Evidence for an involvement of the brain cholecystokinin B receptor in anxiety.

The effect of neuropeptide cholecystokinin (CCK) receptor agonists and antagonists was examined in the rat elevated X-maze model of anxiety. The selective CCK-B receptor antagonists CI-988 (PD 134308) and L-365,260 produced anxiolytic-like effects, whereas MK-329, a CCK-A receptor antagonist, was respectively less potent by factors of 313 and 200. The intracerebroventricular administration of the nonselective CCK receptor agonist caerulein or the selective CCK-B receptor agonist pentagastrin increased dose dependently the level of anxiety. CI-988 dose dependently antagonized the anxiogenic response to pentagastrin but not that induced by pentylenetetrazol. These results strongly suggest that activation of the brain CCK-B receptor induces anxiety and that selective antagonists of this receptor represent a separate class of anxiolytic agents.     

Evolution of the vacuolar H+-ATPase: implications for the origin of eukaryotes.

Active transport across the vacuolar components of the eukaryotic endomembrane system is energized by a specific vacuolar H+-ATPase. The amino acid sequences of the 70- and 60-kDa subunits of the vacuolar H+-ATPase are approximately equal to 25% identical to the beta and alpha subunits, respectively, of the eubacterial-type F0F1-ATPases. We now report that the same vacuolar H+-ATPase subunits are approximately equal to 50% identical to the alpha and beta subunits, respectively, of the sulfur-metabolizing Sulfolobus acidocaldarius, an archaebacterium (Archaeobacterium). Moreover, the homologue of an 88-amino acid stretch near the amino-terminal end of the 70-kDa subunit is absent from the F0F1-ATPase beta subunit but is present in the alpha subunit of Sulfolobus. Since the two types of subunits (alpha and beta subunits; 60- and 70-kDa subunits) are homologous to each other, they must have arisen by a gene duplication that occurred prior to the last common ancestor of the eubacteria, eukaryotes, and Sulfolobus. Thus, the phylogenetic tree of the subunits can be rooted at the site where the gene duplication occurred. The inferred evolutionary tree contains two main branches: a eubacterial branch and an eocyte branch that gave rise to Sulfolobus and the eukaryotic host cell. The implication is that the vacuolar H+-ATPase of eukaryotes arose by the internalization of the plasma membrane H+-ATPase of an archaebacterial-like ancestral cell.     

Angiotensin II increases H+-ATPase B1 subunit expression in medullary collecting ducts

Metabolic alkalosis is a common feature of hypokalemic hypertensive syndromes associated with angiotensin II excess. The alkalosis-generating effect of angiotensin II is usually ascribed to its stimulatory effect on aldosterone secretion, a hormone that upregulates collecting duct hydrogen ion secretion. We studied the effect of angiotensin II infusions on the expression of B1 and a4 protein, subunits of the renal H+-ATPase in adrenalectomized rats. Adrenalectomized rats were given either angiotensin II or vehicle for 7 days via osmotic mini-pumps. H+-ATPase B1 protein expression was evaluated by Western blot analysis in isolated medulla and cortex plasma membrane preparations from one kidney, whereas the contralateral kidney was used for immunostaining. By Western blotting, the relative abundance of B1 protein was 2-fold higher in renal medulla membranes from rats with intact adrenal glands (sham surgery) than from adrenalectomized rats (219+/-47%, n=12; P<0.05). In contrast to renal medulla, adrenalectomy did not significantly alter the relative abundance of B1 protein in renal cortex. Angiotensin II also did not significantly alter the relative levels of B1 protein in the cortex, but it increased it significantly in renal medullary membranes (231+/-56%, n=8; P<0.005). Moreover, enhanced H+-ATPase B1 subunit protein immunoreactivity was found in medullary collecting duct segments of rats infused with angiotensin II. In contrast to B1, expression of a4, another subunit of the H+-ATPase was not altered by adrenalectomy or angiotensin II. We conclude that adrenalectomy decreases whereas angiotensin II increases H+-ATPase B1 subunit expression in medullary, but not in cortical collecting ducts. By increasing the relative abundance of the B1 subunit of H+-ATPase in the collecting duct, angiotensin II excess may lead to increased hydrogen ion secretion and thus metabolic alkalosis-a common feature of hypertensive syndromes associated with angiotensin II overactivity.     

Evidence for the involvement of the submandibular gland epidermal growth factor in mouse mammary tumorigenesis.

The submandibular gland is a rich source of epidermal growth factor (EGF) in mice. The concentration of EGF in the gland of virgin female mice of C3H/HeN strain increased as much as 9-fold from the age of 30 to 52 weeks. During this period, the incidence of mammary tumor in virgin females increased markedly to a maximal level of 62.5% (n = 48) at 52 weeks of age. Removal of the submandibular gland (sialoadenectomy) of virgin mice 14-22 weeks old reduced the tumor incidence to 12.8% (n = 39) at the age of 52 weeks and also increased the latency period of mammary tumor development as much as 14 weeks when compared to that of normal mice. Long-term treatment of sialoadenectomized virgin mice with EGF (5 micrograms per mouse every other day) increased the tumor incidence to 33.3%. Moreover, sialoadenectomy of mammary tumor-bearing animals caused a rapid and sustained cessation of tumor growth, but EGF administration (5 micrograms per mouse per day) quickly restored the rate of tumor growth to a normal level. These results indicate that submandibular gland EGF plays a crucial role in mouse mammary tumorigenesis.     

Evidence for regulation of mitochondrial function by the L-type Ca channel in ventricular myocytes

The L-type Ca²⁺ channel is responsible for initiating contraction in the heart. Mitochondria are responsible for meeting the cellular energy demands and calcium is required for the activity of metabolic intermediates. We examined whether activation of the L-type Ca²⁺ channel alone is sufficient to alter mitochondrial function. The channel was activated directly with the dihydropyridine agonist BayK(−) or voltage-clamp of the plasma membrane and indirectly by depolarization of the membrane with high KCl. Activation of the channel increased superoxide production (assessed as changes in dihydroethidium fluorescence), NADH production and metabolic activity (assessed as formation of formazan from tetrazolium) in a calcium-dependent manner. Activation of the channel also increased mitochondrial membrane potential assessed as changes in JC-1 fluorescence. The response was reversible upon inactivation of the channel during voltage-clamp of the plasma membrane and did not appear to require calcium. We examined whether the response may be mediated through movement of cytoskeletal proteins. Depolymerization of actin or exposing cells to a peptide directed against the alpha-interacting domain of the α_1C-subunit of the channel (thereby preventing movement of the β-subunit) attenuated the increase in mitochondrial membrane potential. We conclude that activation of the L-type Ca²⁺ channel can regulate mitochondrial function and the response appears to be modulated by movement through the cytoskeleton.     

Plasma mitochondrial coupling factor 6 in patients with acute myocardial infarction.

Previous studies have shown that mitochondrial coupling factor 6 (CF6) is an endogenous peptide that inhibits prostacyclin (PGI2) synthesis in vascular endothelial cells. In this study, we measured the plasma CF6 level of patients with acute myocardial infarction (AMI) to observe dynamic changes of CF6. All patients showed elevated plasma CF6 levels upon admission for treatment of AMI. Their CF6 levels peaked approximately 72 h after the onset of AMI and remained high for 7 days. At 7 days, their CF6 levels decreased to the level seen upon admission, but not to within a normal range. Hyperlipidemic patients had significantly greater CF6 levels at 24 h after onset of AMI than patients with a normal lipid profile. On admission, the plasma CF6 level in patients with a cardiac function of Killip class > or =II was higher than that in patients with a Killip class I cardiac function. At 3 days after the onset of AMI, the plasma CF6 levels of patients with a creatinine kinase (CK) peak value > or =1,500 units/l were significantly higher than those of patients with a CK peak value <1,500 units/l (p =0.05). At 7 days after the onset of AMI, the plasma CF6 levels of patients who received no reperfusion were significantly higher than those of patients who received a successful reperfusion. The plasma CF6 levels of AMI patients at admission, at 24 h, and at 3 days after onset of symptoms correlated positively with the cardiac function by Killip classification, respectively. At 24 h after onset of AMI, the plasma CF6 levels correlated positively with plasma total cholesterol levels and low-density lipoprotein levels. At 3 days, the plasma level of CF6 correlated positively with the plasma CK peak value and correlated negatively with left ventricular ejection fraction. These results suggest that the plasma CF6 level was elevated in patients with AMI.     

ATP-sensitive K+ channels in a plasma membrane H+-ATPase mutant of the yeast Saccharomyces cerevisiae.

A mutant in the plasma membrane H+-ATPase gene of the yeast Saccharomyces cerevisiae with a reduced H+-ATPase activity, when examined at the single-channel level with the patch-clamp technique, was found to exhibit K+ channels activated by intracellular application of ATP. In the parent strain, the same channel, identified by its conductance and selectivity, is not activated by ATP. This activity in the mutant is blocked by the ATPase inhibitor N,N'-dicyclohexylcarbodiimide. ADP and the ATP analog adenosine 5'-[gamma-[35S]thio]triphosphate do not activate the channel. These findings suggest a tight physical coupling between the plasma membrane ATPase and the K+ channel.     

联系方式：周涛 chowtao@sina.com. Tel:13985024647

目的 探讨浅低温心脏不停跳心内直视手术对体外循环瓣膜置换患者线粒体耦联因子6（CF6）表达的影响.方法 选择2010年1月至2011年11月广西医科大学第一附属医院择期行人工机械瓣膜置换手术患者50例,使用随机数字表法分为心脏停跳组（停跳组,在中度低温心脏停跳下完成心脏瓣膜置换手术）和浅低温心脏不停跳组（不停跳组,在浅低温心脏跳动下完成手术）,每组25例,分别于体外循环转机前（T1）,转机30 min（T2）,开放升主动脉时（T3）,停体外循环后6 h（T4）、24 h（T5）、72 h（T6）、120 h（T7）7个时间点取静脉血,使用放射免疫分析法测定CF6、6-酮-前列环素F1a（6-Keto-PGF1a）的表达.结果 在T2～T5时间点,停跳组CF6浓度（pg/ml）依次为574.3±103.7、855.3±175.8、665.1±95.6、398.9±74.5,较T1时浓度244.5±52.6升高（P＜0.05）;不停跳组CF6浓度（pg/ml）依次为317.1±93.3、673.9±115.1、452.6±81.2、296.2±61.4,较T1时浓度238.4±49.3升高（P＜0.05）;停跳组变化更明显（P＜0.05）.两组均于T6恢复至T1水平.停跳组T2 ～T4时6-Keto-PGF1a浓度（pg/ml）依次为330.7±67.9、435.6±75.8、235.7±35.0,较T1浓度64.3±18.4升高（P＜0.05）;不停跳组T2～T4时浓度（pg/ml）依次为467.4±43.5、573.9±33.1、356.2±41.9,较T1浓度68.3±19.3升高（P＜0.05）;不停跳组升高明显（P＜0.05）.两组均于T5恢复至T1水平.结论 心脏不停跳手术对心肌包括内皮细胞损伤较小是体外循环手术CF6表达上调相对较小的重要原因.CF6的变化可以影响机体前列环素的表达.     

Evidence for mitochondrial K+ channels and their role in cardioprotection

Twenty years after the discovery of sarcolemmal ATP-sensitive K+ channels and 12 years after the discovery of mitochondrial K(ATP) (mitoK(ATP)) channels, progress has been remarkable, but many questions remain. In the case of the former, detailed structural information is available, and it is well accepted that the channel couples bioenergetics to cellular electrical excitability; however, in the heart, a clear physiological or pathophysiological role has yet to be defined. For mitoK(ATP), structural information is lacking, but there is abundant evidence linking the opening of the channel to protection against ischemia-reperfusion injury or apoptosis. This review updates recent progress in understanding the physiological role of mitoK(ATP) and highlights outstanding questions and controversies, with the intent of stimulating additional investigation on this topic.     

Evidence for H+ secretion by the in vivo canine gallbladder

In humans and most other species, a decline in pH of gallbladder contents occurs during the concentration of bile. Recent in vitro studies in rabbit, guinea pig, and Necturus gallbladders have strongly suggested mucosal H+ secretion during sodium reabsorption, presumably representing a Na+/H+ exchange. The present in vivo studies are the first attempt to determine whether H+ secretion by the gallbladder can be demonstrated in the living animal. Gallbladder bile was obtained from 27 anesthetized dogs after 12-24-h fasts; 12 samples of common duct bile were also obtained in 3 dogs during variable taurocholate infusion. In common duct bile, observed ranges were as follows: pH, 7.37-7.85; CO2 partial pressure (PCO2), 21-32 mmHg; total CO2 concentration ([TCO2]), 16.4-41.4 mM; total bile salt concentration ([TBS]), 16-93 mM; and [Na], 153-192 mM. In gallbladder bile, respective ranges were as follows: pH, 5.72-7.29; PCO2, 36-101 mmHg; [TCO2], 1.21-15.5 mM; [TBS], 150-305 mM; and [Na], 199-266 mM. In all samples [Na] was linearly related to [TBS]. Carbon dioxide partial pressure increased from a mean of 27.3 mmHg in common duct bile to greater than 100 mmHg in gallbladder bile at [TBS] = 180 mM, then declined to approximately 36 mmHg as [TBS] increased to greater than 300 mM. Peak PCO2 occurred at pH approximately 6.4-6.6, then declined as pH decreased to approximately 5.7. Bile to plasma PCO2 ratios increased from a mean of 1.08 in common duct samples to greater than 2.0 in gallbladder samples at pH approximately 6.3, then declined to approximately 1.0 in fully concentrated bile. If the high PCO2 values in bile were solely due to tissue CO2 production, a sustained increase in PCO2 throughout Na+ reabsorption might be expected. The results strongly suggest H+ secretion (HCO3- neutralization), as peak PCO2 occurred when [TBS] was only about 180 mM, long before sodium absorption was complete. It is hypothesized that H+ secretion may have important favorable effects on calcium lithogenicity, reducing the likelihood of the formation of CaCO3- containing gallstones.     

Evidence for the involvement of corticotropin-releasing factor in the inhibition of gonadotropin release induced by hyperprolactinemia

The hypothesis was tested that corticotropin-releasing factor (CRF) is involved in the inhibition of gonadotropin secretion during chronic hyperprolactinemia. Two models of hyperprolactinemia were used, namely inoculation with the prolactin (PRL)-secreting tumor 7315b and implantation of isogenic pituitary glands. Gonadectomized, adrenalectomized male rats received a testosterone capsule and a corticosterone pellet and were inoculated subcutaneously with tumor 7315b. Similar rats without tumor served as controls. The rats were studied 3-4 weeks later while anesthetized with urethane. Plasma testosterone and corticosterone were similar in the two groups of rats. Compared to controls, the tumor-bearing rats had significantly higher plasma levels of PRL (100-fold increase) and adrenocorticotropin (ACTH; 3-fold increase), whereas plasma luteinizing hormone (LH) and follicle-stimulating hormone (FSH) had significantly decreased to 15 and 40%, respectively. CRF release into hypophysial stalk plasma was higher in rats with tumor 7315b than in controls (298 +/- 23 vs. 197 +/- 28 pg/h), and hypothalamic CRF content had increased from 3.0 +/- 0.3 to 4.3 +/- 0.3 ng. Male rats received 3 pituitary glands under the kidney capsule. Sham-operated rats served as controls. They were studied 5-7 weeks later while anesthetized with urethane. Compared to controls, pituitary-grafted rats had larger adrenals (49 +/- 4 vs. 34 +/- 2 mg), higher plasma PRL (156 +/- 18 vs. 52 +/- 8 ng/ml), ACTH (0.46 +/- 0.05 vs. 0.22 +/- 0.02 ng/ml) and corticosterone (455 +/- 39 vs. 268 +/- 14 ng/ml), and lower plasma levels of LH (21 +/- 2 vs. 41 +/- 6 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for the involvement of endogenous opioids in the inhibition of luteinizing hormone by corticotropin-releasing factor

Experiments were carried out in castrate adult male rats to further examine whether endogenous opioids are involved in CRF-induced suppression of LH secretion. Serum LH levels in rats castrated 5 days earlier were significantly reduced by intracerebroventricular administration of homologous (rat) CRF (0.02-2 nmol) within 30 min posttreatment; the effects of 0.02 nmol CRF lasted for at least 2 h, whereas those of 0.2 and 2 nmol CRF were evident for up to 6 h. Rats that received sc infusions of the opioid receptor antagonist naloxone (9.6 mg/kg.day) for 48 h before testing with 0.2 nmol CRF showed a significant reduction of the LH response to CRF. Rats that received two acute injections of naloxone (2 mg/kg, iv, 30 min apart) also showed an attenuated response to the LH-suppressive effects of CRF. In another experimental model where the opioidergic control of LH secretion is absent or masked, the long term castrate rat, there was also a marked attenuation of the LH-suppressing effects of CRF. Transient replacement of testosterone in long term castrates reinstated the inhibitory effects of CRF on LH secretion. A third experiment, in which short term castrates were pretreated with an opioid antibody and then with CRF, resulted in a significant reduction of the CRF-induced reduction of serum LH levels. These observations indicate that opioid receptor-mediated events play an important part in the actions of CRF on LH secretion. On the basis of our previous experiments in vitro, we propose that CRF stimulates the release of hypothalamic opioid peptides, which in turn inhibits the activity of LHRH neurons and, thus, LH secretion.     

Immunocytochemical studies of gastric H+,K+-ATPase in the developing rat

The appearance of the enzyme H+,K+-ATPase was studied in the gastric mucosa of rats during the perinatal period. By means of monoclonal antibodies against the enzyme, immunoreactivity was regularly detected in parietal cells 1 day before birth. The intensity of the staining and the frequency of stained cells increased up to 10-12 days after birth, when adult levels were approached. Electron microscopy showed that the initial staining occurred at the apical surface of the parietal cells, with only faint or no staining at the secretory canaliculi. From 5 days after birth immunoreactivity was observed also at the tubulovesicular membranes.     

Plasma level of mitochondrial coupling factor 6 increases in patients with coronary heart disease.

BACKGROUND: The aim of the present study was to investigate alterations in the plasma level of coupling factor 6 (CF6), a novel endogenous inhibitor of prostacyclin, in patients with coronary heart disease. METHODS AND RESULTS: In total, 35 patients with coronary heart disease and 20 age-matched healthy subjects were examined. Plasma levels of CF6 and 6-keto-prostaglandin (PG)F(1a) (a stable metabolite of prostacyclin) were measured using radioimmunoassay. The plasma level of CF6 was significantly increased in patients (254.1+/-29.8 pg/ml vs 219.4 +/-36.7 pg/ml in controls, p<0.0001), whereas that of 6-keto-PGF(1a) was significantly decreased (23.4 +/-2.3 pg/ml vs 26.1+/-4.5 pg/ml in controls, p=0.001). Moreover, after percutaneous transluminal coronary angioplasty (PTCA) and stent therapy, the level of CF6 was further increased by 30% to 330.4+/-26.0 pg/ml, and that of 6-keto-PGF (1a) was decreased by 42% to 13.5+/-2.0 pg/ml, compared with baseline (all p<0.01). Univariate analysis showed a significant result that the plasma level of CF6 was inversely correlated with that of 6-keto-PGF(1a) in the patients. The plasma ratio of CF6 to 6-keto-PGF(1a) was 8.4 in the control group and that in patients with coronary heart disease was increased to 24.4 after the therapy from 10.9 before therapy. CONCLUSIONS: The results suggest that an increased CF6 level may be responsible in part for the decreased prostacyclin level observed in patients with coronary heart disease, in particular after PTCA and stent therapy. As a potential risk factor for coronary heart disease, CF6 might have important clinical significance.     

Control of gastric acid secretion. Histamine H2-receptor antagonists and H+K(+)-ATPase inhibitors.

Gastric acid secretion is regulated by an intricate interplay of neural (acetylcholine), hormonal (gastrin), and paracrine (histamine, somatostatin) mechanisms. Receptors for each of these agents and the signal transduction pathways to which these receptors are coupled have been identified on the parietal cell. The stimulatory effect of acetylcholine and gastrin is mediated by an increase in cytosolic calcium, whereas that of histamine is mediated by activation of adenylate cyclase and generation of cAMP. Strong potentiation between histamine and either gastrin or acetylcholine reflects postreceptor interaction between the distinct pathways as well as the ability of acetylcholine and gastrin to release histamine from mucosal ECL cells. The inhibitory effects of somatostatin on acid secretion are mediated by receptors coupled by guanine nucleotide-binding proteins to inhibition of adenylate cyclase activity. All the pathways converge on and modulate the activity of the luminal enzyme, H+K(+)-ATPase, the proton pump of the parietal cell. Precise information on the mechanisms involved in gastric acid secretion has led to the development of potent drugs capable of inhibiting acid secretion. These include competitive antagonists that interact with stimulatory receptors (e.g., histamine H2-receptor antagonists) as well as noncompetitive inhibitors of H+K(+)-ATPase (e.g., omeprazole). The histamine H2-receptor antagonists (cimetidine, ranitidine, famotidine, and nizatidine) continue as first-line therapy for peptic ulcer disease and are effective in preventing relapse. Although they are generally well tolerated, histamine H2-receptor antagonists may cause untoward CNS, cardiac, and endocrine effects as well as interference with the absorption, metabolism, and elimination of various drugs. Omeprazole is a weak base that reaches the parietal cell through the bloodstream, diffuses through the cytoplasm, and becomes activated and trapped as a sulfenamide in the acidic canaliculus of the parietal cell. It covalently binds to H+K(+)-ATPase, thereby irreversibly blocking acid secretion in response to all modes of stimulation. The main drawback to its use is its extreme potency, which leads to virtual anacidity, gastrin and ECL cell hyperplasia, hypergastrinemia, and, in rats, to the development of carcinoid tumors.     

Resolution of the membrane moiety of the H+-ATPase complex into two kinds of subunits.

The H+-translocating ATPase complex from the thermophilic bacterium PS3 (TF0-F1) is composed of a water-soluble part with ATP-hydrolyzing activity (TF1) and a membrane moiety with H+-conducting activity (TF0). TF0 was obtained by treating TF0-F1 with urea and removing contaminations on a carboxymethyl-cellulose column. This TF0 contained only two kinds of subunits, band 6 protein (13,500 daltons) and band 8 protein (5400 daltons), and it was active in H+ conduction and TF1 binding when reconstituted into proteoliposomes (TF0 vesicles). The binding of TF1 to TF0 present in vesicles restored energy-transducing activities, such as ATP-32Pi exchange, dicyclohexylcarbodiimide-sensitive ATPase, and ATP-dependent enhancement of 8-anilinonaphthalene-1-sulfonate fluorescence. Treatments such as protease digestion and chemical modification with acetic anhydride, succinic anhydride, or diazobenzenesulfonic acid destroyed the TF1-binding activity, which was caused by band 6 protein. Band 8 protein was a proteolipid that reacted specifically with dicylcohexyl-carbodiimide and seemed to play a central role in H+ conduction through the membrane.     

Paired moving charges in mitochondrial energy coupling. II. Universality of the principles for energy coupling in biological systems.

The thesis is developed that an acceptable model of biological energy coupling must have universal application. The paired moving charge model of mitochondrial energy coupling is examined from the standpoint of this thesis. Fundamental to this model is the notion that energy coupling involves interaction between paired uncompensated charged species in two vectorially aligned and spatially separated reaction centers. The two charge-separating devices are assumed to be the electron transfer chain (in chloroplast and mitochondria) and intrinsic ionophores (in all transducing organelles and kinases). The universality of the ionophore principle becomes then the crucial test of the validity of the paired moving charge model. The multiple facets of ionophore-mediated couples processes are explored, e.g., coupled hydrolysis of ATP, hormonal control of ion movements, and active transport.     

Evidence for the direct involvement of the rhinovirus canyon in receptor binding.

Evidence is presented that indicates a deep crevice located on the surface of human rhinovirus type 14 is involved in virion attachment to cellular receptors. By using mutagenesis of an infectious cDNA clone, 11 mutants were created by single amino acid substitutions or insertions at positions 103, 155, 220, 223, and 273 of the structural protein VP1. Seven of the recovered mutants had a small plaque phenotype and exhibited binding affinities significantly lower than wild-type virus. One mutant, in which glycine replaced proline at amino acid position 155, showed a greatly enhanced binding affinity. Single-cycle growth kinetics suggested that 5 of the mutants had delayed growth cycles due to intracellular deficiencies apart from receptor binding.