Ca2+ flux through voltage-gated channels with flow cessation in pulmonary microvascular endothelial cells

Objective: To investigate the role of voltage-gated Ca2+ channels in Ca2+ influx with flow cessation in flow-adapted rat pulmonary microvascular endothelial cells. Methods: Cells were evaluated for mRNA and protein levels for major components of the voltage-gated Ca2+ channels. Ca2+ influx with flow cessation and cell membrane potential were measured in real time with fluorescent dyes. Mibefradil and nifedipine were used as inhibitors of Ca2+ channel activity. Results: Voltage-gated Ca2+ channel protein and mRNA for the T-type channel were expressed at a relatively low level in endothelial cells cultured under static conditions and expression was induced significantly during flow adaptation. Flow-adapted but not control cells showed Ca2+ influx during flow cessation that was blocked by mibefradil but not by nifedipine. Ca2+ influx also was blocked by cromakalim, a KATP channel agonist. Cell membrane depolarization with flow cessation was unaffected by mibefradil. Conclusions: Rat pulmonary microvascular endothelial cells express T-type voltage-gated Ca2+ channels that are induced during adaptation to flow and are responsible for Ca2+ influx that occurs as a result of flow cessation-mediated membrane depolarization.     

Role of membrane depolarization and T-type Ca2+ channels in angiotensin II and K+ stimulated aldosterone secretion

The hypothesis that Ca2+ influx necessary for angiotensin II (AngII) and K+ stimulation of aldosterone secretion is primarily mediated by membrane depolarization and activation of T-type Ca2+ channels was examined in isolated rat adrenal glomerulosa cells. Perforated-patch clamp recordings of membrane potential (Vm) demonstrated that AngII and K+ induce concentration-dependent depolarizations capable of activating T channels and, at high K+ and AngII concentrations, activating L channels and inactivating T channels. K+-induced depolarizations were stable and readily reversible. Vm was proportional to K+ concentration, exhibiting a linear slope of 53.7 mV per 10-fold increase in K+. AngII-induced depolarizations were complex, consisting of a slow maintained component superimposed with small amplitude depolarizing fluctuations. Slow oscillations in Vm were occasionally observed in response to 10(-9) M AngII or greater. The slow, maintained component of depolarization coincided with inhibition of K+ conductance. Neither rapid fluctuations nor slow oscillations in Vm were blocked by mibefradil or other treatments that inhibit voltage-gated Ca2+ channels. Perforated-patch clamp experiments also demonstrated that AngII (10(-8) M) inhibited L channels by 45.6% without affecting T channels. Thus AngII activates T channels by depolarization rather than T channel modulation in rat cells. The concentration dependencies of mibefradil inhibition of T channels and AngII- and K+-induced aldosterone secretion were compared. Under whole-cell patch clamp mibefradil induced a concentration-dependent inhibition of T channels, exhibiting a K(app) of 0.62 microM. Mibefradil inhibition was use-dependent but mibefradil neither acted as an open channel blocker nor significantly affected T channel inactivation or activation. Mibefradil inhibited K+- and AngII-induced secretion at concentrations similar to that for T channel inhibition; at high concentrations (10 microM) mibefradil inhibited AngII-induced secretion by 88% and completely inhibited K+-induced secretion. The IC50 for K+-induced secretion was dependent on K+ concentration, increasing from 0.2 microM for 6 mM K+ to 2.5 microM for 10 mM K+ or greater. Mibefradil exhibited an IC50 of 1.1 microM for inhibition of secretion at all AngII concentrations examined (0.1, 1.0, and 10 nM). Mibefradil also exhibited multiple nonspecific effects, which complicated the assessment of T channel function, including; inhibition of leak and voltage-dependent K+ conductances, inhibition of Ca2+-independent aldosterone secretion, and inhibition of secretion under conditions expected to completely inactivate T channels (10 nM AngII or 20 mM K+). In summary, these results indicate that voltage-gated T channels represent the primary Ca2+ influx pathway activated by physiological concentrations of AngII and K+ but other Ca2+ influx pathways must mediate aldosterone secretion induced by high K+ or AngII concentrations.     

Possible role for calmodulin and the Ca2+/calmodulin-dependent protein kinase II in postsynaptic neurotransmission.

The theory presented here is based on results from in vitro experiments and deals with three proteins in the postsynaptic density/membrane-namely, calmodulin, the Ca2+/calmodulin-dependent protein kinase, and the voltage-dependent Ca2+ channel. It is visualized that, in vivo in the polarized state of the membrane, calmodulin is bound to the kinase; upon depolarization of the membrane and the intrusion of Ca2+, Ca2(+)-bound calmodulin activates the autophosphorylation of the kinase. Calmodulin is visualized as having less affinity for the phosphorylated form of the kinase and is translocated to the voltage-dependent Ca2+ channel. There, with its bound Ca2+, it acts as a Ca2+ sensor, to close off the Ca2+ channel of the depolarized membrane. At the same time, it is thought that the configuration of the kinase is altered by its phosphorylated states; by interacting with Na+ and K+ channels, it alters the electrical properties of the membrane to regain the polarized state. Calmodulin is moved to the unphosphorylated kinase to complete the cycle, allowing the voltage-dependent Ca2+ channel to be receptive to Ca2+ flux upon the next cycle of depolarization. Thus, the theory tries to explain (i) why calmodulin and the kinase reside at the postsynaptic density/membrane site, and (ii) what function autophosphorylation of the kinase may play.     

Biophysical basis of pituitary cell type-specific Ca2+ signaling-secretion coupling.

All secretory pituitary cells exhibit spontaneous and extracellular Ca2+-dependent electrical activity. Somatotrophs and lactotrophs fire plateau-bursting action potentials, which generate Ca2+ signals of sufficient amplitude to trigger hormone release. Gonadotrophs also fire action potentials spontaneously, but as single, high-amplitude spikes with limited ability to promote Ca2+ influx and secretion. However, Ca2+ mobilization in gonadotrophs transforms single spiking into plateau-bursting-type electrical activity and triggers secretion. Patch clamp analysis revealed that somatotrophs and lactotrophs, but not gonadotrophs, express BK (big)-type Ca2+-controlled K+ channels, activation of which is closely associated with voltage-gated Ca2+ influx. Conversely, pituitary gonadotrophs express SK (small)-type Ca2+-activated K+ channels that are colocalized with intracellular Ca2+ release sites. Activation of both channels is crucial for plateau-bursting-type rhythmic electrical activity and secretion.     

Ca2+ signaling in mouse pancreatic polypeptide cells

Ca2+ signaling was studied in pancreatic polypeptide (PP)-secreting cells isolated from mouse islets of Langerhans. After measuring the cytoplasmic Ca2+ concentration ([Ca2+]i), the cells were identified by immunocytochemistry. Most PP-cells reacted to carbachol and epinephrine with prompt and reversible elevation of [Ca2+]i, often manifested as slow oscillations. The carbachol effect was muscarinic, because it was inhibited by atropine. Beta-adrenergic elevation of cAMP explains the epinephrine stimulation, which was mimicked by an activator of adenylate cyclase and blocked by an inhibitor of protein kinase A. The responses to carbachol and epinephrine apparently involve depolarization with opening of voltage-dependent Ca2+ channels, because the effects were prevented by the Ca2+ channel antagonist methoxyverapamil and by diazoxide, which activates ATP-dependent K+ (K(ATP)) channels. Being equipped with K(ATP) channels, the PP-cells often responded to tolbutamide or high concentrations of glucose with elevation of [Ca2+]i. Somatostatin reversed the [Ca2+]i elevation obtained by carbachol, epinephrine, tolbutamide, and glucose. These preliminary studies support the idea that glucose has a direct stimulatory effect on the PP-cells, which can be masked by locally released somatostatin. Expressing both K(ATP) channels and voltage-dependent Ca2+ channels, the PP-cells share fundamental regulatory mechanisms with other types of islet cells.     

Dual modulation of K channels by thyrotropin-releasing hormone in clonal pituitary cells.

Transmembrane electrical activity in pituitary tumor cells can be altered by substances that either stimulate or inhibit their secretory activity. Using patch recording techniques, we have measured the resting membrane potentials, action potentials, transmembrane macroscopic ionic currents, and single Ca2+-activated K channel currents of GH3 and GH4/C1 rat pituitary tumor cells in response to thyrotropin-releasing hormone (TRH). TRH, which stimulates prolactin secretion, causes a transient hyperpolarization of the membrane potential followed by a period of elevated action potential frequency. In single cells voltage clamped and internally dialyzed with solutions containing K+, TRH application results in a transient increase in Ca2+-activated K currents and a more protracted decrease in voltage-dependent K currents. However, in cells internally dialyzed with K+-free solutions, TRH produces no changes in inward Ca2+ or Ba2+ currents through voltage-dependent Ca channels. The time courses of the effects on Ca2+-activated and voltage-dependent K currents correlate with the phases of hyperpolarization and hyperexcitability, respectively. During application of TRH to whole cells, single Ca2+-activated K channel activity increases in cell-attached patches not directly exposed to TRH. In contrast, TRH applied directly to excised membrane patches produces no change in single Ca2+-activated K channel behavior. We conclude that TRH (i) triggers intracellular Ca2+ release, which opens Ca2+-activated K channels, (ii) depresses voltage-dependent K channels during the hyperexcitable phase, which further elevated intracellular Ca2+, and (iii) does not directly modulate Ca channel activity.     

Role of Ca2+ channels in the ability of membrane depolarization to prevent neuronal death induced by trophic-factor deprivation: evidence that levels of internal Ca2+ determine nerve growth factor dependence of sympathetic ganglion cells.

Sympathetic neurons depend on nerve growth factor (NGF) for their survival both in vivo and in vitro; these cells die upon acute deprivation of NGF. We studied the effects of agents that cause membrane depolarization on neuronal survival after NGF deprivation. High-K+ medium (greater than or equal to 33 mM) prevented cell death; the effect of K+ was dose-dependent (EC50 = 21 mM). The protection by high K+ was abolished either by withdrawal of extracellular Ca2+ or by preloading the cells with a Ca2+ chelator. The involvement of Ca2+ flux across membranes in high-K+ saving of NGF-deprived neurons was also supported by experiments using Ca2+-channel antagonists and agonists. The Ca2+ antagonists nimodipine and nifedipine effectively blocked the survival-promoting effect of high K+. The Ca2+ agonists Bay K 8644 and (S)-202-791 did not by themselves save neurons from NGF deprivation but did strongly augment the effect of high K+; EC50 was shifted from 21 mM to 13 mM. These data suggest that dihydropyridine-sensitive L-type Ca2+ channels play a major role in the high-K+ saving. The depolarizing agents choline (EC50 = 1 mM) and carbamoylcholine (EC50 = 1 microM), acting through nicotinic cholinergic receptors, also rescued NGF-deprived neurons. The saving effect of nicotinic agonists was not blocked by withdrawal of extracellular Ca2+ but was counteracted by a chelator of intracellular Ca2+, suggesting the possible involvement of Ca2+ release from internal stores. Based on these findings we propose a "Ca2+ set-point hypothesis" for the degree of trophic-factor dependence of sympathetic neurons in vitro.     

Ca2+ permeability in deoxygenated sickle cells

Deoxygenation of sickle cells is known to increase cation permeabilities (Na+, K+, and Ca2+). The possible mechanisms involved in the increased uptake of Ca2+ were investigated: activation of Ca2+ channels, involvement of the anion channel, and the formation of endocytic vacuoles. The Ca2+ channel blocker nifedipine reduced the deoxy-stimulated Ca2+ uptake by about 30% to 40%. The anion channel inhibitor DIDS (4,4' diisothiocyanate stilbene 2,2' disulfonate) inhibited the deoxy-stimulated Ca2+ uptake by approximately 50%. Maximal possible endocytic uptake, measured by using an impermeant marker ([3H] inuline), accounted for 6% to 9% of the total Ca2+ uptake. These data indicate that the deoxygenation-induced increase in Ca2+ permeability could result from both the activation of a Ca2+ channel and of a transport system for cations involving interactions between polymerized hemoglobin S, band 3 and other membrane components. Endocytosis appears to play only a minor role in the Ca2+ uptake of deoxygenated sickle cells.     

Ca2+-activated synexin forms highly selective, voltage-gated Ca2+ channels in phosphatidylserine bilayer membranes.

Synexin, a cytosolic protein that mediates Ca2+-dependent membrane fusion, was incorporated into acidic phospholipid bilayers, formed at the tip of a patch pipet. The pipet was filled with a high-Ca2+ solution (50 mM) and immersed in a chamber containing a low-Ca2+ solution (1 mM). Brief exposures of the bilayer to synexin increased the capacitance of the bilayer by a factor of 10 and decreased the membrane resistance by a factor of 20. Reduction of Ca2+ in the chamber to 1 microM caused an abrupt increase in the current required to hold the pipet potential at 0 mV. Under certain conditions channel events could be detected, often occurring in bursts. Consistently, open-time histograms were found to be voltage-dependent and to exhibit one time constant in the time range examined here. The slope conductance for the synexin channel was estimated as 10.2 +/- 2.1 pS for the large Ca2+ gradient with low chamber Ca2+. However, for symmetrical, low-Cl- solutions containing 25 mM Ca2+ the conductance was 26.5 +/- 5.2 pS. Ion-replacement studies showed the synexin channel to much prefer Ca2+ over Ba2+ or Mg2+. Cd2+, a potent blocker of other voltage-gated Ca2+ channels at 100 microM, blocked synexin channels only at very high concentrations (greater than or equal to 10 mM). Similarly, nifedipine, an inhibitor of the nonactivating Ca2+ channel, was effective only at extremely high concentrations (greater than 300 microM). The high selectivity for Ca2+ and the lack of response of the channel to various drugs known to block Ca2+ channels thus distinguish the synexin channel from other types of Ca2+ channels hitherto reported.     

Ca2+ influx through stretch-activated cation channels activates maxi K+ channels in porcine endocardial endothelium.

The endocardial endothelium is an important modulator of myocardial function. The present study demonstrates the existence of a stretch-activated Ca(2+)-permeable cation channel and of a Ca(2+)-activated K+ channel in the endocardial endothelium of the porcine right atrium. The stretch-activated channel is permeable for K+, Na+, Ca2+, and Ba2+, with mean conductances of approximately 32 pS for the monovalent cations and approximately 13 pS for divalent cations. The Ca(2+)-activated K+ channel has a mean conductance of 192 pS in symmetrical KCl. solution. Channel activity is strongly dependent on membrane potential and the cytosolic Ca2+ concentration. Half-maximal activation occurs at a cytosolic Ca2+ concentration of approximately 5 microM. The influx of Ca2+ through the stretch-activated channel is sufficient to activate the Ca(2+)-activated K+ channel in cell-attached patches. Upon activation of the stretch-activated channel, the cytosolic Ca2+ concentration increases, at least locally, to values of approximately 0.5 microM, as deduced from the open probability of the Ca(2+)-dependent K+ channel that was activated simultaneously. The stretch-activated channels are capable of inducing an intracellular Ca2+ signal and may have a role as mechanosensors in the atrial endothelium, possibly activated by atrial overload.     

Crosstalk between voltage-independent Ca2+ channels and L-type Ca2+ channels in A7r5 vascular smooth muscle cells at elevated intracellular pH: evidence for functional coupling between L-type Ca2+ channels and a 2-APB-sensitive cation channel

This study was designed to investigate the role of voltage-independent and voltage-dependent Ca2+ channels in the Ca2+ signaling associated with intracellular alkalinization in A7r5 vascular smooth muscle cells. Extracellular administration of ammonium chloride (20 mmol/L) resulted in elevation of intracellular pH and activation of a sustained Ca2+ entry that was inhibited by 2-amino-ethoxydiphenyl borate (2-APB, 200 micromol/L) but not by verapamil (10 micro;mol/L). Alkalosis-induced Ca2+ entry was mediated by a voltage-independent cation conductance that allowed permeation of Ca2+ (PCa/PNa approximately 6), and was associated with inhibition of L-type Ca2+ currents. Alkalosis-induced inhibition of L-type Ca2+ currents was dependent on the presence of extracellular Ca2+ and was prevented by expression of a dominant-negative mutant of calmodulin. In the absence of extracellular Ca2+, with Ba2+ or Na+ as charge carrier, intracellular alkalosis failed to inhibit but potentiated L-type Ca2+ channel currents. Inhibition of Ca2+ currents through voltage-independent cation channels by 2-APB prevented alkalosis-induced inhibition of L-type Ca2+ currents. Similarly, 2-APB prevented vasopressin-induced activation of nonselective cation channels and inhibition of L-type Ca2+ currents. We suggest the existence of a pH-controlled Ca2+ entry pathway that governs the activity of smooth muscle L-type Ca2+ channels due to control of Ca2+/calmodulin-dependent negative feedback regulation. This Ca2+ entry pathway exhibits striking similarity with the pathway activated by stimulation of phospholipase-C-coupled receptors, and may involve a similar type of cation channel. We demonstrate for the first time the tight functional coupling between these voltage-independent Ca2+ channels and classical voltage-gated L-type Ca2+ channels.     

Ontogenesis and localization of Ca2+ channels in mammalian skeletal muscle in culture and role in excitation-contraction coupling.

The mechanism of excitation-contraction (E-C) coupling in skeletal muscle is not yet well established. Cultured mouse skeletal muscle cells have been used to study the relationships between triad formation, Ca2+ channel activities, and contractions. The ontogenesis of voltage-dependent Ca2+ channels and their localization in relation to the ability of muscle to contract and the ultrastructural organization of sarcomeres and triads have been investigated by using an electrophysiological approach together with an electron microscope study. At an early stage of development, both fast (Ifast) and slow (Islow) types of Ca2+ channels are found at the surface membrane. At later stages of development, fast Ca2+ channels remain at the surface membrane, while slow Ca2+ channels migrate to the transverse-tubule membrane. The voltage dependence of fast Ca2+ channels compared to the voltage dependence of contraction clearly shows that these Ca2+ channels have no direct role in E-C coupling. Detubulation at all stages of development has confirmed that T tubules contain essential elements for E-C coupling. However, this work also shows that Ca2+ flowing through slow Ca2+ channels situated in the T-tubular system is not important for contraction. Myotubes lacking slow Ca2+ channels or having no slow Ca2+ channel transport activity (jumps to high membrane potentials, no external Ca2+, block of Islow by Co2+) still retain contraction.     

Inhibition of Ca2+ conductance in identified leech neurons by benzodiazepines.

Benzodiazepines (BZs) in micromolar concentrations inhibit Mn2+- and Co2+-sensitive regenerative divalent cation potentials, which are revealed in the presence of tetraethylammonium ion, in leech nociceptive neurons (N cells). This BZ effect is reversible and dose-dependent. The BZs, like Mn2+ and Co2+, inhibit the maximum rate of depolarization (Vmax) and duration of divalent cation potentials at concentrations that do not significantly affect resting membrane potential or Vmax of the Na+-dependent action potential. Ultraviolet-induced BZ binding to micromolar-affinity sites in ganglia and isolated cells irreversibly blocks Ca2+ conductance in neurons without significantly affecting resting membrane potentials. BZ binding studies with leech neuronal membrane show saturable, specific binding in the micromolar concentration range that was similar to BZ binding to synaptosomal membrane fractions. The apparent Kd obtained from the micromolar-affinity BZ binding curve for leech ganglionic membrane preparations agrees well with the apparent Ki estimated from the dose-response curve measuring BZ inhibition of Vmax of the divalent cation potentials. These findings indicate that BZs act like Ca2+-channel antagonists in intact neuronal preparations and are consistent with the hypothesis that BZ binding to micromolar-affinity receptors modulates voltage-gated Ca2+ channels.     

Galanin inhibits a dihydropyridine-sensitive Ca2+ current in the RINm5f cell line.

Mechanisms of action of the neuropeptide galanin, a putative neuromodulator in the central and peripheral nervous systems, have been evaluated extensively in insulin-secreting cells isolated from pancreas and cell lines derived from pancreatic tumors. Galanin inhibits insulin secretion from these cells through several mechanisms, including activation of ATP-dependent K+ channels and inhibition of adenylyl cyclase leading to a decrease in cAMP. Here we report that galanin also inhibits a dihydropyridine-sensitive Ca2+ current. Both electrophysiological actions by galanin would result in less Ca2+ entry, as the action to increase K+ current would hyperpolarize the cells and the decrease in voltage-gated Ca2+ current would decrease Ca2+ influx at depolarized potentials where these channels are activated. These galanin actions would directly counter the two opposing electrophysiological responses to carbohydrate stimulation in RINm5f cells, which are to inhibit K+ current and to stimulate Ca2+ current. Given that stimulation of presynaptic nerve terminals in pancreas releases galanin, these results suggest that Ca(2+)-dependent insulin release from native pancreatic beta cells may also be regulated by similar neuropeptide effects.     

Coordinate regulation of gonadotropin-releasing hormone neuronal firing patterns by cytosolic calcium and store depletion

Elevation of cytosolic free Ca2+ concentration ([Ca2+]i) in excitable cells often acts as a negative feedback signal on firing of action potentials and the associated voltage-gated Ca2+ influx. Increased [Ca2+]i stimulates Ca2+-sensitive K+ channels (IK-Ca), and this, in turn, hyperpolarizes the cell and inhibits Ca2+ influx. However, in some cells expressing IK-Ca the elevation in [Ca2+]i by depletion of intracellular stores facilitates voltage-gated Ca2+ influx. This phenomenon was studied in hypothalamic GT1 neuronal cells during store depletion caused by activation of gonadotropin-releasing hormone (GnRH) receptors and inhibition of endoplasmic reticulum (Ca2+)ATPase with thapsigargin. GnRH induced a rapid spike increase in [Ca2+]i accompanied by transient hyperpolarization, followed by a sustained [Ca2+]i plateau during which the depolarized cells fired with higher frequency. The transient hyperpolarization was caused by the initial spike in [Ca2+]i and was mediated by apamin-sensitive IK-Ca channels, which also were operative during the subsequent depolarization phase. Agonist-induced depolarization and increased firing were independent of [Ca2+]i and were not mediated by inhibition of K+ current, but by facilitation of a voltage-insensitive, Ca2+-conducting inward current. Store depletion by thapsigargin also activated this inward depolarizing current and increased the firing frequency. Thus, the pattern of firing in GT1 neurons is regulated coordinately by apamin-sensitive SK current and store depletion-activated Ca2+ current. This dual control of pacemaker activity facilitates voltage-gated Ca2+ influx at elevated [Ca2+]i levels, but also protects cells from Ca2+ overload. This process may also provide a general mechanism for the integration of voltage-gated Ca2+ influx into receptor-controlled Ca2+ mobilization.     

Pharmacological basis of Ca2+ channels and Ca2+ channel antagonists

Ca2+ plays multiple roles in muscle E-C coupling, secretion, and neural transmission, in addition to survival, proliferation, and death of cells. The voltage-dependent L-type Ca2+ channel is a transmembrane protein that selectively permeates Ca2+ on activation by membrane depolarization. Ca2+ channel blockers (or Ca2+ antagonists) selectively block this channel. The blocking action is exerted in a tissue-specific manner, which underlies the unique pharmacological properties of Ca2+ channel blockers. The later generation of slowly-acting and long-lasting Ca2+ channel blockers has been designed to overcome the side effects of classical Ca2+ channel blockers. The pharmacological and molecular basis for the unique action of Ca2+ channel blockers will be discussed.     

Cytokinin stimulates dihydropyridine-sensitive calcium uptake in moss protoplasts.

Ca2+ influx through dihydropyridine (DHP)-sensitive Ca2+ channels is thought to be an early event in cytokinin-induced bud formation in moss protonema because DHP antagonists inhibit bud formation in the presence of cytokinin and DHP agonists stimulate bud formation in the absence of cytokinin [Conrad, P. A. & Helper, P. K. (1988) Plant Physiol. 86, 684-687]. In the present study, we established the presence of a DHP-sensitive Ca2+ transport system by measuring 45Ca2+ influx into moss protoplasts. Ca2+ influx was stimulated by external KCl (up to 5 mM), indicating that transport is voltage-dependent. K(+)-induced Ca2+ influx was DHP-sensitive with > 50% inhibition at 500 nM nifedipine. Ca2+ influx was stimulated by increasing concentrations of the DHP Ca2+ channel agonist Bay K8644 with half-maximal effects at 25 nM; this stimulation was seen only in the absence of K+, suggesting that the agonist works preferentially on polarized membranes. Ca2+ influx was also inhibited by phenylalkylamines (verapamil) and benzothiazepines (diltiazem). The phytohormone 6-benzylaminopurine consistently stimulated Ca2+ influx with a Km value of 1 nM, whereas adenine, indoleacetic acid, and gibberellic acid had no effect on Ca2+ transport. The cytokinins kinetin and trans-zeatin caused a greater stimulation of Ca2+ influx and induced more bud formation than did 6-benzylaminopurine. These results indicate that Ca2+ is taken up into moss protoplasts through voltage-dependent DHP-sensitive Ca2+ channels on the plasma membrane and that one of the cytokinin effects in the induction of bud formation is regulation of this plasma membrane Ca2+ channel.     

Cav3.1 (alpha1G) T-type Ca2+ channels mediate vaso-occlusion of sickled erythrocytes in lung microcirculation

In the present study, we demonstrate that lung microvascular endothelial cells express a Cav3.1 (alpha1G) T-type voltage-gated Ca2+ channel, whereas lung macrovascular endothelial cells do not express voltage-gated Ca2+ channels. Voltage-dependent activation indicates that the Cav3.1 T-type Ca2+ current is shifted to a positive potential, at which maximum current activation is -10 mV; voltage-dependent conductance and inactivation properties suggest a "window current" in the range of -60 to -30 mV. Thrombin-induced transitions in membrane potential activate the Cav3.1 channel, resulting in a physiologically relevant rise in cytosolic Ca2+. Furthermore, activation of the Cav3.1 channel induces a procoagulant endothelial phenotype; eg, channel inhibition attenuates increased retention of sickled erythrocytes in the inflamed pulmonary circulation. We conclude that activation of the Cav3.1 channels selectively induces phenotypic changes in microvascular endothelial cells that mediate vaso-occlusion by sickled erythrocytes in the inflamed lung microcirculation.     

The effects of maitotoxin on 45Ca2+ flux and hormone release in GH3 rat pituitary cells

Maitotoxin has been reported to activate calcium channels and stimulate calcium-dependent functions in several tissues, but a thorough investigation of 45Ca2+ fluxes is lacking. To characterize the influence of maitotoxin on 45Ca2+ flux in greater detail, we incubated dispersed GH3 pituitary tumor cells in 45Ca2+ with maitotoxin and other agents affecting calcium channels. Within 10 sec of exposure, maitotoxin induced a net calcium influx in cells at isotopic equilibrium. Calcium uptake was concentration dependent between 0.4 and 40 ng/ml maitotoxin and was inhibited by antagonists of voltage-dependent calcium channels but not by inhibitors of sodium channels. PRL and GH release from perifused GH3 cells was stimulated within 1 min by maitotoxin. We conclude that maitotoxin causes a rapid, concentration-dependent influx of calcium through presumed voltage-dependent endogenous calcium channels, culminating in enhanced hormone release. This potent toxin may provide a more precise understanding of the role of calcium in the stimulus-secretion coupling process.     

Regulation of vascular L-type Ca2+ channels by phosphatidylinositol 3,4,5-trisphosphate

Modulation of voltage-gated L-type Ca2+ channels by phosphoinositide 3-kinase (PI3K) regulates Ca2+ entry and plays a crucial role in vascular excitation-contraction coupling. Angiotensin II (Ang II) activates Ca2+ entry by stimulating L-type Ca2+ channels through Gbeta-sensitive PI3K in portal vein myocytes. Moreover, PI3K and Ca2+ entry activation have been reported to be necessary for receptor tyrosine kinase-coupled and G protein-coupled receptor-induced DNA synthesis in vascular cells. We have previously shown that tyrosine kinase-regulated class Ia and G protein-regulated class Ib PI3Ks are able to modulate vascular L-type Ca2+ channels. PI3Ks display 2 enzymatic activities: a lipid-kinase activity leading to the formation of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3 or PIP3] and a serine-kinase activity. Here we show that exogenous PIP3 applied into the cell through the patch pipette is able to reproduce the Ca2+ channel-stimulating effect of Ang II and PI3Ks. Moreover, the Ang II-induced PI3K-mediated stimulation of Ca2+ channel and the resulting increase in cytosolic Ca2+ concentration are blocked by the anti-PIP3 antibody. Mutants of PI3K transfected into vascular myocytes also revealed the essential role of the lipid-kinase activity of PI3K in Ang II-induced Ca2+ responses. These results suggest that PIP3 is necessary and sufficient to activate a Ca2+ influx in vascular myocytes stimulated by Ang II.