Regulation of Ca2+-dependent K+-channel activity in tracheal myocytes by phosphorylation

Isoprenaline is a beta-adrenergic agonist of clinical importance as a remedy for asthma. In airway smooth muscle its relaxant action is accompanied by hyperpolarization of the membrane and elevation of the level of intracellular cyclic AMP. Hyperpolarization and relaxation are also induced by drugs such as forskolin, theophylline and dibutyryl cAMP, indicating that cAMP-dependent phosphorylation is involved in producing the electrical response. Cyclic AMP-dependent protein kinase (protein kinase A) has been reported to activate Ca2+-dependent K+ channels in cultured aortic smooth muscle cells and snail neurons. The membrane of tracheal smooth-muscle cells is characterized by a dense distribution of Ca2+-dependent K+-channels. We have now examined the effect of isoprenaline and protein kinase A on Ca2+-dependent K+-channels in isolated smooth muscle cells of rabbit trachea, using the patch-clamp technique. Our results show that the open-state probability of Ca2+-dependent K+-channel of tracheal myocytes is reversibly increased by either extracellular application of isoprenaline or intracellar application of protein kinase A. We also show that this effect is significantly enhanced and prolonged in the presence of a potent protein phosphatase inhibitor, okadaic acid.     

Cyclic nucleotides control a system which regulates Ca2+ sensitivity of platelet secretion

Cellular responses to extracellular signals are mediated by changes in the intracellular concentrations of one or more second messengers. In platelets, inhibitory agonists increase intracellular cyclic-3',5'-AMP [( cyclic AMP]i (refs 2, 3] whereas excitatory agonists increase [Ca2+]i and/or [1,2-diacylglycerol]i (refs 4-9), and in some cases decrease [cyclic AMP]i (refs 10, 11). Both activation and inhibition of platelet responses have been attributed to an increase in [cyclic-3',5'-GMP]i (refs 8, 12). The activity of protein kinase C, which is associated with the platelet secretory response, is increased by both 1,2-diacylglycerol and Ca2+ (refs 4, 7, 8). The role of cyclic AMP may involve either inhibition of Ca2+ mobilization to the cytosol or stimulation of intracellular Ca2+ uptake, and in addition inhibition of 1,2-diacylglycerol formation. The relationship between cyclic-3',5'-GMP (cyclic GMP) and other second messengers in platelet activation has not been defined. Using platelets made permeable by exposure to an intense electric field, we demonstrate here modulation of the Ca2+ sensitivity of platelet secretion by thrombin, and by 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1-oleyl-2- acetylglycerol ( OAG ), both potent activators of protein kinase C. The effect of thrombin is selectively modified by cyclic GMP and cyclic AMP. The response to OAG and TPA is also modulated by cyclic AMP but to a much lesser extent.     

Renal cytochrome P450-related arachidonate metabolite inhibits (Na+ + K+)ATPase

The function of the nephron, the anatomical unit of the kidney, is segmented; at least 12 segments have been identified that differ in their morphology, transport properties and hormonal responsiveness. The medullary portion of the thick ascending limb of the loop of Henle (mTALH) has one of the highest concentrations of (Na+ + K+)ATPase found in mammalian tissues, reflecting the importance of this nephron segment in the regulation of extracellular fluid volume, as active sodium transport is driven by (Na+ + K+)ATPase. Here, in cells derived primarily from the mTALH of the outer medulla of rabbit kidney, we have identified a cytochrome P450-dependent monooxygenase system which metabolizes arachidonic acid to two biologically active oxygenated products; one of the products inhibits (Na+ + K+)ATPase and the other relaxes blood vessels. We report that formation of these oxygenated arachidonate metabolites is stimulated by arginine vasopressin (AVP) and salmon calcitonin (SCT).     

Role of intracellular Ca2+ sequestration in beta-adrenergic relaxation of a smooth muscle.

Various mechanisms have been proposed for beta-adrenergically mediated relaxation of smooth muscle. All theories suggest the involvement of cyclic AMP as a second messenger: beta-agonists stimulate adenylate cyclase which converts ATP to cyclic AMP and protein kinase, activated by cyclic AMP, is then thought to catalyse a protein phosphorylation that leads to a reduction in free Ca2+, thus effecting relaxation. How this last step is accomplished is much debated, but the following possibilities are currently considered as the mechanisms responsible for cyclic AMP-induced reduction of cytoplasmic Ca2+: activation of a Ca2+-ATPase in the plasma and/or sarcoplasmic reticulum membranes which lowers cytoplasmic [Ca2+] in a direct manner or stimulation of (Na+-K+)ATPase in the cell membrane which may indirectly effect Ca2+ extrusion. Among the hypotheses suggested, those of Ca2+ sequestration by the sarcoplasmic reticulum and of Ca2+ extrusion across the cell membrane are consistent with each other if it is assumed that both processes are effected by a cyclic AMP-sensitive Ca2+-ATPase. However, quite a different mechanism is implied by involving the Na+-K+ pump and Na+-Ca2+ exchange carrier. In this report, we present evidence that suggests intracellular Ca2+ sequestration is the mechanism involved.     

Amino-acid sequence of the beta-subunit of the (Na+ + K+)ATPase deduced from a cDNA

The sodium/potassium-dependent ATPase [(Na+ + K+)ATPase], which establishes and maintains the Na+ and K+ gradients across the plasma membrane of animal cells, consists of two subunits, alpha and beta. Complementary DNA clones encoding the catalytic (alpha) subunit of sheep kidney and Torpedo californica electroplax enzymes have previously been isolated and characterized. However, there is little information concerning the primary structure of the beta-subunit, a glycoprotein of unknown function and relative molecular mass (Mr) approximately 55,000 (ref. 3). Here we describe the isolation and characterization of a cDNA clone containing the entire coding region of the beta-subunit of the sheep kidney (Na+ + K+)ATPase. We also discuss structural aspects of the protein and present evidence for a possible evolutionary relationship with the KdpC subunit of the Escherichia coli K+-ATPase.     

Somatostatin stimulates Ca(2+)-activated K+ channels through protein dephosphorylation.

The neuropeptide somatostatin inhibits secretion from electrically excitable cells in the pituitary, pancreas, gut and brain. In mammalian pituitary tumour cells somatostatin inhibits secretion through two distinct pertussis toxin-sensitive mechanisms. One involves inhibition of adenylyl cyclase, the other an unidentified cyclic AMP-independent mechanism that reduces Ca2+ influx by increasing membrane conductance to potassium. Here we demonstrate that the predominant electrophysiological effect of somatostatin on metabolically intact pituitary tumour cells is a large, sustained increase in the activity of the large-conductance Ca(2+)- and voltage-activated K+ channels (BK). This action of somatostatin does not involve direct effects of Ca2+, cAMP or G proteins on the channels. Our results indicate instead that somatostatin stimulates BK channel activity through protein dephosphorylation.     

Combined effects of adrenaline and insulin on active electrogenic Na+-K+ transport in rat soleus muscle.

Both beta 2-adrenoreceptor stimulants (such as adrenaline and salbutamol) and insulin can increase active Na+-K+ transport and hyperpolarise skeletal cells. Thus, adrenaline and insulin, which are otherwise antagonistic regulators of several metabolic processes, have one action in common, namely, stimulation of active ion translocation. This is especially interesting as cyclic AMP stimulates Na+-K+ transport, whereas a lowering of the cytoplasmic concentration of cyclic AMP has been proposed as an early signal in the action of insulin. Here we report the results of experiments in which the active Na+-K+ transport and membrane potential (EM) of rat soleus muscles were studied during the action of supramaximal doses of insulin and beta 2-adrenoreceptor stimulants, alone and in combination. We conclude that the stimulant action of insulin on active electrogenic Na+-K+ transport is unlikely to be evoked by a lowering of the intracellular concentration of cyclic AMP.     

Nerve growth factor induces adrenergic neuronal differentiation in F9 teratocarcinoma cells

Teratocarcinoma cells have been used as a model to study differentiation and development in vertebrates. Treatment with retinoic acid (RA) and dibutyryl cyclic AMP can in some embryonal carcinoma (EC) cell lines lead to neural differentiation, as judged by neurofilament expression and by the induction of enzymes involved in cholinergic transmission. Short-term culture of F9 line cells with RA and dibutyryl cyclic AMP results in a biochemically demonstrable rise in acetylcholinesterase (AChE) activity. We now report that long-term culture of F9 cells with RA and dibutyryl cyclic AMP induces neurofilament expression, demonstrated by immunofluorescence with specific antibodies. Furthermore, if nerve growth factor (NGF) is also added, the developing neurone-like cells exhibit immunoreactivity to tyrosine hydroxylase, a rate-limiting enzyme of catecholamine synthesis specific for adrenergic neurones. Immunoreactivity for Leu-enkephalin-like peptides is also induced. These results suggest that F9 cells can differentiate into cells with adrenergic characteristics.     

Yeast plasma membrane ATPase is essential for growth and has homology with (Na+ + K+), K+- and Ca2+-ATPases

The plasma membrane ATPase of plants and fungi is a hydrogen ion pump. The proton gradient generated by the enzyme drives the active transport of nutrients by H+-symport. In addition, the external acidification in plants and the internal alkalinization in fungi, both resulting from activation of the H+ pump, have been proposed to mediate growth responses. This ATPase has a relative molecular mass (Mr) similar to those of the Na+-, K+- and Ca2+-ATPases of animal cells and, like these proteins, forms an aspartylphosphate intermediate. We have cloned, mapped and sequenced the gene encoding the yeast plasma membrane ATPase (PMA1) and report here that it maps to chromosome VII adjacent to LEU1. The strong homology between the amino-acid sequence encoded by PMA1 and those of (Na+ + K+), Na+-, K+- and Ca2+- ATPases is consistent with the notion that the family of cation pumps which form a phosphorylated intermediate evolved from a common ancestral ATPase. The function of the PMA1 gene is essential because a null mutation is lethal in haploid cells.     

External Na dependence of ouabain-sensitive ATP:ADP exchange initiated by photolysis of intracellular caged-ATP in human red cell ghosts

Coupled active transport of Na+ and K+ across cellular plasma membranes is mediated by (Na+ + K+)-stimulated Mg2+-dependent ATPase. Active cation transport by this Na pump involves a cyclic Na-dependent phosphorylation of the enzyme by intracellular ATP and hydrolytic dephosphorylation of the phosphoenzyme, stimulated by K+ (ref. 1). In human red blood cells, skeletal muscle and squid axons, replacement of extracellular K by Na results in a ouabain-sensitive efflux of Na coupled to an influx of extracellular Na. There is apparently no net Na movement nor net hydrolysis of ATP. The rate of Na:Na exchange is stimulated by increased levels of ADP and exchange transport is not observed in cells totally depleted of intracellular ATP. These characteristics suggest that the biochemical mechanism underlying the Na exchange mode of the Na pump involves phosphorylation of the enzyme by ATP (which requires intracellular Na) followed by its dephosphorylation by ADP. Such a reaction has been observed in partially purified (Na+ + K+) ATPase from a variety of sources and its dependence on Na concentration has been described (although not previously for the red cell enzyme). In the present work, intracellular ATP:ADP exchange reaction was initiated by photoreleased ATP following brief irradiation at 350 nm of ghosts containing caged-ATP. The ouabain-sensitive component of the ensuing ATP:ADP exchange reaction shows a biphasic response to extracellular Na. External Na in the range 0--10 mM has an inhibitory effect whilst increasing concentrations beyond this range stimulate the rate of exchange in a roughly linear fashion up to 100 mM Na. These results represent the first direct demonstration of the sidedness of the effects of Na on this partial sequence in the overall enzyme cycle and bear a qualitative resemblance to the Na effects on the Na-ATPase which occur in the absence of intracellular ADP in human red blood cells.     

Inositol trisphosphate-dependent periodic activation of a Ca(2+)-activated K+ conductance in glucose-stimulated pancreatic beta-cells.

Glucose-stimulated insulin secretion is associated with the appearance of electrical activity in the pancreatic beta-cell. At intermediate glucose concentrations, beta-cell electrical activity follows a characteristic pattern of slow oscillations in membrane potential on which bursts of action potentials are superimposed. The electrophysiological background of the bursting pattern remains unestablished. Activation of Ca(2+)-activated large-conductance K+ channels (KCa channel) has been implicated in this process but seems unlikely in view of recent evidence demonstrating that the beta-cell electrical activity is unaffected by the specific KCa channel blocker charybdotoxin. Another hypothesis postulates that the bursting arises as a consequence of two components of Ca(2+)-current inactivation. Here we show that activation of a novel Ca(2+)-dependent K+ current in glucose-stimulated beta-cells produces a transient membrane repolarization. This interrupts action potential firing so that action potentials appear in bursts. Spontaneous activity of this current was seen only rarely but could be induced by addition of compounds functionally related to hormones and neurotransmitters present in the intact pancreatic islet. K+ currents of the same type could be evoked by intracellular application of GTP, the effect of which was mediated by mobilization of Ca2+ from inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores. These observations suggest that oscillatory glucose-stimulated electrical activity, which is correlated with pulsatile release of insulin, results from the interaction between the beta-cell and intraislet hormones and neurotransmitters. Our data also provide evidence for a close interplay between ion channels in the plasma membrane and InsP3-induced mobilization of intracellular Ca2+ in an excitable cell.     

Serotonin and cyclic AMP close single K+ channels in Aplysia sensory neurones

We have identified a serotonin-sensitive K+ channel with novel properties. The channel is active at the testing potential; its gating is moderately affected by membrane potential and is not dependent on the activity of intracellular calcium ions. Application of serotonin to the cell body or intracellular injection of cyclic AMP causes prolonged and complete closure of the channel, thereby reducing the effective number of active channels in the membrane. The closure of the channel can account for the increases in the duration of the action potential, Ca2+ influx, and transmitter release which underlie behavioural sensitization, a simple form of learning.     

Induction of increased calcium uptake in mouse T lymphocytes by concanavalin A and its modulation by cyclic nucleotides.

The binding of concanavqlin A to T but not B mouse spleen lymphocytes increases Ca-2+ uptake in these cells which is measurable by 45 s and complete by 1 min. Dibutyrl cyclic AMP, but not sodium azide inhibits induced Ca-2+ uptake, wheras dibutyryl cyclic GMP enhances it. B cell mitogens do not cause a similar Ca-2+ uptake in mouse B lymphocytes. The induction of increased Ca-2+ uptake by T cells is discussed in terms of gated membrane channels for Ca-2+.     

The apical surface of canine chief cell monolayers resists H+ back-diffusion

The resistance of the gastric mucosa to acid and peptic injury is reflected by a resistance to the back-diffusion of H+ from gastric lumen to blood. The nature of this 'barrier', however, remains undefined. Using Ussing chambers, we have now studied the acid-barrier function of monolayers prepared from dispersed canine fundic chief cells. These monolayers secrete pepsinogen in response to stimulation. We found that, on acidification of the apical solution to pH 2, transepithelial resistance (R) increased 2.6-fold and the monolayers maintained this 1:100,000 H+ concentration gradient for more than 4 h. The addition of aspirin to the acidified apical solution caused a rapid decay in R, as did acidification of the basolateral solution to a pH less than 5.5. Ouabain-treated monolayers displayed the rise in R expected with apical acidification, while potential difference (V) and short-circuit current (Isc) decreased essentially to zero, indicating impermeability to H+. However, if the integrity of the ouabain-treated monolayers was disrupted by low apical pH, H+ permeation occurred, reflected by an Isc that was dependent on the H+ gradient across monolayers. These data indicate that the apical surface of chief cells is a very tight barrier to H+ diffusion and may be an important element resisting acid-peptic injury.     

Inhibition by dopamine of (Na(+)+K+)ATPase activity in neostriatal neurons through D1 and D2 dopamine receptor synergism

The (Na(+)+K+)ATPase, an integral membrane protein located in virtually all animal cells, couples the hydrolysis of ATP to the countertransport of Na+ and K+ ions across the plasma membrane. In neurons, a large portion of cellular energy is expended by this enzyme to maintain the ionic gradients that underlie resting and action potentials. Although neurotransmitter regulation of the enzyme in brain has been reported, such regulation has been characterized either as a nonspecific phenomenon or as an indirect effect of neurotransmitter-induced changes in ionic gradients. We report here that the neurotransmitter dopamine, through a synergistic effect on D1 and D2 receptors, inhibits the (Na(+)+K+)ATPase activity of isolated striatal neurons. Our data provide unequivocal evidence for regulation by a neurotransmitter of a neuronal ion pump. They also demonstrate that synergism between D1 and D2 receptors, which underlies many of the electrophysical and behavioural effects of dopamine in the mammalian brain, can occur on the same neuron. In addition, the results support the possibility that dopamine and other neurotransmitters can regulate neuronal excitability through the novel mechanism of pump inhibition.     

猪抑胃肽/葡萄糖依赖性促胰岛素释放肽(GIP)基因的克隆与组织表达分析

本研究采用RT-PCR方法,克隆长白猪(Landrace)GIP基因全长cDNA序列,并对其在家猪组织中的表达情况进行分析.结果显示:长白猪GIP(pGIP)cDNA全长435bp,编码144个氨基酸GIP的前体蛋白;该前体蛋白含有信号肽序列,经蛋白酶水解后产生GIP成熟肽.与人、小鼠GIP表达图谱类似,GIP mRNA也在长白猪小肠及肾脏中有较高水平表达.     

Neuronal inhibition by the peptide FMRFamide involves opening of S K+ channels

Neurotransmitters modulate the activity of ion channels through a variety of second messengers, including cyclic AMP, cyclic GMP and the products of phosphatidylinositol breakdown. Little is known about how different transmitters acting through different second-messenger systems interact within a cell to regulate single ion channels. We here describe the reciprocal actions of serotonin and the molluscan neuropeptide, FMRFamide, on individual K+ channels in Aplysia sensory neurons. In these cells, serotonin causes prolonged all-or-none closure of a class of background conductance K+ channels (the S channels) through cAMP-dependent protein phosphorylation. Using single-channel recording, we have found that FMRFamide produces two actions on the S channels; it increases the probability of opening of the S channels via a cAMP-independent second-messenger system and it reverses the closures of S channels produced by serotonin or cAMP.     

Altered Gs and adenylate cyclase activity in human GH-secreting pituitary adenomas

Gs and Gi are guanine nucleotide-binding, heterotrimer proteins that regulate the activity of adenylate cyclase, and are responsible for transferring stimulatory and inhibitory hormonal signals, respectively, from cell surface receptors to the enzyme catalytic unit. These proteins can be directly activated by agents such as GTP and analogues, fluoride and magnesium. Decreased amounts of Gs and Gi, and even the absence of Gs, have been described, whereas an altered Gs has been reported in a cultured cell line (UNC variant of S49 lymphoma cells), but has never been observed in human disease states. We have found a profoundly altered Gs protein in a group of human growth hormone-secreting pituitary adenomas, characterized by high secretory activity and intracellular cyclic AMP levels. In the membranes from these tumours no stimulation of adenylate cyclase activity by growth hormone-releasing hormone, by GTP or by fluoride was observed. Indeed, the last two agents caused an inhibition, probably mediated by Gi. In contrast, adenylate cyclase stimulation by Mg2+ was enormously increased. This altered pattern of adenylate cyclase regulation was reproduced when a cholate extract of the tumour membranes (which contains G proteins) was reconstituted with Gs-free, cyc- S49 cell membranes. Inasmuch as secretion from somatotrophic cells is known to be a cAMP-dependent function, the alteration of Gs could be the direct cause of the high secretory activity of the tumours in which it occurs.     

2-烷基-取代苯并咪唑的合成及结构表征

根据Ｌａｄｅｎｂｕｒｇ方法，以邻苯二胺类化合物、脂肪酸为原料，合成２烷基取代苯并咪唑，通过选择反应物配比等，获得了一条较满意的合成路线。通过熔点测定、红外光谱、质谱等方法，对产物进行了结构认证。