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PubMed1998

Stimulation of pepsinogen release from chief cells by Helicobacter pylori: evidence for a role of calcium and calmodulin.

To define the mechanisms by which Helicobacter pylori stimulates pepsinogen secretion, the in vitro release of pepsinogen was studied using a preparation of pig chief cell monolayers. Helicobacter pylori induced a time- and concentration-dependent release of pepsinogen into the medium, with about a three-fold increase in pepsinogen secretion over controls found after 45 min of incubation. 3x10(7) H. pylori produced 50% of the maximal response found at a H. pylori count of 2x10(8). The action of H. pylori did not depend on the presence of the vacuolating toxin (vacA) and the cytotoxin-associated protein (cagA). Dibutyryl-cAMP and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate also markedly stimulated pepsinogen secretion and enhanced the stimulatory effect of H. pylori. Helicobacter pylori-stimulated pepsinogen release was inhibited by lanthanum and the calmodulin antagonist W-7, but not by the L-type Ca2+ channel blocker nifedipine, TMB-8, an agent that blocks the release of Ca2+ from intracellular stores, the protein kinase C inhibitor staurosporine and the protein kinase A inhibitor H-8. It is suggested that H. pylori directly stimulates pepsinogen release from gastric chief cells and that this effect is mediated via the calcium/calmodulin messenger branch.

Animals↗
PubMed2001

Regulation of nitric oxide production by murine peritoneal macrophages treated in vitro with chemokine monocyte chemoattractant protein 1.

Monocyte chemoattractant protein 1 (MCP-1) is an important mediator of monocyte/macrophage recruitment and activation at the sites of chronic inflammation and neoplasia. In the current study, the role of nitrogen monoxide (NO) in the activation of murine peritoneal macrophages to the tumoricidal state in response to in vitro MCP-1 treatment and the regulatory mechanisms involved therein were investigated. Murine peritoneal macrophages upon activation with MCP-1 showed a dose- and time-dependent production of NO together with increased tumoricidal activity against P815 mastocytoma cells. N-monomethyl-l-arginine (L-NMMA), a specific inhibitor of the l-arginine pathway, inhibited the MCP-1-induced NO secretion and generation of macrophage-mediated tumoricidal activity against P815 (NO-sensitive, TNF-resistant) cells but not the L929 (TNF-sensitive, NO-resistant) cells. These results indicated l-arginine-dependent production of NO to be one of the effector mechanisms contributing to the tumoricidal activity of MCP-1-treated macrophages. Supporting this fact, expression of iNOS mRNA was also detected in the murine peritoneal macrophages upon treatment with MCP-1. Investigating the signal transduction pathway responsible for the NO production by the MCP-1-activated murine peritoneal macrophages, it was observed that the pharmacological inhibitors wortmannin, H-7 (1-(5-isoquinoline sulfonyl)-2-methyl piperazine dihydrochloride), and PD98059 blocked the MCP-1-induced NO production, suggesting the probable involvement of phosphoinositol-3-kinase, protein kinase C, and p42/44 MAPkinases in the above process. Various modulators of calcium and calmodulin (CaM) such as EGTA, nifedipine, TMB-8 (3,4,5-trimethoxybenzoic acid-8-(diethylamino)octyl ester), A23187, and W-7 (N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide) were also found to modulate the in vitro macrophage NO release in response to MCP-1. This observation indicated the regulatory role of calcium/CaM in the process of MCP-1-induced macrophage NO production. Similarly, the role of serine/threonine and protein tyrosine phosphatases in the above pathway was suggested using the specific inhibitors of these phosphatases, okadaic acid and sodium orthovanadate.

Animals↗
PubMed1994

Sodium cyanide increases cytosolic free calcium: evidence for activation of the reversed mode of the Na+/Ca2+ exchanger and Ca2+ mobilization from inositol trisphosphate-insensitive pools.

This study characterized the cytosolic free Ca2+ concentration ([Ca2+]i) in NaCN-treated human A-431 cells. The resting [Ca2+]i was 85 +/- 8 nM (n = 141) in untreated cells at 37 degrees C, determined with the fura-2 fluorescence probe. When cells were treated with NaCN, [Ca2+]i increased in a time- and NaCN concentration-dependent manner. When cells were exposed to 10 mM NaCN for 10 min, [Ca2+]i increased 278 +/- 28% (n = 5) but returned to normal within 45 min after treatment. The [Ca2+]i increase depended on the presence of external Ca2+. La3+ and Cd2+, but not verapamil or nifedipine, inhibited the NaCN-induced [Ca2+]i increase. The NaCN-induced [Ca2+]i increase also depended on external Na+ (K1/2 = 85 mM). The intracellular Na+ concentration, measured with the fluorescence probe SBFI, increased 267 +/- 16% after NaCN treatment. The NaCN-induced [Ca2+]i increase was modulated by treatment with ouabain or veratridine and was completely blocked by tetrodotoxin, amiloride (K1/2 = 5.4 microM), and dichlorobenzamil (K1/2 = 0.28 microM). These results suggest NaCN activates the Na+/Ca2+ exchange system. TMB-8 and ryanodine both partially blocked the increase in [Ca2+]i in the presence of external Ca2+, indicating that Ca2+ release from intracellular pools also occurred after the initial Ca2+ influx. NaCN decreased inositol trisphosphates production. U-73122, bradykinin, or monensin did not prevent the NaCN-induced increase in [Ca2+]i. However, the magnitude of the [Ca2+]i increase caused by NaCN was abolished in ionomycin-treated the [Ca2+]i increase caused by NaCN was abolished in ionomycin-treated cells, indicating that intracellular Ca2+ release induced by NaCN is derived from an ionomycin-sensitive Ca2+ pool. The results suggest that NaCN initially increased Na+ influx, which activated the reverse mode of a Na+/Ca2+ exchanger, leading to an increase in Ca2+ influx. The Ca2+ influx induced a Ca2+ mobilization from only an ionomycin-sensitive intracellular Ca2+ pool containing ryanodine receptors.

Amiloride↗
PubMed1995

Neutrophil degranulation and superoxide production induced by polychlorinated biphenyls are calcium dependent.

2,2',4,4'-Tetrachlorobiphenyl (TCB), a polychlorinated biphenyl (PCB), alters neutrophil functions in vitro by unknown mechanisms. The role of calcium in mediating PCB stimulation of neutrophil function was investigated. Peritoneal neutrophils were isolated from retired breeder, male, Sprague-Dawley rats. Neutrophils were incubated with 10 micrograms/ml 2,2',4,4'-TCB prior to stimulation with phorbol myristate acetate (PMA), and superoxide anion (O2-) production was measured. 2,2',4,4'-TCB alone elicited O2- production and potentiated the response to PMA. When neutrophils were incubated in calcium-free medium, the ability of TCB to stimulate O2- production and to potentiate the response to PMA was abolished. The absence of extracellular calcium did not alter the response to PMA alone. TMB-8, an antagonist of the mobilization of intracellular calcium, inhibited O2- production in response to 2,2',4,4'-TCB stimulation but not to PMA. Degranulation of neutrophils, measured by release of myeloperoxidase, occurred upon exposure to 10 micrograms/ml 2,2',4,4'-TCB alone. This response was increased by cotreatment with the calcium ionophore A23187. 2,3,4,5-TCB, a mono-ortho-substituted PCB congener, potentiated O2- production in response to PMA stimulation by a mechanism that was partly dependent on the presence of extracellular calcium. This congener also stimulated neutrophils to release myeloperoxidase. 3,3',4,4'-TCB, a coplanar congener with high affinity for the Ah receptor, did not elicit neutrophil degranulation. These results indicate that TCBs affect neutrophil function in vitro through signal transduction pathways that appear to be calcium dependent.

Animals↗
PubMed1997

Effects of antioxidants and Ca2+ in cisplatin-induced cell injury in rabbit renal cortical slices.

Effects of antioxidants, reactive oxygen species (ROS) scavengers, and Ca2+ on cisplatin-induced renal cell injury were studied in rabbit renal cortical slices in vitro. Cisplatin induced LDH release and lipid peroxidation, inhibition of PAH uptake, and GSH depletion. These changes were significantly prevented by thiols (DTT and GSH), antioxidants (DPPD and BHA), and an iron chelator (deferoxamine). Superoxide dismutase partially reduced the cisplatin-induced LDH release without affecting the lipid peroxidation and the GSH depletion. Catalase did not affect the LDH release and the lipid peroxidation induced by cisplatin. Hydroxyl radical scavengers prevented the lipid peroxidation, whereas they did not alter the LDH release, the inhibition of PAH uptake, and the GSH depletion induced by cisplatin. Removal of Ca2+ or addition of EGTA to the incubation medium did not alter cisplatin effects on LDH release and lipid peroxidation. Buffering intracellular Ca2+ with quin-2/AM or inhibition of intracellular Ca2+ release with TMB-8 significantly reduced the cisplatin effect on LDH release without any effect on the lipid peroxidation and the GSH depletion. Ruthenium red attenuated the LDH release, the lipid peroxidation, and the inhibition of PAH uptake mediated by cisplatin. La3+ prevented the cisplatin effect on the LDH release, whereas it did not affect the lipid peroxidation, the inhibition of PAH uptake, and the GSH depletion by cisplatin. These results suggest that cisplatin induces a lethal cell injury by lipid peroxidation-dependent and -independent mechanisms and that the cell injury and the lipid peroxidation by cisplatin are iron-dependent. In addition, the data indicate that the Ca2+ released from intracellular stores, but not the Ca2+ moved from extracellular space, plays a role in the cisplatin-induced cell injury independent of lipid peroxidation.

Animals↗
PubMed2000

The effects of aliphatic (n-nonane), naphtenic (1,2, 4-trimethylcyclohexane), and aromatic (1,2,4-trimethylbenzene) hydrocarbons on respiratory burst in human neutrophil granulocytes.

This study investigates the effects of aliphatic (n-heptane, n-nonane), naphtenic (methylcyclohexane, 1,2,4-trimethylcyclohexane (TMCH)), and aromatic (methylbenzene, 1,2,4-trimethylbenzene (TMB)) hydrocarbons on respiratory burst in human granulocytes. The free radical formation was measured as 2,7-dichlorofluorescein diacetate-amplified (DCF) fluorescence, by electron paramagnetic resonance (EPR) spectroscopy and by hydroxylation of 4-hydroxybenzoate. The chemotactic peptide N-formyl-met-leu-phe (fMLP) and phorbol 12-myristate 13-acetate (PMA), a diacylglycerol analogue, were included as positive controls. DCF fluorescence was elevated in a concentration-dependent manner by C9 hydrocarbons. The C7 hydrocarbons did not stimulate respiratory burst in the concentration range examined. The naphtenic hydrocarbon TMCH showed the strongest effect on respiratory burst and was therefore selected for mechanistic studies of this free radical formation. In the absence of extracellular Ca(2+), fluorescence in response to TMCH and fMLP was reduced by 77 and 90%, respectively. Preincubation of the granulocytes with the protein kinase C inhibitor bisindolylmaleimide reduced the DCF fluorescence stimulated with TMCH, fMLP, and PMA by 82, 56, and 90%, respectively. The phospholipase C inhibitor U73122 lowered the TMCH- and fMLP-activated DCF fluorescence by 87 and 76%. In addition, the TMCH- and fMLP-induced DCF fluorescence, after the preincubation with the phospholipase D modulator n-butanol, was lowered by 83 and 52%, respectively. The importance of protein kinase C, phospholipase C, and phospholipase D for elevation of respiratory burst was also demonstrated by the EPR experiments using the spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO). Preincubation with the NADPH oxidase inhibitor diphenyleneiodonium and diethyldithiocarbamate, which inhibits superoxide dismutase, led to an almost complete reduction of DCF fluorescence in response to TMCH, fMLP, and PMA. Preincubation with diethyldithiocarbamate led to the elevation of superoxide adducts of DEPMPO. The hydrocarbons stimulated formation of mainly the superoxide (O(*-)(2)) adduct of DEPMPO (DEPMPO-OOH) but also small amounts of the hydroxyl adduct ((*)OH) (DEPMPO-OH). Using 4-hydroxybenzoate as a hydroxyl radical trap confirmed formation of (*)OH after stimulation with the hydrocarbons. In conclusion, our findings indicate that TMCH-activated respiratory burst is dependent on the Ca(2+)-dependent phospholipase C, phospholipase D, and protein kinase C prior to activation of the NADPH oxidase.

1-Butanol↗
PubMed1989

The inhibition of alpha 1-adrenoceptor-mediated contractions of rabbit pulmonary artery by Ca2+-withdrawal, pertussis toxin and N-ethylmaleimide is dependent on agonist intrinsic efficacy.

Contractions were induced in rings of rabbit pulmonary artery with the preferential alpha 1-adrenoceptor agonists, phenylephrine, methoxamine and St 587 [2-(2-chloro-trifluoromethyl-phenylimino)imidazolidine and the preferential alpha 2-adrenoceptor agonists, clonidine and B-HT 920 [6-allyl-2-amino-5,6,7,8-tetrahydro-4H-thiazolo-(4,5-d) azepine] [corrected]. Phenylephrine and methoxamine acted as full agonists whereas St 587, clonidine and B-HT 920 were partial agonists (intrinsic activities 0.62, 0.38 and 0.42, respectively). Experiments with alpha 1- and alpha 2-adrenoceptor antagonists indicated that the receptors involved are of the alpha 1 type only. Removal of extracellular Ca2+ inhibited maximal contractions to phenylephrine and methoxamine by 30% and 49%, respectively. The remaining contraction components of the full agonists were abolished by the "intracellular Ca2+ antagonist" TMB-8 [8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate]. Contractions to St 587, clonidine and B-HT 920 were virtually abolished in Ca2+-free medium. Pretreatment of the donor rabbits with pertussis toxin (2.5 micrograms/kg i.v., 5-6 days before sacrifice) attenuated the efficacies of the full agonists, phenylephrine and methoxamine by only 24% and 17%, respectively, whereas maximal contractions to the partial agonists, St 587, clonidine and B-HT 920, were inhibited by 46%, 61% and 75%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic alpha-Agonists↗
PubMed1991

Angiotensin II-induced increase in slowly exchanging 45Ca2+ in relation to contractile responses of rat and guinea-pig aorta.

To gain more information about sources of activator Ca2+ involved in the contraction of rat and guinea-pig aorta evoked by angiotensin II and their sensitivity to Ca2+ entry blockers, measurement of slowly exchanging 45Ca2+ was established. A more physiological procedure was used, replacing La(3+)- and EGTA-containing solutions by a normal Ca(2+)-containing buffer. It was demonstrated that the angiotensin II-induced increase in slowly exchanging 45Ca2+ in rat aorta was incompletely (by approximately 60%-70%) inhibited by the organic Ca2+ entry blockers nifedipine, verapamil and diltiazem and by other Ca2+ entry blocking compounds like CoCl2 and chlorpromazine. 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) was able to inhibit the angiotensin II-induced increase in 45Ca2+ content completely, but this may be an intracellular storage effects. By contrast, the organic Ca2+ entry blockers completely inhibited that part of the angiotensin II-induced contraction of rat aorta which was dependent upon extracellular Ca2+. In guinea-pig aorta, the increase in 45Ca2+ content elicited by angiotensin II could be completely suppressed by all compounds under study. The results of these experiments correlated well with data from the functional experiments in guinea-pig aorta. In both preparations the release of Ca2+ from a rapidly as well as a slowly exchanging intracellular pool appears to contribute to the contractile response elicited by angiotensin II.

Angiotensin II↗
PubMed1990

Comparison of the calcium entry and calcium overload blocking properties of R71811 and flunarizine.

The effects of several calcium antagonists on cell death induced by A23187 were studied. Furthermore, R71811, 1-[Bis(4-fluorophenyl)methyl-4-[(4-methoxyphenyl)- carbamoylmethyl-trans-2,5-dimethyl-piperazine has been evaluated as a calcium overload blocker and compared to flunarizine. The viability of cultured glial cells was decreased by incubation with the calcium ionophore, A23187. The cytotoxicity of A23187 was reduced by flunarizine and cinnarizine at 10 mumol/l; nicardipine, nifedipine, and verapamil, but not diltiazem, reduced cytotoxicity at 100 mumol/l. N-(6-aminohexyl)-5-chloro-1- naphthalenesulfonamide (W-7) and trifluoperazine did not reduce cytotoxicity and trifluoperazine enhanced cytotoxicity at 100 mumol/l. Leupeptin reduced cytotoxicity at 100 mumol/l. [8-(N,N-dimethylamino)-octyl-3,4,5- trimethoxybenzoate] (TMB-8) showed no effect. The results indicate that flunarizine, a recognized calcium overload blocker, was most effective in inhibiting the A23187-induced cytotoxicity and that calcium entry blockade does not appear to be implicated in the cytoprotective effect. R71811 inhibited A23187 cytotoxicity at similar concentrations to flunarizine. R71811, as well as flunarizine, also inhibited erythrocyte crenation induced by A23187. Although R71811 showed a relaxant effect in isolated rat aorta contracted by high potassium, its activity was less than that of flunarizine (IC50 values, 4.1 and 0.045 mumol/l, respectively). On the other hand, R71811 and flunarizine showed similar inhibitory effects on A23187-induced contractions (IC50 values, 14 and 11 mumol/l, respectively). The results indicate that R71811 has a similar protective effect against A23187-induced cytotoxicity but only weak calcium entry blocking action in comparison to flunarizine.

Animals↗
PubMed1992

Types of callosally projecting nonpyramidal neurons in rat visual cortex identified by lysosomal HRP retrograde labeling.

Callosally projecting neurons, labeled following injection of horseradish peroxidase (HRP) into the 17/18a border of the contralateral hemisphere, have been examined by light and electron microscopy. These neurons exhibit two types of horseradish peroxidase labeling: either a diffuse, Golgi-like labeling, or a granular, punctate labeling. The punctate type of HRP-labeling is the predominant form in nonpyramidal neurons, while pyramidal neurons frequently display either diffuse or punctate labeling. Only punctately labeled neurons have been examined in this study. Light microscopic analyses of 1-microns sections show that in the heavily labeled zone at the area 17/18a border approximately 9% of all of the cells in layer II/III are callosally projecting nonpyramidal cells, and 70% of them are callosally projecting pyramidal cells. Light and electron microscopic examinations indicate that the nonpyramidal neurons are a heterogeneous group which consists of small multipolar neurons, large multipolar neurons, small bipolar neurons, and large bipolar neurons. To investigate the ultrastructural appearance of the punctate HRP labeling, selected neurons have been examined in thin sections. In the electron microscope, the tetramethylbenzidine (TMB) reaction product appears as electron-dense crystals, while the diaminobenzidine (DAB) reaction product appears as dark, electron-dense material which fills the lysosomes. These lysosomes occasionally have a halo of reaction product, but often they are not morphologically distinguishable from dark lysosomes present within neurons from control animals in which the darkening results from staining the thin sections with lead citrate and uranyl acetate. However, labeled neurons possess more dark lysosomes than neurons from control animals. These additional dark lysosomes presumably contain the HRP reaction product visible by light microscopy.

Animals↗
PubMed1990

External calcium is required for activation of phospholipase C by angiotensin II in adrenal glomerulosa cells.

Previous studies have shown that external calcium (Ca2+) is required for the effects of angiotensin II (AII) on aldosterone secretion in adrenal glomerulosa zone. Using bovine adrenal glomerulosa cells prepared by collagenase dispersion, we examined whether external Ca2+ is required for the activation of phospholipase C by AII. Adrenal glomerulosa cells were exposed to Ca-EGTA buffered media to provide accurate estimates of external free Ca2+ concentrations. Phospholipase C activation was evaluated by measurement of inositol phosphates production. At 0.1 microM Ca2+ and less, sustained AII effects on inositol monophosphate (IP), inositol bisphosphate (IP2) and inositol trisphosphate (IP3) were markedly inhibited. Increasing the Ca2+ concentration to 50 microM or greater fully restored AII-induced inositol phosphates production. AII-induced increases in cytosolic Ca2+ measured by Quin-2 fluorescence, were diminished at lower external Ca2+ concentrations. Treating adrenal glomerulosa cells with Chelex-100, a strong Ca2+ binding resin, blocked early activation of phospholipase C by AII. Inhibition of IP3 production was also observed when inhibitors of Ca2+ movement across the plasma membrane were used, viz., La2+, TMB-8 and nifedipine. The requirement for Ca2+ during AII-induced activation of phospholipase C may be explained, at least partly by a requirement for Ca2+ at a site between the AII receptor and Phospholipase C.

Aldosterone↗
PubMed1990

The histochemical reaction of horseradish peroxidase in rodent 'whole-brain' preparations.

Subsequent to eye injections of horseradish peroxidase (HRP) 'whole-brain' preparations with only the cortex removed were reacted for HRP using tetramethylbenzidine (TMB) as the chromogen and ammonium heptamolybdate (AHM) as a stabilizer. Retinal projections could be photographed and visualized globally coursing across the surface of the thalamus and midbrain. The brains were then sectioned, and when necessary re-reacted, enabling a 3-D reconstruction of retinofugal pathways to be made.

Animals↗
PubMed1985

Origin in the medial accessory olive of climbing fibres to the x and lateral c1 zones of the cat cerebellum: a combined electrophysiological/WGA-HRP investigation.

The climbing fibres to the x and lateral c1 zones of the anterior lobe of the cerebellum arise as branches of common stem olivary axons. Anatomical studies have shown that the c1 zone receives its climbing fibres from the dorsal accessory olive (DAO). It has, therefore, been assumed that the x-zone also receives its climbing fibres from this olivary subnucleus. The present study demonstrates that both the x-zone and the lateral part of the c1 zone in fact receive their climbing fibre input from the middle portion of the medial accessory olive (MAO) (approximate antero-postero-levels P10-13). Electrophysiological techniques were used to define the extent of these cerebellar zones and small volumes (15-50 nl) of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) were pressure injected into the defined zone. These small pressure injections resulted in injection sites with minimal spread to adjacent zones. The sensitive tetramethylbenzidine (TMB) reaction was used to visualize both the injection site and retrogradely labelled cells in the inferior olive. This combination of electrophysiological and neuroanatomical techniques gave extremely reproducible results. The results suggest that the zone previously named lateral c1 would be better designated cx.

Animals↗
PubMed1985

Effects of neonatal intraocular colchicine on synaptogenesis and on the retention of the ipsilateral retinofugal projection within the superior colliculus.

A normally transient ipsilateral retinofugal projection exists in the rat but is retained following eye removal because of the loss of competitive interaction between crossed and uncrossed fibers. To further explore this phenomenon, colchicine (10(-3)M) was injected into the right eye of newborn albino rats to partially suppress axonal transport in optic fibers, alter the developmental time course of retinofugal synaptic terminals and determine if this would in turn extend the period of survival of the ipsilateral projection. Measurements of the number of fibers in the nerve were also made to insure that colchicine was not lethal to the retinofugal projection. Projections into the superior colliculus were demonstrated by anterograde movement of HRP from the left eye. TMB histochemistry revealed dense labeling of the contralateral retino-recipient layers at 5 dpn in untreated or saline-injected controls. The ipsilateral projection was seen as a lighter band of activity across the colliculus which was most concentrated in the antero-medial quadrant. This pathway was transient and degenerated by 10 dpn, except for a few antero-medial fibers. Animals treated with colchicine demonstrated a retention of this pathway through 20 dpn. A concomitant quantitative analysis of synaptic development within the superior colliculus revealed populations of boutons with round (R) and flattened (F) vesicles, as well as multiple junctional (MJ) and serial (S) complexes, most of which were specialized R boutons. The various synaptic categories displayed specific ratios unique to the different stages of maturation. Intraocular colchicine reduced the ratio of R, MJ and S boutons to F terminals between 5-15 dpn (P less than 0.01). By 20 dpn, the proportions of MJ and S boutons remained depressed but the normal ratio of R to F boutons was restored. Areal determinations of each synaptic profile included in the counts revealed a significant reduction in the size of MJ synaptic profiles examined in colchicine-treated animals and this may have been reflected in the slight loss of tectal volume (6-9%). Removal of the left eye and assessment of degenerating boutons showed that the expanded ipsilateral projection was not sufficiently dense to produce such a restoration. It thus appears that colchicine delayed the growth of the R population, but the effect was reversible and this category of boutons continued to develop, albeit on a later time course.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1982

A demonstration of hippocampal mossy fiber axon morphology using the anterograde transport of horseradish peroxidase.

A modified horseradish peroxidase (HRP) technique that capitalizes on the anterograde transport of this glycoprotein was used to visualize the unique morphology of the hippocampal mossy fiber axon. Small iontophoretic injections of HRP in a solution containing dimethylsulfoxide (DMSO) and polyvinylpyrrolidone (PVP) were made in the dentate gyrus of adult rats. The tissue was then processed for the localization of HRP using tetramethyl benzidine (TMB) as a chromogen. Small injections, coupled with short survival times (15-22 h) were found to optimally label small numbers of individual mossy fiber axons including the characteristic swellings that occur periodically along their extent. The HRP-labeled mossy fiber axons, which heretofore at the light-microscopic level could only be visualized using Golgi methods, could be traced for long distances when the hippocampus was sectioned perpendicular to the septo-temporal axis. Injections restricted to either blade of the dentate gyrus labeled both infra- and suprapyramidal mossy fiber axons. Both Golgi-stained and HRP-labeled mossy fiber swellings were observed to increase in size as the distance from the granule cell somata increased.

Animals↗
PubMed1983

The distribution of displaced ganglion cells in the retina of the pigeon.

Displaced ganglion cells in the pigeon's retina, at the inner margin of the inner nuclear layer, were labelled by retrograde axonal transport of horseradish peroxidase (HRP). Large HRP injections were made in order to fill all the retinal projection sites in the thalamus and midbrain. The distribution of labelled cells was studied in retinal whole mounts incubated with tetramethyl benzidine (TMB) substrate for HRP. A maximum of 5,300 HRP labelled displaced ganglion cells was found. They were concentrated in a band of retina centred on the horizontal meridian, with high density areas (of about 110 cells/mm2) near the area centralis and in the mid-temporal retina. This is a different distribution to that of ganglion and inner nuclear layer cells; these are concentrated in the area centralis and red field. The orientation of retinal maps was checked by ophthalmoscopic measurements of the angle of the pecten to the horizontal in alert pigeons; this was found to be approximately 70 degrees. The array of displaced ganglion cells, studied by nearest neighbour distributions, was irregular and nearly random, which is consistent with a system of low spatial acuity. In the central retina only the cell bodies and not the dendrites of small displaced ganglion cells (7.5 microns diameter) were labelled; towards the periphery large displaced ganglion cells (16 microns diameter) with 2-5 radially arranged primary dendrites were found.

Animals↗
PubMed1985

Retrograde HRP study of neurons in the cervical enlargement projecting to the cerebellum in the cat.

After cerebellar HRP injections in kittens labeled neurons were found in laminae V-VIII in the cervical enlargement. Most of the labeled neurons were localized in two groups, one in laminae V-VI, the other centrally in lamina VII. Labeled neurons were also observed in the medial part of lamina VII of C5 and T1 and a few in lamina VIII. Neurons in the cervical enlargement seem to terminate largely in cerebellar lobules IV-V of the anterior lobe. Some neurons in laminae V-VI terminated in the ipsilateral paramedian lobule. Neurons in laminae V-VI and central lamina VII of C5-T1 had uncrossed axons. Neurons in medial lamina VII of C5, in lamina VIII and neighbouring parts of lamina VII of C6-T1 had crossed axons. The ramifications of proximal dendrites and axons of the labeled neurons are described using the tetramethylbenzidine (TMB) method for HRP histochemistry. The neurons in the various laminae differed in their characteristic morphology. In conclusion, the findings of Matsushita et al. (1979) concerning the localization and axonal course of cerebellar projecting neurons in the cervical enlargement are confirmed. In addition new data concerning the morphology of the labeled neurons are presented.

Animals↗
PubMed1985

Reversible and irreversible inhibition of rat brain muscarinic receptors is related to different substitutions on bisquaternary pyridinium oximes.

The role of the functional substituents on the pyridinium ring of bisquaternary pyridinium compounds, mostly oximes, in exerting reversible and irreversible inhibition of binding of [3H]-N-methyl-4-piperidyl benzilate [( 3H]-4NMPB) to rat brain stem muscarinic receptors was studied. The drugs tested, i.e. HGG-42, HGG-12, HGG-52, HI-6, obidoxim, SAD-128 and TMB-4, could reversibly inhibit binding of [3H]-4NMPB, with the highest potency (KI = 1.7 - 6 microM) exhibited by analogs possessing hydrophobic substituents at position 3 or 4 of the pyridinium ring. Bisquaternary drugs possessing an oxime moiety at position 2, but not at position 4 of the pyridinium ring, could also induce about 30% reduction of maximal binding capacity (Bmax) (loss of muscarinic receptors) in addition to their reversible effect. Thus the structural correlates of the reversible and the irreversible effects of these drugs are different.

Animals↗