Catecholamine exocytosis during low frequency stimulation in mouse adrenal chromaffin cells is primarily asynchronous and controlled by the novel mechanism of Ca2+ syntilla suppression.

Adrenal chromaffin cells (ACCs), stimulated by the splanchnic nerve, generate action potentials (APs) at a frequency near 0.5 Hz in the resting physiological state, at times described as 'rest and digest'. How such low frequency stimulation in turn elicits sufficient catecholamine exocytosis to set basal sympathetic tone is not readily explained by the classical mechanism of stimulus-secretion coupling, where exocytosis is synchronized to AP-induced Ca(2+) influx. By using simulated action potentials (sAPs) at 0.5 Hz in isolated patch-clamped mouse ACCs, we show here that less than 10% of all catecholaminergic exocytosis, measured by carbon fibre amperometry, is synchronized to an AP. The asynchronous phase, the dominant phase, of exocytosis does not require Ca(2+) influx. Furthermore, increased asynchronous exocytosis is accompanied by an AP-dependent decrease in frequency of Ca(2+) syntillas (i.e. transient, focal Ca(2+) release from internal stores) and is ryanodine sensitive. We propose a mechanism of disinhibition, wherein APs suppress Ca(2+) syntillas, which themselves inhibit exocytosis as they do in the case of spontaneous catecholaminergic exocytosis.

Type 1 ryanodine receptor knock-in mutation causing central core disease of skeletal muscle also displays a neuronal phenotype.

The type 1 ryanodine receptor (RyR1) is expressed widely in the brain, with high levels in the cerebellum, hippocampus, and hypothalamus. We have shown that L-type Ca(2+) channels in terminals of hypothalamic magnocellular neurons are coupled to RyRs, as they are in skeletal muscle, allowing voltage-induced Ca(2+) release (VICaR) from internal Ca(2+) stores without Ca(2+) influx. Here we demonstrate that RyR1 plays a role in VICaR in nerve terminals. Furthermore, in heterozygotes from the Ryr1(I4895T/WT) (IT/+) mouse line, carrying a knock-in mutation corresponding to one that causes a severe form of human central core disease, VICaR is absent, demonstrating that type 1 RyR mediates VICaR and that these mice have a neuronal phenotype. The absence of VICaR was shown in two ways: first, depolarization in the absence of Ca(2+) influx elicited Ca(2+)syntillas (scintilla, spark, in a nerve terminal, a SYNaptic structure) in WT, but not in mutant terminals; second, in the presence of extracellular Ca(2+), IT/+ terminals showed a twofold decrease in global Ca(2+) transients, with no change in plasmalemmal Ca(2+) current. From these studies we draw two conclusions: (i) RyR1 plays a role in VICaR in hypothalamic nerve terminals; and (ii) a neuronal alteration accompanies the myopathy in IT/+ mice, and, possibly in humans carrying the corresponding RyR1 mutation.

Individual calcium syntillas do not trigger spontaneous exocytosis from nerve terminals of the neurohypophysis.

Recently, highly localized Ca(2+) release events, similar to Ca(2+) sparks in muscle, have been observed in neuronal preparations. Specifically, in murine neurohypophysial terminals (NHT), these events, termed Ca(2+) syntillas, emanate from a ryanodine-sensitive intracellular Ca(2+) pool and increase in frequency with depolarization in the absence of Ca(2+) influx. Despite such knowledge of the nature of these Ca(2+) release events, their physiological role in this system has yet to be defined. Such localized Ca(2+) release events, if they occur in the precise location of the final exocytotic event(s), may directly trigger exocytosis. However, directly addressing this hypothesis has not been possible, since no method capable of visualizing individual release events in these CNS terminals has been available. Here, we have adapted an amperometric method for studying vesicle fusion to this system which relies on loading the secretory granules with the false transmitter dopamine, thus allowing, for the first time, the recording of individual exocytotic events from peptidergic NHT. Simultaneous use of this technique along with high-speed Ca(2+) imaging has enabled us to establish that spontaneous neuropeptide release and Ca(2+) syntillas do not display any observable temporal or spatial correlation, confirming similar findings in chromaffin cells. Although these results indicate that syntillas do not play a direct role in eliciting spontaneous release, they do not rule out indirect modulatory effects of syntillas on secretion.

Suppression of Ca2+ syntillas increases spontaneous exocytosis in mouse adrenal chromaffin cells.

A central concept in the physiology of neurosecretion is that a rise in cytosolic [Ca(2+)] in the vicinity of plasmalemmal Ca(2+) channels due to Ca(2+) influx elicits exocytosis. Here, we examine the effect on spontaneous exocytosis of a rise in focal cytosolic [Ca(2+)] in the vicinity of ryanodine receptors (RYRs) due to release from internal stores in the form of Ca(2+) syntillas. Ca(2+) syntillas are focal cytosolic transients mediated by RYRs, which we first found in hypothalamic magnocellular neuronal terminals. (scintilla, Latin for spark; found in nerve terminals, normally synaptic structures.) We have also observed Ca(2+) syntillas in mouse adrenal chromaffin cells. Here, we examine the effect of Ca(2+) syntillas on exocytosis in chromaffin cells. In such a study on elicited exocytosis, there are two sources of Ca(2+): one due to influx from the cell exterior through voltage-gated Ca(2+) channels, and that due to release from intracellular stores. To eliminate complications arising from Ca(2+) influx, we have examined spontaneous exocytosis where influx is not activated. We report here that decreasing syntillas leads to an increase in spontaneous exocytosis measured amperometrically. Two independent lines of experimentation each lead to this conclusion. In one case, release from stores was blocked by ryanodine; in another, stores were partially emptied using thapsigargin plus caffeine, after which syntillas were decreased. We conclude that Ca(2+) syntillas act to inhibit spontaneous exocytosis, and we propose a simple model to account quantitatively for this action of syntillas.

Dihydropyridine receptors and type 1 ryanodine receptors constitute the molecular machinery for voltage-induced Ca2+ release in nerve terminals.

Ca2+ stores were studied in a preparation of freshly dissociated terminals from hypothalamic magnocellular neurons. Depolarization from a holding level of -80 mV in the absence of extracellular Ca2+ elicited Ca2+ release from intraterminal stores, a ryanodine-sensitive process designated as voltage-induced Ca2+ release (VICaR). The release took one of two forms: an increase in the frequency but not the quantal size of Ca2+ syntillas, which are brief, focal Ca2+ transients, or an increase in global [Ca2+]. The present study provides evidence that the sensors of membrane potential for VICaR are dihydropyridine receptors (DHPRs). First, over the range of -80 to -60 mV, in which there was no detectable voltage-gated inward Ca2+ current, syntilla frequency was increased e-fold per 8.4 mV of depolarization, a value consistent with the voltage sensitivity of DHPR-mediated VICaR in skeletal muscle. Second, VICaR was blocked by the dihydropyridine antagonist nifedipine, which immobilizes the gating charge of DHPRs but not by Cd2+ or FPL 64176 (methyl 2,5 dimethyl-4[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylate), a non-dihydropyridine agonist specific for L-type Ca2+ channels, having no effect on gating charge movement. At 0 mV, the IC50 for nifedipine blockade of VICaR in the form of syntillas was 214 nM in the absence of extracellular Ca2+. Third, type 1 ryanodine receptors, the type to which DHPRs are coupled in skeletal muscle, were detected immunohistochemically at the plasma membrane of the terminals. VICaR may constitute a new link between neuronal activity, as signaled by depolarization, and a rise in intraterminal Ca2+.

Syntillas release Ca2+ at a site different from the microdomain where exocytosis occurs in mouse chromaffin cells.

Spontaneous, short-lived, focal cytosolic Ca2+ transients were found for the first time and characterized in freshly dissociated chromaffin cells from mouse. Produced by release of Ca2+ from intracellular stores and mediated by type 2 and perhaps type 3 ryanodine receptors (RyRs), these transients are quantitatively similar in magnitude and duration to Ca2+ syntillas in terminals of hypothalamic neurons, suggesting that Ca2+ syntillas are found in a variety of excitable, exocytotic cells. However, unlike hypothalamic nerve terminals, chromaffin cells do not display syntilla activation by depolarization of the plasma membrane, nor do they have type 1 RyRs. It is widely thought that focal Ca2+ transients cause "spontaneous" exocytosis, although there is no direct evidence for this view. Hence, we monitored catecholamine release amperometrically while simultaneously imaging Ca2+ syntillas, the first such simultaneous measurements. Syntillas failed to produce exocytotic events; and, conversely, spontaneous exocytotic events were not preceded by syntillas. Therefore, we suggest that a spontaneous syntilla, at least in chromaffin cells, releases Ca2+ into a cytosolic microdomain distinct from the microdomains containing docked, primed vesicles. Ryanodine (100 microM) reduced the frequency of Ca2+ syntillas by an order of magnitude but did not alter the frequency of spontaneous amperometric events, suggesting that syntillas are not involved in steps preparatory to spontaneous exocytosis. Surprisingly, ryanodine also increased the total charge of individual amperometric events by 27%, indicating that intracellular Ca2+ stores can regulate quantal size.

Ca(2+) spark sites in smooth muscle cells are numerous and differ in number of ryanodine receptors, large-conductance K(+) channels, and coupling ratio between them.

Ca(2+) sparks are highly localized Ca(2+) transients caused by Ca(2+) release from sarcoplasmic reticulum through ryanodine receptors (RyR). In smooth muscle, Ca(2+) sparks activate nearby large-conductance, Ca(2+)-sensitive K(+) (BK) channels to generate spontaneous transient outward currents (STOC). The properties of individual sites that give rise to Ca(2+) sparks have not been examined systematically. We have characterized individual sites in amphibian gastric smooth muscle cells with simultaneous high-speed imaging of Ca(2+) sparks using wide-field digital microscopy and patch-clamp recording of STOC in whole cell mode. We used a signal mass approach to measure the total Ca(2+) released at a site and to estimate the Ca(2+) current flowing through RyR [I(Ca(spark))]. The variance between spark sites was significantly greater than the intrasite variance for the following parameters: Ca(2+) signal mass, I(Ca(spark)), STOC amplitude, and 5-ms isochronic STOC amplitude. Sites that failed to generate STOC did so consistently, while those at the remaining sites generated STOC without failure, allowing the sites to be divided into STOC-generating and STOC-less sites. We also determined the average number of spark sites, which was 42/cell at a minimum and more likely on the order of at least 400/cell. We conclude that 1) spark sites differ in the number of RyR, BK channels, and coupling ratio of RyR-BK channels, and 2) there are numerous Ca(2+) spark-generating sites in smooth muscle cells. The implications of these findings for the organization of the spark microdomain are explored.

Spontaneous mitochondrial depolarizations are independent of SR Ca2+ release.

The mitochondrial membrane potential (DeltaPsi(m)) underlies many mitochondrial functions, including Ca(2+) influx into the mitochondria, which allows them to serve as buffers of intracellular Ca(2+). Spontaneous depolarizations of DeltaPsi(m), flickers, have been observed in isolated mitochondria and intact cells using the fluorescent cationic lipophile tetramethylrhodamine ethyl ester (TMRE), which distributes across the inner mitochondrial membrane in accordance with the Nernst equation. Flickers in cardiomyocytes have been attributed to uptake of Ca(2+) released from the sarcoplasmic reticulum (SR) via ryanodine receptors in focal transients called Ca(2+) sparks. We have shown previously that an increase in global Ca(2+) in smooth muscle cells causes an increase in mitochondrial Ca(2+) and depolarization of DeltaPsi(m). Here we sought to determine whether flickers in smooth muscle cells are caused by uptake of Ca(2+) released focally in Ca(2+) sparks. High-speed three-dimensional imaging was used to monitor DeltaPsi(m) in freshly dissociated myocytes from toad stomach that were simultaneously voltage clamped at 0 mV to ensure the cytosolic TMRE concentration was constant and equal to the low level in the bath (2.5 nM). This approach allows quantitative analysis of flickers as we have previously demonstrated. Depletion of SR Ca(2+) not only failed to eliminate flickers but rather increased their magnitude and frequency somewhat. Flickers were not altered in magnitude or frequency by ryanodine or xestospongin C, inhibitors of intracellular Ca(2+) release, or by cyclosporin A, an inhibitor of the permeability transition pore. Focal Ca(2+) release from the SR does not cause flickers in the cells employed here.

Ca2+ syntillas, miniature Ca2+ release events in terminals of hypothalamic neurons, are increased in frequency by depolarization in the absence of Ca2+ influx.

Localized, brief Ca2+ transients (Ca2+ syntillas) caused by release from intracellular stores were found in isolated nerve terminals from magnocellular hypothalamic neurons and examined quantitatively using a signal mass approach to Ca2+ imaging. Ca2+ syntillas (scintilla, L., spark, from a synaptic structure, a nerve terminal) are caused by release of approximately 250,000 Ca ions on average by a Ca2+ flux lasting on the order of tens of milliseconds and occur spontaneously at a membrane potential of -80 mV. Syntillas are unaffected by removal of extracellular Ca2+, are mediated by ryanodine receptors (RyRs) and are increased in frequency, in the absence of extracellular Ca2+, by physiological levels of depolarization. This represents the first direct demonstration of mobilization of Ca2+ from intracellular stores in neurons by depolarization without Ca2+ influx. The regulation of syntillas by depolarization provides a new link between neuronal activity and cytosolic [Ca2+] in nerve terminals.

Quantitative analysis of spontaneous mitochondrial depolarizations.

Spontaneous transient depolarizations in mitochondrial membrane potential (DeltaPsi(m)), mitochondrial flickers, have been observed in isolated mitochondria and intact cells using the fluorescent probe, tetramethylrhodamine ethyl ester (TMRE). In theory, the ratio of [TMRE] in cytosol and mitochondrion allows DeltaPsi(m) to be calculated with the Nernst equation, but this has proven difficult in practice due to fluorescence quenching and binding of dye to mitochondrial membranes. We developed a new method to determine the amplitude of flickers in terms of millivolts of depolarization. TMRE fluorescence was monitored using high-speed, high-sensitivity three-dimensional imaging to track individual mitochondria in freshly dissociated smooth muscle cells. Resting mitochondrial fluorescence, an exponential function of resting DeltaPsi(m), varied among mitochondria and was approximately normally distributed. Spontaneous changes in mitochondrial fluorescence, indicating depolarizations and repolarizations in DeltaPsi(m), were observed. The depolarizations were reversible and did not result in permanent depolarization of the mitochondria. The magnitude of the flickers ranged from <10 mV to >100 mV with a mean of 17.6 +/- 1.0 mV (n = 360) and a distribution skewed to smaller values. Nearly all mitochondria flickered, and they did so independently of one another, indicating that mitochondria function as independent units in the myocytes employed here.

Site of action of fatty acids and other charged lipids on BKCa channels from arterial smooth muscle cells.

Fatty acids and other negatively charged single-chain lipids increase large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel activity, whereas sphingosine and other positively charged single-chain lipids suppress activity. Because these molecules are effective on both inside-out and outside-out patches and because they can flip across the bilayer, the location of their site of action is unclear. To identify the site of action of charged lipids on this channel, we used two compounds that are unlikely to flip across the lipid bilayer. Palmitoyl coenzyme A (PCoA) was used to identify the site of action of negatively charged lipids, and a positively charged myristoylated pentapeptide (myr-KPRPK) was used to investigate the site of action of positively charged lipids. The effect of these compounds on channel activity was studied in excised patches using patch-clamp techniques. In "normal" ionic strength solutions and in experiments where high-ionic strength solutions were used to shield membrane surface charge, PCoA increased channel activity only when applied to outside-out patches, suggesting that the site of action of negatively charged lipids is located on the outer surface of the membrane. A decrease in activity, similar to that of other positively charged lipids, was observed only when myr-KPRPK was applied to outside-out patches, suggesting that positively charged lipids suppress activity by also acting on the outer membrane surface. Some channel blockade effects of myr-KPRPK and KPRPK are also described. The sidedness of action suggests that modulation of channel activity by single-chain lipids can occur by their interaction with the channel protein.

Modulation of BK(Ca) channel activity by fatty acids: structural requirements and mechanism of action.

To determine the mechanism of fatty acid modulation of rabbit pulmonary artery large-conductance Ca2+ -activated K+ (BK(Ca)) channel activity, we studied effects of fatty acids and other lipids on channel activity in excised patches with patch-clamp techniques. The structural features of the fatty acid required to increase BK(Ca) channel activity (or average number of open channels, NP(o)) were identified to be the negatively charged head group and a sufficiently long (C > 8) carbon chain. Positively charged lipids like sphingosine, which have a sufficiently long alkyl chain (C >or= 8), produced a decrease in NP(o). Neutral and short-chain lipids did not alter NP(o). Screening of membrane surface charge with high-ionic-strength bathing solutions (330 mM K+ or 130 mM K+, 300 mM Na+) did not alter the modulation of the BK(Ca) channel NP(o) by fatty acids and other charged lipids, indicating that channel modulation is unlikely to be due to an alteration of the membrane electric field or the attraction of local counterions to the channel. Fatty acids and other negatively charged lipids were able to modulate BK(Ca) channel activity in bathing solutions containing 0 mM Ca2+, 20 mM EGTA, suggesting that calcium is not required for this modulation. Together, these results indicate that modulation of BK(Ca) channels by fatty acids and other charged lipids most likely occurs by their direct interaction with the channel protein itself or with some other channel-associated component.

Spontaneous transient outward currents arise from microdomains where BK channels are exposed to a mean Ca(2+) concentration on the order of 10 microM during a Ca(2+) spark.

Ca(2+) sparks are small, localized cytosolic Ca(2+) transients due to Ca(2+) release from sarcoplasmic reticulum through ryanodine receptors. In smooth muscle, Ca(2+) sparks activate large conductance Ca(2+)-activated K(+) channels (BK channels) in the spark microdomain, thus generating spontaneous transient outward currents (STOCs). The purpose of the present study is to determine experimentally the level of Ca(2+) to which the BK channels are exposed during a spark. Using tight seal, whole-cell recording, we have analyzed the voltage-dependence of the STOC conductance (g((STOC))), and compared it to the voltage-dependence of BK channel activation in excised patches in the presence of different [Ca(2+)]s. The Ca(2+) sparks did not change in amplitude over the range of potentials of interest. In contrast, the magnitude of g((STOC)) remained roughly constant from 20 to -40 mV and then declined steeply at more negative potentials. From this and the voltage dependence of BK channel activation, we conclude that the BK channels underlying STOCs are exposed to a mean [Ca(2+)] on the order of 10 microM during a Ca(2+) spark. The membrane area over which a concentration > or =10 microM is reached has an estimated radius of 150-300 nm, corresponding to an area which is a fraction of one square micron. Moreover, given the constraints imposed by the estimated channel density and the Ca(2+) current during a spark, the BK channels do not appear to be uniformly distributed over the membrane but instead are found at higher density at the spark site.

Natural bile acids and synthetic analogues modulate large conductance Ca2+-activated K+ (BKCa) channel activity in smooth muscle cells.

Bile acids have been reported to produce relaxation of smooth muscle both in vitro and in vivo. The cellular mechanisms underlying bile acid-induced relaxation are largely unknown. Here we demonstrate, using patch-clamp techniques, that natural bile acids and synthetic analogues reversibly increase BK(Ca) channel activity in rabbit mesenteric artery smooth muscle cells. In excised inside-out patches bile acid-induced increases in channel activity are characterized by a parallel leftward shift in the activity-voltage relationship. This increase in BK(Ca) channel activity is not due to Ca(2+)-dependent mechanism(s) or changes in freely diffusible messengers, but to a direct action of the bile acid on the channel protein itself or some closely associated component in the cell membrane. For naturally occurring bile acids, the magnitude of bile acid-induced increase in BK(Ca) channel activity is inversely related to the number of hydroxyl groups in the bile acid molecule. By using synthetic analogues, we demonstrate that such increase in activity is not affected by several chemical modifications in the lateral chain of the molecule, but is markedly favored by polar groups in the side of the steroid rings opposite to the side where the methyl groups are located, which stresses the importance of the planar polarity of the molecule. Bile acid-induced increases in BK(Ca) channel activity are also observed in smooth muscle cells freshly dissociated from rabbit main pulmonary artery and gallbladder, raising the possibility that a direct activation of BK(Ca) channels by these planar steroids is a widespread phenomenon in many smooth muscle cell types. Bile acid concentrations that increase BK(Ca) channel activity in mesenteric artery smooth muscle cells are found in the systemic circulation under a variety of human pathophysiological conditions, and their ability to enhance BK(Ca) channel activity may explain their relaxing effect on smooth muscle.