5-Hydroxytryptamine stimulates 86Rb+ efflux from pancreatic β-cells

The effects of 5-hydroxytryptamine and 5-hydroxytryptophan on ⁸⁶Rb⁺ efflux from prelabelled ob/ob-mouse islets were studied to better understand the cellular mechanisms underlying the effects of 5-hydroxytryptamine and 5-hydroxytryptophan on insulin release. 5-Hydroxytryptophan (4 mM) had no effect on ⁸⁶Rb⁺ efflux either at a low (3 mM) or at a high (20 mM) d-glucose concentration, whereas 5-hydroxytryptamine (4 mM) stimulated ⁸⁶Rb⁺ efflux at both glucose concentrations. These results indicate that 5-hydroxytryptamine may reduce glucose-induced insulin release by inhibiting early steps in the β-cell stimulus-secretion coupling.     

Glucose inhibits Ca efflux from pancreatic β-cells also in the absence of Na-Ca countertransport

During perifusion with medium deprived of Ca²⁺, addition of glucose or omission of Na⁺ resulted in prompt and quantitatively similar inhibitions of ⁴⁵Ca efflux from β-cell rich pancreatic islets microdissected from ob / ob mice. Glucose had no additional inhibitory effect when Na⁺ was isoosmotically replaced by sucrose or choline⁺. When K⁺ was used as a substitute for Na⁺, the inhibitory effect of Na⁺ removal on ⁴⁵Ca efflux became additive to that of glucose. The observation that glucose can be equally effective in inhibiting ⁴⁵Ca efflux in the presence or absence of Na⁺ is difficult to reconcile with the postulate that the Na⁺-Ca²⁺ countertransport mechanism is a primary site of action for glucose.     

Microfluorometric analysis of Cl permeability and its relation to oscillatory Ca signalling in glucose-stimulated pancreatic β-cells

The cytoplasmic concentrations of Cl⁻([Cl⁻]_i) and Ca²⁺ ([Ca²⁺]_i) were measured with the fluorescent indicators N-(ethoxycarbonylmethyl)-6-methoxyquinilinum bromide (MQAE) and fura-2 in pancreatic β-cells isolated from ob/ob mice. Steady-state [Cl⁻]_i in unstimulated β-cells was 34 mM, which is higher than expected from a passive distribution. Increase of the glucose concentration from 3 to 20 mM resulted in an accelerated entry of Cl⁻ into β-cells depleted of this ion. The exposure to 20 mM glucose did not affect steady-state [Cl⁻]_i either in the absence or presence of furosemide inhibition of Na⁺, K⁺, 2 Cl⁻ co-transport. Glucose-induced oscillations of [Ca²⁺]_i were transformed into sustained elevation in the presence of 4,4′ diisothiocyanato-dihydrostilbene-2,2′-disulfonic acid (H₂DIDS). A similar effect was noted when replacing 25% of extracellular Cl⁻ with the more easily permeating anions SCN⁻, I⁻, NO₃⁻ or Br⁻. It is concluded that glucose stimulation of the β-cells is coupled to an increase in their Cl⁻ permeability and that the oscillatory Ca²⁺ signalling is critically dependent on transmembrane Cl⁻ fluxes.     

Sodium efflux from perfused giant algal cells

Internodal cells of the giant alga Chara corallina were perfused internally to replace the native cytoplasm, tonoplast and vacuole with artificial cytoplasm. Sodium efflux from perfused cells, measured by including ²²Na in the perfusion media, was increased by increasing the internal sodium concentration and by decreasing the external pH, and was inhibited by external application of the renal diuretic amiloride. The sodium efflux was markedly ATP-dependent, with a 50-fold decrease in efflux observed after perfusion with media lacking ATP. Efflux in the presence of ATP was reduced by 33% by inclusion of 10 M N,N-dicyclohexylcarbodiimide in the perfusion medium. The membrane potential of the perfused cells approximated that of intact cells from the same culture. It is suggested that sodium efflux in perfused Chara cells proceeds via a secondary antiporter with protons, regulated by ATP in a catalytic role and with the proton motive force acting as the energy source.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Mes 2-(N-morpholino)ethanesulphonic acid - Mops 3(N-morpholino)propanesulphonic acid - Taps tris(hydroxymethyl)methylaminopropanesulphonic acid     

Influence of surface charge and transmembrane potential on rubidium-86 efflux of human red blood cells

Summary The dependence of the rate constant of Rb⁺ efflux on extracellular cation concentration was measured. At low ionic strengths Rb⁺ efflux increased strongly. Permeability coefficients were calculated from the rate constants measured, using the Goldman flux equation, with and without making allowance for surface potentials. Only when allowance was made for surface potentials and the associated differences beween ion concentrations in the bulk solutions and at the membrane surface, the permeability coefficient remained constant. Best agreement between experimental data and theoretically calculated values was obtained when an interior surface potential of – 110 mV was assumed.When the surface charge of erythrocytes is reduced by neuraminidase, the rate constants for Rb⁺ efflux decreased, indicating a significant influence of surface potential.     

Role of Na+/Ca2+ exchange and the plasma membrane Ca2+ pump in hormone-mediated Ca2+ efflux from pancreatic acini

Summary The relative contributions of the Na⁺/Ca²⁺ exchange and the plasma membrane Ca²⁺ pump to active Ca²⁺ efflux from stimulated rat pancreatic acini were studied. Na⁺ gradients across the plasma membrane were manipulated by loading the cells with Na⁺ or suspending the cells in Na⁺-free media. The rates of Ca²⁺ efflux were estimated from measurements of [Ca²⁺] _i using the Ca²⁺-sensitive fluorescent dye Fura 2 and⁴⁵Ca efflux. During the first 3 min of cell stimulation, the pattern of Ca²⁺ efflux is described by a single exponential function under control, Na⁺-loaded, and Na⁺-depleted conditions. Manipulation of Na⁺ gradients had no effect on the hormone-induced increase in [Ca²⁺] _i . The results indicate that Ca²⁺ efflux from stimulated pancreatic acinar cells is mediated by the plasma membrane Ca²⁺ pump. The effects of several cations, which were used to substitute for Na⁺, on cellular activity were also studied. Choline⁺ and tetramethylammonium⁺ (TMA⁺) released Ca²⁺ from intracellular stores of pancreatic acinar, gastric parietal and peptic cells. These cations also stimulated enzyme and acid secretion from the cells. All effects of these cations were blocked by atropine. Measurements of cholecystokinin-octapeptide (CCK-OP)-stimulated amylase release from pancreatic acini, suspended in Na⁺, TMA⁺, choline⁺, or N-methyl-d-glucamine⁺ (NMG⁺) media containing atropine, were used to evaluate the effect of the cations on cellular function. NMG⁺, choline⁺, and TMA⁺ inhibited amylase release by 55, 40 and 14%, respectively. NMG⁺ also increased the Ca²⁺ permeability of the plasma membrane. Thus, to study Na⁺ dependency of cellular function, TMA⁺ is the preferred cation to substitute for Na⁺. The stimulatory effect of TMA⁺ can be blocked by atropine.     

Human pancreatic islets develop through fusion of distinct β and α/δ islets

Human pancreatic islets show unique architecture in which α and δ cells are mostly at the peripheral and perivascular areas. It has remained unknown how such prototype is realized in every islet. Here, I report that fetal islets develop first in two distinct types consisting of β or α/δ cells, respectively. The α/δ islets are variable in shape, composed of α and δ cells evenly intermixed. They are vascularized better but encapsulated poorer than β islets in general. During the development, the β and α/δ islets adjoin and fuse with each other in such a way that α and δ cells form a crescent on β cells and, then, progress to encompass and encroach into β cells. Most mature‐form islets appear to develop through the fusion. Islets at various stages of fusion are present concurrently until late gestation, suggesting that the islet fusion is an ongoing developmental process. The α/δ islets appear to play a primary role for the process, approaching toward the fusion partner actively. Direct connection is present between the α/δ islets and neural ganglia undergoing active neurogenesis, suggesting an organ‐wide neuroendocrine network development. The fusion of precursor islets appears to be a principle of human pancreatic development providing the prototype of mature islets. The complex development might be a reference for in vitro reproduction of biologically competent islets.     

Ouabain-resistant Na+, K+ transport system in mouse NIH 3T3 cells

Summary It is shown that the ouabain-resistant (OR) furosemide-sensitive K⁺(Rb⁺) transport system performs a net efflux of K⁺ in growing mouse 3T3 cells. This conclusion is based on the finding that under the same assay conditions the furosemidesensitive K⁺(Rb⁺) efflux was found to be two- to threefold higher than the ouabain-resistant furosemide-sensitive K⁺(Rb⁺) influx. The oubain-resistant furosemide-sensitive influxes of both²²Na and⁸⁶Rb appear to be Cl^– dependent, and the data are consistent with coupled unidirectional furosemide-sensitive influxes of Na⁺, K⁺ and Cl^– with a ratio of 1 1 2. However, the net efflux of K⁺ performed by this transport system cannot be coupled to a ouabain-resistant net efflux of Na⁺ since the unidirectional ouabain-resistant efflux of Na⁺ was found to be negligible under physiological conditions. This latter conclusion was based on the fact that practically all the Na⁺ efflux appears to be ouabainsensitive and sufficient to balance the Na⁺ influx under such steady-state conditions. Therefore, it is suggested that the ouabain-resistant furosemide-sensitive transport system in growing cells performs a facilitated diffusion of K⁺ and Na⁺, driven by their respective concentration gradients: a net K⁺ efflux and a net Na⁺ influx.     

Rb fluxes in Chinese hamster ovary cells as a function of membrane cholesterol content

Steady-state fluxes of ⁸⁶Rb⁺ (as a tracer for K⁺) were measured in Chinese hamster ovary cells (CHO-K1) and a mutant (CR1) defective in the regulation of cholesterol biosynthesis; the membrane cholesterol content of this mutant was varied by growing it on a range of cholesterol supplements to lipid-free medium (Sinensky, M. (1978) Proc. Natl. Acad. Sci. U.S. 75, 1247–1249).Analogous to previous findings in ascites tumor cells, ⁸⁶Rb⁺ influx in the parent strain was differentiated into a ouabain-inhibitable ‘pump’ flux, furosemide-sensitive, chloride-dependent exchange diffusion, and a residual ‘leak’ flux.On the basis of this flux characterization, ⁸⁶Rb⁺ pump and leak fluxes were measured in the mutant as a function of membrane cholesterol content. Pump and leak fluxes, when expressed per ml cell water, were independent of the cholesterol content of the mutant. Moreover, ⁸⁶Rb⁺ fluxes in the mutant were equal to those in the parent strain. Our data imply that the flux behavior of K⁺ in the steady state is independent of the ordering of membrane lipid acyl chains.     

Distinct effects of acetylcholine and glucose on 45calcium and 86rubidium efflux from mouse pancreatic islets

The similarities between the effects of acetylcholine and glucose on phospholipid metabolism in pancreatic islet cells prompted the comparison of their effects on ionic fluxes. Acetylcholine (1 μM) consistently increased ⁴⁵Ca²⁺ efflux from mouse islets, whereas glucose increased it in the presence, but decreased it in the absence of extracellular Ca²⁺. Acetylcholine consistently accelerated ⁸⁶Rb⁺ efflux, and this effect was augmented by Ca²⁺ omission. On the other hand, glucose markedly inhibited ⁸⁶Rb⁺ efflux, except when its concentration was raised from 10 to 15 mM in the presence of Ca²⁺. Unlike their effects on phospholipid metabolism, the ionic effects of the two insulin-secretagogues are thus very different.     

Mammalian NHE2 Na(+)/H+ exchanger mediates efflux of potassium upon heterologous expression in yeast

Na(+)/H+exchangers form a broad family of transporters that mediate opposing fluxes of alkali metal cations and protons across cell membranes. They play multiple roles in different organisms (protection from toxic cations, regulation of cell volume or pH). Rat NHE2 exchanger was expressed in a Saccharomyces cerevisiae mutant strain lacking its own exporters of alkali metal cations. Though most of the overexpressed NHE2 remained entrapped in the secretory pathway, part of it reached the plasma membrane and mediated K+ efflux from the yeast. We demonstrate for the first time that a mammalian Na(+)/H+ exchanger transports alkali metal cations in yeast in the opposite direction than in mammalian cells, and that the substrate specificity of the rat NHE2 exchanger is limited only to potassium cations upon expression in yeast cells.     

Immunoreactivity of subunits of the (Na + K)-ATPase Cross-reactivity of the α,α+ and β forms in different organs and species

The immunologic cross-reactivity of the α and α+ forms of the large subunit and the β subunit of the (Na⁺ + K⁺)-ATPase from brain and kidney preparations was examined using rabbit antiserum prepared against the purified holo lamb kidney enzyme. As previously reported by Sweadner ((1979) J. Biol. Chem. 254, 6060–6067) phosphorylation of the large subunit of the (Na⁺ + K⁺)-ATPase in the presence of Na⁺, Mg²⁺, and [γ-³²P]ATP revealed that dog and, very likely, rat brain contain two forms of the large subunit (designated α and α+) while dog, rat, and lamb kidney contain only one form (α). The cross-reactivity of the α and α+ forms in these preparations was investigated by resolving the subunits by SDS-polyacrylamide gel electrophoresis. The separated polypeptides were transferred to unmodified nitrocellulose paper, and reacted with rabbit anti-lamb kidney serum, followed by detection of the antigen-antibody complex with ¹²⁵I-labeled protein A and autoradiography. By this method, the α and α+ forms of rat and dog brain, as well as the α form found in kidney, were shown to cross-react. In addition, membranes from human cerebral cortex were shown to contain two immunoreactive bands corresponding to the α and α+ forms of dog brain. In contrast, the brain of the insect Manduca sexta contains only one immunoreactive polypeptide with a molecular weight intermediate to the α and α+ forms of dog brain. The β subunit from lamb, dog and rat kidney and from dog and rat brain cross-reacts with anti-lamb kidney (Na⁺ + K⁺)-ATPase serum. The mobility of the β subunit from dog and rat brain on SDS-polyacrylamide electrophoresis gels is greater than the mobility of the β subunit from lamb, rat or dog kidney.     

Mechanisms involved in α6β1-integrin-mediated Ca signalling

Contact of Jurkat T-lymphocytes with the extracellular matrix (ECM) protein laminin resulted in long-lasting α6β1-integrin-mediated Ca²⁺ signalling. Both Ca²⁺ release from thapsigargin-sensitive Ca²⁺ stores and capacitative Ca²⁺ entry via Ca²⁺ channels sensitive to SKF 96365 constitute important parts of this process. Inhibition of α6β1-integrin-mediated Ca²⁺ signalling by (1) the src kinase inhibitor PP2, (2) the PLC inhibitor U73122, and (3) the cyclic adenosine diphosphoribose (cADPR) antagonist 7-deaza-8-Br-cADPR indicate the involvement of src tyrosine kinases and the Ca²⁺-releasing second messengers d-myo-inositol 1,4,5-trisphosphate (InsP₃) and cADPR.     

5-Hydroxytryptamine2 and 5-hydroxytryptamine3 receptors mediate serotonin-induced short-circuit current in pig jejunum

The purpose of this study was to determine the effect of methysergide, ketanserin, granisetron, cisapride, and renzapride on serotonin 5-hydroxytryptamine-evoked short-circuit current in muscle and myenteric plexus-stripped pig jejunum using the Ussing chamber technique. Ketanserin, granisetron, cisapride, and renzapride all reduced the 5-hydroxytryptamine-induced increase in short-circuit current by about 50%. Combination of ketanserin and granisetron only reduced the 5-hydroxytryptamine-induced peak increase in short-circuit current by 25%. Cisapride caused a small concentration-dependent increase in short-circuit current. Atropine and hexamethonium both almost completely suppressed the cisapride-induced peak increase in short-circuit current. Ketanserin, granisetron, methysergide, and renzapride did not alter the basal short-circuit current. These results suggest that 5-hydroxytryptamine elicits an increase in short-circuit current by activating epithelial and submucosal 5-hydroxytryptamine₂ and 5-hydroxytryptamine₃ receptor subtypes. Furthermore, the short-circuit current-increasing effect of cisapride, is due to activation of at least muscarinic and nicotinic receptors.Abbreviations 5-HT 5-hydroxytryptamine, serotonin - AUC area under the curve - EC enterochromaffin - ENS enteric nervous system - GI gastrointestinal - MW molecular weight - 5-HTP-DP N-acetyl-5-hydroxytryptophyl-5-hydroxytrytophan amide - SSC short-circuit current - TTX tetrodotoxin     

Diurnal Rb+ transport inPhaseolus pulvinus

Transport of Rb⁺ from the roots to the pulvinus and its diurnal movement in the pulvinus were investigated. Rb⁺ was given to the roots as its chloride or nitrate. The results showed that (1) Rb⁺ was transported from the roots to the pulvinus through the hypocotyl, epicotyl and petiole, (2) Rb⁺ was absorbed into the pulvinus in exchange for K⁺, (3) the absorbed Rb⁺ moved diurnally in the same phase as the remaining K⁺. Namely, Rb⁺ moved in the pulvinus diurnally just like K⁺.     

Role of the choline exchanger in Na-independent Mg efflux from rat erythrocytes

Two types of Na⁺-independent Mg²⁺ efflux exist in erythrocytes: (1) Mg²⁺ efflux in sucrose medium and (2) Mg²⁺ efflux in high Cl⁻ media such as KCl-, LiCl- or choline Cl-medium. The mechanism of Na⁺-independent Mg²⁺ efflux in choline Cl medium was investigated in this study. Non-selective transport by the following transport mechanisms has been excluded: K⁺,Cl⁻- and Na⁺,K⁺,Cl⁻-symport, Na⁺/H⁺-, Na⁺/Mg²⁺-, Na⁺/Ca²⁺- and K⁺(Na⁺)/H⁺ antiport, Ca²⁺-activated K⁺ channel and Mg²⁺ leak flux. We suggest that, in choline Cl medium, Na⁺-independent Mg²⁺ efflux can be performed by non-selective transport via the choline exchanger. This was supported through inhibition of Mg²⁺ efflux by hemicholinum-3 (HC-3), dodecyltrimethylammonium bromide (DoTMA) and cinchona alkaloids, which are inhibitors of the choline exchanger. Increasing concentrations of HC-3 inhibited the efflux of choline and efflux of Mg²⁺ to the same degree. The K_d value for inhibition of [¹⁴C]choline efflux and for inhibition of Mg²⁺ efflux by HC-3 were the same within the experimental error. Inhibition of choline efflux and of Mg²⁺ efflux in choline medium occurred as follows: quinine>cinchonine>HC-3>DoTMA. Mg²⁺ efflux was reduced to the same degree by these inhibitors as was the [¹⁴C]choline efflux.     

Calcium-dependent K efflux from rat submandibular gland: The effects of trifluoperazine and quinidine

The Ca²⁺-dependent K⁺ efflux from rat submandibular gland was studied using a K⁺-sensitive electrode. A K⁺ efflux was induced by either adrenalin or by using the divalent cation ionophore A23187 plus added Ca²⁺ to bypass the receptor mechanism. Trifluoperazine, which was used to investigate the role of calmodulin, was found to block the adrenalin-induced K⁺ efflux but not the A23187/Ca²⁺-induced K⁺ efflux. The adrenalin-induced K⁺ efflux was abolished by quinidine and the A23187/Ca²⁺-induced K⁺ efflux was significantly reduced by quinidine. In other experiments, the presence of indomethacin did not inhibit the adrenalin-induced K⁺ efflux, and exogenously added arachidonic acid did not induce a K⁺ efflux. It is concluded that neither prostaglandin synthesis, nor a cytosolic Ca²⁺-calmodulin complex is involved in the agonist-induced K⁺ efflux from rat submandibular gland. A similarity between the Ca²⁺-dependent K⁺ efflux mechanism of erythrocyte ghosts and submandibular tissue is indicated by their common response to quinidine.     

Properties of the Ca-activated K channel in pancreatic β-cells

The existence of [Ca²⁺]_i-activated K⁺-channels in the pancreatic β-cell membrane is based in two observations: quinine inhibits K⁺-permeability and, increasing intracellular Ca²⁺ stimulates it. The changes in K⁺-permeability of the β-cell have been monitored electrically by combining measurements of the dependence of the membrane potential on external K⁺ concentration and input resistance. The changes in the passive ⁴²K and ⁸⁶Rb efflux from the whole islet have been measured directly. Intracellular Ca²⁺ has been increased by various means, including increasing extracellular Ca²⁺, addition of the Ca²⁺-ionophore A23187 or noradrenaline and application of mitochondrial uncouplers and blockers. In addition to quinine, many other substances have been found to inhibit or modulate the [Ca²⁺]_i-activated K⁺-channel. The most important of these is the natural stimulus for insulin secretion, glucose. Glucose may inhibit K⁺-permeability by lowering intracellular Ca²⁺. Glibenclamide, a hypoglycaemic sulphonylurea, is about 25 times more active than quinine in blocking the K⁺-channel in β-cells. The methylxanthines, c-AMP, various calmodulin inhibitors and Ba²⁺ also inhibit K⁺-permeability. Genetically diabetic mice have been studied and show an alteration in the [Ca²⁺]_i-activated K⁺-channel.It is concluded that the [Ca²⁺]_i-activated K⁺-channel plays a major role in the normal function of the pancreatic β-cell. The study of its properties should prove valuable for the understanding and treatment of diabetes.     

NAD+ ameliorates endotoxin‐induced acute kidney injury in a sirtuin1–dependent manner via GSK‐3β/Nrf2 signalling pathway

Acute kidney injury (AKI) is a substantial worldwide public health concern with no specific and effective therapies in clinic. NAD⁺ is a pivotal determinant of cellular energy metabolism involved in the progression of AKI; however, its mechanism in kidney injury remains poorly understood. Sirtuin 1 (SIRT1) is an NAD⁺‐dependent deacetylase associated with renal protection and acute stress resistance. In this study, we have investigated the role of NAD⁺ in AKI and the potential mechanism(s) involved in its renoprotective effect. NAD⁺ was notably decreased and negatively correlated with kidney dysfunction in AKI, restoring NAD⁺ with NMN significantly ameliorates LPS‐induced oxidative stress and apoptosis and attenuates renal damage. We also found that the protection of NAD⁺ is associated with SIRT1 expressions and performs in a SIRT1‐dependent manner. Inhibition of SIRT1 blunted the protective effect of NAD⁺ and up‐regulated the activity of glycogen synthase kinase‐3β (GSK‐3β) that was concomitant with mitigated Nrf2 nuclear accumulation, thereby exacerbates AKI. These findings suggest that NAD⁺/SIRT1/GSK‐3β/Nrf2 axis is an important mechanism that can protect against AKI which might be a potential therapeutic target for the treatment of AKI.     

Inhibitors of sterol synthesis: effects of a 7α-alkyl analog of 3β-hydroxy-5α-cholest-8(14)-en-15-one on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured mammalian cells and on serum cholesterol levels and other parameters in rats

The 7α-methyl analog (II) of 3β-hydroxy-5α-cholest-8(14)-en-15-one (I) was prepared by chemical synthesis and evaluated with respect to its effects on HMG-CoA reductase activity in CHO-K1 cells and on serum cholesterol levels in rats. The 7α-methyl substitution had no detectable effect on the potency of I in lowering HMG-CoA reductase activity in the cultured cells. In contrast, the 7α-methyl substitution had a marked effect on the action of I in the suppression of food consumption in rats. Whereas II was less potent than I in lowering serum cholesterol levels in rats, it did so at dosage levels at which only slight or moderate effects on food consumption were observed. Full ¹H and ¹³C-NMR assignments for II and intermediates in its synthesis have been presented. Conformational analysis, based on ¹H-¹H coupling constants, NMR shieldings and force-field calculations, indicated that the 7α-methyl substitution had virtually no effect on the conformation of the 15-ketosterol apart from minor distortions of ring B.