H+ -mediated coupling of transmembrane Ca2+ fluxes in vegetative Trichoderma viride mycelia suggested by the study of ageing and adaptation to extreme Ca2+ concentrations

The adaptation to extreme concentrations of Ca(2+) and its consequence on the properties of the (45)Ca(2+) transport were studied in submerged mycelia of Trichoderma viride. The adaptation to low [Ca(2+)](o) did not cause changes in kinetic parameters of the (45)Ca(2+) influx but the adaptation to high [Ca(2+)](o) increased the K(M(Ca2+)). The V(max) of the (45)Ca(2+) influx decreased with the age of (non-adapted) mycelia with concomitant decrease of the K(M(Ca2+)) these changes were prevented in mycelia adapted to high Ca(2+). High [Ca(2+)](o) decreased the stimulation by the uncoupler, 3, 3', 4', 5-tetrachloro salicylanilide (TCS) (30 muM), as compared to the control, whereas the Ca(2+) chelator, EGTA, stimulated it. In the aged mycelia, the stimulation by TCS of the (45)Ca(2+) influx faded away, in parallel with the activity of the H(+)-ATPase. The (45)Ca(2+) efflux from mycelia was affected by TCS in a similar way as the (45)Ca(2+) influx. The results demonstrate the adaptive responses of transport processes participating in the mycelial Ca(2+) homeostasis and ageing are in agreement with a notion that both Ca(2+)-influx and-efflux are coupled by the H(+)-homeostasis at the plasma membrane.     

Reverse Na(+)/Ca(2+)-exchange mediated Ca(2+)-entry and noradrenaline release in Na(+)-loaded peripheral sympathetic nerves

[(3)H]noradrenaline ([(3)H]NA) released from sympathetic nerves in the isolated main pulmonary artery of the rabbit was measured in response to field stimulation (2Hz, 1ms, 60V for 3min) in the presence of uptake blockers (cocaine, 3 x10(-5)M and corticosterone, 5 x10(-5)M). The [(3)H]NA-release was fully blocked by the combined application of the selective and irreversible 'N-type' voltage-sensitive Ca(2+)-channel (VSCC)-blocker omega-conotoxin (omega-CgTx) GVIA (10(-8)M) and the 'non-selective' VSCC-blocker aminoglycoside antibiotic neomycin (3x10(-3)M). Na(+)-loading (Na(+)-pump inhibition by K(+)-free perfusion) was required to elicit further NA-release after blockade of VSCCs (omega-CgTx GVIA+neomycin). In K(+)-free solution, in the absence of functioning VSCCs (omega-CgTx GVIA+neomycin), the fast Na(+)-channel activator veratridine (10(-5)M) further potentiated the nerve-evoked release of [(3)H]NA. This NA-release was significantly inhibited by KB-R7943, and fully blocked by Ca(o)(2+)-removal. However, Li(+)-substitution was surprisingly ineffective. The non-selective K(+)-channel blocker 4-aminopyridine (4-AP, 10(-4)M) also further potentiated the nerve-evoked release of NA in K(+)-free solution. This potentiated release was concentration-dependently inhibited by KB-R7943, significantly inhibited by Li(+)-substitution and abolished by Ca(o)(2+)-removal. It is concluded that in Na(+)-loaded sympathetic nerves, in which the VSCCs are blocked, the reverse Na(+)/Ca(2+)-exchange-mediated Ca(2+)-entry is responsible for transmitter release on nerve-stimulation. Theoretically we suppose that the fast Na(+)-channel and the exchanger proteins are close to the vesicle docking sites.     

Calbindin-D28K, a 1 alpha,25-dihydroxyvitamin D3-induced calcium-binding protein, binds five or six Ca2+ ions with high affinity

Calbindin-D28K is a 1 alpha,25-dihydroxyvitamin D3-dependent protein that belongs to the superfamily of high affinity calcium-binding proteins which includes parvalbumin, calmodulin, and troponin C. All of these proteins bind Ca2+ ligands by an alpha-helix-loop-alpha-helix domain that is termed an EF-hand. Calbindin-D28K has been reported previously to have four high affinity Ca2(+)-binding sites (KD less than 10(-7)) as quantitated by equilibrium dialysis. With the determination of the amino acid sequence, it was clear that there are in fact six apparent EF-hand domains, although the Ca2(+)-binding functionality of the two additional domains was unclear. It was of interest to quantitate the Ca2(+)-binding ability of chick intestinal calbindin-D28K utilizing several different Ca2+ titration methods that cover a range of macroscopic binding constants for weak or strong Ca2+ sites. Titrations with the Ca2+ chelator dibromo-1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (5,5'-Br2BAPTA), a Ca2+ selective electrode, and as followed by 1H NMR, which measure KD values of 10(-6)-10(-8) M, 10(-4)-10(-7) and 10(-3)-10(-5) M, respectively, gave no evidence for the presence of weak Ca2(+)-binding sites. However, Ca2+ titration of the fluorescent Ca2+ chelator Quin 2 in the presence of calbindin-D28K yielded a least squares fit optimal for 5.7 +/- 0.8 Ca2(+)-binding sites with macroscopic dissociation constants around 10(-8) M. The binding of Ca2+ by calbindin was found to be cooperative with at least two of the sites exhibiting positive cooperativity.     

Membrane currents in canine bronchial artery and their regulation by excitatory agonists

The bronchial vasculature plays an important role in airway physiology and pathophysiology. We investigated the ion currents in canine bronchial smooth muscle cells using patch-clamp techniques. Sustained outward K(+) current evoked by step depolarizations was significantly inhibited by tetraethylamonium (1 and 10 mM) or by charybdotoxin (10(-6) M) but was not significantly affected by 4-aminopyridine (1 or 5 mM), suggesting that it was primarily a Ca(2+)-activated K(+) current. Consistent with this, the K(+) current was markedly increased by raising external Ca(2+) to 4 mM but was decreased by nifedipine (10(-6) M) or by removing external Ca(2+). When K(+) currents were blocked (by Cs(+) in the pipette), step depolarizations evoked transient inward currents with characteristics of L-type Ca(2+) current as follows: 1) activation that was voltage dependent (threshold and maximal at -50 and -10 mV, respectively); 2) inactivation that was time dependent and voltage dependent (voltage causing 50% maximal inactivation of -26 +/- 22 mV); and 3) blockade by nifedipine (10(-6) M). The thromboxane mimetic U-46619 (10(-6) M) caused a marked augmentation of outward K(+) current (as did 10 mM caffeine) lasting only 10-20 s; this was followed by significant suppression of the K(+) current lasting several minutes. Phenylephrine (10(-4) M) also suppressed the K(+) current to a similar degree but did not cause the initial transient augmentation. None of these three agonists elicited inward current of any kind. We conclude that bronchial arterial smooth muscle expresses Ca(2+)-dependent K(+) channels and voltage-dependent Ca(2+) channels and that its excitation does not involve activation of Cl(-) channels.     

Studies on the microsomal sodium-plus-potassium ion-stimulated adenosine triphosphatase system in rat ventral prostate

A Mg(2+)+Na(+)+K(+)-stimulated adenosine triphosphatase (ATPase) preparation was isolated from rat ventral prostate by flotation of microsomal membranes in high-density sucrose solutions. The reaction medium for optimum Na(+)+K(+)-stimulated ATPase activity was found to be: Na(+), 115mm; K(+), 7-10mm; Mg(2+), 3mm; ATP, 3mm; tris buffer, pH7.4 at 38 degrees , 20mm. The average DeltaP(i) (Mg(2+)+Na(+)+K(+) minus Mg(2+)+Na(+)) was 9mumoles/mg. of protein/hr., representing a 30% increase over the Mg(2+)+Na(+)-stimulated ATPase activity. At high concentrations, K(+) was inhibitory to the enzyme activity. Half-maximal inhibition of Na(+)+K(+)-stimulated ATPase activity was elicited by ouabain at 0.1mm. The preparation exhibited phosphatase activity towards ribonucleoside triphosphates other than ATP. However, stimulation of P(i) release by Na(+)+K(+) was observed only with ATP as substrate. The apparent K(m) for ATP for Na(+)+K(+)-stimulated activity was about 0.3x10(-3)m. Ca(2+) inhibited only the Na(+)+K(+)-stimulated ATPase activity. Mg(2+) could be replaced by Ca(2+) but then no Na(+)+K(+) stimulation of ATPase activity was noticed. The addition of testosterone or dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one) in vitro at 0.1-10mum under a variety of experimental conditions did not significantly increase the Na(+)+K(+)-stimulated ATPase activity. The enzyme preparations from prostates of orchidectomized rats, however, exhibited a drastic decrease in the specific activity of Na(+)+K(+)-stimulated ATPase; these changes were prevented in the orchidectomized rats by injection of testosterone propionate.     

Sodium-dependent calcium efflux from adrenal chromaffin cells following exocytosis. Possible role of secretory vesicle membranes.

Although cytosolic Ca2+ transients are known to influence the magnitude and duration of hormone and neurotransmitter release, the processes regulating the decay of such transients after cell stimulation are not well understood. Na(+)-dependent Ca2+ efflux across the secretory vesicle membrane, following its incorporation into the plasma membrane, may play a significant role in Ca2+ efflux after stimulation of secretion. We have measured an enhanced 45Ca2+ efflux from cultured bovine adrenal chromaffin cells following cell stimulation with depolarizing medium (75 mM K+) or nicotine (10 microM). Such stimulation also causes Ca2+ uptake via voltage-gated Ca2+ channels and secretion of catecholamines. Na+ replacement with any of several substitutes (N-methyl-glucamine, Li+, choline, or sucrose) during cell stimulation inhibited the enhanced 45Ca2+ efflux, indicating and Na(+)-dependent Ca2+ efflux process. Na+ deprivation did not inhibit 45Ca2+ uptake or catecholamine secretion evoked by elevated K+. Suppression of exocytotic incorporation of secretory vesicle membranes into the plasma membrane with hypertonic medium (620 mOsm) or by lowering temperature to 12 degrees C inhibited K(+)-stimulated 45Ca2+ efflux in Na(+)-containing medium but did not inhibit the stimulated 45Ca2+ uptake. Enhancement of exocytotic secretion with pertussis toxin resulted in an enhanced 45Ca2+ efflux without affecting calcium uptake. The combined results suggest that Na(+)-dependent Ca2+ efflux across secretory vesicle membranes, following their incorporation into the plasma membrane during exocytosis, plays a significant role in regulating calcium efflux and the decay of cytosolic Ca2+ in adrenal chromaffin cells and possibly in related secretory cells.     

Positive heterotropic allosteric regulators of dihydropyridine binding increase the Ca2+ affinity of the L-type Ca2+ channel. Stereoselective reversal by the novel Ca2+ antagonist BM 20.1140.

The alpha 1-subunit of the voltage-dependent L-type Ca2+ channel has distinct, allosterically coupled binding domains for drugs from different chemical classes (dihydropyridines, benzothiazepines, phenylalkylamines, diphenylbutylpiperidines). (-)-BM 20.1140 (ethyl-2,2-di-phenyl-4-(1-pyrrolidino)-5-(2-picolyl)- oxyvalerate) is a novel Ca2+ channel blocker which potently stimulates dihydropyridine binding (K0.5 = 2.98 nM) to brain membranes. This property is shared by (+)-cis-diltiazem, (+)-tetrandrine, fostedil and trans-diclofurime, but (-)-BM 20.1140 does not bind in a competitive manner to the sites labeled by (+)-cis-[3H]diltiazem. (+)-cis-Diltiazem and (-)-BM 20.1140 have differential effects on the rate constants of dihydropyridine binding. (+)-BM 20.1140 reverses the stimulation of the positive allosteric regulators (pA2 value for reversal of (-)-BM 20.1140 stimulation = 7.4, slope 0.72). The underlying molecular mechanism of the potentiation of dihydropyridine binding has been clarified. The K0.5 for free Ca2+ to stabilize a high affinity binding domain for dihydropyridines on purified L-type channels from rabbit skeletal muscle is 300 nM. (+)-Tetrandine (10 microM) increases the affinity 8-fold (K0.5 for free Ca2+ = 30.1 nM) and (+)-BM 20.114 (10 microM) inhibits the affinity increase (K0.5 for free Ca2+ = 251 nM). Similar results were obtained with membrane-bound Ca(2+)-channels from brain tissue which have higher affinity for free Ca2+ (K0.5 for free Ca2+ = 132 nM) and for dihydropyridines compared with skeletal muscle. It is postulated that the dihydropyridine and Ca(2+)-binding sites are interdependent on the alpha 1-subunit, that the different positive heterotropic allosteric regulators (by their differential effects on Ca2+ rate constants) optimize coordination for Ca2+ in the channel pore and, in turn, increase affinity for the dihydropyridines.     

Stimulatory pathways of the Calcium-sensing receptor on acid secretion in freshly isolated human gastric glands.

Gastric acid secretion is not only stimulated via the classical known neuronal and hormonal pathways but also by the Ca(2+)-Sensing Receptor (CaSR) located at the basolateral membrane of the acid-secretory gastric parietal cell. Stimulation of CaSR with divalent cations or the potent agonist Gd(3+) leads to activation of the H(+)/K(+)-ATPase and subsequently to gastric acid secretion. Here we investigated the intracellular mechanism(s) mediating the effects of the CaSR on H(+)/K(+)-ATPase activity in freshly isolated human gastric glands. Inhibition of heterotrimeric G-proteins (G(i) and G(o)) with pertussis toxin during stimulation of the CaSR with Gd(3+) only partly reduced the observed stimulatory effect. A similar effect was observed with the PLC inhibitor U73122. The reduction of the H(+)/K(+)-ATPase activity measured after incubation of gastric glands with BAPTA-AM, a chelator of intracellular Ca(2+), showed that intracellular Ca(2+) plays an important role in the signalling cascade. TMB-8, a ER Ca(2+)store release inhibitor, prevented the stimulation of H(+)/K(+)-ATPase activity. Also verapamil, an inhibitor of L-type Ca(2+)-channels reduced stimulation suggesting that both the release of intracellular Ca(2+) from the ER as well as Ca(2+) influx into the cell are involved in CaSR-mediated H(+)/K(+)-ATPase activation. Chelerythrine, a general inhibitor of protein kinase C, and Go 6976 which selectively inhibits Ca(2+)-dependent PKC(alpha) and PKC(betaI)-isozymes completely abolished the stimulatory effect of Gd(3+). In contrast, Ro 31-8220, a selective inhibitor of the Ca(2+)-independent PKCepsilon and PKC-delta isoforms reduced the stimulatory effect of Gd(3+) only about 60 %. On the other hand, activation of PKC with DOG led to an activation of H(+)/K(+)-ATPase activity which was only about 60 % of the effect observed with Gd(3+). Incubation of the parietal cells with PD 098059 to inhibit ERK1/2 MAP-kinases showed a significant reduction of the Gd(3+) effect. Thus, in the human gastric parietal cell the CaSR is coupled to pertussis toxin sensitive heterotrimeric G-Proteins and requires calcium to enhance the activity of the proton-pump. PLC, ERK 1/2 MAP-kinases as well as Ca(2+) dependent and Ca(2+)-independent PKC isoforms are part of the down-stream signalling cascade.     

Calcium-stressed erythrocyte membrane structure and function for assessing glipizide effects on transglutaminase activation.

A proposed mechanism of action of hypoglycemic sulfonylureas is the prevention of transglutaminase-mediated endocytosis of insulin receptors. When activated by high levels of intracellular calcium, transglutaminase (TG) catalyzes the cross-linking of intracellular proteins to membrane proteins and modifies membrane structure and function. This study examined the effects of the sulfonylurea glipizide on TG activity in an erythrocyte model by assessing various membrane ATPase activities and high molecular weight protein polymer formation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. To activate TG, red blood cells were exposed to 1 mM intracellular Ca2+ using 10(-5) M Ca2(+)-ionophore A23187. In Ca2(+)-stressed cells, calmodulin stimulation (0.1 micrograms/ml) of (Ca2+ + Mg2+)-ATPase was decreased to 21.2% of control activity. Increasing concentrations of calmodulin (0.1-3.0 micrograms/ml) could not overcome the inhibitory effects of TG on the (Ca2+ + Mg2+)-ATPase in Ca2(+)-stressed cells with or without glipizide. An increased Ca2+ sensitivity of calmodulin-independent (Ca2+ + Mg2+)-ATPase due to Ca2+ stress was seen in all Ca2(+)-stressed cells even in the presence of 1 mM glipizide. Structural changes were observed in the form of high molecular weight polymer formation. Cells exposed to high Ca2+ and glipizide (3 x 10(-5)-10(-3) M) showed no improvement in ATPase activity or protection from protein cross-linking compared with cells without the drug. We conclude that in this model glipizide fails to inhibit TG induced protein cross-linking and does not prevent the decrease in (Ca2+ + Mg2+)-ATPase activation in Ca2(+)-stressed red blood cells. This finding considerably weakens the proposal that sulfonylureas act by inhibiting TG activity.     

Effects of pilocarpine on the secretory acinar cells in human submandibular glands

Pilocarpine has been used as a choice of drugs for treatment of impaired salivary flow. Although considerable data are available as to the stimulatory effect of pilocarpine on the salivary secretion in human, its underlying mechanism, at the cellular level, has not been rigorously studied. In this experiment, we studied the effect of pilocarpine on the ion channel activity, cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) and aquaporin (AQP)-5 expression, which play key roles in the secretary process and determine the capacity of fluid secretion. In human submandibular gland (SMG) acinar cells, 10(-5) M pilocarpine activated the outward rectifying-current, which was predominantly K(+) selective in the whole cell patch clamp study. The pilocarpine increased [Ca(2+)](i) in a concentration-dependent manner in the range of 10(-6) M to 10(-4) M. We found that both increases of [Ca(2+)](i) and outward rectifying- K(+) current were inhibited by 10(-5) M U-73122, a specific phospholipase C inhibitor. The magnitudes of pilocarpine-induced [Ca(2+)](i) transients were approximately 55% lower than those with the same concentration of carbachol (CCh). Pilocarpine also increased the amount of AQP-5 protein in the apical membrane (APM) in human SMG acinar cells. Our results suggest that pilocarpine induce salivary secretions in human by activating K(+) channels, increasing [Ca(2+)](i) via phospholipase C dependent pathway, and increasing AQP-5 protein expression in the APM of SMG acinar cells.     

Mechanism by which wortmannin and LY294002 inhibit catecholamine secretion in the rat adrenal medullary cells

The effects of wortmannin and LY294002, inhibitors of PI(3)-kinase, in secretagogue-stimulated rat adrenal chromaffin cells loaded with Calcium Green-1 were studied by simultaneously measuring changes in the fluorescence intensity of the indicator (Ca-response) and in the release of catecholamine (secretory response). Before application of these agents, the profile of the secretory response evoked by a 10-min stimulation with 30 mM K(+)] was approximated by the k th (2.6 on average) power of that of the Ca-response. Both agents dose-dependently inhibited the high-K(+)-elicited Ca-response and secretory response in a similar mode to which the k th power relation was preserved despite the occurrence of profound changes in the shapes and sizes of these two responses. The L-type Ca(2+)-channel blocker PN200-110 inhibited the high-K(+)-evoked responses in a similar fashion. Thus, it is likely that wortmannin and LY294002 inhibit high-K(+)-evoked CA secretion by inhibiting a Ca(2+)-influx through voltage-dependent Ca(2+)channels. Although regulation of L-type Ca(2+)channel activity via PI(3)-kinase has been reported in vascular myocytes, this possibility may be limited in the present case since the doses of LY294002 and wortmannin used to inhibit the secretory response are much higher than IC(50)'s for inhibition of PI(3)-kinase with these agents. Compared with the high-K(+)-elicited responses, muscarine-evoked Ca-responses and secretory responses were more strongly inhibited by wortmannin, but less affected by LY294002. The differential effects suggest that the inhibition of the muscarine-evoked secretion by these agents i s not associated with the inhibition of PI(3)-kinase.     

The potential-dependent action of the opioid peptide dermorphin]

Dermorphin action was studied on cross-section strip of frog stomach muscle by a mechanographic recorder. The results show that dermorphin (10(-5)-10(-8) M) blocks acetylcholine effects, spontaneous activity and muscle contractions induced by direct electrical stimulation. All the above effects are hardly reversible. Dermorphin fails to block spontaneous and evoked activities if it is injected into the incubated medium during K(+)-depolarization (KCI--100 mM) of the muscle. Thus, dermorphin has voltage-dependent action. The discussion deals with dermorphin action on voltage-dependent Ca(2+)-channels of muscle cell membrane.     

Blunted IgE-mediated activation of mast cells in mice lacking the Ca2+-activated K+ channel KCa3.1

Mast cell stimulation by Ag is followed by the opening of Ca(2+)-activated K(+) channels, which participate in the orchestration of mast cell degranulation. The present study has been performed to explore the involvement of the Ca(2+)-activated K(+) channel K(Ca)3.1 in mast cell function. To this end mast cells have been isolated and cultured from the bone marrow (bone marrow-derived mast cells (BMMCs)) of K(Ca)3.1 knockout mice (K(Ca)3.1(-/-)) and their wild-type littermates (K(Ca)3.1(+/+)). Mast cell number as well as in vitro BMMC growth and CD117, CD34, and FcepsilonRI expression were similar in both genotypes, but regulatory cell volume decrease was impaired in K(Ca)3.1(-/-) BMMCs. Treatment of the cells with Ag, endothelin-1, or the Ca(2+) ionophore ionomycin was followed by stimulation of Ca(2+)-activated K(+) channels and cell membrane hyperpolarization in K(Ca)3.1(+/+), but not in K(Ca)3.1(-/-) BMMCs. Upon Ag stimulation, Ca(2+) entry but not Ca(2+) release from intracellular stores was markedly impaired in K(Ca)3.1(-/-) BMMCs. Similarly, Ca(2+) entry upon endothelin-1 stimulation was significantly reduced in K(Ca)3.1(-/-) cells. Ag-induced release of beta-hexosaminidase, an indicator of mast cell degranulation, was significantly smaller in K(Ca)3.1(-/-) BMMCs compared with K(Ca)3.1(+/+) BMMCs. Moreover, histamine release upon stimulation of BMMCs with endothelin-1 was reduced in K(Ca)3.1(-/-) cells. The in vivo Ag-induced decline in body temperature revealed that IgE-dependent anaphylaxis was again significantly (by approximately 50%) blunted in K(Ca)3.1(-/-) mice. In conclusion, K(Ca)3.1 is required for Ca(2+)-activated K(+) channel activity and Ca(2+)-dependent processes such as endothelin-1- or Ag-induced degranulation of mast cells, and may thus play a critical role in anaphylactic reactions.     

Defective expression of neutrophil C3b receptors and impaired lymphocyte Na(+)-K(+)-ATPase activity in patients with systemic lupus erythematosus.

In our previous report, we demonstrated that the functions of phagocytes and lymphocytes were defective in patients with systemic lupus erythematosus (SLE). In an attempt to further clarify the defective mechanisms of these cells, 25 active SLE, 10 bronchial asthma patients (BA) on corticosteroids and 25 age and sex-matched normal individuals were investigated for the expression of membraneous C3b receptors, ionophore-induced 45Ca(2+)-uptake, mitochondrial potentials and phagocytic activity of neutrophils. We found decreased expression of C3b receptors on SLE PMN in both resting (37.2 +/- 3.7% of the normal controls) and FMLP-stimulated (68.3 +/- 7.1% of the normal controls) conditions, whereas the C3b receptor expression on BA-PMN receiving long-term steroid treatment was not different from normal controls. This suggests that the defective phagocytosis of SLE PMN is in the recognition, but not in the ingestion phase because of the normal function of Ca(2+)-influx and mitochondrial activity in SLE PMN. On the other hand, hyporesponsiveness to PHA stimulation (stimulation index: 127.4 +/- 46.3 in SLE vs. 311.2 +/- 30.4 in normals, p = 0.0077) was a distinct cell-mediated immune abnormality in our SLE patients. We measured the membrane potential of individual cells using 3,3'-dihexyloxacarbocyanin and found hyperpolarization in resting SLE lymphocytes. However, the membrane polarization of SLE lymphocytes became lower than that of normal cells after PHA stimulation for 3 days. A similar tendency was also found in Na(+)-K(+)-dependent ATPase activity in SLE lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

The phosphatase activity of the plasma membrane Ca2+ pump. Activation by acidic lipids in the absence of Ca2+ increases the apparent affinity for Mg2+

The purified PMCA supplemented with phosphatidylcholine was able to hydrolyze pNPP in a reaction media containing only Mg(2+) and K(+). Micromolar concentrations of Ca(2+) inhibited about 75% of the pNPPase activity while the inhibition of the remainder 25% required higher Ca(2+) concentrations. Acidic lipids increased 5-10 fold the pNPPase activity either in the presence or in the absence of Ca(2+). The activation by acidic lipids took place without a significant change in the apparent affinities for pNPP or K(+) but the apparent affinity of the enzyme for Mg(2+) increased about 10 fold. Thus, the stimulation of the pNPPase activity of the PMCA by acidic lipids was maximal at low concentrations of Mg(2+). Although with differing apparent affinities vanadate, phosphate, ATP and ADP were all inhibitors of the pNPPase activity and their effects were not significantly affected by acidic lipids. These results indicate that (a) the phosphatase function of the PMCA is optimal when the enzyme is in its activated Ca(2+) free conformation (E2) and (b) the PMCA can be activated by acidic lipids in the absence of Ca(2+) and the activation improves the interaction of the enzyme with Mg(2+).     

Activation of Ca(2+)-dependent K(+) channels contributes to rhythmic firing of action potentials in mouse pancreatic beta cells

We have applied the perforated patch whole-cell technique to beta cells within intact pancreatic islets to identify the current underlying the glucose-induced rhythmic firing of action potentials. Trains of depolarizations (to simulate glucose-induced electrical activity) resulted in the gradual (time constant: 2.3 s) development of a small (<0.8 nS) K(+) conductance. The current was dependent on Ca(2+) influx but unaffected by apamin and charybdotoxin, two blockers of Ca(2+)-activated K(+) channels, and was insensitive to tolbutamide (a blocker of ATP-regulated K(+) channels) but partially (>60%) blocked by high (10-20 mM) concentrations of tetraethylammonium. Upon cessation of electrical stimulation, the current deactivated exponentially with a time constant of 6.5 s. This is similar to the interval between two successive bursts of action potentials. We propose that this Ca(2+)-activated K(+) current plays an important role in the generation of oscillatory electrical activity in the beta cell.     

Characterizing voltage-dependent Ca(2+) channels coupled to VIP release and NO synthesis in enteric synaptosomes

In enteric synaptosomes of the rat, the role of voltage-dependent Ca(2+) channels in K(+)-induced VIP release and nitric oxide (NO) synthesis was investigated. Basal VIP release was 39 +/- 4 pg/mg, and cofactor-substituted NO synthase activity was 7.0 +/- 0.8 fmol. mg(-1). min(-1). K(+) depolarization (65 mM) stimulated VIP release Ca(2+) dependently (basal, 100%; K(+), 172.2 +/- 16.2%; P < 0.05, n = 5). K(+)-stimulated VIP release was reduced by blockers of the P-type (omega-agatoxin-IVA, 3 x 10(-8) M) and N-type (omega-conotoxin-GVIA, 10(-6) M) Ca(2+) channels by ~50 and 25%, respectively, but not by blockers of the L-type (isradipine, 10(-8) M), Q-type (omega-conotoxin-MVIIC, 10(-6) M), or T-type (Ni(2+), 10(-6) M) Ca(2+) channels. In contrast, NO synthesis was suppressed by omega-agatoxin-IVA, omega-conotoxin-GVIA, and isradipine by ~79, 70, and 70%, respectively, whereas Ni(2+) and omega-conotoxin-MVIIC had no effect. These findings are suggestive of a coupling of depolarization-induced VIP release primarily to the P- and N-type Ca(2+) channels, whereas NO synthesis is presumably dependent on Ca(2+) influx not only via the P- and N- but also via the L-type Ca(2+) channel. In contrast, none of the Ca(2+) channel blockers affected VIP release evoked by exogenous NO, suggesting that NO induces VIP secretion by a different mechanism, presumably involving intracellular Ca(2+) stores.     

Rapid kinetics of depolarization-induced changes in intrasynaptosomal calcium concentrations.

The rapid kinetics of depolarization-evoked calcium influxes in isolated nerve terminals from rat cortex were monitored by stopped-flow spectrofluorimetry using specific indicators (Fluo-3, Indo-1). A very rapid increase in the intrasynaptosomal Ca(2+)-level was detected within the subsecond time range after depolarizing synaptosomes by mixing with physiological saline containing elevated K(+)-concentrations. About 15 mM [K+]o was determined as threshold concentration for inducing Ca(2+)-influx, which increased with higher concentration and saturated at [K+]o-concentrations of about 40 mM [K+]o.     

Influence of monovalent cation identity on parvalbumin divalent ion-binding properties

Rat alpha- and beta-parvalbumins have distinct monovalent cation-binding properties [Henzl et al. (2000) Biochemistry 39, 5859-5867]. Beta binds two Na(+) or one K(+), and alpha binds one Na(+) and no K(+). Ca(2+) abolishes these binding events, suggesting that the monovalent ions occupy the EF-hand motifs. This study compares alpha and beta divalent ion affinities in Na(+) and K(+) solutions. Solvent cation identity seriously affects alpha. In Hepes-buffered NaCl, at 5 degrees C, the macroscopic Ca(2+)-binding constants are 2.6 x 10(8) and 6.4 x 10(7) M(-1) and the Mg(2+) constants, 1.8 x 10(4) and 4.3 x 10(3) M(-1). In Hepes-buffered KCl, the Ca(2+) values increase to 2.9 x 10(9) and 6.6 x 10(8) M(-1) and the Mg(2+) values to 2.2 x 10(5) and 3.7 x 10(4) M(-1). Monte Carlo simulation of alpha binding data-employing site-specific constants and explicitly considering Na(+) binding-yields a K(Na) of 630 M(-1) and indicates that divalent ion-binding is positively cooperative. NMR data suggest that the lone Na(+) ion occupies the CD loop. Solvent cation identity has a smaller impact on beta. In Na(+), the Ca(2+) constants for the EF and CD sites are 2.3 x 10(7) and 1.5 x 10(6) M(-1), respectively; the Mg(2+) constants are 9.2 x 10(3) and 1.7 x 10(2) M(-1). In K(+), these values shift to 3.1 x 10(7) and 3.8 x 10(6) M(-1) and the latter to 1.4 x 10(4) and 2.9 x 10(2) M(-1). These data suggest that parvalbumin divalent ion affinity, particularly that of rat alpha, can be significantly attenuated by increased intracellular Na(+) levels.     

Glutamate release from adult primary sensory neurons in culture is modulated by growth factors.

The aim of this study was to examine possible modulatory effects of some trophic molecules, i.e. nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF), on potassium (K(+))-, bradykinin (BK)- or capsaicin (CAPS)-evoked release of glutamate (GLU) from dorsal root ganglion (DRG) neurons in vitro. BK (0.5 and 1 microM) induced a dramatic and significant increase in glutamate release. Neither CAPS nor K(+) (60 mM) produced any significant increase of GLU release vs. basal levels during a 5-min stimulation. The BK-evoked release of GLU was almost completely blocked by HOE 140, a selective BK2-receptor antagonist at high doses. Basal release of GLU was significantly reduced in cultures grown in the presence of bFGF, whereas BDNF and NGF had no significant effect. Incubation with growth factors generally decreased the BK-stimulated GLU release, an effect most pronounced for bFGF, which completely blocked BK-stimulated release. The rise in intracellular [Ca(2+)] following stimulation with BK (100 nM-1 microM), potassium (60 mM) or ATP (10 microM) was also studied using a Ca(2+)-sensitive indicator, Fura-2, in cultures grown in basal medium with or without bFGF. None of the bFGF-treated cells exhibited strong Ca(2+) responses to BK or ATP stimulation, while 10-20% of the responding cells grown in basal medium exhibited strong responses. The K(+)-induced increase of [Ca(2+)] did not vary between the different groups.The present findings suggest that sensory neurotransmission involving glutamate may be modulated by growth factors and that regulation of intracellular Ca(2+) homeostasis may be a contributing factor.