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A traditional Chinese mineral medicine, cinnabar, naturally occurring mercuric sulfide (HgS), is still occasionally prescribed, but the neurotoxic effects of HgS have not been elucidated. In this paper, an animal model of the purified HgS intoxication was established in guinea pigs in order to study neurotoxicity and pathophysiology of the vestibular ocular reflex system (VOR). Guinea pigs were dosed with HgS by gastric gavage (0.01, 0.1 and 1.0 g/kg per day) for 7 consecutive days. By means of caloric testing coupled with the electronystagmographic (ENG) recording in guinea pigs, we have found that HgS at a dose of 0.1 g/kg induced reversible caloric hypofunction pattern and at a higher dose of 1.0 g/kg induced irreversible hypofunction of caloric test. Monitoring the mercury contents of various tissues (blood, kidney, liver and cerebellum) by continuous flow and cold vapor atomic absorption spectrometry (AAS) revealed that a certain amount of HgS could be absorbed from the gastrointestinal tract and was detectable in these tissues. In addition to the induced dysfunction of VOR system, HgS also caused disturbance of motor performance in guinea pigs. In enzyme assay, Na+/K+-ATPase activity of cerebellum was also significantly inhibited by HgS. Morphological studies showed partial cell loss only in the cerebellar Purkinje cell layer, but not in the granule cell layer, nor in the vestibular labyrinth. All of these findings suggest that cerebellar Purkinje cells are the sensitive target site responsible for HgS-inducing dysfunctions of both VOR system and the motor performance in guinea pigs. Thus, it is concluded that caloric test coupled with ENG recording in VOR system is certainly a sensitive biomarker for monitoring the neurotoxicity of HgS.

Na+,K+-ATPase and Ca2+-ATPase activities have not been studied quantitatively in the cochlea affected by endolymphatic hydrops. The present study was designed to measure quantitatively the Na+,K+-ATPase and Ca2+-ATPase activities in the cochlear lateral wall and the threshold of auditory brainstem response (ABR) for guinea pigs in the early stages (=2 months) of experimentally induced endolymphatic hydrops. A significant negative association was demonstrated between Ca2+-ATPase activity and the change in ABR threshold for hydropic cochleae (P=0.014), but not for control cochleae (P=0.123), although no such significant association was revealed between Na+,K+-ATPase activity and any change in ABR threshold for both hydropic cochleae (P=0.751) and control cochleae (P=0.352). A significant increase in Ca2+-ATPase activity in the cochlear lateral wall was observed for the hydropic ear, in which normal ABR thresholds were maintained, as compared to the control ear. On the contrary, a mild decrease in Ca2+-ATPase activity in the cochlear lateral wall was observed for the hydropic ear, in which ABR thresholds increased significantly. The present findings suggest that alterations of Ca2+-ATPase activity in the cochlear lateral wall may implicate disturbed calcium-homeostasis in the inner ear, resulting in hearing dysfunction in the early stages of experimentally induced endolymphatic hydrops.

This study compared the neurobehavioral toxicities of three mercurial compounds: methyl mercury (MeHg) which is soluble and organic. and mercuric sulfide (HgS) and cinnabar (naturally occurring HgS), which are insoluble and inorganic. Cinnabar, a Chinese mineral medicine, is still used as a sedative in some Asian countries, but there is relatively little toxicological information about it. These mercurial compounds were administered intraperitoneally (MeHg, 2 mg/ kg) or orally (HgS and cinnabar, 1.0 g/kg) to male rats once every day for 13 consecutive days with assays conducted during or after discontinuous administration for 1 h, 2, 8 and 33 weeks. Neurotoxicity was assessed based on the active avoid-ance response and locomotor activity. The results obtained showed that MeHg and cinnabar prominently and irreversibly caused a decrease in body weight, prolongation of latency for escape from electric shock, a decrease in the percentage for the conditioned avoidance response (CAR) to electric shock, impairment of spontaneous locomotion and inhibition of Na+/K+-ATPase activity of the cerebral cortex. In contrast. HgS reversibly inhibited spontaneous locomotion and Na+/K+-ATPase activity. It was noted that HgS significantly decreased the latency of escape from electric shock during the ad-ministration period, which lasted for 33 weeks after discontinuous administration. In fact that pretreatment with arecoline (a cholinergic receptor agonist) but not fipexide (a dopaminergic receptor agonist) could significantly shorten the prolonged latency for escape caused by MeHg and cinnabar, suggested that the deficit in the active avoidance response was perhaps, at least in part, mediated by the dysfunction of the cholinergic rather than the dopaminergic system. Determination of the Hg levels of the whole blood and cerebral cortex revealed that the tissue mercury content was highly correlated with the degree of neurobehavioral toxicity of these Hg compounds. These findings suggest that insoluble HgS and cinnabar can be absorbed from the G-I tract and distributed to the brain. The possibility that contamination due to other minerals in the cinnabar is responsible for the greater neurotoxic effects compared to HgS is under investigation.

Na(+),K(+)-ATPase and Ca(2+)-ATPase are well known participants in the active transport of ions in the inner ear. These two enzymes play an important role in maintaining cochlear function. Although changes in these enzymes' activities in the cochlea have been implicated in noise-induced hearing loss, no evidence of quantitative alteration of Na(+),K(+)-ATPase or Ca(2+)-ATPase activities has ever been shown. The present study was undertaken to determine the quantitative alterations of their activities by microcolorimetric assay in the cochlear lateral wall after acoustic trauma. Adult albino guinea pigs were exposed to white noise at 105+/-2 dB A for 10 min or 40 h. The age-matched control animals were not exposed to noise. Noise exposure resulted in a significant threshold shift of the auditory brainstem response (P<0.001). Significant decreases in activities of Na(+),K(+)-ATPase and Ca(2+)-ATPase were found in the cochlear lateral wall after noise exposure (P<0.001). Statistical analysis indicated that a good correlation held not only between the decline of these enzyme activities and noise-induced hearing loss, but also between the gradual partial recovery of these parameters during the first 10-day recovery period. The present findings suggest that metabolic damage and ionic disturbance may contribute, at least partially, to noise-induced hearing threshold shift.

The in vivo effect of methylcyclopentadienyl manganese tricarbonyl (MMT), an organic manganese-containing compound, on the mouse motor nerve was studied. The motor nerve conduction velocity was markedly decreased in MMT-treated mice. The Na(+),K(+)-ATPase activity of sciatic nerve isolated from MMT-treated mice was decreased; however, the sciatic nerve Na(+),K(+)-ATPase activity was not affected by the in vitro treatment of MMT. Moreover, [(3)H]ouabain binding of sciatic nerve isolated from MMT-treated mice was decreased. Using Western blot analysis, the amount of Na(+),K(+)-ATPase catalytic alpha1 subunit polypeptide in sciatic nerve of MMT-treated mice was also decreased. These results indicate that a causal relationship may exist between reduced nerve Na(+),K(+)-ATPase activity and motor nerve conduction velocity in MMT-treated mice and that a measurable decrease in alpha1 catalytic subunit isoform of Na(+),K(+)-ATPase may be necessary for the development of peripheral neuropathy by MMT., (Copyright 2000 Academic Press.)

Penta-O-galloyl-beta-D-glucose is structurally related to (-)-epigallocatechin gallate and is isolated from hydrolyzed tannin. Penta-O-galloyl-beta-D-glucose can inhibit tumor promotion by teleocidin. We investigated the effects of penta-O-galloyl-beta-D-glucose and various tea polyphenols on cell viability in human leukemia HL-60 cells. In this study, we demonstrated that penta-O-galloyl-beta-D-glucose was able to induce apoptosis in a concentration- and time-dependent manner; however, other polyphenols were less effective. We further investigated the molecular mechanisms of penta-O-galloyl-beta-D-glucose-induced apoptosis. Treatment with penta-O-galloyl-beta-D-glucose caused induction of caspase-3/CPP32 activity in dose- and time-dependent manner, but not caspase-1 activity, and induced the degradation of poly-(ADP-ribose) polymerase. Pretreatment with acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) and Z-Val-Ala-Asp-fluoromethyl-ketone (Z-VAD-FMK) inhibited penta-O-galloyl-beta-D-glucose-induced DNA fragmentation. Furthermore, treatment with penta-O-galloyl-beta-D-glucose (50 microM) caused a rapid loss of mitochondrial transmembrane potential, release of mitochondrial cytochrome c into cytosol, and subsequent induction of procaspase-9 processing. Our results indicate that penta-O-galloyl-beta-D-glucose allows caspase-activated deoxyribonuclease to enter the nucleus and degrade chromosomal DNA, and induces DFF-45 (DNA fragmentation factor) degradation. These results lead to a working hypothesis that penta-O-galloyl-beta-D-glucose-induced apoptosis is triggered by the release of cytochrome c into the cytosol, procaspase-9 processing, activation of caspase-3, degradation of poly-(ADP-ribose) polymerase, and DNA fragmentation caused by the caspase-activated deoxyribonuclease through the digestion of DFF-45. The induction of apoptosis by penta-O-galloyl-beta-D-glucose may provide a pivotal mechanism for its cancer chemopreventive action.

Objectives: The purpose of this study was to explore the effect of high dietary cholesterol on the lipid composition, Na(+)-K(+)-ATPase activity and ouabain receptor property of the myocardial sarcolemma., Methods: Male New Zealand white rabbits were fed with standard chow or standard chow supplemented with 0.5% (w/w) cholesterol and 10% (w/w) coconut oil to induce hypercholesterolemia. After 8 weeks, the rabbits were sacrificed; a myocardial sarcolemma fraction was then prepared from the left ventricular myocardium and analyzed for lipid composition. Assay of Na(+)-K(+)-ATPase activity and 3H-ouabain binding studies were performed in the myocardial sarcolemma from the control and cholesterol-fed rabbits., Results: The cholesterol content, but not the phospholipid content, of the sarcolemma was significantly greater in the cholesterol-fed group, thus, resulting in an increased cholesterol/phospholipid molar ratio in the cholesterol-fed group. In addition, a decrease in Na(+)-K(+)-ATPase activity was also found in this group. The decrease in Na(+)-K(+)-ATPase activity was selective, since the Mg(++)-ATPase and 5'-nucleotidase activities remained unchanged. In the 3H-ouabain binding study, a decrease in the number of maximum binding sites, but not the binding affinity, for 3H-ouabain was found in the cholesterol-fed group., Conclusions: High dietary cholesterol induces higher levels of cholesterol not only in the plasma, but also in the myocardial sarcolemma. These changes result in decreased myocardial Na(+)-K(+)-ATPase activity mediated by a reduction in the maximum number of binding sites for ouabain but not a change in binding affinity.

The purpose of this study was to explore the effect of ischemia on the Na(+)-K(+)-ATPase activity and ouabain receptor of the myocardial sarcolemma in hypercholesterolemic rabbits. Male New Zealand white rabbits were fed with either standard chow or standard chow supplemented with 0.5% (w/w) cholesterol and 10% (w/w) coconut oil. After an 8 week feeding period, the rabbits underwent a thoracotomy and myocardial ischemia was induced by occlusion of the coronary artery. Myocardial samples from the ischemic and non-ischemic regions of the left ventricle of control and cholesterol-fed rabbits were taken for study. The cholesterol-fed group showed a decrease in both Na(+)-K(+)-ATPase activity and [3H]ouabain binding sites as compared to the control group. Ischemia caused a reduction in both Na(+)-K(+)-ATPase activity [3H]ouabain bindings sites in both control and cholesterol-fed rabbits. The combination of ischemia and hypercholesterolemia produced an additive effect, with a further decrease in both Na(+)-K(+)-ATPase activity and [3H]ouabain binding sites. Neither the activity of Mg+(+)-ATPase nor the binding affinity for [3H]ouabain was affected by either hypercholesterolemia or ischemia. These findings indicate that hypercholesterolemia may exaggerate certain aspects of functional deterioration arising during myocardial ischemia.

The effects of melittin from bee venom, cardiotoxin from Formosan cobra venom, and ouabain on Na+-K+-ATPase activity of the synaptic membrane isolated from rat cerebral cortex were studied. Melittin was the most potent in inhibiting Na+-K+-ATPase activity. Mg2+-ATPase was less susceptible than Na+-K+-ATPase to the inhibitory action of toxins. High K+ (30 mM) reversed the inhibitory action of melittin on Na+-K+-ATPase but did not affect that of cardiotoxin. A comparison between the effects of ouabain and melittin was studied, using double-reciprocal plots of Na+-K+-ATPase activity against K+. It was shown that both were competitive with K+ for binding to the K+ site. Moreover, a median-effect plot revealed that ouabain and melittin antagonized each other when inhibiting Na+-K+-ATPase. Phosphatidylcholine (PC) was the only one of the phospholipids tested capable of protecting Na+-K+-ATPase from the inhibitory action of melittin but not that of ouabain. However, the inhibitory action of cardiotoxin on this enzyme was decreased by phosphatidylserine and sphingomyelin, in addition to PC. All of these findings suggest that the melittin polypeptide potently inhibits Na+-K+-ATPase, possibly by binding to the K+ site.

Na+-K+-ATPase activity and [3H]ouabain binding were studied in cardiac ventricles of single wrapped kidney and DOCA-NaCl hypertensive rats. It was found that the total Na+-K+-ATPase activity decreased in the DOCA-NaCl and kidney wrapped hypertensive rats. The decrease of enzyme activity in DOCA-NaCl hypertensive rats was due to extracellular fluid expansion induced by NaCl loading, as DOCA itself had no effect on the enzyme. All these alterations were specific for Na+-K+-ATPase, since Mg2+-ATPase and 5'-nucleotidase activities were unaffected. Binding studies with [3H]ouabain showed that the decrease in Na+-K+-ATPase activity was due to a reduction in the number of binding sites for ouabain rather than to a change of binding affinity. The reduced myocardial Na+-K+-ATPase activity observed in these two types of low renin hypertension, coupled with the observation of reduced vascular Na+ pump activity by others, suggests a common underlying defect in the cardiovascular Na+-K+ transport system of these hypertensive rats.

(Na+ -K+)ATPase activity in erythrocyte membranes of spontaneously (SHR), one kidney-one wrapped, and deoxycorticosterone acetate (DOCA)-NaCl hypertensive rats was studied. (Na+ -K+) ATPase activity decreased in both prehypertensive (6 weeks old) and hypertensive (14 weeks old) stages of SHR, suggesting that the alteration of this enzymic activity may be due to a pre-existing defect in the membrane rather than being a consequence of hypertension. By contrast, (Na+ -K+)ATPase activity remained unchanged in the one kidney-one wrapped hypertensive rats, whereas that of one kidney-one wrapped normotensive rats as well as that of DOCA-NaCl hypertensive rats was increased significantly (P less than 0.05). These changes were specific for (Na+ -K+) ATPase, since Mg2+-ATPase activity was not altered. The susceptibility of (Na+ -K+)ATPase to the inhibitory action of ouabain was not changed significantly. These findings indicate that (Na+ -K+)ATPase activities of erythrocyte membranes isolated from the different types of hypertensive rats were subject to different changes. Whether this phenomenon applies to the clinical distinctions among the various types of hypertension remains a subject for further investigation.

Na+-K+-ATPase activity and [3H]ouabain binding were studied in cerebral cortex, kidney and heart isolated from spontaneously hypertensive rats (SHR) in both the prehypertensive (6 week old) and the hypertensive stages (14 week old). Na+-K+-ATPase activity of heart and kidney was found to be decreased by about 38 and 16% in the prehypertensive and hypertensive stages of SHR respectively; that of cerebral cortex decreased by 23.5% only in the hypertensive stages. Similar results were obtained by pretreatment of membranes with either 0.001% Triton X-100 or by increasing the K concentration from 4.7 to 12.7 mM in the Krebs solution. No significant differences in microsomal protein yield were noted between prehypertensive or hypertensive SHR and the age-matched WKY rats. The study of binding of [3H]ouabain to cerebral cortex, kidney and heart showed that the decreased Na+-K+-ATPase in hypertensive SHR was due to a 31.6, 21.8 and 41.3% reduction in the number of high affinity binding sites respectively, while the affinity constants (Kd) of ouabain binding sites on this enzyme in cerebral cortex, kidney and heart of the normotensive WKY rats were 26.5, 455.9 and 74.7 nM respectively and those from the hypertensive SHR were not altered. The plasma K concentration of the SHR in the prehypertensive and hypertensive stages was 4.07 and 4.13 mM, respectively, significantly less than that of the age-matched WKY rats. It appears that the decrease of plasma K and Na+-K+-ATPase activity in heart and kidney in SHR is derived from a genetic defect and may be related to the abnormal Na handling in this genetically hypertensive strain.