrites; and (ii) the neurohypophysis-in pituicytes and subpopulation of neurosecretory axons. In both the hypothalamic nuclei examined, labelled asymmetric axo-dendritic synapses were commonly observed. These synapses involved either P2X(2)-labelled axon terminals (synaptic buttons) and unlabelled dendrites or labelled dendrites and unlabelled axon terminals. Axo-somatic synapses established by P2X(2)-positive axons on P2X(2)-positive endocrine cell bodies as well as on P2X(2)-negative somata were also observed. The functional significance of these findings is discussed.

Electron-immunocytochemical localization of P2X1 receptors in the rat cerebellum.

The distribution of the P2X1 subtype of purinoceptors associated with the extracellular activities of ATP was studied in the rat cerebellum at the electron-microscope level. Receptors were labelled with peroxidase-antiperoxidase and the avidin-biotin-peroxidase complex for immunocytochemistry. Immunoreactivity to P2X1 receptors was localized in subpopulations of synapses between varicosities of parallel fibres of granule cells and dendritic spines of Purkinje cells. Unlabelled varicosities of parallel fibres formed asymmetric synapses with labelled dendritic spines, whereas labelled varicosities of parallel fibres formed asymmetric synapses with unlabelled dendritic spines. P2X1 immunoreactivity was also localized in some astrocyte processes. The functional significance of these findings is discussed.

CYFRA 21-1 serum levels in women with adnexal masses and inflammatory diseases.

The aim of the present study was to evaluate the clinical usefulness of the cytokeratin marker CYFRA 21-1 as a screening marker for ovarian cancer, as a predictive marker in patients with adnexal masses and as a prognostic marker in women suffering from ovarian cancer. In order to determine the specificity of the CYFRA 21-1 test, we have investigated CYFRA 21-1 serum levels in several benign conditions. This retrospective study comprises 37 patients suffering from ovarian cancer FIGO stages Ia-III. Sera from patients with benign ovarian cysts, endometriosis, pelvic inflammatory disease, inflammatory bowel disease and liver cirrhosis were evaluated in 90, 10, 38, 10 and 20 cases respectively. With a sensitivity of 41% and a specificity of 95%, CYFRA 21-1 was not suitable as a screening marker for ovarian cancer. Although CYFRA 21-1 was able to discriminate between ovarian cancer and benign adnexal tumours (univariate regression model, P = 0.0001), CYFRA 21-1 did not reveal additional information to CA 125 in a multivariate regression analysis (P = 0.06). CYFRA 21-1 serum levels were elevated in benign conditions such as liver cirrhosis, but not in endometriosis and inflammatory diseases. In ovarian cancer patients, elevated CYFRA 21-1 serum levels before therapy were associated with a poor overall and disease-free survival (log-rank test, P = 0.02 and log-rank test, P = 0.005 respectively). CYFRA 21-1, while obviously not suitable for screening or differential diagnosis of adnexal masses, could be useful as an additional prognostic factor in ovarian cancer patients.