A Role for the Tyrosine Kinase Pyk2 in Depolarization-induced Contraction of Vascular Smooth Muscle

Depolarization of the vascular smooth muscle cell membrane evokes a rapid (phasic) contractile response followed by a sustained (tonic) contraction. We showed previously that the sustained contraction involves genistein-sensitive tyrosine phosphorylation upstream of the RhoA/Rho-associated kinase (ROK) pathway leading to phosphorylation of MYPT1 (the myosin-targeting subunit of myosin light chain phosphatase (MLCP)) and myosin regulatory light chains (LC₂₀). In this study, we addressed the hypothesis that membrane depolarization elicits activation of the Ca²⁺-dependent tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2). Pyk2 was identified as the major tyrosine-phosphorylated protein in response to membrane depolarization. The tonic phase of K⁺-induced contraction was inhibited by the Pyk2 inhibitor sodium salicylate, which abolished the sustained elevation of LC₂₀ phosphorylation. Membrane depolarization induced autophosphorylation (activation) of Pyk2 with a time course that correlated with the sustained contractile response. The Pyk2/focal adhesion kinase (FAK) inhibitor PF-431396 inhibited both phasic and tonic components of the contractile response to K⁺, Pyk2 autophosphorylation, and LC₂₀ phosphorylation but had no effect on the calyculin A (MLCP inhibitor)-induced contraction. Ionomycin, in the presence of extracellular Ca²⁺, elicited a slow, sustained contraction and Pyk2 autophosphorylation, which were blocked by pre-treatment with PF-431396. Furthermore, the Ca²⁺ channel blocker nifedipine inhibited peak and sustained K⁺-induced force and Pyk2 autophosphorylation. Inhibition of Pyk2 abolished the K⁺-induced translocation of RhoA to the particulate fraction and the phosphorylation of MYPT1 at Thr-697 and Thr-855. We conclude that depolarization-induced entry of Ca²⁺ activates Pyk2 upstream of the RhoA/ROK pathway, leading to MYPT1 phosphorylation and MLCP inhibition. The resulting sustained elevation of LC₂₀ phosphorylation then accounts for the tonic contractile response to membrane depolarization.     

Blockade by nickel ions of phasic contraction to K+ and high affinity calcium of ileal longitudinal muscle of guinea-pig

1. Preincubation with 1 or 2mM Ni²⁺ inhibited dose-dependently the ileal phasic response to K⁺ (60 mM) without appreciable effects on the tonic response. Ni²⁺ above 3mM inhibited the tonic response.2. Ni²⁺ inhibited the high affinity Ca²⁺ sites than the low affinity sites during K⁺ contraction.3. After treatment with Ni²⁺, the K⁺ response was fairly restored by a wash with normal medium. The nickel bound to the ileal cells was almost eliminated with the washing.4. This probably indicates that Ni²⁺ mainly inhibited the K⁺-induced phasic tension by reducing Ca²⁺ release rather than Ca²⁺ influx.     

Influence of ryanodine receptor agonist and antagonist on development of potassium contracture in phasic (twitch) and tonic fibers of frog skeletal muscle

We compared the influence of external calcium and the inhibitor (dantrolene) and activator (4-chloro-m-cresol) of ryanodine-sensitive Ca channels of the sarcoplasmic reticulum on the characteristics of potassium contracture in phasic and tonic frog skeletal muscle fibers. The duration of contracture in tonic fibers, as contrasted to the phasic ones, is not limited by the presence of Ca²⁺. The tonic contractile response is virtually indifferent to dantrolene and is much less sensitive to chlorocresol than the phasic one (1 mM vs. 0.25 mM). In phasic fibers, the K⁺ contracture on the chlorocresol background is quite similar in amplitude and dynamics to that in control, whereas tonic fibers exhibit response summation without relaxation upon removal of excessive K⁺. One can suggest that in phasic fibers the Ca²⁺ influx can directly create a level sufficient to sustain contraction, while in tonic fibers its effect is mediated by Ca-dependent activation of the beta isoform of the ryanodine-sensitive channel.     

Influence of monovalent cations on the Ca2+-ATPase of sarcoplasmic reticulum isolated from rabbit skeletal and dog cardiac muscles. An interpretation of transient-state kinetic data

Transient-state kinetics of phosphorylation and dephosphorylation of the Ca²⁺-ATPase of sarcoplasmic reticulum vesicles from rabbit skeletal and dog cardiac muscles were studied in the presence of varying concentrations of monovalent and divalent cations. Monovalent cations affect the two types of sarcoplasmic reticulum differently. When the rabbit skeletal sarcoplasmic reticulum was Ca²⁺ deficient, preincubation with K⁺ (as compared with preincubation with choline chloride) did not affect initial phosphorylation at various concentrations of Ca²⁺, added with ATP to phosphorylate the enzyme. This is in contrast to preincubation with K⁺ of the Ca²⁺-deficient dog cardiac sarcoplasmic reticulum, which resulted in an increase in the phosphoenzyme level. When Ca²⁺ was bound to the rabbit skeletal sarcoplasmic reticulum, K⁺ inhibited E ~ P formation; but under the same conditions, E ~ P formation of dog cardiac sarcoplasmic reticulum was activated by K⁺ at 12 μM Ca²⁺ and inhibited at 0.33 and 1.3 μM Ca²⁺. Li⁺, Na⁺ and K⁺ also have different effects on E ~ P decomposition of skeletal and cardiac sarcoplasmic reticulum. The latter responded less to these cations than the former. Studies with ADP revealed differences between the two types of sarcoplasmic reticulum. For rabbit skeletal sarcoplasmic reticulum, 40% of the phosphoenzyme formed was ‘ADP sensitive’, and the decay of the remaining E ~ P was enhanced by K⁺ and ADP. Dog cardiac sarcoplasmic reticulum yielded about 40–48% ADP-sensitive E ~ P, but the decomposition rate of the remaining E ~ P was close to the rate measured in the absence of ADP. Thus, these studies showed certain qualitative differences in the transformation and decomposition of phosphoenzymes between skeletal and cardiac muscle which may have bearing on physiological differences between the two muscle types.     

Mg2+ and ATP effects on K+ activation of the Ca2+-transport ATpase of cardiac sarcoplasmic reticulum

ATP and the divalent cations Mg²⁺ and Ca²⁺ regulated K⁺ stimulation of the Ca²⁺-transport ATPase of cardiac sarcoplasmic reticulum vesicles. Millimolar concentrations of total ATP increased the K⁺-stimulated ATPase activity of the Ca²⁺ pump by two mechanisms. First, ATP chelated free Mg²⁺ and, at low ionized Mg²⁺ concentrations, K⁺ was shown to be a potent activator of ATP hydrolysis. In the absence of K⁺ ionized Mg²⁺ activated the enzyme half-maximally at approximately 1 mM, whereas in the presence of K⁺ the concentration of ionized Mg²⁺ required for half-maximal activation was reduced at least 20-fold. Second MgATP apparently interacted directly with the enzyme at a low affinity nucleotide site to facilitate K⁺-stimulation. With a saturating concentration of ionized Mg²⁺, stimulation by K⁺ was 2-fold, but only when the MgATP concentration was greater than 2 mM. Hill plots showed that K⁺ increased the concentration of MgATP required for half-maximal enzymic activation approx. 3-fold.Activation of K⁺-stimulated ATPase activity by Ca²⁺ was maximal at anionized Ca²⁺ concentration of approx. 1 μM. At very high concentrations of either Ca²⁺ or Mg²⁺, basal Ca²⁺-dependent ATPase activity persisted, but the enzymic response to K⁺ was completely inhibited. The results provide further evidence that the Ca²⁺-transport ATPase of cardiac sarcoplasmic reticulum has distinct sites for monovalent cations, which in turn interact allosterically with other regulatory sites on the enzyme.     

INFLUENCE OF DIVALENT CATIONS ON REGULATION OF CYCLIC GMP AND CYCLIC AMP LEVELS IN BRAIN TISSUE

—Depolarizing concentrations of K⁺ elevate levels of both adenosine 3′,5′monophosphate (cyclic AMP) and guanosine 3′,5′monophosphate (cyclic GMP) in incubated slices of mouse cerebellum. Calcium is an essential requirement for the K⁺ -induced accumulation of cyclic GMP. Barium and Sr²⁺, but not Mn²⁺ or Co²⁺, can substitute for Ca²⁺ in this process. Relatively high concentrations of Mg²⁺ inhibit the effect of Ca²⁺ on K⁺-induced accumulation of cyclic GMP. In contrast, depolarizing concentrations of K⁺ are capable of elevating cyclic AMP levels in brain slices suspended in media containing Mg²⁺ and no other divalent cations. High concentrations of Ca²⁺ (1 mm or greater) augment this Mg²⁺ -dependent, K⁺-induced accumulation of cyclic AMP, however. Strontium and Mn²⁺, but not Ba²⁺ or Co²⁺, can substitute for Ca²⁺ in this process, and high concentrations of Mg²⁺ are not inhibitory. The divalent cation ionophore, A-23187 (10 μm ), in the presence of extracellular Ca²⁺ elevates the level of cyclic GMP, but not cyclic AMP, in incubated mouse cerebellum slices. The results of this study indicate that intracellular Ca²⁺ concentration is a major factor regulating cyclic GMP levels in brain. In addition the present results suggest that, in brain tissue, depolarization-induced accumulation of cyclic GMP, but not cyclic AMP, is closely linked to some Ca²⁺-dependent mechanism(s) mediating release of intracellular substances.     

Silver ions induce a rapid Ca2+ release from isolated intact bovine rod outer segments by a cooperative mechanism

Summary Micromolar concentrations of silver ion activate large Ca²⁺ fluxes across the plasma membrane of intact rod outer segments isolated from bovine retinas (intact ROS). The rate of Ag⁺-induced Ca²⁺ efflux from intact ROS depended on the Ag⁺ concentration in a sigmoidal manner suggesting a cooperative mechanism with a Hill coefficient between 2 and 3. At a concentration of 50 m Ag⁺ the rate of Ca²⁺ efflux was 7×10⁶ Ca²⁺/outer segment/sec; this represents a change in total intracellular Ca²⁺ by 0.7mm/outer segment/sec. Addition of the nonselective ionophore gramicidin in the absence of external alkali cations greatly reduced the Ag⁺-induced Ca²⁺ efflux from intact ROS, apparently by enabling internal alkali cations to leak out. Adding back alkali cations to the external medium restored Ag⁺-induced Ca²⁺ efflux when gramicidin was present. In the presence of gramicidin, Ag⁺-induced Ca²⁺ efflux from intact ROS was blocked by 50 m tetracaine orl-cis diltiazem, whereas without gramicidin both blockers were ineffective. Bothl-cis diltiazem and tetracaine are blockers of one kinetic component of cGMP-induced Ca²⁺ flux across ROS disk membranes. The ion selectivity of the Ag⁺-induced pathway proved to be broad with little discrimination between the alkali cations Li⁺, Na⁺, K⁺, and Cs⁺ or between Ca²⁺ and Mg²⁺. The properties of the Ag⁺-induced pathway(s) suggest that it may reflect the cGMP-dependent conductance opened in the absence of cGMP by silver ions.     

Binding of cations by microsomes from rabbit skeletal muscle

Fragmented sarcoplasmic reticulum and transverse tubular system, as isolated in the microsomal fraction from rabbit skeletal muscle, bind H⁺, Na⁺, K⁺, Ca⁺⁺, Mg⁺⁺, and Zn⁺⁺. The binding depends on a cation exchange type of interaction between these cations and the chemical components of the membranous systems of the muscle cell. The monovalent and divalent cations exchange quantitatively for each other at the binding sites on an equivalent basis. Scatchard plots of the H⁺ binding data indicate that the binding groups can be resolved into two major components in terms of their pK values. Component 1 has a pK value of 6.6 and a capacity for H⁺ binding of 2.2 meq/g N . The second component has a much higher H⁺ binding capacity (7–8 meq/g N ), but its pK value, 3.4, is non-physiological. The binding of cations other than H⁺ at a neutral pH occurs at the binding sites making up component 1. The order of affinity of the cations for the microsome binding sites is H⁺ » Zn⁺⁺ > Ca⁺⁺ > Mg⁺⁺ » Na⁺ = K⁺ as reflected by the apparent respective pK_M values: 6.6, 5.2, 4.7, 4.2, 1.3, 1.3. Caffeine, which causes contracture and potentiates the twitch of skeletal muscle, does not interfere with the binding of Ca⁺⁺ by the microsomes at neutral pH.     

Schistosoma mansoni: Calcium efflux and effects of calcium-free media on responses of the adult male musculature to praziquantel and other agents inducing contraction

When male Schistosoma mansoni were incubated in a Ca²⁺-free medium their responsiveness to the contracture inducing agents, praziquantel (PZ), dinitrophenol (DNP), 60 mM K⁺ (high K⁺), ouabain, and low temperature, was rapidly attenuated. After 5 min in a zero Ca²⁺ medium the responsiveness to PZ was reduced by 60% but a residual response of 20% remained even after 40 min in a calcium-free medium. The contracture induced by ouabain or low temperature was totally lost within 1 min exposure to a zero Ca²⁺ medium. The efflux of ⁴⁵Ca²⁺ from parasites incubated in a medium containing no Ca²⁺ closely parallels the drop in responsiveness of their musculature to high K⁺, DNP, and PZ. The total amount of Ca²⁺ in the parasite was reduced by only 30% after 60 min in zero Ca²⁺ medium. A relatively rapid exponential decline in muscle tension was noted when parasites, previously treated with PZ, high K⁺, or DNP, were transferred to a medium containing these agents but with no Ca²⁺. Parasites that had been contracted with ouabain or low temperature showed no significant relaxation 16 min after transfer to a zero Ca²⁺ medium. The ⁴⁵Ca²⁺ efflux from worms bathed in zero Ca²⁺ medium was not significantly altered by the presence of ouabain. These results suggest the presence of active Ca²⁺ transport at the level of the parasites' muscle membranes.     

The calculation of partial specific volumes of proteins in guanidine hydrochloride

The effects of various modifiers on the ATPase activity of bovine platelet actomyosin has been studied. The order of activation by monovalent cations was NH₄⁺? K⁺ > Li⁺ > Na⁺. The order of activation by divalent cations was Ca²⁺ > Mn²⁺ = Sr²⁺ > Ba²⁺> Co²⁺ > Mg²⁺ > Zn²⁺. Ethylenediaminetetraacetate inhibits. Activity increased with increasing concentrations of monovalent cations, except for inhibition by increasing concentrations of NH₄⁺ in the presence of Ca²⁺. Adenosine triphosphatase activity was increased by low concentrations of urea and trypsin, but was unaffected by low concentrations of N-ethylmaleimide. For all enzymatic properties where direct comparisons are possible, actomyosin from platelets is unlike that from skeletal muscle, but is similar to that from smooth muscle and non-muscle sources.     

Phasic Contractions of the Mouse Vagina and Cervix at Different Phases of the Estrus Cycle and during Late Pregnancy

Background/Aims

The pacemaker mechanisms activating phasic contractions of vaginal and cervical smooth muscle remain poorly understood. Here, we investigate properties of pacemaking in vaginal and cervical tissues by determining whether: 1) functional pacemaking is dependent on the phase of the estrus cycle or pregnancy; 2) pacemaking involves Ca²⁺ release from sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) -dependent intracellular Ca²⁺ stores; and 3) c-Kit and/or vimentin immunoreactive ICs have a role in pacemaking.

Methodology/Principal Findings

Vaginal and cervical contractions were measured in vitro, as was the distribution of c-Kit and vimentin positive interstitial cells (ICs). Cervical smooth muscle was spontaneously active in estrus and metestrus but quiescent during proestrus and diestrus. Vaginal smooth muscle was normally quiescent but exhibited phasic contractions in the presence of oxytocin or the K⁺ channel blocker tetraethylammonium (TEA) chloride. Spontaneous contractions in the cervix and TEA-induced phasic contractions in the vagina persisted in the presence of cyclopiazonic acid (CPA), a blocker of the SERCA that refills intracellular SR Ca²⁺ stores, but were inhibited in low Ca²⁺ solution or in the presence of nifedipine, an inhibitor of L-type Ca²⁺channels. ICs were found in small numbers in the mouse cervix but not in the vagina.

Conclusions/Significance

Cervical smooth muscle strips taken from mice in estrus, metestrus or late pregnancy were generally spontaneously active. Vaginal smooth muscle strips were normally quiescent but could be induced to exhibit phasic contractions independent on phase of the estrus cycle or late pregnancy. Spontaneous cervical or TEA-induced vaginal phasic contractions were not mediated by ICs or intracellular Ca²⁺ stores. Given that vaginal smooth muscle is normally quiescent then it is likely that increases in hormones such as oxytocin, as might occur through sexual stimulation, enhance the effectiveness of such pacemaking until phasic contractile activity emerges.     

Potassium channel activators decrease endogenous glutamate release from rat cerebellar slices

Summary The effects of the sulphonylurea activators of ATP-sensitive potassium channels (K⁺ _ATP), cromakalim and pinacidil, on the evoked-release of endogenous glutamate from superfused slices of rat cerebellum was examined. K⁺-stimulated release was Ca²⁺-dependent, whereas tetrapentylammonium (TPeA)-evoked release occurred both in the presence and absence of Ca²⁺, but was significantly greater in Ca²⁺-free medium. The Ca²⁺-dependent TPeA and K⁺-evoked release of glutamate was inhibited by both cromakalim and pinacidil in a concentration-dependent fashion. However, although cromakalim markedly reduced Ca²⁺-independent TPeA-evoked release, pinacidil was ineffective. In addition, the vehicle for cromakalim, ethanol, markedly potentiated both Ca²⁺-dependent and -independent TPeA-evoked release, but not K⁺-evoked release. Despite a high concentration of sulphonylurea binding sites and a dense glutamatergic innervation, the concentrations of K⁺ _ATP channel activators required to inhibit stimulus-evoked release from the cerebellum are higher than those reported to inhibit glutamate release or reduce neuronal activity in other parts of the CNS.     

Schistosoma mansoni: effect of some cations on the proteolytic enzymes of cercariae

The effects of various salts on the proteolytic activity of extracts from Schistosoma mansoni cercariae were tested. Using an Azocoll substrate, stimulation (2 to 2.5-fold) of activity by the monovalent cations Na⁺ and K⁺ was demonstrated, with maximum stimulation at 20–40 mM concentrations. The divalent cations Mg²⁺ and Ca²⁺ stimulated proteolytic activity at low concentrations (between 0 and 10 mM) but inhibited activity at higher concentrations. The divalent cations Zn²⁺, Cu²⁺, Fe²⁺, and Co²⁺ were inhibitory even at very low concentrations. The results presented here are discussed in relation to previously described ion effects on cercarial infectivity.     

Evidence for the involvement of calmodulin in the operation of Ca-activated K channels in mouse fibroblasts

Summary The oscillation of membrane potential in fibroblastic L cells is known to result from periodic stimulation of Ca²⁺-activated K⁺ channels due to the oscillatory increase in the intracellular Ca²⁺ concentration. These repeated hyperpolarizations were inhibited by putative calmodulin antagonists, trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and promethazine (PMZ), and the concentrations required for half-maximal inhibition were 25, 30 and 300 m, respectively. These doses were lower than those for reducing the membrane resistance due to nonspecific cell damages. Another calmodulin antagonist, chlorpromazine (CPZ), was also effective, but CPZ-sulfoxide was not. Intracellular pressure injections of calmodulin-interacting divalent cations, Ca²⁺, Sr²⁺, Mn²⁺ and Ni²⁺, elicited slow hyperpolarizations, whereas Mg²⁺ and Ba²⁺, which are known to be essentially inert for calmodulin, failed to evoke any responses. The injection of purified calmodulin also brought about a similar hyperpolarization. Quinine, an inhibitor of Ca²⁺-activated K⁺ channels, abolished both Ca²⁺-and calmodulin-induced hyperpolarizations. TFP prevented Ca²⁺-induced hyperpolarizations. The TFP effect was partially reversed by the calmodulin injection. It is concluded that calmodulin is involved in the operation of Ca²⁺-activated K⁺ channels in fibroblasts.     

Effect of antihistamines and chlorpromazine on the calcium-induced hyperpolarization of the Amphiuma red cell membrane

1. 1. It has previously been demonstrated that an increase in extracellular Ca²⁺ concentration induces a transient increase in K⁺ permeability and associated hyperpolarization of the red cell membrane of the giant salamander, Amphiuma means. This phenomenon is analogous to the Ca²⁺-induced KCl loss observed in ATP-depleted human red cells and red cell ghosts.

2. 2. Histamine, which enhances the Ca²⁺-induced K⁺ loss from depleted human red cells, is without effect on this Ca²⁺-induced hyperpolarization of Amphiuma red cells.

3. 3. Promethazine (10 μM) and mepyramine (1 mM), which inhibit the Ca²⁺-induced K⁺ loss in depleted human red cells, also block the Ca²⁺-related hyperpolarization of Amphiuma erythrocytes.

4. 4. Chlorpromazine (25 μM), despite being a weak antihistamine, is equally effective in blocking the Ca²⁺-induced hyperpolarization of Amphiuma red cells.

5. 5. Ionophore A23187 causes a large and sustained Ca²⁺/K⁺-dependent hyperpolarization even in the presence of normal (1.8 mM) concentrations of Ca²⁺. This hyperpolarization is relatively insensitive to chlorpromazine and promethazine.

6. 6. The inhibition of the Ca²⁺-induced hyperpolarization of the Amphiuma red cell membrane by chlorpromazine and promethazine may be related to their properties as local anaesthetics.

Alteration of Ca2+ Fluxes in Brain Microsomes by K+ and Na+: Modulation by Sulfated Polysaccharides and Trifluoperazine

Abstract: Rat brain microsomes accumulate Ca²⁺ at the expense of ATP hydrolysis. The rate of transport is not modulated by the monovalent cations K⁺, Na⁺, or Li⁺. Both the Ca²⁺ uptake and the Ca²⁺-dependent ATPase activity of microsomes are inhibited by the sulfated polysaccharides heparin, fucosylated chondroitin sulfate, and dextran sulfate. Half-maximal inhibition is observed with sulfated polysaccharide concentrations ranging from 0.5 to 8.0 µg/ml. The inhibition is antagonized by KCl and NaCl but not by LiCl. As a result, Ca²⁺ transport by the native vesicles, which in the absence of polysaccharides is not modulated by monovalent cations, becomes highly sensitive to these ions. Trifluoperazine has a dual effect on the Ca²⁺ pump of brain microsomes. At low concentrations (20–80 µM) it stimulates the rate of Ca²⁺ influx, and at concentrations >100 µM it inhibits both the Ca²⁺ uptake and the ATPase activity. The activation observed at low trifluoperazine concentrations is specific for the brain Ca²⁺-ATPase; for the Ca²⁺-ATPases found in blood platelets and in the sarcoplasmic reticulum of skeletal muscle, trifluoperazine causes only a concentration-dependent inhibition of Ca²⁺ uptake. Passive Ca²⁺ efflux from brain microsomes preloaded with Ca²⁺ is increased by trifluoperazine (50–150 µM), and this effect is potentiated by heparin (10 µg/ml), even in the presence of KCl. It is proposed that the Ca²⁺-ATPase isoform from brain microsomes is modulated differently by polysaccharides and trifluoperazine when compared with skeletal muscle and platelet isoforms.     

Divalent Cation Interactions with Na,K-ATPase Cytoplasmic Cation Sites: Implications for the <Emphasis Type="Italic">para</Emphasis>-Nitrophenyl Phosphatase Reaction Mechanism

The interactions of divalent cations with the adenosine triphosphatase (ATPase) and para-nitrophenyl phosphatase (pNPPase) activity of the purified dog kidney Na pump and the fluorescence of fluorescein isothiocyanate (FITC)-labeled pump were determined. Sr²⁺ and Ba²⁺ did not compete with K⁺ for ATPase (an extracellular K⁺ effect). Sr²⁺ and Ba²⁺ did compete with Na⁺ for ATPase (an intracellular Na⁺ effect) and with K⁺ for pNPPase (an intracellular K⁺ effect). These results suggest that Ba²⁺ or Sr²⁺ can bind to the intracellular transport site, yet neither Ba²⁺ nor Sr²⁺ was able to activate pNPPase activity; we confirmed that Ca²⁺ and Mn²⁺ did activate. As another measure of cation binding, we observed that Ca²⁺ and Mn²⁺, but not Ba²⁺, decreased the fluorescence of the FITC-labeled pump; we confirmed that K⁺ substantially decreased the fluorescence. Interestingly, Ba²⁺ did shift the K⁺ dose-response curve. Ethane diamine inhibited Mn²⁺ stimulation of pNPPase (as well as K⁺ and Mg²⁺ stimulation) but did not shift the 50% inhibitory concentration (IC₅₀) for the Mn²⁺-induced fluorescence change of FITC, though it did shift the IC₅₀ for the K⁺-induced change. These results suggest that the Mn²⁺-induced fluorescence change is not due to Mn²⁺ binding at the transport site. The drawbacks of models in which Mn²⁺ stimulates pNPPase by binding solely to the catalytic site vs. those in which Mn²⁺ stimulates by binding to both the catalytic and transport sites are presented. Our results provide new insights into the pNPPase kinetic mechanism as well as how divalent cations interact with the Na pump.     

Calcium-induced potassium pathway in sided erythrocyte membrane vesicles

We have characterized the asymmetric effect of Ca²⁺ on passive K⁺ permeability in erythrocyte membranes, using inside out and right-side out vesicles. Ca²⁺, but not Mg²⁺, can induce an increase in K⁺ uptake in inside out vesicles. The half-maximal concentration of Ca²⁺ required to induce the K⁺ uptake is 0.2 mM, and the permeability increase is not specific for K⁺. Thus, the Ca²⁺-induced permeation process in inside out vesicles is changed from that in the energy-depleted intact cell which requires only micromolar concentrations of Ca²⁺ and is specific for K⁺. Removal of spectrin had no effect on the vesicle permeability increase due to Ca²⁺. Studies with $\text{N-}\text{ethylmaleimide}$ show that the vesicle channel opening is mediated by a protein and passage is controlled by sulfhydryl groups; furthermore, the Ca²⁺-induced vesicle pathway is distinct from the normal channel for passive K⁺ leak in the absence of Ca²⁺. The protein is sensitive to its phospholipid environment since removal of easily accessible phospholipid head groups on the cytoplasmic face of the vesicles inhibits the Ca²⁺-stimulated channel opening.     

Stimulating effects of monovalent cations on activator-dissociated and activator-associated states of Ca2+-ATPase in human erythrocytes

The additional activation by monovalent cations of the $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-dependent}$ ATPase (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes was studied.The Ca²⁺-ATPase occurs in two different states. In the A-state the enzyme is virtually free of protein activator and the kinetics of Ca²⁺ activation is characterized by low apparent Ca²⁺ affinity and low maximum activity. In the B-state the enzyme is associated with activator and the kinetics is characterized by high Ca²⁺ affinity and high maximum activity.At optimum concentrations of Ca²⁺ the additional activation of the B-state by K⁺, NH₄⁺, Na⁺ and Rb⁺ exceeded the corresponding activations of the A-state, and half-maximum activations by K⁺, NH₄⁺, and Na⁺ were achieved at lower concentrations in the B-state than in the A-state. Li⁺ and Cs⁺ activated the two states almost equally but maximum activation was obtained at lower cation concentrations in the B-state than in the A-state.The activation of the B-state by the various cations decreased in the order $\text{K}{}^{\mathrm{+}}\text{> NH}{}_4{}^{\mathrm{+}}\text{> Na}{}^{\mathrm{+}}\text{= Rb}{}^{\mathrm{+}}\text{> Li}{}^{\mathrm{+}}\text{= Cs}{}^{\mathrm{+}}$. The A-state was activated almost equally by K⁺, Na⁺, NH₄⁺, and Rb⁺ and to a smaller extent by Li⁺ and Cs⁺.At sub-optimum concentrations of Ca²⁺ high concentrations of monovalent cations (100 mM) activated the Ca²⁺-ATPase equally in the A-state and the B-state. In the absence of Ca²⁺ the monovalent cations inhibited the Mg²⁺-dependent ATPase in both types of membranes. This dependence on Ca²⁺ indicates that the monovalent cations interact with the Ca²⁺ sites in the B-state.The results suggest that K⁺ or Na⁺, or both, contribute to the regulation of the Ca²⁺ pump in erythrocytes.     

DHEA attenuates catecholamine secretion from bovine adrenal chromaffin cells

Dehydroepiandrosterone (DHEA) is a putative anti-stress agent and stress is associated with the secretion of catecholamine from the adrenal gland, but the effects of DHEA on catecholamine secretion are not fully understood. Using bovine chromaffin cells, we found that DHEA inhibited catecholamine secretion and cytosolic Ca²⁺ ([Ca²⁺]_i) rise coupled with nicotinic acetylcholine receptor (nAChR) without exerting an effect on³H-nicotine binding. In the case of high K⁺ stimulation, DHEA effectively suppressed secretion without affecting [Ca²⁺]₁ rise. Trifluoperazine (TFP), a calmodulin inhibitor, was capable of counteracting the inhibition of DHEA on high K⁺-induced secretions. In permeabilized cells, DHEA suppressed the Ca²⁺-induced secretion. These results suggest that DHEA (a) acts as a channel blocker that suppresses Ca²⁺ influx and subsequent secretions associated with nAChR, or (b) affects the intracellular secretion machinery to suppress high K⁺-induced secretions without affecting the high K⁺-induced [Ca²⁺]_i rise.