Single-cell fura-2 microfluorometry reveals different purinoceptor subtypes coupled to Ca2+ influx and intracellular Ca2+ release in bovine adrenal chromaffin and endothelial cells

ATP and adenosine(5)tetraphospho(5)adenosine (Ap₄A), released from adrenal chromaffin cells, are potent stimulators of endothelial cell function. Using single-cell fura-2 fluorescence recording techniques to measure free cytosolic Ca²⁺ concentration ([Ca²⁺]_i), we have investigated the role of purinoceptor subtypes in the activation of cocultured chromaffin and endothelial cells. ATP evoked concentration-dependent [Ca²⁺]_i rises (EC₅₀=3.8 M) in a subpopulation of chromaffin cells. Both ATP-sensitive and -insensitive cells were potently activated by nicotine, bradykinin and muscarine. Reducing extracellular free Ca²⁺ concentration to around 100 nM suppressed the [Ca²⁺]_i transient evoked by ATP but not the [Ca²⁺]_i response to bradykinin. ATP-sensitive chromaffin cells were also potently stimulated by 2-methylthioadenosine triphosphate (2MeSATP; EC₅₀= 12.5 M) and UTP, but did not respond to either adenosine 5-[-thio]diphosphate (ADP[S]), a P_2Y receptor agonist, adenosine 5-[,-methylene]triphosphate (pp[CH₂]pA), a P_2X agonist or AMP. Adrenal endothelial cells displayed concentration-dependent [Ca²⁺]_i responses when stimulated with ATP (EC₅₀=0.86 M), UTP (EC₅₀=1.6 M) and 2MeSATP (EC₅₀= 0.38 M). 2MeSATP behaved as a partial agonist. Ap₄A and ADP[S] also raised the [Ca²⁺]_i in endothelial cells, whereas AMP and pp[CH₂]pA were ineffective. Lowering extracellular free Ca²⁺ to around 100 nM did not affect the peak ATP-evoked [Ca²⁺]_i rise in these cells. It is concluded that different purinoceptor subtypes are heterogeneously distributed among the major cell types of the adrenal medulla. An intracellular Ca²⁺-releasing P_2U-type purinoceptor is specifically localized to adrenal endothelial cells, while a subpopulation of chromaffin cells expresses a non-P_2X, non-P_2Y subtype exclusively coupled to Ca²⁺ influx.     

Effects of aerobic endurance training status and specificity on oxygen uptake kinetics during maximal exercise

The main purpose of this study was to analyze the effects of exercise mode, training status and specificity on the oxygen uptake (O₂) kinetics during maximal exercise performed in treadmill running and cycle ergometry. Seven runners (R), nine cyclists (C), nine triathletes (T) and eleven untrained subjects (U), performed the following tests on different days on a motorized treadmill and on a cycle ergometer: (1) incremental tests in order to determine the maximal oxygen uptake (O_2max) and the intensity associated with the achievement of O_2max (IO_2max); and (2) constant work-rate running and cycling exercises to exhaustion at IO_2max to determine the effective time constant of the O₂ response (O₂). Values for O_2max obtained on the treadmill and cycle ergometer [R=68.8 (6.3) and 62.0 (5.0); C=60.5 (8.0) and 67.6 (7.6); T=64.5 (4.8) and 61.0 (4.1); U=43.5 (7.0) and 36.7 (5.6); respectively] were higher for the group with specific training in the modality. The U group showed the lowest values for O_2max, regardless of exercise mode. Differences in O₂ (seconds) were found only for the U group in relation to the trained groups [R=31.6 (10.5) and 40.9 (13.6); C=28.5 (5.8) and 32.7 (5.7); T=32.5 (5.6) and 40.7 (7.5); U=52.7 (8.5) and 62.2 (15.3); for the treadmill and cycle ergometer, respectively]; no effects of exercise mode were found in any of the groups. It is concluded that O₂ during the exercise performed at IO_2max is dependent on the training status, but not dependent on the exercise mode and specificity of training. Moreover, the transfer of the training effects on O₂ between both exercise modes may be higher compared with O_2max.     

Modulation of calcium current by ATP in guinea-pig adrenal chromaffin cells

The effects of extracellular adenosine 5-triphosphate (ATP) on voltage-dependent Ca²⁺ currents were examined using the whole-cell voltage-clamp technique in guinea-pig isolated adrenal chromaffin cells. ATP (500 M) reversibly suppressed Ca²⁺ currents in the presence of 5 mM Ca²⁺ in the extracellular solution. The inhibitory effect of ATP on Ca²⁺ currents tended to increase with increases in the peak amplitude of ATP-evoked current when the intracellular solution contained 0.1 or 1 mM ethylenebis(oxonitrilo)tetraacetate(EGTA). Using the intracellular solution containing 10 mM EGTA, on the other hand, the inhibitory efftect did not change regardless of the amplitude of current responses to ATP In the presence of 10 mM Ba²⁺, ATP (100 mol/l). reduced Ba²⁺ currents in a manner similar to Ca²⁺ currents. This reduction was decreased by dialysis of cells with the internal solution containing guanosine 5-O-(2-thiodiphosphate) (GDP [-S]; 1 mM) or guanosine 5-O-(3-thiotriphos-phate) (GTP [-S]; 100 mol/l). A depolarizing prepulse channels. In addition, ATP seems to modulate Ca²⁺ channels via the pathway related to G-protein. Adenine nucleotides and adenosine may play a role in controlling secretory activity in guinea-pig adrenal chromaffin cells.     

ADP exerts a protective effect against rundown of the Ca2+ current in bovine chromaffin cells

In isolated chromaffin cells, the high-voltage-activated Ca²⁺ current, recorded using 5 mM Ca²⁺ as the divalent charge carrier, exhibits rundown within 10 min, which is delayed for 1 h at least by the addition of 1 mM adenosine 5-triphosphate (ATP) to the pipette medium. The mechanism of this stabilizing action of ATP has been examined. ATP action is dose dependent; the rundown process, which was delayed at concentrations below 0.4 mM, was totally abolished at higher concentrations. The requirement for ATP was shown to be quite strict: 2 mM inosine 5-triphosphate (ITP) could not replace ATP, whereas guanosine 5-triphosphate (GTP) could, but at higher concentrations. This effect of ATP was shown to require the presence of MgCl₂ and the liberation of a phosphate group since the ATP analogue 5-adenylyl-imidodiphosphate (AMP-PNP) could not act as a substitute for ATP, suggesting an action through either adenosine 5-diphosphate (ADP) or a phosphorylation step. ADP, in the presence of Mg²⁺ only, could replace ATP in the same concentration range. This effect was shown to be specific to ADP; it was maintained after blocking the pathways which convert ADP into ATP, and could not be mimicked by guanosine 5-diphosphate (GDP). Similarly, ATP and ADP effects were abolished at an increased internal Ca²⁺ concentration (pCa 6 instead of pCa 7.7, where pCa = –log₁₀[Ca²⁺]). Nevertheless, the presence of 1 mM Mg-ADP in the bathing solution did not prevent the rundown of the Ca²⁺ channels when going to the inside-out patch recording configuration. In conclusion, the stabilizing effect of ATP may be interpreted by a Mg²⁺-ADP binding site present on high-voltage-activated Ca²⁺ channels. A localization of such an ADP regulatory site on the L-type Ca²⁺ channel itself cannot be excluded, though with an additional requirement since Mg-ADP alone is not able to maintain the corresponding activity on excised patches.     

ω-Grammotoxin blocks action-potential-induced Ca2+ influx and whole-cell Ca2+ current in rat dorsal-root ganglion neurons

Field-potential stimulation of rat dorsal-root ganglion (DRG) neurons evoked action-potential-mediated transient increases in intracellular free calcium concentration ([Ca²⁺]_i) as measured by indo-1-based microfluorimetry. Field-potential-evoked [Ca²⁺]_i transients were abolished by tetrodotoxin, and their dependence on stimulus intensity exhibited an abrupt threshold. -Conotoxin GVIA (-CgTx, 100 nM) inhibited action-potential-mediated Ca²⁺ influx by 79%, while nitrendipine (1 M) had little effect. -Grammotoxin SIA (-GsTx, 267 nM), a peptide toxin purified from the venom of the tarantula spider, Grammostola spatulata, blocked action-potential-mediated Ca²⁺ influx as effectively as did -CgTx, suggesting that -GsTx blocks N-type Ca²⁺ channels. In contrast to block by -CgTx, the block produced by -GsTx reversed upon washout of the peptide. -GsTx (270 nM) blocked 80%, and -CgTx (1 M) blocked 64%, of whole-cell Ca²⁺ current (I _Ca) elicited by step depolarization to 0 mV from a holding potential of –80 mV. -GsTx completely occluded inhibition of I _Ca by -CgTx. However, when applied after -CgTx, -GsTx produced an additional inhibition of 27%, indicating that -GsTx also blocked a non-N-type Ca²⁺ channel. BayK8644 (1 M) elicited an increase in I _Ca in the presence of maximally effective concentrations of -GsTx, suggesting that -GsTx does not block L-type channels. Thus, -GsTx displays a selectivity for Ca²⁺ channel subtypes which should prove useful for studying Ca²⁺ channels and Ca²⁺-channel-mediated processes.     

Ca2+- and GTP[γS]-induced translocation of the glucose transporter,GLUT-4, to the plasma membrane of permeabilized cardiomyocytes determined using a novel immunoprecipitation method

In cardiomyocytes glucose transport is activated not only by insulin but also by contractile activity that causes translocation of the glucose transporter, GLUT-4, from intracellular vesicles to the plasma membrane. The latter effect may possibly be mediated by intracellular Ca²⁺, as suggested by previous studies. To investigate the role of Ca²⁺, we permeabilized neonatal rat myocytes with -toxin and incubated them for 1 h either at a pCa (i.e.–log₁₀ [Ca²⁺]) of 8 (control) or at a pCa of 5 in the presence of adenosine 5-triphosphate (ATP). Translocation of GLUT-4 was then monitored by a novel immunoprecipitation method using a peptide antibody directed against an exofacial (extracellular) loop of GLUT-4 (residues 58–80). Incorporation of GLUT-4 into the plasmalemma was stimulated 1.8-fold by 10 M Ca²⁺ and 1.7-fold by insulin (as in the case of intact cells). The insulin effect was Ca²⁺ independent, i.e. it was identical in the absence and presence of Ca²⁺ (10 M). Guanosine 5-O-(3-thiotriphosphate) (GTP[S]), which was inactive in intact cells, also caused translocation of GLUT-4 in permeabilized cardiomyocytes. Thus, incorporation of GLUT-4 into the plasma membrane was enhanced 2.5-fold by 200 M GTP[S] in the virtual absence of Ca²⁺ (pCa 8) and even 3.5-fold at 10 M free Ca²⁺. We conclude that an increase in intracellular Ca²⁺ concentration increases GLUT-4 translocation of (permeabilized) cardiomyocytes to a similar extent as do insulin and GTP[S] in the absence of Ca²⁺, but that the effects of Ca²⁺ and GTP[S] may be additive.     

Effects of tyramine and calcium on the kinetics of secretion in intact and electroporated chromaffin cells superfused at high speed

Fast superfusion of electroporated bovine adrenal chromaffin cells with a K⁺ glutamate-based solution containing 50 nM free Ca²⁺ and 2 mM adenosine 5-triphosphate, dipotassium salt (K₂ATP), produced a steady-state low catecholamine secretion, measured on-line with an electrochemical detector (about 20 nA). Rapid switching to electroporation solutions containing increasing Ca²⁺ concentrations ([Ca²⁺]) produced a rapid increase in the rate and peak secretion, followed by a decline. At intermediate [Ca²⁺] (3–100 M), a fast peak and a slow secretory plateau were distinguished. The fast secretory peak identifies a readily releasable catecholamine pool consisting of about 200–400 vesicles per cell. Pretreatment of cells with tyramine (10 M for 4 min before electroporation) supressed the initial fast secretory peak, leaving intact the slower phase of secretion. With [Ca²⁺] in the range of 0.1–3 M, the activation rate of secretion increased from 2.3 to 35.3 nA · s^–1, reached a plateau between 3–30 M and rose again from 100 to 1000 M [Ca²⁺] to a maximum of 91.9 nA · s^–1. In contrast, total secretion first increased (0.1–1 M Ca²⁺), then plateaud (1–100 M Ca²⁺) and subsequently decreased (100–1000 M Ca²⁺). At 30 and 1000 M extracellular [Ca²⁺] or [Ca²⁺]_o, the activation rates of secretion from intact cells depolarised with 70 mM K⁺were close to those obtained in electroporated cells. However, secretion peaks were much lower in intact (93 nA at 30 M Ca²⁺) than in electroporated cells (385 nA). On the other hand, inactivation of secretion was much faster in intact than in electroporated cells; as a consequence, total secretion in a 5-min period was considerably smaller in intact (10.6 A · s at 1000 M Ca²⁺) than in electroporated cells (42.4 A,s at 1 M Ca²⁺). Separation of the time-courses of changes in intracellular [Ca²⁺] or [Ca²⁺]_i and secretion in intact chromaffin cells depolarised with 70 mM K⁺was demonstrated at different [Ca²⁺]_o. The increase in the rate of catecholamine release was substantially higher than the increase of the average [Ca²⁺]_i. In contrast, the decline of secretion was faster than the decline of the peak [Ca²⁺]_i. The results are compatible with the idea that the peak and the amount of catecholamine released from depolarised intact cells is determined essentially by plasmalemmal factors, rather than by vesicle supply from reserve pools. These plasmalemmal factors limit the supply of Ca²⁺ by the rates of opening and closing of voltage-dependent Ca²⁺ channels of the L-and Q-subtypes, which control the local [Ca²⁺]_i near to exocytotic sites.     

Properties of two calcium transport systems of isolated rat ileal epithelial cells: effects of Ca2+ channel modulators and membrane potential examined with fluorescent dye,fura-2

Calcium transport systems of isolated ileal epithelial cells were investigated. The concentration of cytosolic free calcium ions, [Ca²⁺]_i, was monitored with a fluorescent Ca²⁺ dye, fura-2. The fluorescence intensity ratio (I ₃₄₀/I ₃₈₀) was used as an index of [Ca²⁺]_i. [Ca²⁺]_i of the cells suspended in the nominally Ca²⁺-free solution was estimated at 52±3 nM. Ca²⁺ uptake was followed for as long as 5 min in the presence of 100–1000 M added CaCl₂. Most of the experiments were performed at 200 M CaCl₂. The Ca²⁺ uptake was abolished by 0.8 mM Ni²⁺ and 50 M Mn²⁺ and partitally antagonized by 50 M verapamil and 50 M diltiazem but not affected by 20 M nifedipine. The Ca²⁺ entry was reduced by increasing concentrations of extracellular K⁺ in the presence of valinomycin, suggesting a voltage-dependent nature of the uptake. On the other hand, the Ca²⁺ transport doubled in the presence of Bay K8644 (8 M), a Ca²⁺ channel agonist. The Bay-K-8644-induced uptake was inhibited by either 10 M nifedipine, 10 M verapamil or 10 M diltiazem and was relatively independent of extracellular K⁺ concentration. These results suggest that there are at least two distinct Ca²⁺ transport systems in the rat ileal epithelial cells, one resistant to organic Ca²⁺ channel blockers but relatively sensitive to membrane potential (basal uptake) and another inducible by Bay K 8644 and sensitive to the channel blockers but relatively independent of membrane potential.     

The release of intracellular Ca2+ in lacrimal acinar cells by α-, β-adrenergic and muscarinic cholinergic stimulation: the roles of inositol trisphosphate and cyclic ADP-ribose

Stimulation of rat lacrimal acinar cells with acetylcholine (ACh) and the -adrenergic agonist isoprenaline causes a rapid increase in inositol phosphates with 1–4 phosphate groups, resulting in release of Ca²⁺ from intracellular stores. Stimulation with the -adrenergic agonist phenylephrine, however, causes a release of Ca²⁺ from internal stores which is 36% of that observed with ACh stimulation, but without inositol phosphate production. This Ca²⁺ rise was completely inhibited by 100 M ryanodine. Adrenaline (causing activation of both - and -adrenergic receptors) induces a Ca²⁺ release with inositol phosphate synthesis identical to that occuring in the -adrenergic response. Thus, the signalling pathway for -adrenergic stimulation occurs via a path different from that which releases Ca²⁺ via muscarinic cholinergic and -adrenergic stimulation. In permeabilized lacrimal acinar cells cyclic adenosine 5-diphosphoribose (cADP-ribose) and inositol 1,4,5-trisphosphate [Ins(1,4,5)P ₃] cause release of Ca²⁺ from intracellular stores. The Ca²⁺ release evoked by cADP-ribose, but not by Ins(1,4,5)P ₃, was abolished by 100 M ryanodine, implicating a possible involvement of cADP-ribose in phenylephrine-induced Ca²⁺ signalling. When the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) is raised by application of ionomycin, inositol phosphates are synthesized with a half-maximal effect seen at 425 nM. In contrast, loading cells with the Ca²⁺ chelator 1,2-bis(2-aminophenoxy) ethane-N,N,N,N-tetraacetic acid (BAPTA) reduced the adrenaline-induced inositol phosphate synthesis by 27%. The stimulation-induced rise in [Ca²⁺]_i, therefore, appears to cause further synthesis of inositol phosphates, thereby amplifying the receptor-mediated response.     

On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation

The Ca²⁺ channel subunits _1C-a and _1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba²⁺ current (I _Ba) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 M cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 M PKA and 1 M okadaic acid. The activity of the _1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 M H 89 and was not increased by superfusion with 5 M forskolin plus 20 M isobutylmethylxanthine (IBMX). The _1C-a·₂·₂/ complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 M H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the _1C-a·₂·₂/ channel with 10 M PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca²⁺ current (I _Ca) of cardiac myocytes increased threefold during internal dialysis with 5 M PKA or 25 M microcystin and during external superfusion with 0.1 M isoproterenol or 5 M forskolin plus 50 M IBMX. These results indicate that the L-type Ca²⁺ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.     

Modulation of Ca2+-activated K+ channels by Mg2+ and ATP in frog oxyntic cells

Ca²⁺-activated K⁺ channels in the basolateral plasma membrane of bullfrog oxynticopeptic cells are intimately involved in the regulation of acid secretion. Patch-clamp techniques were applied to study the regulating mechanism of these channels. In the excised inside-out configuration, intracellular Mg²⁺ decreased channel activity in a dose-dependent manner. In the absence of Mg²⁺, administration of adenosine 5 triphosphate (ATP) to the cytoplasmic side also inhibited channel activity. On the other hand, in the presence of Mg²⁺, addition of ATP markedly increased channel activity. At a fixed concentration of free Mg²⁺ the Mg-ATP complex caused channel activation and shifted the dose response relationship between channel activity and the intracellular Ca²⁺ concentration to the left. A nonhydrolysable ATP analogue, adenosine 5-[,-imido]triphosphate (AMP-PNP) adenylyl [,-methylene]diphosphate (AMP-PCP), could not substitute for ATP in channel activation, but a hydrolysable ATP analogue, adenosine 5-O-(3-thiotriphosphate) (ATP[S]) could do so. Furthermore, application of alkaline phosphatase to the cytoplasmic side inhibited channel activity. These results demonstrate that Ca²⁺-activated K⁺ channels are regulated by Mg²⁺ and ATP, and suggest that a phosphorylation reaction may be involved in the regulation mechanism of these channels.     

Modulation of ionic currents in smooth muscle balls of the rabbit intestine by intracellularly perfused ATP and cyclic AMP

The effects of intracellularly perfused ATP and cyclic-AMP (c-AMP) on ionic currents recorded from fragmented smooth muscle cells (smooth muscle ball; SMB) were investigated, using the single electrode whole cell voltage clamp method. The Ca²⁺ current was distinguished from K⁺ currents, using pipette solution containing Cs⁺, TEA⁺ and 4 mM EGTA. ATP enhanced the Ca²⁺ current, dose-dependently between 0.3 and 10. mM, and slightly slowed the slow component of the decay of the Ca²⁺ current, while the steady-state inactivation curve remained unaffected. Intracellular application of 5-adenylyl-imidodiphosphate (AMP-PNP; 1mM) inhibited the Ca²⁺ current by competitionwith ATP, but c-AMP (up to 300 M) had no effect. With a high-K⁺ solution containing 0.3 mM EGTA and ATP in the pipette and physiological salt solution in the bath, a net inward current with transient (Ca²⁺ dependent) and delayed (Ca²⁺ independent) K⁺ outwart currents were evoked. Increased concentrations of ATP (above 1 mM) but not c-AMP (up to 100 M) in the pipette enhanced the transient K⁺ outward current Neither agent had any effect on the delayed outward current. When repetitive stimulations of intervals shorter than 5 s were applied, the amplitude of the transient outward current was markedly reduced, and 100 M c-AMP partially prevented this attenuation. ATP may act on the Ca²⁺ channel either by phosphorylating the channel protein or by other ATP requiring mechanisms, independently from those induced by the action of c-AMP. Thus, the different responses of cardiac and visceral smooth muscles induced by -adrenoceptor stimulation may be explained in part by the different natures of the Ca²⁺ channel in response to c-AMP.     

Intracellular nucleotides and polyamines inhibit the Ca2+-activated cation channel TRPM4b

TRPM4b (in contrast to the short splice variant TRPM4a) is a Ca²⁺-activated but Ca²⁺-impermeable cation channel. We have studied TRPM4 currents in inside-out patches. Supramicromolar Ca²⁺ concentrations applied at the inner side, [Ca²⁺]_i, activated TRPM4 with an EC₅₀ value of 0.37 mM, a value that is much higher than that of whole-cell currents. Current amplitudes decreased above 1 mM [Ca²⁺]_i, (IC₅₀ 9.3 mM). Sr²⁺ but not Ba²⁺could partially substitute for Ca²⁺. ATP, ADP, AMP and AMP-PNP all quickly and reversibly inhibited TRPM4 with IC₅₀ values between 2 and 19 M (at +100 mV). Adenosine also blocked TRPM4 at 630 M. The block at high ATP concentrations was incomplete and was not affected by the presence of free Mg²⁺. ADP induced the most sensitive block with an IC₅₀ of 2.2 M. For inhibition of TRPM4 by free ATP^4–, an IC₅₀ value of 1.7±0.3 M was calculated. GTP, UTP and CTP at concentrations up to 1 mM did not induce a similar block. Spermine blocked TRPM4 currents with an IC₅₀ of 61 M. In conclusion, TRPM4 is a channel that can be effectively modulated by intracellular nucleotides and polyamines.     

Mechanism of the ATP-induced rise in cytosolic Ca2+ in freshly isolated smooth muscle cells from human saphenous vein

Effects of exogenous adenosine 5-triphosphate (ATP) were studied by measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and membrane currents in myocytes freshly isolated from the human saphenous vein. At a holding potential of –60 mV, ATP (10 M) elicited a transient inward current and increased [Ca²⁺]_i. These effects of ATP were inhibited by ,-methylene adenosine 5-triphosphate (AMPCPP, 10 M). The ATP-gated current corresponded to a non-selective cation conductance allowing Ca²⁺ entry. The ATP-induced [Ca²⁺]_i rise was abolished in Ca²⁺-free solution and was reduced to 30.1±5.5% (n=14) of the control response when ATP was applied immediately after caffeine, and to 23.7±3.8% (n=11) in the presence of thapsigargin. The Ca²⁺-induced Ca²⁺ release blocker tetracaine inhibited the rise in [Ca²⁺]_i induced by both caffeine and ATP, with apparent inhibitory constants of 70 M and 100 M, respectively. Of the ATP-induced increase in [Ca²⁺]_i 29.3±3.9% (n=8) was tetracaine resistant. It is concluded that the effects of ATP in human saphenous vein myocytes are only mediated by activation of P_2x receptor channels. The ATP-induced [Ca²⁺]_i rise is due to both Ca²⁺ entry and Ca²⁺ release activated by Ca²⁺ ions that enter the cell through P_2x receptor channels.     

α 2-Adrenoceptors activate dihydropyridine-sensitive calcium channels via Gi-proteins and protein kinase C in rat portal vein myocytes

The presence of functional ₂-adrenoceptors was investigated in isolated smooth muscle cells from rat portal vein using the nystatin-perforated patch-clamp technique. The free cytoplasmic calcium concentration ([Ca²⁺]_i) was estimated using emission from the dye Fura-2. Activation of ₂-adrenoceptors by clonidine (an ₂-adrenoceptor agonist) or noradrenaline (a non-selective -adrenoceptor agonist), both in the presence of 0.1 M prazosin to block ₁-adrenoceptors, caused a slow and sustained increase in [Ca²⁺]_i which was inhibited by 0.1 M rauwolscine (an ₂-adrenoceptor antagonist). A similar Ca²⁺ response was obtained with oxymetazoline (a selective _2A-adrenoceptor agonist) suggesting that the increase in [Ca²⁺]_i resulted from activation of the _2A-adrenoceptor subtype. The increase in [Ca²⁺]_i did not occur in calcium-free solution or in the presence of oxodipine (a voltage-dependent calcium channel blocker), indicating that it depended on a calcium influx. The _2A-adrenoceptor-activated calcium influx was unchanged after complete release of the stored calcium induced by applications of ryanodine and caffeine. In addition, no accumulation of inositol trisphosphate was detected in the presence of 0.1 M prazosin. Taken together, these results indicate that _2A-adrenoceptor activation does not stimulate phosphoinositide turnover and subsequent calcium release from intracellular stores. Wholecell patch-clamp experiments showed that _2A-adrenoceptor activation promoted calcium influx through voltage-dependent L-type channels. Concomitant with calcium influx, _2A-adrenoceptor activation induced a 10- to 15-mV depolarization. Similar effects on both calcium channel current and [Ca²⁺]_i were obtained with mastoparan, an activator of Gi-proteins. Activation of calcium influx by both _2A-adrenoceptors and mastoparan was reduced by treatment with pertussis toxin and GF 109203X (a protein kinase C inhibitor). These data suggest that activation of protein kinase C through a transduction pathway involving Gi-proteins phosphorylates voltage-activated L-type calcium channels and thus, increases their opening probability.     

Ca2+-dependent actin remodeling in the contracting A7r5 cell

Previous work has shown that stimulation of contraction in A7r5 smooth muscle cells with phorbol ester (PDBu) results in the disassembly and remodeling of the -actin component of the cytoskeleton (Fultz et al., 2000, J Mus Res Cell Motil 21: 775–781). In the present study, we evaluated the effect of increasing intracellular calcium ion concentration [Ca²⁺]i by A₂₃₁₈₇ and thapsigargin on - and -actin remodeling. The effects of A₂₃₁₈₇ and thapsigargin on cell contraction and actin remodeling were effectively identical. The two compounds caused contraction of A7r5 cells that was earlier in onset and more quickly completed than PDBu-induced contractions. Both the - and -actin isoforms were incorporated into stress cables in the resting cell. During the interval of contraction, -actin cables shortened without evidence of disassembly. By comparison, the increase of [Ca²⁺]i resulted in partial or complete dissolution of -actin cables without further remodeling. In addition, PDBu-mediated -actin remodeling was blocked in the presence of A₂₃₁₈₇. Increased [Ca²⁺]i also caused dispersal of -actinin but had no effect on the cellular distribution of talin suggesting the effect was selective for -actin cytoskeletal structure. The incubation of cells in calcium-free media prevented -actin dissolution by A₂₃₁₈₇/thapsigargin and also blocked PDBu-mediated remodeling. Finally, of six kinase inhibitors investigated, only ML-7 partially blocked the dissolution of -actin cables by increased [Ca²⁺]i. The results suggest that the sustained elevation of [Ca²⁺]i beyond a threshold level initiates depolymerization of -actin but not -actin. It further appears that PDBu-induced -actin remodeling requires Ca²⁺ but increases of [Ca²⁺]i beyond a threshold level may inhibit this activity. The finding that ML-7 partially inhibits -actin dissolution in the presence of A₂₃₁₈₇/thapsigargin may be suggesting that myosin light chain kinase (MLCK) plays a role in destabilizing -actin structure in the activated cell.     

The hypocalciuric effect of thiazides: subcellular localization of the action

The acute administration of thiazides results in a decrease in the urinary Ca²⁺/Na⁺ ratio, whereas chronic administration of these diuretics decreases calciuria. In both situations, Ca²⁺ transport is enhanced in the early part of the distal tubule. The purpose of our study was to determine whether the hypocalciuric action of thiazides was due to a change in the active transport of Ca²⁺ through the basolateral membrane of the nephron or to an effect (direct or indirect) on the permeability of the distal tubule luminal membrane to calcium. In order to detect intrinsic differences between membranes of the proximal and distal tubules, the effect of the diuretic was examined in proximal and distal tubule preparations, and in basolateral and luminal membranes from the two segments separately.Preincubation of microdissected distal tubules in hypotonic solution containing 500 M hydrochlorothiazide (HCTZ) did not influence the Ca²⁺-dependent ATP hydrolysis (Ca²⁺=1 M) nor the Mg²⁺-dependent ATP hydrolysis (Mg²⁺=100 M). Similarly 100 M HCTZ did not change the Ca²⁺ ATPase activity in intact proximal and distal tubule suspensions, at Ca²⁺ concentrations ranging from 0.05 M to 1 M.ATP-dependent Ca²⁺ transport was present in basolateral membrane vesicles from proximal and distal tubule suspensions. Preincubation of the membranes with 100 M HCTZ did not influence this transport. A Na⁺/ Ca²⁺ exchanger, present in the basolateral membranes from the distal tubule, was also insensitive to HCTZ. In contrast, preincubation of luminal membranes from the distal tubules (but not proximal tubules) with 500 M HCTZ significantly increased the Ca²⁺ uptake by these membranes. This increase in Ca²⁺ uptake, in the presence of Na⁺, was dose-dependent; the minimal and the maximal effects of the diuretic were observed at concentrations of 25 M and 100 M respectively. HCTZ increased the V _maxCa²⁺ from2.5±0.3 pmol g^–1 (10 s^–1) to 3.7±0.6 pmol g^–1 (10 s^–1) (P<0.01), but did not influence the K _m (1.43±0.25 mM and 1.37±0.1 mM Ca²⁺ in experimental and control membranes, respectively). Na⁺ was necessary for this effect. Na⁺ per se decreased Ca²⁺ uptake in a concentration-dependent manner and HCTZ partially reestablished Ca²⁺ uptake to the levels observed in a Na⁺-free medium. The anion of the Na⁺ salt also modulated the effect of HCTZ on Ca²⁺ transport. While Cl^– and SCN^– permitted HCTZ to enhance Ca²⁺ uptake, the SO ₄ ^2– anion did not. It is therefore concluded that (a) the hypocalciuric effect of thiazides is primarily due to an increase in the Ca²⁺ uptake of the luminal membrane from the distal tubule, (b) Na⁺ and Ca²⁺ transports are tightly related in the distal luminal membrane, (c) HCTZ modulates this interrelationship by decreasing the inhibitory effect of Na⁺ on Ca²⁺ uptake. Whether the Ca²⁺ and Na⁺ carriers are the same molecule or different entities needs further investigation.     

Activation of calcium influx by ATP and store depletion in primary cultures of renal proximal cells

Cytoplasmic calcium changes and calcium influx evoked by adenosine triphosphate (ATP) were investigated in primary cultures of rabbit proximal convoluted tubule cells. Extracellular ATP (50 M) induced a biphasic increase of [Ca²⁺]_i measured with the calcium probe fura-2. In the early phase, the mobilization of intracellular pools resulted in a transient increase of [Ca²⁺]_i from 106±11 nM (n=36) to 1059±115% (n=29) of the resting level within 10 s. In the presence of external calcium, [Ca²⁺]_i then decreased within 3 min to a sustained level (398±38%,n=8). Measurements of fura-2 quenching by external manganese revealed that this phase was the result of an increased Ca²⁺ uptake, blocked by lanthanum (10 M) and verapamil (100 M) but not by the nifedipin (25 M). Internal calcium store depletion by ATP induced an increased calcium influx through lanthanum- and verapamil-sensitive, nifedipininsensitive calcium channels, located on the apical membrane of the cells. As indicated by⁸⁶Rb⁺ efflux measurements, ATP activated a potassium efflux that was blocked by barium andLeiurus quinquestriatus hebraeus (LQH) venom (containing charybdotoxin) indicating the involvement of Ca²⁺-sensitive K⁺ channels. Moreover, in the presence of the LQH venom, the internal calcium stores were not replenished after being depleted by ATP. Our results indicate that an ATPevoked hyperpolarization of the plasma membrane leads to increased Ca²⁺ influx, which facilitates the replenishment of the internal stores.     

Inhibition of ω-conotoxin-sensitive Ca2+ channel currents by internal Mg2+ in cultured rat cerebellar granule neurones

The effects of changing the intracellular concentrations of either free Mg²⁺ ions ([Mg²⁺]_i) or Mg²⁺-bound adenosine triphosphate ([Mg · ATP]_i) on Ca²⁺ channel currents were assessed in cultured rat cerebellar granule neurones using the whole-cell patch-clamp technique. Raising [Mg²⁺]_i from 0.06 mM to 1.0 mM inhibited Ca²⁺ channel currents by approximately 50%. The action of -conotoxin GVIA (-CgTX), a selective inhibitor of N-type Ca²⁺ channels was also investigated. With increasing [Mg²⁺]_i, the proportion of current irreversibly blocked by -CgTX was reduced, and was negligible (approximately 5 pA of current) in the presence of [Mg²⁺]_i values of 0.5 mM or greater. Block of the -CgTX-sensitive current accounted for the reduction in total current by concentrations of [Mg²⁺]_i to 0.5 mM. Raising [Mg²⁺]_i had no effect on the rate of decay of Ca²⁺ currents, but did produce a negative shift in current activation, possibly due to a non-specific interaction with negative surface charge. Altering [Mg · ATP]_i from 0.3 to 5.0 mM caused a twofold increase in the size of currents without affecting the proportion of current sensitive to -CgTX. [Mg²⁺]_i was also effective in inhibiting the Ca²⁺ channel current following potentiation by increasing [Mg · ATP]_i. These data suggest that -CgTX-sensitive current in these cells is selectively inhibited by internal Mg²⁺ whereas both -CgTX-sensitive and -resistant components of current are potentiated by internal Mg · ATP. The mechanism by which Mg²⁺ inhibits N-type channels is unclear, but may involve an open channel block.     

Ca2+- and swelling-induced activation of ion conductances in bronchial epithelial cells

The present study was performed to examine Ca²⁺-dependent and cell-swelling-induced ion conductances in a polarized bronchial epithelial cell line (16HBE14o-). Whole-cell currents were measured in fast and slow whole-cell patch-clamp experiments in cells grown either on filters or on coated plastic dishes. In addition the transepithelial voltage (V _te) and resistance (R _te) were measured in confluent monolayers. Resting cells had a membrane voltage (V _m) of –36±1.1 mV (n=137) which was mainly caused by K⁺ and Cl^– conductances and to a lesser extent by a Na⁺ conductance. V _te was apical-side-negative after stimulation. Equivalent short-circuit current (I _sc = V _te/R _te) was increased by the secretagogues histamine (0.1 mmol/l), bradykinin (0.1–10 mol/l) and ATP (0.1–100 mol/l). The histamine-induced I _sc was blocked by either basolateral diphenhydramine (0.1 mmol/l, n=4) or apical cimetidine (0.1 mmol/l, n=4). In fast and slow whole-cell recordings ATP and bradykinin primarily activated a transient K⁺ conductance and hyperpolarized V _m. This effect was mimicked by the Ca²⁺ ionophore ionomycin (1 mol/l, n=11). Inhibition of the bradykinin-induced I _sc by the blocker HOE140 (1 mol/l, n=3) suggested the presence of a BK₂ receptor. The potency sequence of different nucleotide agonists on the purinergic receptor was UTP ATP > ITP > GTP CTP [,-methylene] ATP 2-methylthio-ATP = 0 and was obtained in I _sc measurements and patch-clamp recordings. This suggests the presence of a P_2u receptor. Hypotonic cell swelling activated both Cl^– and K⁺ conductances. The Cl^– conductance was only slightly inhibited by 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (0.5 mmol/ l, n=3). These data indicate that 16HBE140- bronchial epithelial cells, which are known to express high levels of cystic fibrosis transmembrane conductance regulator protein, form a secretory epithelium. While hypotonic cell swelling activates both K⁺ and Cl^– channels, the Ca²⁺-induced Cl^– secretion is due mainly to activation of basolateral K⁺ channels.