腺苷A1受体在离体延髓脑片上对节律性呼吸的调制

实验旨在探讨腺苷A1受体在对基本呼吸节律调制中的可能作用。制作新生大鼠离体延髓脑片标本,主要包含面神经后核内侧区(themedial region of the nucleus retrofacialis,mNRF),并保留完整的舌下神经根。以改良Kreb‘s液灌流脑片,记录mNRF吸气神经元的电活动,并同步记录舌下神经根呼吸节律性放电(respiratory rhythmical discharge activity,RRDA)。在灌流液中先分别单独给予腺苷A1受体的特异性拮抗剂8-环戊-1,3-二丙基黄嘌呤(8-cyclopenty 1-1,3-dipropylxanthine,DPCPX)和特异性激动剂R-苯异丙基-腺苷(R-phenylisopropyl-adenosine,R-PIA);再分别先后给予R-PIA和R-PIA DPCPX,观察RRDA和吸气神经元电活动的变化。结果显示,给予腺苷A1受体拮抗剂DPCPX后,呼气时程和呼吸周期明显缩短,吸气神经元中期放电的频率和峰频率显著增大;给予腺苷Al受体激动剂R-PIA后,吸气时程、积分幅度和吸气神经元中期放电的频率和峰频率均显著降低,呼吸周期明显延长,且R-PIA的呼吸抑制作用可部分地被DPCPX逆转。实验结果提示,腺苷A1受体可能通过介导吸气神经元的抑制性突触输入参与节律性呼吸的调制。     

Hypercalcemic effect of insulin in thyroparathyroidectomized alloxan-treated rats

The acute effect of a hypoglycaemic dose of 0.5 U/100 g BW insulin administered intramuscularly on calcium metabolism was investigated in fasted alloxan-treated rats. It was found that the hypercalcaemic effect of insulin was evident only in thyroparathyroidectomized (TPTX) and not in parathyroidectomized (PTX) rats. A subcutaneous administration of 180 MRC mU/100 g BW calcitonin abolished the calcium raising effect of insulin in TPTX rats suggesting a protective role of calcitonin against insulin action in intact rats. In an attempt to elucidate the mechanism of the calcium raising effect of insulin 45Ca administered intravenously was used to indicate the movement of calcium from the plasma pool. Insulin administration delayed the plasma 45Ca disappearance rate but had no effect on bone 45Ca uptake within 120 min. In contrast, insulin administration resulted in a 31% reduction of urinary 45Ca excretion while the urine volume remained unchanged. However, the insulin-induced reduction of urinary calcium excretion could not totally account for the calcium raising effect of insulin in TPTX animals.     

Effect of aspartame on circadian oscillations of calcium and inorganic phosphorus in rats

The effect of aspartame on circadian rhythms of calcium and inorganic phosphorus levels was studied in rats. Acrophase delays in calcium rhythms and advances in inorganic phosphorus rhythms and alteration in mesor values in both rhythms were observed in aspartame-treated rats. However, no change in amplitude values was observed. Oral administration of aspartame leads to increased levels of aspartate in the brain, which could alter the characteristics of calcium and inorganic phosphorus rhythms, possibly by modulating transmission in several areas/nuclei in brain, including retinohypothalamic tract (RHT) and suprachiasmatic nuclei (SCN).     

The effects of glucagon and epinephrine on two preparations of cardiac mitochondria

The effects of $\text{in}$$\text{vivo}$ administration in epinephrine on calcium uptake were measured in two preparations of heart mitochondria, intermyofibrillar (IMF) and subsarcolemmal (SSL) using either ⁴⁵Ca²⁺ or murexide to follow calcium movement. The administration of either hormones resulted in an increased calcium uptake in both preparation of mitochondria subsequently isolated. This increase might be the consequence of the increased State 3 respiration, also evoked by hormones. The possibility is raised that the inotropic actions of glucagon and epinephrine might be partially mediated by mitochondria.     

Effects of chronic ethanol feeding and acetaldehyde metabolism on calcium transport by rat liver mitochondria

The prolonged feeding of ethanol to rats alters in vitro mitochondrial transport of calcium. Hepatic mitochondria isolated from rats fed ethanol for 7 weeks exhibited decreased retention of calcium in the presence of 4mM-Pi. This defect was associated with enhanced efflux of calcium when mitochondria were incubated with EGTA. Acetaldehyde at low, "physiological" concentrations (100 microM) enhanced calcium retention by mitochondria but this response was blunted after chronic ethanol administration. The in vitro actions of acetaldehyde appear to be mediated, in part, by its metabolism in mitochondria since pretreatment of rats with cyanamide (an aldehyde dehydrogenase inhibitor) prevents this effect.     

Calcium as a counteractive agent to streptomycin induced respiratory depression: an in vivo electrophysiological observation.

Effect of streptomycin on respiratory function in cats was studied. It was observed that streptomycin at a dose of 40 mg/kg body weight intravenously (i.v.) caused respiratory failure or streptomycin induced respiratory depression (SIRD). This respiratory failure is not linked with Herring-Breuer stretch receptors because the effect remained unaltered in artificially ventilated cats. The involvement of central structures in SIRD can be discarded since intracarotid and intraventricular administration of streptomycin failed to produce any change in respiration. Studies on monosynaptic reflex, dorsal and ventral root activities of spinal phrenic and intercostal nerves, and on fusimotor and alpha-motor neuron activities of spinal intercostal and phrenic nerves in decerebrated cats indicated clearly that respiratory depression is not only due to blockade at neuromuscular junction but due to functional depression at the level of muscle receptors and spinal cord motor neurons. The respiratory depression induced by streptomycin was more or less completely reversed when calcium was administered intravenously from external source. It is speculated that streptomycin induced respiratory depression may be mediated through calcium inhibition which can be treated with external calcium in conjunction with artificial respiration.     

Disruption of learned timing in P/Q calcium channel mutants

To optimize motor performance, both the amplitude and temporal properties of movements should be modifiable by motor learning. Here we report that the modification of movement timing is highly dependent on signaling through P/Q-type voltage-dependent calcium channels. Two lines of mutant mice heterozygous for P/Q-type voltage-dependent calcium channels exhibited impaired plasticity of eye movement timing, but relatively intact plasticity of movement amplitude during motor learning in the vestibulo-ocular reflex. The results thus demonstrate a distinction between the molecular signaling pathways regulating the timing versus amplitude of movements.     

Calcium transport from blood into the bile in normal and regenerating rat liver

We have studied calcium movement from blood into the bile by injecting 45Ca2+ intravenously and measuring the radioactivity appearing in the bile. 45Ca2+ started to appear in the bile at 3 min and maximum values were observed at 5 min after its administration. The amount of calcium secreted into the bile was proportional to the blood calcium concentration indicating that the main pathway involved in calcium movement behaved as a non-saturable system. We have also studied the 45Ca2+ circulation from blood into the bile in rats subjected to a partial hepatectomy. Thereafter, the calcium transported into the bile per gram of liver increased by about 50 per cent. Since bile flow behaved in a similar way, the biliar calcium concentration remained unmodified after hepatectomy. Determination of the activities of the Ca2+ transporting systems in isolated plasma membrane fractions from regenerating livers showed no modification in these activities suggesting that the elevation in calcium movement observed after hepatectomy is not due to an increase in the circulation of Ca2+ through the transhepatocyte pathway, an observation compatible with the absence of saturation in the transport.     

Changes in [3H]Nitrendipine Binding and γ-Aminobutyric Acid Release in Rat Hippocampus Following Repeated Morphine Administration

An antagonistic effect of calcium on the action of morphine was studied in rat hippocampal slices. The effect of repeated administration of morphine on gamma-aminobutyric acid (GABA) release and binding of [3H]nitrendipine, a calcium antagonist, was also examined. (1) In rat brain hippocampal slices, morphine enlarged the amplitude of the field potentials evoked in pyramidal neurons, disinhibiting them through basket cells. When the calcium concentration was elevated, potentiation of the field potentials by morphine was reduced. Decrease of the calcium concentration, on the other hand, enhanced the potentiating effect of morphine. Following repeated administration of morphine, its enhancing effect on the field potentials in slices was not observed. (2) In hippocampal membrane fractions obtained from rats repeatedly treated with morphine, enhancement of [3H]nitrendipine binding was observed. (3) In hippocampal slice preparations from rats receiving morphine repeatedly, K+ (45 mM)-stimulated [3H]GABA efflux was enhanced. The above results indicate that morphine antagonizes calcium, thereby reducing the release of transmitters. Furthermore, increase in calcium channels following repeated treatment of rats with morphine may explain the mechanism underlying development of tolerance.     

Development of hypothermia in rats at increase of [Ca2+] in the blood

In rats, data on influence of i. v. administration of calcium chloride on the level of [Ca2+] in the blood and on process of oppression ofthermoregulatory and respiratory functions in rats in hypothermia. 0.18 or 0.135 mmol Ca2+ on the 3rd minute from beginning of the administration increased [Ca2+] in the blood from 1.01 +/- 0.03 to 2.56 +/- 0.08 mM (or 2.27 +/- 0.06 mM). Then [Ca2+] was reduced gradually, in 20 minutes from administration, solution of CaCh [Ca2+] exceeded the initial level by 20-30 %. The increase of concentration of ionized calcium in the rat blood strengthened the cold oppression of breathing and cold shivering as compared with the control (administration of physiological solution). Arrest of breathing in rats after administration of CaCl2 solution occurred at higher rectal temperatures (21 +/- 0.03 degrees C) as compared with control experiments (18 +/- 0.4 degrees C), p < 0.05. It is suggested that increase of [Ca2+] in the blood strengthens effects of cold in the form of oppression of thermoregulatory and respiratory functions.     

Effect of verapamil on ventilation and chemical control of breathing in anesthetized rats

The calcium channel blocker, verapamil (0.1-1.0 mg/kg, i.v.) was administered to anesthetized rats to determine its effects on ventilation and on ventilatory responses to hypoxia and CO2. Verapamil produced a dose-dependent increase in tidal volume (VT) and a decrease in respiration rate (f). The bradypnea due to verapamil was characterized by an increase in expiratory duration (TE) and no change of inspiratory duration (TI). Verapamil produced similar changes in VT and f in vagotomized rats. The increase in respiration rate and minute volume due to hypoxia were inhibited by verapamil (0.5 and 1.0 mg/kg) but the increase in tidal volume due to hypoxia was depressed only with the 1.0 mg/kg dose. On the other hand, the increase in VT due to breathing CO2 was not changed by verapamil (0.1-1.0 mg/kg), but depression of the respiratory frequency response to CO2 occurred with 1.0 mg/kg of verapamil. These results indicate that verapamil produced slow, deep breathing and these responses were not mediated by vagal mechanisms. Ventilatory responses to hypoxia were depressed by verapamil. However, since the calcium blocker demonstrated no effect on the VT-CO2 relationship, verapamil did not change ventilatory chemosensitivity to CO2. The data also suggest that mechanisms governing the control of respiratory frequency are more sensitive to verapamil than tidal volume responses.     

Harmonic analysis of nerve impedance in situ

A novel technique to assess electric activity of the whole nerve in situ is advanced based on harmonic analysis of impedance microvariations of femoral nerve in narcotized rats recorded with extracellular electrodes. The amplitude spectrum of nerve bioimpedance (Z-spectrum) made it possible to determine the level of neural asynchronous activity in the frequency band of 20–600 Hz with amplitude resolution of 1% and to reveal the rhythmic activity at Mayer (0.1 Hz) and respiration frequencies with amplitude resolution of 0.1%. Systemic administration of atropine used to shift the sympathovagal balance in favor of sympathetic system increased both rhythmic and unrhythmic (asynchronous) neural activity. The study demonstrated that harmonic analysis of nerve bioimpedance is a novel and potent tool to assess neural activity in the frequency domain.     

Calcium, carbonic anhydrase and gastric acid secretion

Previous data concerning the action of calcium (Ca) on gastric acid secretion (GAS) indicated that calcium ions increase GAS elicited by gastrin released through a vagal mechanism, and also by a direct effect on parietal cells. Our research showed that the stimulating effect of calcium on gastric acid secretion can be antagonized by verapamil administration, which reduces gastric acid secretion . In the present study we followed the effect induced by administration of calcium and Ca-chelating agents (disodium EDTA) on gastric acid secretion and on carbonic anhydrase (CA) activity. We selected two groups of healthy volunteers: Group I (n=21) received a single i.v. dose of CaCl2 (15 mg/kg b.w.), whereas Group II (n=22) received a single i.v. dose of disodium EDTA (5 mg/kg b.w.). We determined blood calcium before and after treatment, gastric acid secretion at 2 hours. erythrocyte CA II activity, and CA IV activity in membrane parietal cells, which were isolated from gastric mucosa obtained by endoscopic biopsy. Assessment of carbonic anhydrase activity was achieved by the stopped-flow method. In Group I calcium administration increased blood calcium, HCl output, CA II and CA IV activity as compared to initial values. In Group II, disodium EDTA reduced blood calcium, HCl output, CA II and CA IV activity as compared to initial values. The results demonstrated that increased blood calcium and GAS values after calcium administration correlated with the increase of erythrocyte CA II and parietal cell CA IV activity, while disodium EDTA induced a reversed process. Our results also show that cytosolic CA II and membrane CA IV values are sensitive to calcium changes and they directly depend on these levels. Our data suggest that intra- and extracellular pH changes induced by carbonic anhydrase might account for the modulation of the physiological and pathological secretory processes in the organism.     

Duodenal calcium transporter mRNA expression in stressed male rats treated with diazepam, fluoxetine, reboxetine, or venlafaxine

Chronic stress has been reported to decrease bone density and intestinal calcium absorption, but its underlying mechanism remains elusive. Since long-term exposure to glucocorticoids, major stress hormones from adrenal gland, is known to downregulate the mRNA expression of intestinal calcium transporter TRPV6, the present study aimed to demonstrate whether decreases in mRNA expressions of duodenal calcium transporter genes were observed in male rats subjected to restraint stress for 4?weeks. The results from quantitative real-time PCR showed that restraint stress significantly downregulated the mRNA expressions of apical calcium channels (TRPV6 and Ca(v)1.3), cytoplasmic calcium-binding protein (calbindin-D(9k)), and basolateral calcium pump (PMCA(1b)), but not the expression of TRPV5 or NCX1. The mRNA expressions of paracellular genes, ZO-1, occludin, and claudin-3, were not altered by restraint stress. Since several antidepressant or anxiolytic drugs effectively alleviate stress-induced depressive and anxiety symptoms, we further hypothesized that these drugs may also enhance calcium transporter gene expression in stressed rats. As expected, 4-week daily administration of 10?mg/kg fluoxetine, 10?mg/kg reboxetine, or 10?mg/kg venlafaxine differentially increased calcium transporter mRNA expression in stressed rats, whereas 2?mg/kg diazepam had no such effect. It could, therefore, be concluded that 4-week restraint stress downregulated some important calcium transporter mRNA expression in the duodenal epithelial cells of male rats, which could be prevented by oral administration of fluoxetine, reboxetine, and venlafaxine. The present findings may be applied to help alleviate the stress-induced bone loss and osteoporosis by restoring intestinal calcium absorption to provide calcium for bone formation.     

In Vitro Dose-Dependent Inhibition of the Intracellular Spontaneous Calcium Oscillations in Developing Hippocampal Neurons by Ketamine

Spatial and temporal abnormalities in the frequency and amplitude of the cytosolic calcium oscillations can impact the normal physiological functions of neuronal cells. Recent studies have shown that ketamine can affect the growth and development and even induce the apoptotic death of neurons. This study used isolated developing hippocampal neurons as its study subjects to observe the effect of ketamine on the intracellular calcium oscillations in developing hippocampal neurons and to further explore its underlying mechanism using Fluo-4-loaded laser scanning confocal microscopy. Using a semi-quantitative method to analyze the spontaneous calcium oscillatory activities, a typical type of calcium oscillation was observed in developing hippocampal neurons. In addition, the administration of NMDA (N-Methyl-D-aspartate) at a concentration of 100 µM increased the calcium oscillation amplitude. The administration of MK801 at a concentration of 40 µM inhibited the amplitude and frequency of the calcium oscillations. Our results demonstrated that an increase in the ketamine concentration, starting from 30 µM, gradually decreased the neuronal calcium oscillation amplitude. The inhibition of the calcium oscillation frequency by 300 µM ketamine was statistically significant, and the neuronal calcium oscillations were completely eliminated with the administration of 3,000 µM Ketamine. The administration of 100, 300, and 1,000 µM NMDA to the 1 mM ketamine-pretreated hippocampal neurons restored the frequency and amplitude of the calcium oscillations in a dose-dependent manner. In fact, a concentration of 1,000 µM NMDA completely reversed the decrease in the calcium oscillation frequency and amplitude that was induced by 1 mM ketamine. This study revealed that ketamine can inhibit the frequency and amplitude of the calcium oscillations in developing hippocampal neurons though the NMDAR (NMDA receptor) in a dose-dependent manner, which might highlight a possible underlying mechanism of ketamine toxicity on the rat hippocampal neurons during development.     

Pressor, tachycardic and feeding responses in conscious rats following i.c.v. administration of dynorphin. Central blockade by opiate and alpha 1-receptor antagonists

Experiments were designed to determine the hemodynamic responses of conscious, unrestrained rats given intracerebroventricular (i.c.v.) injections of dynorphin A-(1-13) and the possible central receptor mechanisms mediating those changes. Male Sprague-Dawley rats (300 gb. wt.) received i.c.v. injections (by gravity flow in a total volume of 3 or 5 microliter) of control solutions of sterile saline (SS) or dimethylsulfoxide (DMSO) or 1.5, 3.0 or 6.1 nmol of dynorphin A-(1-13). Blood pressure and heart rate changes were monitored over 2 h after administration; as well, feeding activity was visually assessed and scored over this period. Other groups of conscious rats were pretreated i.c.v. with equimolar doses (3.0-24.4 nmol) of specific receptor antagonists (naloxone HCl, phentolamine HCl, propranolol HCl, yohimbine HCl or prazosin HCl) 10 min before subsequent i.c.v. administration of SS or DMSO/SS or 6.1 nmol of dynorphin A-(1-13). I.c.v. injection of dynorphin A-(1-13) caused a dose-related pressor response, associated temporally with tachycardia. As well, dynorphin evoked feeding activity and some grooming, which occurred when the rats were hypertensive and tachycardic and decreased as heart rate and blood pressure returned to control levels. I.c.v. pretreatment studies indicated that naloxone HCl (12.2 nmol), phentolamine HCl (12.2 nmol) and prazosin HCl (6.1 nmol) blocked the pressor response, tachycardia as well as feeding activity of rats subsequently given dynorphin. The results suggest the pressor and tachycardic effects of conscious rats following i.c.v. dynorphin administration may, in part, be due to behavioral activation (feeding). As well, these data indicate that both opioid as well as alpha 1-adrenergic receptors within the CNS are involved in mediating the pressor, tachycardic and feeding responses of conscious rats given i.c.v. injections of dynorphin A.     

Characterization of calcium accumulation in the brain of rats administered orally calcium: The significance of energy-dependent mechanism

The characterization of calcium accumulation in the brain of rats administered orally calcium chloride solution was investigated. Rats received a single oral administration of calcium (15–50 mg/100 g body weight), and they were sacrificed by bleeding-between 15 and 120 min after the administration. The administration of calcium (50 mg/100 g) produced a significant increase in serum calcium concentration and a corresponding elevation of brain calcium content, indicating that the transport of calcium into the brain is associated with the elevation of serum calcium levels. The increase in brain calcium content by calcium administration was not appreciably altered by the pretreatment with Ca²⁺ channel blockers (verapamil or diltiazem with the doses of 1.5 and 3.0 mg/100 g). In thyroparathyroidectomized rats, the administration of calcium (50 mg/100 g) caused a significant increase in brain calcium content, indicating that calcium-regulating hormones do not participate in the brain calcium transport. Now, brain calcium content was clearly elevated by fasting (overnight), although serum calcium level was not significantly altered. Calcium administration to fasted rats induced a further elevation of brain calcium content as compared with that of control (fasted) rats. The fasting-induced increase in brain calcium content was appreciably restored by refeeding. This restoration was also seen by the oral administration of glucose (0.4 g/100 g) to fasted rats. The present study demonstrates that serum calcium is transported to brain, and that the increased brain calcium is released promptly. The release of calcium from brain may be involved in energy metabolism, and this release may be weakened by the reduction of glucose supply into brain. The finding suggests a physiological significance of energy-dependent mechanism in the regulation of brain calcium.     

Combined Oral Administration of Bovine Collagen Peptides with Calcium Citrate Inhibits Bone Loss in Ovariectomized Rats

Purpose

Collagen peptides (CPs) and calcium citrate are commonly used as bone health supplements for treating osteoporosis. However, it remains unknown whether the combination of oral bovine CPs with calcium citrate is more effective than administration of either agent alone.

Methods

Forty 12-week-old Sprague-Dawley rats were randomly divided into five groups (n = 8) for once-daily intragastric administration of different treatments for 3 months at 3 months after ovariectomy (OVX) as follows: sham + vehicle; OVX + vehicle; OVX + 750 mg/kg CP; OVX + CP-calcium citrate (75 mg/kg); OVX + calcium citrate (75 mg/kg). After euthanasia, the femurs were removed and analyzed by dual energy X-ray absorptiometry and micro-computed tomography, and serum samples were analyzed for bone metabolic markers.

Results

OVX rats supplemented with CPs or CP-calcium citrate showed osteoprotective effects, with reductions in the OVX-induced decreases in their femoral bone mineral density. Moreover, CP-calcium citrate prevented trabecular bone loss, improved the microarchitecture of the distal femur, and significantly inhibited bone loss with increased bone volume, connectivity density, and trabecular number compared with OVX control rats. CP or CP-calcium citrate administration significantly increased serum procollagen type I N-terminal propeptide levels and reduced serum bone-specific alkaline phosphatase, osteocalcin, and C-telopeptide of type I collagen levels.

Conclusions

Our data indicate that combined oral administration of bovine CPs with calcium citrate inhibits bone loss in OVX rats. The present findings suggest that combined oral administration of bovine CPs with calcium citrate is a promising alternative for reducing bone loss in osteopenic postmenopausal women.     

Hepatocyte mitochondrion electron-transport chain alterations in CCl<Subscript>4</Subscript> and alcohol induced hepatitis in rats and their correction with simvastatin

The goal of this study was to examine the state of hepatocyte mitochondrial respiratory chain of rats with toxic hepatitis induced by CCl₄ and ethanol. Oxygen consumption by hepatocytes and mitochondria was determined. Endogenous oxygen consumption by pathological hepatocytes was 1.3-fold higher compared with control. Rotenone resulted in 27% suppression of respiration by pathological hepatocytes whereas 2,4-dinitrophenol produced a 1.4-fold increase of respiration. States 3 and 4 of mitochondrial respiration with malate and glutamate were found to be higher as compared to control. State dinitrophenol and state 3 respirations were similar within every group of animals when being tested with malate and glutamate or succinate. Cytochrome c oxidase activity in hepatitis was 1.8-fold higher compared with control. Simvastatin administration resulted in a decrease in hepatocyte endogenous respiration in hepatitis. The presented data lead to the assumption that the increased oxygen consumption by the respiratory chain of pathological mitochondria to be linked mainly with the altered function of complex I.     

Control of respiration and oxidative phosphorylation in isolated rat liver cells

NAD(P)H fluorescence, mitochondrial membrane potential and respiration rate were measured and manipulated in isolated liver cells from fed and starved rats in order to characterize control of mitochondrial respiration and phosphorylation. Increased mitochondrial NADH supply stimulated respiration and this accounted for most of the stimulation of respiration by vasopressin and extracellular ATP. From the response of respiration to NADH it was estimated that the control coefficient over respiration of the processes that supply mitochondrial NADH was about 0.15-0.3 in cells from fed rats. Inhibition of the ATP synthase with oligomycin increased the mitochondrial membrane potential and decreased respiration in cells from fed rats, while the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone had the opposite effect. There was a unique relationship between respiration and membrane potential irrespective of the ATP content of the cells indicating that phosphorylation potential controls respiration solely via phosphorylation (rather than by controlling NADH supply). From the response of respiration to the mitochondrial membrane potential (delta psi M) it was estimated that the control coefficients over respiration rate in cells from fed rats were: 0.29 by the processes that generate delta psi M, 0.49 by the process of ATP synthesis, transport and consumption, and 0.22 by the processes that cycle protons across the inner mitochondrial membrane other than via ATP synthesis (e.g. the passive proton leak). Control coefficients over the rate of mitochondrial ATP synthesis were 0.23, 0.84 and -0.07, respectively, by the same processes. The control distribution in cells from starved rats was similar.