Ca2+ regulation in detrusor smooth muscle from developing fetal sheep bladders

Sheep fetus is a useful model to study in utero bladder outflow obstruction but little is known about cell physiology of fetal bladders. To remedy this defect we have characterised intracellular Ca(2+) regulation in fetal sheep myocytes of different developmental ages. Fetal detrusor myocytes had a similar resting [Ca(2+)](i) to adult cells and exhibited transient [Ca(2+)](i) increases in response to carbachol, ATP, high-K, caffeine and low-Na. The carbachol transients were abolished by atropine and caffeine; the ATP response was blocked by alpha,beta-methylene ATP; high-K-evoked [Ca(2+)](i) rises were antagonised by verapamil. The maximal responses to carbachol, high-K, caffeine and low-Na in fetal cells were similar to those of adult counterparts, whilst the ATP response was smaller (p < 0.05). These variables were largely similar between the three gestational groups with the exception of ATP-induced response between early fetal and adult bladders (p < 0.05). Dose-response curves to carbachol demonstrated an increase of potency between mid-gestation and early adulthood (p < 0.05). These data show that muscarinic receptors coupled to intracellular Ca(2+) release, P2X receptor-linked Ca(2+) entry, depolarisation-induced Ca(2+) rise via L-type Ca(2+) channels, Na(+)/Ca(2+) exchange and functional intracellular Ca(2+) stores are all operational in fetal bladder myocytes. Whilst most of Ca(2+) regulators are substantially developed and occur at an early fetal age, a further functional maturation for cholinergic sensitivity and purinergic efficacy continues throughout to adulthood.     

Extracellular ATP increases free cytosolic calcium in rat parotid acinar cells. Differences from phospholipase C-linked receptor agonists.

The effects of extracellular ATP on intracellular free calcium concentration [( Ca2+]i), phosphatidylinositol (PtdIns) turnover, amylase release and Ca2+-activated membrane currents were examined in isolated rat parotid acinar cells and contrasted with the effects of receptor agonists known to activate phospholipase C. ATP was more effective than muscarinic and alpha-adrenergic agonists and substance P as a stimulus for elevating [Ca2+]i (as measured with quin2). The ATP effect was selectively antagonized by pretreating parotid cells with the impermeant anion-exchange blocker 4,4'-di-isothiocyano-2,2'-stilbenedisulphonate (DIDS), which also inhibited binding of [alpha-32P]ATP to parotid cells. By elevating [Ca2+]i, ATP and the muscarinic agonist carbachol both activated Ca2+-sensitive membrane currents, which were measured by whole-cell and cell-attached patch-clamp recordings. However, there were marked contrasts between the effects of ATP and the receptor agonists linked to phospholipase C, as follows. (1) Although the combination of maximally effective concentrations of carbachol, substance P and phenylephrine had no greater effect on [Ca2+]i than did carbachol alone, there was some additivity between maximal ATP and carbachol effects. (2) Intracellular dialysis with guanosine 5'-[beta-thio]diphosphate did not block activation of ion channels by ATP, but did block channel activation by the muscarinic agonist carbachol. This suggests that a G-protein is involved in the muscarinic response, but not in the response to ATP. (3) Despite its pronounced effect on [Ca2+]i, ATP had little effect on PtdIns turnover in these cells, in contrast with the effects of carbachol and other Ca2+-mobilizing agents. (4) Although ATP was able to stimulate amylase release from parotid acinar cells, the stimulation was only 33 +/- 9% of that obtained with phospholipase C-linked receptor agonists. These differences suggest that ATP increases [Ca2+]i through specific activation of a pathway which is distinct from that shared by the classical phospholipase C-linked receptor agonists.     

Store-operated Ca2+ entry suppresses distention-induced ATP release from the urothelium

Epithelial cells in the urinary bladder (urothelium) trigger sensory signals in micturition by releasing ATP in response to distention of the bladder wall. Our previous study revealed the distinct roles of extracellular Ca(2+) and the Ca(2+) stores in the endoplasmic reticulum (ER) in urothelial ATP release. In the present study, we investigated the regulation of urothelial ATP release by Ca(2+) influx from the extracellular space and Ca(2+) release from the ER using a distention assay of the mouse bladder wall in a small Ussing chamber. Stimulation of Ca(2+) release from the ER in the mucosal side of the bladder induced significant ATP release without distention. Blockade of the inositol 1,4,5-triphosphate receptor reduced distention-induced ATP release, suggesting that Ca(2+) release from the ER is essential for the induction of urothelial ATP release. On the other hand, blockade of store-operated Ca(2+) entry (SOCE) from the extracellular space significantly enhanced distention-induced ATP release. Thus Ca(2+) release from the ER causes urothelial ATP release and depletion of Ca(2+) stores in the ER, which in turn causes the depletion-inducing SOCE to suppress the amount of urothelial ATP released.     

Effects of Muscarinic Agonists and Depolarizing Agents on Inositol Monophosphate Accumulation in the Rabbit Vagus Nerve

The effects of muscarinic agonists and depolarizing agents on inositol phospholipid hydrolysis in the rabbit vagus nerve were assessed by the measurement of [3H]inositol monophosphate production in nerves that had been preincubated with [3H]inositol. After 1 h of drug action, carbachol, oxotremorine, and arecoline increased the inositol monophosphate accumulation, though the maximal increase induced by these agonists differed. Addition of the muscarinic antagonists atropine or pirenzepine shifted the carbachol dose-response curves to the right, without decreasing the carbachol maximal stimulatory effects. The KB for pirenzepine was 35 nM, which is characteristic of muscarinic high-affinity binding sites coupled to phosphoinositide turnover and often associated with the M1 receptor subtype. On the other hand, agents known to depolarize or to increase the intracellular Ca2+ concentration, e.g., elevated extracellular K+, ouabain, Ca2+, and the Ca2+ ionophore A23187, also increased inositol monophosphate accumulation. These effects were not mediated by the release of acetylcholine, as suggested by the fact that they could not be potentiated by the addition of physostigmine nor inhibited by the addition of atropine. The Ca(2+)-channel antagonist Cd2+, also known to inhibit the Na+/Ca2+ exchanger, was able to block the effects of K+ and ouabain, but did not alter those of carbachol. These results suggest that depolarizing agents increase inositol monophosphate accumulation in part through elevation of the intracellular Ca2+ concentration and that muscarinic receptors coupled to phosphoinositide turnover are present along the trunk of the rabbit vagus nerve.     

Effects of extracellular ATP on ion transport systems and [Ca2+]i in rat parotid acinar cells. Comparison with the muscarinic agonist carbachol

The effects of extracellular ATP on ion fluxes and the intracellular free Ca2+ concentration ([Ca2+]i) were examined using a suspension of rat parotid acinar cells and were contrasted with the effects of the muscarinic agonist carbachol. Although ATP and carbachol both rapidly increased [Ca2+]i about threefold above the resting level (200-250 nM), the effect of ATP was due primarily to an influx of Ca2+ across the plasma membrane, while the initial response to carbachol was due to a release of Ca2+ from intracellular stores. Within 10 s, ATP (1 mM) and carbachol (20 microM) reduced the cellular Cl- content by 39-50% and cell volume by 15-25%. Both stimuli reduced the cytosolic K+ content by 57-65%, but there were marked differences in the rate and pattern of net K+ movement as well as the effects of K+ channel inhibitors on the effluxes initiated by the two stimuli. The maximum rate of the ATP-stimulated K+ efflux (approximately 2,200 nmol K+/mg protein per min) was about two-thirds that of the carbachol-initiated efflux rate, and was reduced by approximately 30% (vs. 60% for the carbachol-stimulated K+ efflux) by TEA (tetraethylammonium), an inhibitor of the large conductance (BK) K+ channel. Charybdotoxin, another K+ channel blocker, was markedly more effective than TEA on the effects of both agonists, and reduced the rate of K+ efflux initiated by both ATP and carbachol by approximately 80%. The removal of extracellular Ca2+ reduced the ATP- and the carbachol-stimulated rates of K+ efflux by 55 and 17%, respectively. The rate of K+ efflux initiated by either agonist was reduced by 78-95% in cells that were loaded with BAPTA to slow the elevation of [Ca2+]i. These results indicated that ATP and carbachol stimulated the efflux of K+ through multiple types of K(+)-permeable channels, and demonstrated that the relative proportion of efflux through the different pathways was different for the two stimuli. ATP and carbachol also stimulated the rapid entry of Na+ into the parotid cell, and elevated the intracellular Na+ content to 4.4 and 2.6 times the normal level, respectively. The rate of Na+ entry through Na(+)-K(+)-2Cl- cotransport and Na(+)-H+ exchange was similar whether stimulated by ATP, carbachol, or ionomycin, and uptake through these two carrier-mediated transporters accounted for 50% of the ATP-promoted Na+ influx. The remainder may be due to a nonselective cation channel and an ATP-gated cation channel that is also permeable to Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)     

Extracellular ATP elevates intracellular free calcium in rat parotid acinar cells

The effect of extracellular ATP on intracellular free Ca2+ was characterized in quin2-loaded parotid acinar cells. ATP specifically increased the intracellular Ca2+ concentration six-fold above a basal level of 180 nM. Of other purine nucleotides tested, only adenylylthiodiphosphate (ATP gamma S) had significant activity. ATP and the muscarinic agonist carbachol increased intracellular Ca2+ even in the absence of extracellular Ca2+. Both agonists stimulated K+ release, which was followed by reuptake of K+, even in the continued presence of agonist. In the absence of Mg2+, ATP was much more potent but no more efficacious in elevating intracellular Ca2+, suggesting that ATP4- is the active species. The effect of ATP was reversed by removal with hexokinase, arguing against a role for an active contaminant of ATP and against a non-specific permeabilizing effect of extracellular ATP. Lactate dehydrogenase release was unaffected by a maximally effective concentration of ATP. These observations are consistent with a possible neurotransmitter role for ATP in the rat parotid gland.     

Signaling pathways underlying muscarinic receptor-induced [Ca2+]i oscillations in HEK293 cells

We have investigated the signaling pathways underlying muscarinic receptor-induced calcium oscillations in human embryonic kidney (HEK293) cells. Activation of muscarinic receptors with a maximal concentration of carbachol (100 microm) induced a biphasic rise in cytoplasmic calcium ([Ca2+]i) comprised of release of Ca2+ from intracellular stores and influx of Ca2+ from the extracellular space. A lower concentration of carbachol (5 microm) induced repetitive [Ca2+]i spikes or oscillations, the continuation of which was dependent on extracellular Ca2+. The entry of Ca2+ with 100 microm carbachol and with the sarcoplasmic-endoplasmic reticulum calcium ATPase inhibitor, thapsigargin, was completely blocked by 1 microm Gd3+, as well as 30-100 microm concentrations of the membrane-permeant inositol 1,4,5-trisphosphate receptor inhibitor, 2-aminoethyoxydiphenyl borane (2-APB). Sensitivity to these inhibitors is indicative of capacitative calcium entry. Arachidonic acid, a candidate signal for Ca2+ entry associated with [Ca2+]i oscillations in HEK293 cells, induced entry that was inhibited only by much higher concentrations of Gd3+ and was unaffected by 100 microm 2-APB. Like arachidonic acid-induced entry, the entry associated with [Ca2)]i oscillations was insensitive to inhibition by Gd3+ but was completely blocked by 100 microm 2-APB. These findings indicate that the signaling pathway responsible for the Ca2+) entry driving [Ca2+]i oscillations in HEK293 cells is more complex than originally thought, and may involve neither capacitative calcium entry nor a role for PLA2 and arachidonic acid.     

Intercellular calcium signaling in astrocytes via ATP release through connexin hemichannels.

Astrocytes are capable of widespread intercellular communication via propagated increases in intracellular Ca(2+) concentration. We have used patch clamp, dye flux, ATP assay, and Ca(2+) imaging techniques to show that one mechanism for this intercellular Ca(2+) signaling in astrocytes is the release of ATP through connexin channels ("hemichannels") in individual cells. Astrocytes showed low Ca(2+)-activated whole-cell currents consistent with connexin hemichannel currents that were inhibited by the connexin channel inhibitor flufenamic acid (FFA). Astrocytes also showed molecular weight-specific influx and release of dyes, consistent with flux through connexin hemichannels. Transmembrane dye flux evoked by mechanical stimulation was potentiated by low Ca(2+) and was inhibited by FFA and Gd(3+). Mechanical stimulation also evoked release of ATP that was potentiated by low Ca(2+) and inhibited by FFA and Gd(3+). Similar whole-cell currents, transmembrane dye flux, and ATP release were observed in C6 glioma cells expressing connexin43 but were not observed in parent C6 cells. The connexin hemichannel activator quinine evoked ATP release and Ca(2+) signaling in astrocytes and in C6 cells expressing connexin43. The propagation of intercellular Ca(2+) waves in astrocytes was also potentiated by quinine and inhibited by FFA and Gd(3+). Release of ATP through connexin hemichannels represents a novel signaling pathway for intercellular communication in astrocytes and other non-excitable cells.     

Modulation of Ca2+ channel-gated Ca2+ release by W-7 in cardiac myocytes.

Cardiac muscle excitation-contraction coupling is controlled by the Ca(2+)-induced Ca2+ release mechanism. The present study examines the effects of a calmodulin antagonist W-7 on Ca2+ current (ICa)-induced Ca2+ release in whole cell-clamped rat ventricular myocytes. Exposure of cells to W-7 suppressed ICa, but the intracellular Ca(2+)-transients showed a lesser degree of reduction, suggesting possible enhancement of Ca(2+)-induced Ca2+ release. The effects of W-7 on the efficacy of Ca2+ release were most prominent at negative potentials. At test potentials of -30 mV, 20 microM W-7 almost completely blocked ICa, but significant Ca(2+)-transients remained, thus causing a four to six-fold increase in the efficacy of Ca(2+)-induced Ca2+ release. The depolarization-dependent Ca(2+)-transients were eliminated in absence of extracellular Ca2+, blocked by Cd2+, and were absent when the sarcoplasmic reticulum was depleted of Ca2+, implicating dependency on Ca(2+)-signaling between the L-type channel and the ryanodine receptor. W-7 mediated increase in the efficacy of Ca(2+)-induced Ca2+ release was eliminated when myocytes were dialyzed with the internal solution containing gluathione (5 mM), suggesting the possible role of cellular redox state in the regulation of Ca2+ release by the calmodulin antagonist.     

Parathyroid hormone controls the size of the intracellular Ca(2+) stores available to receptors linked to inositol trisphosphate formation

In HEK 293 cells stably expressing type 1 parathyroid (PTH) receptors, PTH stimulated release of intracellular Ca(2+) stores in only 27% of cells, whereas 96% of cells responded to carbachol. However, in almost all cells PTH potentiated the response to carbachol by about 3-fold. Responses to carbachol did not desensitize, but only the first challenge in Ca(2+)-free medium caused an increase in [Ca(2+)](i), indicating that the carbachol-sensitive Ca(2+) stores had been emptied. Subsequent addition of PTH also failed to increase [Ca(2+)](i), but when it was followed by carbachol there was a substantial increase in [Ca(2+)](i). A similar potentiation was observed between ATP and PTH but not between carbachol and ATP. Intracellular heparin inhibited responses to carbachol and PTH, and pretreatment with ATP and carbachol abolished responses to PTH, suggesting that the effects of PTH involve inositol trisphosphate (IP(3)) receptors. PTH neither stimulated detectable IP(3) formation nor affected the amount formed in response to ATP or carbachol. PTH stimulated cyclic AMP formation, but this was not the means whereby PTH potentiated Ca(2+) signals. We suggest that PTH may regulate Ca(2+) mobilization by facilitating translocation of Ca(2+) between discrete intracellular stores and that it thereby regulates the size of the Ca(2+) pool available to receptors linked to IP(3) formation.     

Attenuation of inositol trisphosphate generation and cytosolic Ca2+ elevation in dispersed rat parotid acini stimulated simultaneously at muscarinic and alpha 1-adrenergic receptors

We have examined intracellular signalling events, peak cytosolic [Ca2+] and inositol trisphosphate levels, in rat parotid acini simultaneously stimulated with two Ca2+ mobilizing agonists, carbachol (muscarinic-cholinergic) and epinephrine (alpha 1-adrenergic). When the agonists were added together, either at sub-maximal (200 nM each, i.e. 400 nM total agonist concentration) or maximal (10 uM each, i.e. 20 uM total) stimulatory concentrations, the resulting elevations in both cytosolic [Ca2+] and inositol trisphosphate levels were not greater than those achieved when each agonist was added individually. However, with 400 nM carbachol these responses were significantly greater than those seen with either 200 nM carbachol or 200 nM carbachol + 200 nM epinephrine. The data indicate that when muscarinic and alpha 1-adrenergic receptors of rat parotid acini are simultaneously stimulated a novel regulatory mechanism is induced, which attenuates inositol trisphosphate generation and, consequently, intracellular Ca2+ release.     

Cytosolic heparin inhibits muscarinic and alpha-adrenergic Ca2+ release in smooth muscle. Physiological role of inositol 1,4,5-trisphosphate in pharmacomechanical coupling

In order to test the physiological significance of inositol 1,4,5-trisphosphate (InsP3) in pharmacomechanical coupling, we have utilized two near-physiological systems, in which relatively high molecular weight solutes can be applied intracellularly and receptor coupling is retained: beta-escin permeabilization and reversible permeabilization. We showed that in smooth muscle permeabilized with beta-escin, one of the saponin esters, alpha 1-adrenergic (phenylephrine) and muscarinic (carbachol) agonists, as well as caffeine and InsP3, cause contractions mediated by Ca2+ release. These contractions were calmodulin-dependent and blocked by depletion of Ca2+ stored in the sarcoplasmic reticulum. Intracellular heparin (Mr = about 5000), a blocker of InsP3 binding to its receptor and a specific inhibitor of InsP3-induced Ca2+ release in smooth muscles, inhibited the responses to the agonists and to InsP3, but not those to caffeine, nor did it block the enhanced contractile response to cytoplasmic Ca2+ induced by agonists and by GTP gamma S. Neomycin blocked Ca2+ release induced by carbachol, but not by caffeine. In reversibly permeabilized ileum smooth muscle cells, loaded with Fura-2 acid and heparin, the intracellular heparin inhibited Ca2+ release and contractions induced by carbachol in Ca2+-free, high K+ solution. Heparin did not inhibit the high K+ contractions (with 1.2 mM Ca2+) and had no significant inhibitory effects on carbachol-induced responses in the presence of extracellular Ca2+. These results, obtained under near-physiological conditions, support the conclusion that InsP3 is the major physiological messenger of the Ca2+ release component of pharmacomechanical coupling, but not of the components mediated by Ca2+ influx or by potentiation of the contractile response to Ca2+.     

Spatial microenvironment defines Ca2+ entry and Ca2+ release in salivary gland cells

The difference of Ca(2+) mobilization induced by muscarinic receptor activation between parotid acinar and duct cells was examined. Oxotremorine, a muscarinic-cholinergic agonist, induced intracellular Ca(2+) release and extracellular Ca(2+) entry through store-operated Ca(2+) entry (SOC) and non-SOC channels in acinar cells, but it activated only Ca(2+) entry from non-SOC channels in duct cells. RT-PCR experiments showed that both types of cells expressed the same muscarinic receptor, M3. Given that ATP activated the intracellular Ca(2+) stores, the machinery for intracellular Ca(2+) release was intact in the duct cells. By immunocytochemical experiments, IP(3)R2 colocalized with M3 receptors in the plasma membrane area of acinar cells; in duct cells, IP(3)R2 resided in the region on the opposite side of the M3 receptors. On the other hand, purinergic P2Y2 receptors were found in the apical area of duct cells where they colocalized with IP(3)R2. These results suggest that the expression of the IP(3)Rs near G-protein-coupled receptors is necessary for the activation of intracellular Ca(2+) stores. Therefore, the microenvironment probably affects intracellular Ca(2+) release and Ca(2+) entry.     

Muscarinic receptors modulate intracellular Ca2+ concentration in hyaline cells of the chicken basilar papilla

Nonsensory hyaline cells border the sensory epithelium of the auditory end-organ (basilar papilla) in birds and reptiles. Their innervation by cochlear cholinergic efferent fibers and the presence of contractile proteins suggest that hyaline cells may actively regulate basilar membrane mechanics. The cholinergic pharmacology of hyaline cells was studied by measuring the intracellular calcium concentration ([Ca(2+)](i)) of fura-2-loaded cells in the chicken cochlea in vitro. Superfusion of the cholinergic agonist carbachol produced a dose-dependent increase in hyaline cell [Ca(2+)](i) (EC(50)=1.05 micromol l(-1)) and small responses in short hair cells. Calcium increases in hyaline cells were evoked by the muscarinic agonists oxotremorine (10 micromol l(-1)) and muscarine (100 micromol l(-1)) whereas nicotine (100 micromol l(-1), 200 micromol l(-1)) was without effect. Carbachol-evoked responses were blocked by the muscarinic antagonist atropine (>or=10(-13) mol l(-1)) and were unaffected by the nicotinic antagonists d-tubocurare (100 micromol l(-1), 1 mmol l(-1)) and hexamethonium (100 micromol l(-1)). Responses persisted in the absence of extracellular Ca(2+) and were abolished by thapsigargin (1 micromol l(-1)). These results indicate that the cholinergic-stimulated increase in hyaline cell [Ca(2+)](i) is due to a muscarinic-mediated release of Ca(2+) from intracellular stores. This is the first evidence that hyaline cells possess a muscarinic receptor whose activation causes mobilization of intracellular Ca(2+).     

Control of ion transport in mouse proximal and distal colon by prolactin.

The lactogenic hormone prolactin (PRL) has been known to affect Ca(2+) and electrolyte transport in the intestinal epithelium. In the present study we analyzed ion transport in mouse proximal and distal colon, and acute changes induced by PRL. In the proximal colon, carbachol activated a Ca(2+) dependent Cl(-) secretion that was sensitive to DIDS and NFA. In the distal colon, both ATP and carbachol activated K(+) secretion. Ca(2+) -activated KCl transport in proximal and distal colon was inhibited by PRL (200 ng/ml), while amiloride sensitive Na(+) absorption and cAMP induced Cl(-) secretion remained unaffected. Luminal large conductance Ca(2+) -activated K(+) (BK) channels were largely responsible for Ca(2+) -activated K(+) secretion in the distal colon, and basolateral BK channels supported Ca(2+) -activated Cl(-) secretion in the proximal colon. Ca(2+) chelating by BAPTA-AM attenuated effects of carbachol and abolished effects of PRL. Both inhibition of PI3 kinase with wortmannin and blockage of MAP kinases with SB 203580 or U 0126, interfered with the acute inhibitory effect of PRL on ion transport, while blocking of Jak/Stat kinases with AG 490 was without effects. PRL attenuated the increase in intracellular Ca(2+) that was caused by stimulation of isolated colonic crypts with carbachol. Thus PRL inhibits Ca(2+) dependent Cl(-) and K(+) secretion by interfering with intracellular Ca(2+) signaling and probably by activating PI3 kinase and MAP kinase pathways.     

Two modes of secretion in pancreatic acinar cells: involvement of phosphatidylinositol 3-kinase and regulation by capacitative Ca(2+) entry

In pancreatic acinar cells, muscarinic agonists stimulate both the release of Ca(2+) from intracellular stores and the influx of extracellular Ca(2+). The part played by Ca(2+) released from intracellular stores in the regulation of secretion is well established; however, the role of Ca(2+) influx in exocytosis is unclear. Recently, we observed that supramaximal concentrations of acetylcholine (>or=10 microM) elicited an additional component of exocytosis despite reducing Ca(2+) influx. In the present study, we found that supramaximal exocytosis was substantially inhibited (approximately 70%) by wortmannin (100 nM), an inhibitor of phosphatidylinositol 3-kinase. In contrast, exocytosis evoked by a lower concentration of acetylcholine (1 microM) was potentiated (approximately 45%) by wortmannin. Exocytosis stimulated by 1 microM acetylcholine in the absence of extracellular Ca(2+) was, like supramaximal exocytosis, inhibited by wortmannin. The switch to a wortmannin-inhibitable form of exocytosis depended upon a reduction in Ca(2+) entry through store-operated Ca(2+) channels, as the switch in exocytotic mode could also be brought about by the selective blockade of these channels by Gd(3+) (2 microM), but not by inhibition of noncapacitative Ca(2+) entry by SB203580 (10 microM). We conclude that supramaximal doses of acetylcholine lead to a switch in the mode of zymogen granule exocytosis by inhibiting store-dependent Ca(2+) influx.     

Membrane potential modulates divalent cation entry in rat parotid acini

This study examines the effect of membrane potential on divalent cation entry in dispersed parotid acini following stimulation by the muscarinic agonist, carbachol, and during refill of the agonist-sensitive internal Ca2+ pool. Depolarizing conditions (addition of gramicidin to cells in Na(+)-containing medium or incubation of cells in medium with elevated [K+]) prevent carbachol-stimulated hyperpolarization of acini and also inhibit carbachol activation of Ca2+ and Mn2+ entry into these cells. Conditions promoting hyperpolarization (cells in medium with Na+ or with N-methyl-D-glucamine instead of Na+) enhance carbachol stimulation of divalent cation entry. Intracellular Ca2+ release (initial increase in [Ca2+]i) does not appear to be affected by these manipulations. Mn2+ entry into resting and internal Ca2+ pool-depleted cells (10-min carbachol stimulation in a Ca(2+)-free medium) is similarly affected by membrane potential modulations, and refill of the internal pool by Ca2+ is inhibited by depolarization. The inhibitory effects of depolarization on divalent cation entry can be overcome by increasing extracellular [Ca2+] or [Mn2+]. These data demonstrate that the modulation of Ca2+ entry into parotid acini by membrane potential is most likely due to effects on the electrochemical gradient (Em-ECa) for Ca2+ entry.     

Intracellular signaling following myocardial stretch: an autocrine/paracrine loop

The stretch of adult papillary muscle elicits a chain of autocrine/paracrine events in which the Na(+)/H(+) exchanger (NHE-1) activation is the central step. This activation is induced by a sequential angiotensin II-endothelin (Ang II-ET) release and results in an increase in intracellular Na(+) (Na(+)(i)) without significant changes in intracellular pH. The increase in Na(+)(i) negatively shifts the reverse potential of the Na(+)/Ca(2+) exchanger (NCX) thus inducing cell Ca(2+) influx that augments myocardial contractility. This increase in force represents the mechanical counterpart of the autocrine/paracrine mechanism triggered by stretch and has been called the slow force response (SFR) to stretch.     

Epidermal growth factor-induced depletion of the intracellular Ca2+ store fails to activate capacitative Ca2+ entry in a human salivary cell line

Epidermal growth factor (EGF) is a multifunctional factor known to influence proliferation and function of a variety of cells. The actions of EGF are mediated by EGF receptor tyrosine kinase pathways, including stimulation of phospholipase Cgamma and mobilization of intracellular Ca(2+) ([Ca(2+)](i)). Generally, agonist-mediated Ca(2+) mobilization involves both Ca(2+) release from internal stores and Ca(2+) influx activated by store depletion (i.e. capacitative or store-operated Ca(2+) influx). However, the role of capacitative Ca(2+) entry in EGF-mediated Ca(2+) mobilization is still largely unknown. In this study, we compared [Ca(2+)](i) signals elicited by EGF with those induced by agents (the muscarinic receptor agonist carbachol and thapsigargin (Tg)) known to activate capacitative Ca(2+) entry. Unlike carbachol and Tg, EGF (5 nm) elicited a transient [Ca(2+)](i) signal without a plateau phase in the presence of extracellular Ca(2+) and also failed to accelerate Mn(2+) entry. Repletion of extracellular Ca(2+) to cells stimulated with EGF in the absence of Ca(2+) elicited an increase in [Ca(2+)](i), indicating that EGF indeed stimulates Ca(2+) influx. However, the influx was activated at lower EGF concentrations than those required to stimulate Ca(2+) release. Interestingly, the phospholipase C inhibitor completely inhibited Ca(2+) release induced by both EGF and carbachol and also reduced Ca(2+) influx responsive to carbachol but had no effect on Ca(2+) influx induced by EGF. EGF-induced Ca(2+) influx was potentiated by low concentrations (<5 ng/ml) of oligomycin, a mitochondrial inhibitor that blocks capacitative Ca(2+) influx in other systems. Transient expression of the hTRPC3 protein enhanced Ca(2+) influx responsive to carbachol but did not increase EGF-activated Ca(2+) influx. Both EGF and carbachol depleted internal Ca(2+) stores. Our results demonstrate that EGF-induced Ca(2+) release from internal stores does not activate capacitative Ca(2+) influx. Rather, EGF stimulates Ca(2+) influx via a mechanism distinct from capacitative Ca(2+) influx induced by carbachol and Tg.     

温度和胞内钠、ATP及酸碱度对心室肌细胞钠钙交换电流的调节

心肌缺血损伤过程中,胞内Na^＋、ATP及pH都出现明显变化。钠／钙交换对心肌细胞的钙平衡起重要的调节作用。本实验采用膜片钳全细胞记录豚鼠心室肌细胞钠／钙交换电流,研究温度和胞内Na^＋、ATP及pH对钠／钙交换双向电流的影响。结果表明,温度从22℃升至34℃,钠／钙交换电流增大约4倍,而pH值的改变对钠／钙交换双向电流没有明显的影响。在22～24℃时,同时耗竭胞内ATP和胞内酸化对钠／钙交换双向转运功能影响程度小;而在34—37℃时,同时耗竭胞内ATP和胞内酸化能抑制钠／钙交换双向电流的外向和内向成分,且内向成分抑制程度高于外向成分抑制程度。表明同时耗竭胞内ATP和胞内酸化对钠／钙交换的作用具有温度依赖性。胞内Na^＋超载能使钠／钙交换电流的外向成分增加,但不增加或减少内向电流（即正向转运）成分。因此,胞内酸化及耗竭胞内ATP损伤细胞排钙机制和胞内钠超载通过钠／钙反向交换引起钙内流是引起心肌细胞钙超载的两个独立的重要因素。