Regulation and function of Ca2+–calmodulin-dependent protein kinase II of fast-twitch rat skeletal muscle

The activation and function of Ca²⁺–calmodulin-dependent kinase II (CaMKII) in contracting rat skeletal muscle was examined. The increase in autonomous activity and phosphorylation at Thr²⁸⁷ of CaMKII of gastrocnemius muscle in response to contractions in situ was rapid and transient, peaking at 1–3 min, but reversed after 30 min of contractions. There was a positive correlation between CaMKII phosphorylation at Thr²⁸⁷ and autonomous CaMKII activity. In contrast to the rapid and transient increase in autonomous CaMKII activity, the phosphorylation of the putative CaMKII substrate trisk95/triadin was rapid and sustained during contractions. There were no changes in CaMKII activity and phosphorylation or trisk95 phosphorylation in the resting contralateral muscles during stimulation. When fast-twitch muscles were contracted ex vivo , CaMKII inhibition resulted in a greater magnitude of fatigue as well as blunted CaMKII and trisk95 phosphorylation, identifying trisk95 as a physiological CaMKII substrate. In summary, skeletal muscle CaMKII activation was rapid and sustained during exercise/contraction and is mediated by factors within the contracting muscle, probably through allosteric activation via Ca²⁺–CaM. CaMKII may signal through trisk95 to modulate Ca²⁺ release in fast-twitch rat skeletal muscle during exercise/contraction.     

The human tumour suppressor gene SLC5A8 expresses a Na+–monocarboxylate cotransporter

The orphan cotransport protein expressed by the SLC5A8 gene has been shown to play a role in controlling the growth of colon cancers, and the silencing of this gene is a common and early event in human colon neoplasia. We expressed this protein in Xenopus laevis oocytes and have found that it transports small monocarboxylic acids. The electrogenic activity of the cotransporter, which we have named SMCT (sodium monocarboxylate transporter), was dependent on external Na⁺ and was compatible with a 3 : 1 stoichiometry between Na⁺ and monocarboxylates. A portion of the SMCT-mediated current was also Cl⁻ dependent, but Cl⁻ was not cotransported. SMCT transports a variety of monocarboxylates (similar to unrelated monocarboxylate transport proteins) and most transported monocarboxylates demonstrated K _m values near 100 μ m , apart from acetate and d -lactate, for which the protein showed less affinity. SMCT was strongly inhibited by 1 m m probenecid or ibuprofen. In the absence of external substrate, a Na⁺-independent leak current was also observed to pass through SMCT. SMCT activity was strongly inhibited after prolonged exposure to high external concentrations of monocarboxylates. The transport of monocarboxylates in anionic form was confirmed by the observation of a concomitant alkalinization of the cytosol. SMCT, being expressed in colon and kidney, represents a novel means by which Na⁺, short-chain fatty acids and other monocarboxylates are transported in these tissues. The significance of a Na⁺–monocarboxylate transporter to colon cancer presumably stems from the transport of butyrate, which is well known for having anti-proliferative and apoptosis-inducing activity in colon epithelial cells.     

Ca2+–calmodulin-dependent protein kinase expression and signalling in skeletal muscle during exercise

Ca²⁺ signalling is proposed to play an important role in skeletal muscle function during exercise. Here, we examined the expression of multifunctional Ca²⁺–calmodulin-dependent protein kinases (CaMK) in human skeletal muscle and show that CaMKII and CaMKK, but not CaMKI or CaMKIV, are expressed. Furthermore, the effect of exercise duration and intensity on skeletal muscle CaMKII activity and phosphorylation of downstream targets was examined. Eight healthy men exercised at ∼67% of peak pulmonary O₂ uptake     with muscle samples taken at rest and after 1, 10, 30, 60 and 90 min of exercise. Ten other men exercised for three consecutive 10 min bouts at 35%, 60% and 85%     with muscle samples taken at rest, at the end of each interval and 30 min post-exercise. There was a rapid and transient increase in autonomous CaMKII activity and CaMKII phosphorylation at Thr²⁸⁷ in skeletal muscle during exercise. Furthermore, the phosphorylation of phospholamban (PLN) at Thr¹⁷, which was identified as a CaMKII substrate in skeletal muscle, was rapidly (< 1 min) increased by exercise, and remained phosphorylated 5-fold above basal level during 90 min of exercise. The phosphorylation of serum response factor at Ser¹⁰³, a putative CaMKII substrate, was higher after 30 min of exercise. PLN phosphorylation at Thr¹⁷ was higher with increasing exercise intensities. These data indicate that CaMKII is the major multifunctional CaMK in skeletal muscle and its activation occurs rapidly and is sustained during continuous exercise, with the activation being greater during intense exercise.     

Ca2+–calmodulin-dependent myosin light chain kinase is essential for activation of TRPC5 channels expressed in HEK293 cells

Mammalian homologues of Drosophila transient receptor potential (TRP) proteins are responsible for receptor-activated Ca²⁺ influx in vertebrate cells. We previously reported the involvement of intracellular Ca²⁺ in the receptor-mediated activation of mammalian canonical transient receptor potential 5 (TRPC5) channels. Here we investigated the role of calmodulin, an important sensor of changes in intracellular Ca²⁺, and its downstream cascades in the activation of recombinant TRPC5 channels in human embryonic kidney (HEK) 293 cells. Ca²⁺ entry through TRPC5 channels, induced upon stimulation of the G-protein-coupled ATP receptor, was abolished by treatment with W-13, an inhibitor of calmodulin. ML-9 and wortmannin, inhibitors of Ca²⁺–calmodulin-dependent myosin light chain kinase (MLCK), and the expression of a dominant-negative mutant of MLCK inhibited the TRPC5 channel activity, revealing an essential role of MLCK in maintaining TRPC5 channel activity. It is important to note that ML-9 impaired the plasma membrane localization of TRPC5 channels. Furthermore, TRPC5 channel activity measured using the whole-cell patch-clamp technique was inhibited by ML-9, whereas TRPC5 channel activity observed in the cell-excised, inside-out patch was unaffected by ML-9. An antibody that recognizes phosphorylated myosin light chain (MLC) revealed that the basal level of phosphorylated MLC under unstimulated conditions was reduced by ML-9 in HEK293 cells. These findings strongly suggest that intracellular Ca²⁺–calmodulin constitutively activates MLCK, thereby maintaining TRPC5 channel activity through the promotion of plasma membrane TRPC5 channel distribution under the control of phosphorylation/dephosphorylation equilibrium of MLC.     

Functional role of Na+–HCO3− cotransport in migration of transformed renal epithelial cells

Cell migration is crucial for immune defence, wound healing or formation of tumour metastases. It has been shown that the activity of the Na⁺–H⁺ exchanger (NHE1) plays an important role in cell migration. However, so far it is unknown whether Na⁺– HCO₃⁻ cotransport (NBC), which has similar functions in the regulation of intracellular pH (pH_i) as NHE1, is also involved in cell migration. We therefore isolated NHE-deficient Madin-Darby canine kidney (MDCK-F) cells and tested whether NBC compensates for NHE in pH_i and cell volume regulation as well as in migration. Intracellular pH was measured with the fluorescent pH indicator 2'7'-bis(carboxyethyl)-5-carboxyfluorescein (BCECF). The expression of NBC isoforms was determined with semiquantitative PCR. Migration was monitored with time-lapse video microscopy and quantified as the displacement of the cell centre. We found that MDCK-F cells express the isoform NBC1 ( SLCA4A gene product) at a much higher level than the isoform kNBC3 ( SLCA4A8 gene product). This difference is even more pronounced in NHE-deficient cells so that NBC1 is likely to be the major acid extruder in these cells and the major mediator of propionate-induced cell volume increase. NHE-deficient MDCK-F cells migrate more slowly than normal MDCK-F cells. NBC activity promotes migration during an acute intracellular acid load and increases migratory speed and displacement on a short timescale (< 30 min) whereas it has no effect on the long-term behaviour of migrating MDCK-F cells. Taken together, our results show that NBC actvity, despite many functional similarities, does not have the same importance for cell migration as NHE1 activity.     

Effect of endurance exercise training on Ca2+–calmodulin-dependent protein kinase II expression and signalling in skeletal muscle of humans

Here the hypothesis that skeletal muscle Ca²⁺–calmodulin-dependent kinase II (CaMKII) expression and signalling would be modified by endurance training was tested. Eight healthy, young men completed 3 weeks of one-legged endurance exercise training with muscle samples taken from both legs before training and 15 h after the last exercise bout. Along with an ∼40% increase in mitochondrial F₁-ATP synthase expression, there was an ∼1-fold increase in maximal CaMKII activity and CaMKII kinase isoform expression after training in the active leg only. Autonomous CaMKII activity and CaMKII autophosphorylation were increased to a similar extent. However, there was no change in α-CaMKII anchoring protein expression with training. Nor was there any change in expression or Thr¹⁷ phosphorylation of the CaMKII substrate phospholamban with training. However, another CaMKII substrate, serum response factor (SRF), had an ∼60% higher phosphorylation at Ser¹⁰³ after training, with no change in SRF expression. There were positive correlations between the increases in CaMKII expression and SRF phosphorylation as well as F₁ATPase expression with training. After training, there was an increase in cyclic-AMP response element binding protein phosphorylation at Ser¹³³, but not expression, in muscle of both legs. Taken together, skeletal muscle CaMKII kinase isoform expression and SRF phosphorylation is higher with endurance-type exercise training, adaptations that are restricted to active muscle. This may contribute to greater Ca²⁺ mediated regulation during exercise and the altered muscle phenotype with training.     

Inhibition of rat Na+–HCO3– cotransporter (NBCn1) function and expression by the alternative splice domain

The Na⁺–HCO₃^– cotransporter NBCn1 (SLC4A7) has multiple variants depending upon splice domains in the cytoplasmic amino- and carboxy-termini of the protein. In this study, we examined the role of the amino-terminal splice domain containing 123 amino acids (cassette II) in the regulation of NBCn1 function and expression. Polymerase chain reaction detected NBCn1 mRNAs containing cassette II in a variety of tissues. Two variants, NBCn1-B containing cassette II and NBCn1-E lacking cassette II, were expressed in Xenopus oocytes and assessed by two-electrode voltage clamp to measure the ionic current mediated by the transporters. The two variants showed similar current–voltage ( I – V ) relations when measured 3–4 days after RNA injection. Replacment of Cl⁻ with gluconate did not affect the I – V relations. When exposed to solutions containing 20–50 m m Na⁺, the current produced by NBCn1-B was slightly more positive than that produced by NBCn1-E. The two currents were similar at 100 m m Na⁺. The slope conductances for the two variants were progressively increased at higher Na⁺ levels, and the increases were parallel and superimposed. Measured at different time points after RNA injection, NBCn1-B produced lower conductance than NBCn1-E at 24–48 h. Protein expression of NBCn1-B was also low at these time points as determined by immunoblot of oocyte membrane preparation. Expressed in opossum kidney (OK) cells, NBCn1-E caused a 1.5-fold increase in ouabain-sensitive production of p -nitrophenol from p -phenyl phosphate compared with control preparations, whereas NBCn1-B had negligible effect. We conclude that the primary function of cassette II is to reduce NBCn1 protein expression.     

Modulation of Ca2+ release and Ca2+ oscillations in HeLa cells and fibroblasts by mitochondrial Ca2+ uniporter stimulation

The recent availability of activators of the mitochondrial Ca²⁺ uniporter allows direct testing of the influence of mitochondrial Ca²⁺ uptake on the overall Ca²⁺ homeostasis of the cell. We show here that activation of mitochondrial Ca²⁺ uptake by 4,4',4"-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) or kaempferol stimulates histamine-induced Ca²⁺ release from the endoplasmic reticulum (ER) and that this effect is enhanced if the mitochondrial Na⁺–Ca²⁺ exchanger is simultaneously inhibited with CGP37157. This suggests that both Ca²⁺ uptake and release from mitochondria control the ability of local Ca²⁺ microdomains to produce feedback inhibition of inositol 1,4,5-trisphosphate receptors (InsP₃Rs). In addition, the ability of mitochondria to control Ca²⁺ release from the ER allows them to modulate cytosolic Ca²⁺ oscillations. In histamine stimulated HeLa cells and human fibroblasts, both PPT and kaempferol initially stimulated and later inhibited oscillations, although kaempferol usually induced a more prolonged period of stimulation. Both compounds were also able to induce the generation of Ca²⁺ oscillations in previously silent fibroblasts. Our data suggest that cytosolic Ca²⁺ oscillations are exquisitely sensitive to the rates of mitochondrial Ca²⁺ uptake and release, which precisely control the size of the local Ca²⁺ microdomains around InsP₃Rs and thus the ability to produce feedback activation or inhibition of Ca²⁺ release.     

Exercise Increases Ca2+–Calmodulin-Dependent Protein Kinase II Activity in Human Skeletal Muscle

There is evidence in rodents that Ca²⁺-calmodulin-dependent protein kinase II (CaMKII) activity is higher in contracting skeletal muscle, and this kinase may regulate skeletal muscle function and metabolism during exercise. To investigate the effect of exercise on CaMKII in human skeletal muscle, healthy men (   n = 8  ) performed cycle ergometer exercise for 40 min at 76 ± 1 % peak pulmonary O₂ uptake (_O2peak), with skeletal muscle samples taken at rest and after 5 and 40 min of exercise. CaMKII expression and activities were examined by immunoblotting and in vitro kinase assays, respectively. There were no differences in maximal (+ Ca²⁺, CaM) CaMKII activity during exercise compared with rest. Autonomous (- Ca²⁺, CaM) CaMKII activity was 9 ± 1 % of maximal at rest, remained unchanged at 5 min, and increased to 17 ± 1 % (   P < 0.01  ) at 40 min. CaMKII autophosphorylation at Thr²⁸⁷ was 50-70 % higher during exercise, with no differences in CaMKII expression. The effect of maximal aerobic exercise on CaMKII was also examined (   n = 9  ), with 0.7- to 1.5-fold increases in autonomous CaMKII activity, but no change in maximal CaMKII activity. CaMKIV was not detected in human skeletal muscle. In summary, exercise increases the activity of CaMKII in skeletal muscle, suggesting that it may have a role in regulating skeletal muscle function and metabolism during exercise in humans.     

Protein kinase C and A sites on troponin I regulate myofilament Ca2+ sensitivity and ATPase activity in the mouse myocardium

Cardiac troponin I (cTnI) is a phosphoprotein subunit of the troponin-tropomyosin complex that is thought to inhibit cardiac muscle contraction during diastole. To investigate the contributions of cTnI phosphorylation to cardiac regulation, transgenic mice were created with the phosphorylation sites of cTnI mutated to alanine. Activation of protein kinase C (PKC) by perfusion of hearts with phorbol-12-myristate-13-acetate (PMA) or endothelin-1 (ET-1) inhibited the maximum ATPase rate by up to 25 % and increased the Ca²⁺ sensitivity of ATPase activity and of isometric tension by up to 0.15 pCa units. PKC activation no longer altered cTnI phosphorylation, depressed ATPase rates or enhanced myofilament Ca²⁺ sensitivity in transgenic mice expressing cTnI that could not be phosphorylated on serines^43/45 and threonine¹⁴⁴ (PKC sites). Modest changes in myosin regulatory light chain phosphorylation occurred in all mouse lines, but increases in myofilament Ca²⁺ sensitivity required the presence of phosphorylatable cTnI. For comparison, the β-adrenergic agonist isoproterenol caused a 38 % increase in maximum ATPase rate and a 0.12 pCa unit decrease in myofilament Ca²⁺ sensitivity. These β-adrenergic effects were absent in transgenic mice expressing cTnI that could not be phosphorylated on serines^23/24 (protein kinase A, PKA, sites). Overall, the results indicate that PKC and PKA exert opposing effects on actomyosin function by phosphorylating cTnI on distinct sites. A primary role of PKC phosphorylation of cTnI may be to reduce the requirements of the contractile apparatus for both Ca²⁺ and ATP, thereby promoting efficient ATP utilisation during contraction.     

Glucagon activates Ca2+ and Cl− channels in rat hepatocytes

Glucagon is one of the major hormonal regulators of glucose metabolism, counteracting the hepatic effects of insulin when the concentration of glucose in the bloodstream falls below a certain level. Glucagon also regulates bile flow, hepatocellular volume and membrane potential of hepatocytes. It is clear that changes in cell volume and membrane potential cannot occur without significant ion fluxes across the plasma membrane. The effects of glucagon on membrane currents in hepatocytes, however, are not well understood. Here we show, by patch-clamping of rat hepatocytes, that glucagon activates two types of currents: a small inwardly rectifying Ca²⁺ current with characteristics similar to those of the store-operated Ca²⁺ current and a larger outwardly rectifying Cl⁻ current similar to that activated by cell swelling. We show that the mechanism of glucagon action on membrane conductance involves phospholipase C and adenylyl cyclase. Contribution of the adenylyl cyclase-dependent pathway to activation of the currents depended on Epac (exchange protein directly activated by cAMP), but not on protein kinase A. The activation of Ca²⁺ and Cl⁻ channels is likely to play a key role in the mechanisms by which glucagon regulates hepatocyte metabolism and volume.     

Mechanisms underlying β2-adrenoceptor-mediated nitric oxide generation by human umbilical vein endothelial cells

Endothelial β₂-adrenoceptor (β₂AR) stimulation increases nitric oxide (NO) generation, but the underlying cellular mechanisms are unclear. We examined the role of l -arginine transport and of phosphorylation of NO synthase 3 (NOS-3) in β₂AR-mediated NO biosynthesis by human umbilical vein endothelial cells (HUVEC). To this end, we assessed l -arginine uptake, NOS activity (from l -arginine to l -citrulline conversion), membrane potential (using [³H]tetraphenylphosphonium), as well as serine phosphorylation of NOS-3 (by Western blotting and mass spectrometry), in HUVEC treated with βAR agonists or cyclic AMP-elevating agents. β₂AR stimulation increased l -arginine transport, as did cyclic AMP elevation with either forskolin or dibutyryl cyclic AMP, and this increase was inhibitable by N -ethylmaleimide. Blockade of l -arginine uptake by l -lysine inhibited NOS activity and, conversely, blockade of NOS using N ^ω-nitro- l -arginine methyl ester ( l -NAME) inhibited l -arginine transport. β₂AR stimulation also caused a membrane hyperpolarization inhibitable by l -NAME, suggesting that the increase in l -arginine uptake occurred in response to NO-mediated hyperpolarization. β₂AR activation also increased NOS activity and phosphorylation of NOS-3 on serine-1177, and these increases were attenuated by inhibition of protein kinase A (PKA), phosphatidylinositol 3-kinase (PI3K) or Akt, and abolished by coinhibition of PKA and Akt. These findings suggest that β₂AR-mediated NOS-3 activation in HUVEC is mediated through phosphorylation of NOS-3 on serine-1177 through both the PKA and the PI3K/Akt systems, and is sustained by an increase in l -arginine uptake resulting from NO-mediated membrane hyperpolarization.     

AMP-activated protein kinase and the regulation of Ca2+ signalling in O2-sensing cells

All cells respond to metabolic stress. However, a variety of specialized cells, commonly referred to as O₂-sensing cells, are acutely sensitive to relatively small changes in   P _O2  . Within a variety of organisms such O₂-sensing cells have evolved as vital homeostatic mechanisms that monitor O₂ supply and alter respiratory and circulatory function, as well as the capacity of the blood to transport O₂. Thereby, arterial   P _O2  may be maintained within physiological limits. In mammals, for example, two key tissues that contribute to this process are the pulmonary arteries and the carotid bodies. Constriction of pulmonary arteries by hypoxia optimizes ventilation–perfusion matching in the lung, whilst carotid body excitation by hypoxia initiates corrective changes in breathing patterns via increased sensory afferent discharge to the brain stem. Despite extensive investigation, the precise mechanism(s) by which hypoxia mediates these responses has remained elusive. It is clear, however, that hypoxia inhibits mitochondrial function in O₂-sensing cells over a range of   P _O2  that has no such effect on other cell types. This raised the possibility that AMP-activated protein kinase might function to couple mitochondrial oxidative phosphorylation to Ca²⁺ signalling mechanisms in O₂-sensing cells and thereby underpin pulmonary artery constriction and carotid body excitation by hypoxia. Our recent investigations have provided significant evidence in support of this view.     

Modulation of Ca2+-activated K+ currents and Ca2+-dependent action potentials by exocytosis in goldfish bipolar cell terminals

Retinal bipolar cells convey light-evoked potentials from photoreceptors to ganglion cells and mediate the initial stages of visual signal processing. They do not fire Na⁺-dependent action potentials (APs) but the Mb1 class of goldfish bipolar cell exhibits Ca²⁺-dependent APs and regenerative potentials that originate in the axon terminal. I have examined the properties of Ca²⁺-dependent APs in isolated bipolar-cell terminals in goldfish retinal slices. All recorded terminals fired spontaneous or evoked APs at frequencies of up to 15 Hz. When an AP waveform was used as a voltage stimulus, exocytosis was evoked by single APs, maintained throughout AP trains and modulated by AP frequency. Furthermore, feedback inhibition of the Ca²⁺ current ( I _Ca) by released vesicular protons reduced depression of exocytosis during AP trains. In the absence of K⁺ current inhibition, step depolarizations and AP waveforms evoked a rapidly activated outward current that was dependent on Ca²⁺ influx ( I _K(Ca)). I therefore investigated whether proton-mediated feedback inhibition of I _Ca affected the activation of I _K(Ca). A transient inhibition of I _K(Ca) was observed that was dependent on exocytosis, blocked by high-pH extracellular buffer, of similar magnitude to inhibition of I _Ca but occurred with a delay of 2.7 ms. In addition, the amplitude of APs evoked under current clamp was inhibited by the action of vesicular protons released by the APs. Protons released via exocytosis may therefore be a significant modulator of Ca²⁺-dependent currents and regenerative potentials in bipolar-cell terminals.     

Calmodulin kinase modulates Ca2+ release in mouse skeletal muscle

Activation of the contractile machinery in skeletal muscle is initiated by the action-potential-induced release of Ca²⁺ from the sarcoplasmic reticulum (SR). Several proteins involved in SR Ca²⁺ release are affected by calmodulin kinase II (CaMKII)-induced phosphorylation in vitro , but the effect in the intact cell remains uncertain and is the focus of the present study. CaMKII inhibitory peptide or inactive control peptide was injected into single isolated fast-twitch fibres of mouse flexor digitorum brevis muscles, and the effect on free myoplasmic [Ca²⁺] ([Ca²⁺]_i) and force during different patterns of stimulation was measured. Injection of the inactive control peptide had no effect on any of the parameters measured. Conversely, injection of CaMKII inhibitory peptide decreased tetanic [Ca²⁺]_i by ≈25 %, but had no significant effect on the rate of SR Ca²⁺ uptake or the force-[Ca²⁺]_i relationship. Repeated tetanic stimulation resulted in increased tetanic [Ca²⁺]_i, and this increase was smaller after CaMKII inhibition. In conclusion, CaMKII-induced phosphorylation facilitates SR Ca²⁺ release in the basal state and during repeated contractions, providing a positive feedback between [Ca²⁺]_i and SR Ca²⁺ release.     

Functional characterization of a Ca2+-activated non-selective cation channel in human atrial cardiomyocytes

Cardiac arrhythmias, which occur in a wide variety of conditions where intracellular calcium is increased, have been attributed to the activation of a transient inward current ( I _ti). I _ti is the result of three different [Ca]_i-sensitive currents: the Na⁺–Ca²⁺ exchange current, a Ca²⁺-activated chloride current and a Ca²⁺-activated non-selective cationic current. Using the cell-free configuration of the patch-clamp technique, we have characterized the properties of a Ca²⁺-activated non-selective cation channel (NSC_Ca) in freshly dissociated human atrial cardiomyocytes. In excised inside-out patches, the channel presented a linear I–V relationship with a conductance of 19 ± 0.4 pS. It discriminated poorly among monovalent cations (Na⁺ and K⁺) and was slightly permeable to Ca²⁺ ions. The channel's open probability was increased by depolarization and a rise in internal calcium, for which the K _d for [Ca²⁺]_i was 20.8 μ m . Channel activity was reduced in the presence of 0.5 m m ATP or 10 μ m glibenclamide on the cytoplasmic side to 22.1 ± 16.8 and 28.5 ± 8.6%, respectively, of control. It was also inhibited by 0.1 m m flufenamic acid. The channel shares several properties with TRPM4b and TRPM5, two members of the 'TRP melastatin' subfamily. In conclusion, the NSC_Ca channel is a serious candidate to support the delayed after-depolarizations observed in [Ca²⁺] overload and thus may be implicated in the genesis of arrhythmias.     

Cyclic AMP-dependent Cl− secretion induced by thromboxane A2 in isolated human colon

Increased release of thromboxane A₂ (TXA₂) has been shown to be involved in inflammatory bowel diseases. In the present study, we have investigated the effect of a stable TXA₂ analogue (STA₂) on the electrical parameters in isolated human colonic mucosa. In the human mucosa set between Ussing chambers, STA₂ stimulated Cl⁻ secretion in a concentration-dependent manner with an EC₅₀ of 0.06 μ m . The STA₂-induced Cl⁻ secretion was significantly inhibited by ONO-3708 (10 μ m ), a specific TXA₂ receptor antagonist. The effect of STA₂ (0.3 μ m ) was independent of the colonic segment from which the tissue was obtained, from caecum to rectum. Chromanol 293B, an inhibitor of the cAMP-dependent KvLQT1 channel, attenuated the STA₂-induced Cl⁻ secretion in the human colonic mucosa (IC₅₀ value 1.18 μ m ). We found that KvLQT1 mRNA and protein were expressed in all the tested segments of the human colon. The STA₂-induced Cl⁻ secretion was significantly inhibited by 8-bromo-2'-monobutyryladenosine-3',5'-cyclic monophosphorothioate (50 μ m ), a membrane-permeant cAMP antagonist. STA₂ (0.3 μ m ) significantly increased the intracellular cAMP levels and the short-circuit current via TXA₂ receptor in a human colonic cell line. These results suggest that the TXA₂-induced Cl⁻ secretion in the colon is mediated via the cAMP pathway in addition to the Ca²⁺–calmodulin pathway which was previously reported.     

Protein tyrosine phosphorylation and the regulation of KCl cotransport in trout erythrocytes

Electroneutral salt transporters are activated and deactivated by changes to the phosphorylation status either of the transporter itself or of other, as yet unidentified, regulatory proteins. We have studied the effects of an inhibitor of protein tyrosine kinase (PTK), genistein, upon KCl cotransport in trout erythrocytes. We show that Cl-dependent K fluxes activated by physiological stimuli, i.e. oxygenation and β-adrenergic agonists, are rapidly and completely blocked by genistein, whilst the inactive analogue of genistein, daidzein, had no effect. By contrast, the protein tyrosine phosphatase (PTP) inhibitor, vanadate (V), caused a slow but strong activation of an inactive cotransporter. This vanadate (V) activated flux was inhibited by genistein as well as by the serine/threonine phosphatase (PSP) inhibitor, calyculin A. However, genistein had no effect upon the activation of the cotransporter by the protein (serine/threonine) kinase (PSK) inhibitor, staurosporine, or by N-ethylmaleimide, which also appears to act by inhibiting a PSK. These results are consistent with a sequential scheme of at least two tyrosine phosphorylation events which lie upstream to the serine/threonine phosphorylation sites in the signal transduction pathway leading from stimulus to transporter activation. The regulation of the activity of KCl cotransporter appears to involve a complex series of phosphorylation reactions. Received: 4 March 1996/Accepted: 1 May 1996