Induction of heme oxygenase in guinea-pig stomach: roles in contraction and in single muscle cell ionic currents.

The role of heme oxygenase reaction products in modulation of stomach fundus excitability was studied. The presence of constitutive heme oxygenase 2 was verified in myenteric ganglia by immunohistochemistry. The role of inducible heme oxygenase isoenzyme was investigated after invivo treatment of animals with CoCl2 (80 mg kg-1 b.w) injected subcutaneously 24 h before they were killed. This treatment resulted in increased production of bilirubin and positive staining for the inducible isoform in stomach smooth muscle and vast induction in the liver. In both control and treated animals haemin, applied to the bath as a substrate of heme oxygenase caused significant decrease of prostaglandin F2alpha-induced tone, and ameliorated the relaxatory response of the fundic strips to electrical field stimulation. Both effects were antagonized by Sn-protoporphyrin IX, competitive heme oxygenase inhibitor, and were found to be neuronally dependent. In single freshly isolated smooth muscle cells from control animals haemin caused a concentration-dependent increase of the whole cell K+ currents, which was not affected by Sn-protoporphyrin IX, cyclic guanosine monophosphate (cGMP)-dependent protein kinase or guanylyl cyclase antagonists, but was reversed by various antioxidants and abolished by an NO scavenger. In cells from treated animals the K+ current increasing effect of haemin did not depend on the presence of antioxidants, but was abolished by protein kinase G and guanylyl cyclase inhibitors, depletors of intracellular Ca2+ pools or Sn-protoporphyrin IX. Biliverdin did not affect contraction or ionic currents. Thus, this is the first study demonstrating that heme oxygenase is an inducible enzyme in guinea-pigs, which exerts a modulatory role on gastric smooth muscle excitability via carbon monoxide production.

Heme oxygenase is the main protective enzyme in rat liver upon 6-day administration of cobalt chloride.

Changes in the activities of rat liver heme oxygenase (HO), superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and glutathione reductase (GR), as well as changes in lipid peroxidation and reduced glutathione (GSH) levels were measured after acute loading and chronic administration of cobalt chloride (CoCl2). Acute loading was achieved by a single subcutaneous injection of 60 mg CoCl2/kg body weight for 24 h. Chronic administration was performed by giving the same total amount of CoCl2 in small doses over longer periods of time: 30 mg CoCl2/kg daily for 2 days, 15 mg CoCl2/kg daily for 4 days, or 10 mg CoCl2/kg daily for 6 days. The results showed that HO activity was increased both after acute loading (7-fold increase) and upon 6-day administration of CoCl2 (5-fold increase). The GSH level, 24 h after a single injection of CoCl2, was lower than that of the control animals. However, upon chronic administration of small doses CoCl2, the level of GSH increased and was accompanied by an increase in GR activity. Chronic administration of CoCl2 produced persistent oxidative stress, which was illustrated with a continuous increase in lipid peroxidation. At the same time, under these conditions, the activities of oxidative-stress-protective enzymes were either inhibited (SOD, catalase) or not significantly changed (GPx). Collectively, these findings suggest that the sustained up-regulation of HO activity in rat liver upon 6 day administration of CoCl2 would be beneficial by providing the cells with antioxidants, biliverdin and bilirubin, and together with the increased levels of GSH would act as a part of the defence mechanisms against the cobalt-induced oxidative stress.

Heme oxygenase--carbon monoxide signalling pathway as a physiological regulator of vascular smooth muscle cells.

Heme oxygenase (HO) is a microsomal enzyme involved in the degradation of heme and biliverdin and carbon monoxide, the former being subsequently converted to bilirubin by the cytosolic biliverdin reductase. Two isoenzymes transcribed from separate genes have been characterized. The HO-2 isoform is constitutively expressed and is present in high concentration in the brain and testes. In contrast, the HO-1 isoform is ubiquitous, found in large quantities in liver and spleen and can be induced by its own substrate, heme and by a variety of stress-associated agents. Both HO-1 and HO-2 mRNA and protein have been detected in endothelial and smooth muscle cells of arterial and venous blood vessels. Carbon monoxide (CO) from HO catalysis has been identified as an endogenous biological messenger and recent studies suggest its important role in the circulation. Similarly to nitric oxide (NO), CO inhibits platelet aggregation and relaxes blood vessels by activating soluble guanylyl cyclase (sGC) and elevating intracellular levels of cyclic guanosine-3',5'-monophosphate (cGMP). CO is a powerful vasodilator and together with NO may serve as an important modulator of vascular cell function.

Nitric oxide synthase (NOS) I during postnatal development in rat and mouse skeletal muscle.

Previous studies on adult rat and mouse skeletal muscles have shown the spatial association of nitric oxide synthase (NOS) I to the dystrophin complex (DC) in the sarcolemma of type II fibers and, in combination with the NMDA receptor-1 (NMDAR-1), an accumulation of the enzyme at the neuromuscular junctions (NMJ) of this fiber type. Using immunohistochemistry, enzyme histochemistry and alpha-bungarotoxin labeling we report here temporal relationships of NOS I, members of the DC, other components of the cortical cytoskeleton in the junctional and non-junctional sarcolemma as well as of molecules involved in NMJ transmission of either type I or II myofibers especially in head and neck muscles during postnatal rat and mouse development. Fiber typing was performed by specific anti-myosin antibodies. Beginning with postnatal day (PD) 1 in both fiber types dystrophin, dystrophin-associated glycoproteins (DAG), beta-dystroglycan, alpha-sarcoglycan (adhalin) and spectrin were present in the junctional and extrajunctional sarcolemma, while utrophin, acetylcholinesterase, alpha-bungarotoxin labeled acetylcholine receptors were concentrated in the NMJ of both fiber types. NOS I activity and immunoreactivity were only found in the NMJ region of type II fibers, where NMDAR-1 appeared around PD 15. Primarily in the tongue there was no strict correlation between muscle fiber type and NOS I behaviour during early postnatal development, and muscle fibers not reactive for myosin antibodies against both fiber types were negative or positive for NOS I but always positive for the other molecules either in both the junctional and extrajunctional sarcolemma or in the NMJ only; later all muscle fibers of the tongue were of type II and NOS I-positive. Maturation of enzyme activities, immunoreactivities and AChR intensity depended on the respective muscle and can last until PD 50; in the tongue and neck muscles they appeared to increase approximately until PD 20 or 25. In conclusion, in type II fibers of rat and mouse skeletal muscle all molecules with the exception of NMDAR-1 and relevant for NOS I targeting and positioning as well as function inside and outside the NMJ are already present at birth, but their concentrations and/or activities increase postnatally, and the adult situation appears to be reached between the third and seventh week of postnatal life. Therefore, initial interactions between NOS I and the other molecules necessary for the formation of the NOS I-DC in and on the way to the sarcolemma presumably take place before birth.

Topographical distribution of NADPH-diaphorase-positive neurons in the cat's claustrum.

The NADPH-diaphorase histochemical technique was used to visualize the morphological features of the NADPH-diaphorase positive cells and fibres, and their distribution in both parts of the cat's claustrum. Taking into account the size and form of the perikaryon and the dendritic and axonal characteristics, the neurons are grouped in different subclasses: large, medium-sized and small. The present data suggest the occurrence of two populations of NADPH-diaphorase neurons in the claustrum. One population consisting of large and medium-sized positive neurons represents the projection neurons while the other population of small positive neurons corresponds to the local circuit neurons.

Differences in the localization of the postsynaptic nitric oxide synthase I and acetylcholinesterase suggest a heterogeneity of neuromuscular junctions in rat and mouse skeletal muscles.

Recently, nitric oxide synthase (NOS) I has been identified in skeletal muscle fibers, where the enzyme is found to be associated to the sarcolemma by the alpha 1-syntrophin-dystrophin complex. It has, however, been proposed that a substantial proportion of NOS I at the neuromuscular junction (NMJ) is of neuronal origin. We have, therefore, investigated the distribution of NOS I in NMJ of normal rats and mice as well as mdx mice which lack dystrophin and, consequently, NOS I in the sarcolemma region by enzyme histochemical and immunohistochemical techniques. Sites of NOS I accumulation, evident at NMJ of healthy animals, were absent in mdx mice, indicating a predominantly, if not exclusively, postsynaptic localization of NOS I at NMJ. Moreover, simultaneous demonstration of acetylcholinesterase (AChE) activity revealed a heterogeneity of NMJ in rat and mouse skeletal muscles: type I showed only AChE activity and was found to predominate; type II was spatially separated from the AChE-positive NMJ, occurred less frequently and contained both AChE activity and NOS I. These data suggest that type II NMJ are provided with additional regulatory mechanisms, such as free radical signaling by the NOS I-derived NO which may exert modulatory effects on the choline acetyltransferase/ACh/AChE pathway. Furthermore, type II may represent those NMJ where recently glutamate-gated NMDA-type Ca2+ channels have been described, which in analogy to those in the nervous system may serve also in skeletal muscle fibers as NOS I activators.

Absence of nitric oxide synthase I despite the presence of the dystrophin complex in human striated muscle.

Recently, it has been shown that in human striated muscle the signalling enzyme, brain-type nitric oxide synthase I (NOS I), is associated with the sarcolemma and complexes with dystrophin and/or members of the dystrophin complex. In order to find out whether there exists a regular association between NOS I and the complex, muscle biopsies from patients with various muscle disorders were analysed by enzyme histochemistry and immunohistochemistry. In patients suffering from Duchenne muscular dystrophy, and to a lesser extent in those with Becker-type dystrophy, NOS I and dystrophin complex components were absent or drastically reduced in the sarcolemma region. In other dystrophies, as well as in metabolic and inflammatory myopathies, NOS I and dystrophin complex constituents were expressed normally, while in the case of neurogenic diseases leading to denervation atrophy and especially congenital idiopathic clubfoot, the immunohistochemical patterns of the distribution of the dystrophin complex constituents were normal, but NOS I activity and protein were deficient or dramatically diminished. The results can be interpreted as indicating that, in general, NOS I targeting to the sarcolemma is dependent on particular members of the dystrophin complex, such as alpha-1 syntrophin, yet the expression and/or positioning of NOS I may be under the control of further factors, probably of neurogenic origin. NOS I-associated diaphorase may thus be a useful complementary tool in the diagnosis of muscle disorders.

Interaction between D-glyceraldehyde-3-phosphate dehydrogenase and calmodulin.

The effect of calmodulin on the associative properties of D-glyceraldehyde-3-phosphate dehydrogenase was investigated by means of a covalently attached fluorescent probe. We found that calmodulin shifts the equilibrium between the different forms of glyceraldehyde-3-phosphate dehydrogenase and binds to the subunits with an apparent dissociation constant of 1.8 microM. Within this heterologous complex calmodulin has no effect on the catalytic activity of the enzyme. The formation of the heterocomplex can be modulated by the specific anti-calmodulin drug, trifluoperazine, as well as by aldolase. The possible role of these associations is that they influence the interaction of both glyceraldehyde-3-phosphate dehydrogenase and calmodulin with other soluble proteins or structural elements.

Adhalin (alpha-sarcoglycan) is not required for anchoring of nitric oxide synthase I (NOS I) to the sarcolemma in non-mammalian skeletal (striated) muscle fibers.

Previous studies have shown the association of NOS I with the sarcolemma in mammalian striated muscle fibers, implicating the dystrophin complex (DC) as a major anchor for the enzyme. The potential role of the sarcoglycan subcomplex, especially of alpha-sarcoglycan (adhalin), as part of the DC in holding of NOS I in the sarcolemmal position was examined by carrying out a comparative study on the distribution of NOS I, dystrophin, dystrophin-associated glycoproteins (DAG) and alpha-sarcoglycan in various skeletal muscles of non-mammals. Rat muscles were included since they reflect the situation in mammals. Catalytic NOS-associated diaphorase (NOSaD) activity as well as NOS I and DAG immunoreactivities were positive in the saracolemma region of skeletal muscle fibers of rats, chicken, and turtles. Adhalin immunoreactivity was present in the rat but absent in the chicken and turtle muscle surface membrane. These data suggest that alpha-sarcoglycan and therefore the entire sarcoglycan subcomplex may not be needed for localizing NOS I to the sarcolemma in these non-mammalian species. This may hold for skeletal muscle fibers in general.

Demonstration of nitric oxide synthase (NOS) in marmosets by NADPH diaphorase (NADPH-d) histochemistry and NOS immunoreactivity.

Since species interdiversity often prevents the extrapolation of laboratory rodent data to man and similar problems may exist for nitric oxide synthase (NOS), NADPH-d activity and immunohistochemistry of NOS were investigated in the New World monkey Callithrix jacchus (marmoset), which has been shown to be close to the human situation in many respects. Using the NADPHd reaction with beta-NADPH and nitroblue tetrazolium (NBT) on acetone-chloroform pretreated cryosections, NBT formazan was found in many neural and non-neural (e.g. diverse epithelia, striated muscle fibers, vascular endothelium) cells in numerous tissues and organs. Prefixation with formaldehyde lowered the number of NADPH-d active sites and the amount of formazan with the exception of neuronal NADPH-d as did incubation of fresh or acetone-chloroform-pretreated sections for NADPH-d in the presence of 0.5% formaldehyde. When 1% formaldehyde or 0.5 mM permanganate were used significant amounts of formazan appeared only in central and peripheral neurons, vasal endothelial cells, small intestinal enterocytes, plasma membrane region of striated muscle fibers as well as arteriolar cells in the kidney; except for enterocytes, these observations were confirmed by NOS-immunohistochemistry which revealed in addition reactive cells in the thymus and intestinal lamina propria.

A clinicomorphological study of pituitary tumors associated with the clinical syndrome of acromegaly.

Transsphenoidal adenomectomy was done on 125 acromegalics. A clinicomorphological study was performed on 113 of these patients. The distribution of morphological types of tumors was similar in both sexes. In the total group it was: sparsely granulated GH cell adenomas 52.8%, densely granulated 25.7%, mixed somatotroph and lactotroph adenomas 15.9%, plurihormonal adenomas 1.8%, acidophil stem cell adenomas 2.6%, and oncocytoma 1.8%. The GH plasma level in male patients was significantly higher than in female patients. In both sexes GH secretion was highest in patients with mixed GH and PRL cell adenomas followed by patients with densely granulated GH cell adenomas. Plasma PRL levels were significantly elevated in all males, except for those with densely granulated GH adenomas where prolactinemia was normal. The mean plasma PRL levels were also elevated in females with highest values in mixed somatotroph and lactotroph adenomas. The PRL levels in female patients with densely granulated GH cell adenomas are more elevated than in female patients with sparsely granulated GH cell adenomas.

Modulation of phosphofructokinase action by macromolecular interactions. Quantitative analysis of the phosphofructokinase-aldolase-calmodulin system.

The simultaneous effect of calmodulin and aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) on the concentration-dependent behaviour of muscle phosphofructokinase (ATP: D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) has been analysed by means of a covalently attached fluorescent probe, gel penetration experiments, and using a kinetic approach. We found that calmodulin-induced inactivation of phosphofructokinase is suspended by addition of an equimolar amount of aldolase. This effect was attributed to an apparent competition of calmodulin and aldolase for the dimeric forms of kinase. Moreover, the direct binding of aldolase to calmodulin has also been demonstrated, which resulted in a significant decrease in the kcat value of the enzyme. The quantitative analysis of these interactions in the system phosphofructokinase-calmodulin-aldolase is presented. A possible molecular model for the modulation of phosphofructokinase action by macromolecular interactions is envisaged.

Functional in vitro test of calmodulin antagonism: effect of drugs on interaction between calmodulin and glycolytic enzymes.

A simple procedure has been elaborated to screen for the calmodulin antagonist effect of drugs. A covalently attached fluorescent probe was used to monitor the binding of enzymes known as target enzymes to calmodulin. Moreover, the probe made it possible to recognize a new target enzyme, aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13), for calmodulin among glycolytic enzymes. The calmodulin antagonist trifluoperazine prevented or eliminated the complex formation between calmodulin and enzymes studied in reconstituted systems; the Ca channel blockers had no effect. The functional consequences of the effect of drugs on calmodulin-phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) interaction were investigated as well. Whereas trifluoperazine suspended the calmodulin-mediated hysteretic inactivation of phosphofructokinase, Ca channel blockers (verapamil and nifedipine) were ineffective. Fendiline (regarded as a Ca channel blocker) seems to act as a functional calmodulin antagonist. Its binding to calmodulin does not prevent the complex formation of phosphofructokinase and calmodulin, but within this ternary complex phosphofructokinase preserves or recovers its original activity measured in the absence of calmodulin. The possible molecular effect of drugs on a calmodulin-enzyme complex is discussed.

Aldolase decreases the dissociation-induced inactivation of muscle phosphofructokinase.

The effect of aldolase on the concentration-dependent kinetic behaviour of phosphofructokinase was investigated by means of covalently attached fluorescent probe and by using a kinetic approach. The dimeric form of kinase in equilibrium with the active tetramer interacts with the native aldolase with an apparent dissociation constant of 2.5 microM. Within this heterologous enzyme complex the phosphofructokinase is catalytically active probably because the aldolase binding to nascent kinase dimers might protect them against inactivation.

On the concanavalin A positive layer on the skeletal muscle fibre.

The localization and distribution of mannosyl and glucosyl residues on the extrajunctional sarcolemma of skeletal muscle fibres of frog, rat and new-born rat was studied by means of concanavalin A labelled with FITC and ferritin-concanavalin A conjugate. A strong fluorescent layer surrounding the muscle fibres was found out. Ultrastructurally concanavalin A binding sites were established on the outer surface of muscle cell membrane, on the basal lamina, on the space between them, as well as on the reticular lamina, which ensheeted the muscle fibre. The width of the concanavalin A positive layer was 0.3 to 0.7 micron. On the basis of intensity of the fluorescence, some differences in the content of mannosyl and glycosyl residues during ontogeny as well between both species was admitted.

Intra-splenic transplantation of allogenic isolated pancreatic islets in nonimmunosuppressed rats. A morphological immunocytochemical study.

Adult rat islets isolated by collagenase technique were injected into the spleen in 10 Alloxantreated allogenic Wistar rats. No immunosuppression was used. Recipients were examined on the 1st, 2nd, 3rd, 4th and 5th after transplantation. Localization of insulin, glucagon and somatostatin-immunoreactive cells were demonstrated in all implants from the 1st to the 5th d. There were considerable differences in topographical arrangement of glucagon- and somatostatin-immunoreactive cells in transplanted islets from the second day.

Immunohistochemical study of an insulinoma.

A typical pancreatic insulinoma is characterized by immunohistochemistry which reflects its histogenesis, morphology and hormonal activity.