Adrenal microvascularization in the common tree shrew (Tupaia glis) as revealed by scanning electron microscopy of vascular corrosion casts.

The blood supply of the adrenal gland in the common tree shrew (Tupaia glis) was studied by use of transmission electron microscopy and vascular corrosion cast/scanning electron microscopy techniques. It was found that the gland receives its blood supply from branches of the inferior phrenic, aorta and renal arteries. Upon reaching the gland, these arteries divide into cortical and medullary arteries. The cortical arteries give rise to the subcapsular capillary plexuses which partially enclose the clusters of cells in the zona glomerulosa (ZG) and appear as lobular-like microvascular networks before running among the cellular cords in the zona fasciculata (ZF) and zona reticularis (ZR). It was noted that the capillaries in ZG and ZR are with more anastomoses than those in the ZF. Capillaries from the ZR become the sinusoidal capillaries in the adrenal medulla before proceeding to the peripheral radicles of the central vein. The medullary arteries penetrate the adrenal cortex and occasionally give off small branches to supply the inner cortex, especially the ZR. Their main branches break up into small or conventional capillaries in the adrenal medulla. These capillaries drain the blood into the peripheral radicles of the central vein and medullary collecting veins which proceed further into a very large central vein. The present findings illustrate that the adrenal medulla receives two blood supplies that yield somewhat different influences upon the adrenal medulla. The portal blood vessel could not be illustrated in the tree shrew adrenal gland.

Studies on the cobra neurotoxin inhibiting activity in an extract of Curcuma sp. (Zingiberaceae) rhizome.

A study was carried on the mode of action and some properties of a cobra neurotoxin inhibitor found in the extract of Curcuma sp. (Zingiberaceae). When the principal postsynaptic neurotoxin (STX) of the Thai cobra (Naja naja siamensis) was mixed with an aqueous extract of Curcuma sp. rhizome, the STX was inactivated as tested in mice or in vitro using a rat hemidiaphragm preparation. The 'neurotoxin inhibitor' ('NTxI') was found only in the water insoluble fraction of the rhizome extract. Using radioactively labeled neurotoxins, 125I-STX and 3H-STX, it was demonstrated that the neurotoxin did not form a stable complex with the 'NTxI'; the inactivated neurotoxin remained in the supernatant of the reaction mixture. After inactivation by 'NTxI', the STX exhibited an unchanged molecular weight as judged by SDS-polyacrylamide gel electrophoresis and an unchanged isoelectric point in isoelectric focusing. Extraction of the Curcuma sp. rhizome with at least 0.2% Triton X-100 resulted in solubilization of a component capable of forming a soluble and stable complex with 3H-STX. By column chromatography on Sephadex G-200 in the presence of 0.1% Triton X-100, the toxin-binding compound was shown to have a molecular weight of about 150 kDa. This 150 kDa component was obtained by Triton extraction of the water-insoluble fraction, and much less from the water soluble fraction, of Curcuma sp. rhizome. It did not possess any carbohydrate side-chain capable of binding the lectin Concanavalin A. The time course of the 150 kDa-3H-STX complex formation was extremely slow (approx 22 hours).(ABSTRACT TRUNCATED AT 250 WORDS)

Cytoarchitecture of the common tree shrew (Tupaia glis) superior cervical ganglion. A scanning electron microscope study on vascular cast/enzyme-digested superior cervical ganglia.

The cytoarchitecture of the superior cervical ganglion of the common tree shrew was investigated by scanning electron microscopy using the vascular cast technique in conjunction with digestion by collagenase-hyaluronidase/HCl. The main cellular constituents were found to be multipolar neurons that were densely distributed throughout the ganglion. These neurons were covered with a smooth cytoplasmic sheath of satellite cells. After the removal of this sheath by digestion of increased duration, the blebs or knobs on the neuronal surfaces became evident. A meshwork of nerve fibers over the surface of neurons was also observed. Preganglionic sympathetic nerve fibers, giving rise to fine branches that ran toward the postganglionic sympathetic neurons before forming synapses, were demonstrated. Groups of neurons surrounded by capillary loops were also frequently observed.

Differential secretion of enkephalin-like peptides from bovine adrenal chromaffin cells.

Stimulation of chromaffin cells in culture with 1,1-dimethyl-4-phenylpiperazinium (DMPP) or depolarizing concentrations of K+ resulted in a significant secretion of high and low molecular weight enkephalin-like peptides (ELPs) into the culture medium. BioGel P-10 column chromatography was used to characterize the ELPs in chromaffin cell extracts and in culture media before and after stimulation with either DMPP or K+. DMPP (50 microM) stimulation produced a significant secretion of primarily low molecular weight (less than 3 kDa) ELPs whereas 56 mM K+ caused a secretion of both high and low molecular weights ELPs. The expected decrease in cellular content of low molecular weight peptides was not observed regardless of stimulation type. Our results support the hypothesis that the precursor/product ratio of secreted ELPs is dependent upon the nature of the chromaffin cell stimulus. Moreover the cellular content of low molecular weight ELPs is not depleted with either type of stimulation.

Binding sites for 125I-neuropeptide Y (NPY) on membranes from bovine adrenal medulla.

Bovine adrenal medulla membranes were examined for the presence of specific 125I-neuropeptide Y (125I-NPY) binding sites using rapid centrifugation to measure the amount of bound ligand. Specific binding was determined from the difference between 125I-NPY bound in the presence and absence of 10(-7) M unlabeled NPY. The binding was saturable and reached equilibrium within 5 min at 0 degrees C. Analysis of specific 125I-NPY binding using the LIGAND computer program indicated a best fit for a two site model with a Kd of 0.26 nM and a Bmax of 12 fmol/mg protein for the high affinity site and a Kd of 170 nM and a Bmax of 6 pmol/mg protein for the low affinity site. The rate of dissociation (k-1) was 0.071/min with a t1/2 of 9 min. Displacement curves for avian or human pancreatic polypeptide revealed that these peptides displaced 125I-NPY from both sites with IC50 values greater than 10 nM.

Characterization of 1,1-dimethyl-4-phenylpiperazinium induced increased proenkephalin processing in bovine chromaffin cells.

Acute stimulation of bovine adrenal chromaffin cells in culture with 1,1-dimethyl-4-phenylpiperazinium (DMPP) gives rise to a significant increase in secretion of [Met5]-enkephalin immunoreactive material (ME-IRM) into the culture medium (1). Following this secretion the cellular ME-IRM levels do not decrease, suggesting the replenishment of the peptides. The repletion of the cellular ME-IRM appears to result from an increase in processing of large molecular weight peptides containing [Met5]-enkephalin and [Leu5]-enkephalin. Gel filtration chromatography on Bio-Gel P-10 was used to fractionate the enkephalin-like peptides (ELPs) present in the culture media and chromaffin cell extracts. Fractionation was done for samples before and after nicotinic receptor stimulation by DMPP to demonstrate the secretion and repletion of the ELPs. Gel chromatographic profiles of ELPs present in the culture media after DMPP stimulation revealed the presence of 4 peaks, representing different molecular forms of these peptides (Peaks 1-4), with a selective increase in secretion of Peaks 3 and 4. The chromatograms of ELPs extracted from cultured chromaffin cells showed similar patterns to those obtained from ELPs present in the culture medium after stimulation. Analyses of individual peaks after fractionation of cell culture extracts showed an increase in the amount of immunoreactive material found in Peak 4 with a concomitant decrease in the immunoreactivity found in the higher molecular weight peaks (Peaks 1-3). Further purification of Peak 4 from cell extracts on reversed-phase HPLC (RP-HPLC) showed a significant amount of ELPs existed as the sulfoxide derivative of [Met5]-enkephalin. The content of [Met5]-enkephalin sulfoxide (ME-O-enk) did not decrease following DMPP stimulation. We conclude that acute stimulation of nicotinic receptors in the chromaffin cells enhances the processing of proenkephalin precursors to keep pace with the secretion of low molecular weight peptides.

Nicotinic receptor stimulation enhances enkephalin-like peptides processing in chromaffin cells.

Acute stimulation of chromaffin cells in cultures with acetylcholine (ACh), 1,1-dimethyl-4-phenylpiperazinium (DMPP), or high potassium gave rise to a significant increase in the release of [Met5]-enkephalin immunoreactive material (ME-IRM) into the assay medium. The cellular content of ME-IRM following the actual release induced by these secretagogues remained constant suggesting the replenishment of the cellular peptides. The repletion of the peptides may occur through an enhancement of the processing rate of the proenkephalin precursor. Furthermore, the increase in secretion as well as the repletion of the cellular ME-IRM were calcium-dependent and were inhibited by the nicotinic receptor antagonist, hexamethonium, but not by atropine. These results indicate that secretion and repletion of the peptides are tightly coupled and activated by nicotinic receptor stimulation.

Proenkephalin-A derived peptides do not modulate cardiovascular effects of epinephrine on the isolated rat atrial preparations.

The catecholamines and the opioid peptides are found to be co-localized in the adrenomedullary chromaffin cells. They are co-secreted from the chromaffin granules in response to various stimuli. The stress-induced released of epinephrine is known to exert its effect on the cardiovascular system resulting in the changes in heart rate and blood pressure. However, the role of the co-released proenkephalin-A derived peptides has not been extensively characterized. Previous work from several investigators suggested that the peptides modulate cardiac functions of the catecholamines. There is considerable conflicting results among these reports. Results from the isolated rat atrial preparation indicated that enkephalins attenuated the increase in atrial rate induced by norepinephrine through restriction of the calcium fluxes. Nonetheless, others reported insensitivity of the enkephalins in similar or different test systems. We further re-examined these discrepancies using the isolated rat atrial preparation to investigate the opioid peptide modulatory effect on the cardiovascular changes induced by exogenous epinephrine. Alterations in rate and force of contraction resulting from epinephrine and the peptides were both studied in parallel. The opioid peptides used in this study were [Met5]-enkephalin (ME), [Leu5]-enkephalin (LE), FMRFamide, [Met5]-enkephalyl-Arg6-Phe7 (MEAP), peptide E, and the non-selective opioid agonist, etorphine. We report here that none of the opioid peptides were effective in alleviating or attenuating the increase in heart rate and developed tension caused by epinephrine. The peptides did not affect the basal beating rate nor the force of contraction. Thus, the present results clearly demonstrate the insensitivity of the enkephalins in modulating the cardiac effects of epinephrine. They further indirectly support the prejunctional synaptic nerve endings as the potential peripheral site of action of the peptides.

Proteolytic-independent cobra neurotoxin inhibiting activity of Curcuma sp. (Zingiberaceae).

The 1: 1 (w/v) aqueous extract of Curcuma sp. (Zingiberaceae) was shown to antagonize the toxic action of Naja naja siamensis neurotoxin possibly via direct inactivation of the toxin. The plant extract possessed proteolytic activity which could be separated from the neurotoxin inhibiting activity. The mechanism of antagonism between the plant extract and the neurotoxin was shown not to be involved with the existence of proteolytic activity in the plant extract.

The absence of antagonism between extracts of Clinacanthus nutans Burm. and Naja naja siamensis venom.

Clinacanthus nutans Burm, a herb reputed in Thailand and Malaysia to be "snakebite antidote" has been tested in vitro and in vivo for antivenin activity. The aqueous extract of C. nutans leaves has been found to have no effect on the inhibition of neuromuscular transmission produced by purified Naja naja siamensis neurotoxin in isolated rat phrenic-nerve diaphragm preparations. The extract of C. nutans, when given orally or intraperitoneally, are ineffective in prolonging the survival time of experimental mice receiving lethal doses of N.n. siamensis crude venom. Oral administrations of the herb extracts pretreated with alpha-amylase or beta-amylase also fail to protect the animal. It is concluded that the extract of C. nutans can not antagonize the action of cobra venom.