Localized, brief Ca 2ϩ transients (Ca 2ϩ 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 Ca 2ϩ imaging. Ca 2ϩ syntillas (scintilla, L., spark, from a synaptic structure, a nerve terminal) are caused by release of ϳ250,000

Ca

A central concept in the physiology of neurosecretion is that a rise in cytosolic [Ca2+] in the vicinity of plasmalemmal Ca2+ channels due to Ca2+ influx elicits exocytosis. Here, we examine the effect on spontaneous exocytosis of a rise in focal cytosolic [Ca2+] in the vicinity of ryanodine receptors (RYRs) due to release from internal stores in the form of Ca2+ syntillas. Ca2+ 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 Ca2+ syntillas in mouse adrenal chromaffin cells. Here, we examine the effect of Ca2+ syntillas on exocytosis in chromaffin cells. In such a study on elicited exocytosis, there are two sources of Ca2+: one due to influx from the cell exterior through voltage-gated Ca2+ channels, and that due to release from intracellular stores. To eliminate complications arising from Ca2+ 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 Ca2+ syntillas act to inhibit spontaneous exocytosis, and we propose a simple model to account quantitatively for this action of syntillas.

Gautier M, Hyvelin J-M, de Crescenzo V, Eder V, Bonnet P. Heterogeneous Kv1 function and expression in coronary myocytes from right and left ventricles in rats. Am J Physiol Heart Circ Physiol 292: H475-H482, 2007. First published May 26, 2006; doi:10.1152/ajpheart.00774.2005.-Coronary blood flow control is not uniform along the vascular tree and particularly between the right coronary artery and the left anterior descending artery. Resting membrane potential that contributes largely to the vascular tone is mainly regulated by K ϩ channels in coronary myocytes. In the present study, we hypothesized that right coronary artery (RCA) and left coronary artery (LCA) exhibited a cell-specific function of K ϩ channels. The net outward current was markedly greater in RCA compared with LCA cells, and this difference was due to a larger 4-aminopyridine (4-AP)-sensitive voltage-gated potssium (Kv) current in RCA cells, whereas the iberiotoxin (IbTx)-sensitive, large conductance Ca 2ϩ -dependent potassium (BK Ca) current was smaller in RCA cells. To go further in the molecular identity of this Kv current, we used 50 nM correolide, which specifically blocked Kv1 family ␣-subunits. Outward currents generated by ramp depolarization protocols were highly sensitive to correolide in both RCA and LCA cells, suggesting that Kv1 contributed for a large part to the net outward current. 4-APinduced contractions in isolated RCA, and LCA were greater than IbTx-induced contraction. Furthermore, the 4-AP-induced contraction in RCA was significantly greater than that in LCA, which is in agreement with the electrophysiological data. Finally, the Kv1.2 ␣-subunit but not the Kv1.5 was detected in both RCA and LCA using primary specific antibody in Western blotting and immunofluorescence assay, and expression of Kv1.2 ␣-subunit was markedly higher in RCA compared with LCA. In summary, we reported for the first time a heterogeneous function and expression of Kv1 ␣-subunits in rat coronary myocytes isolated from RCA or LCA. coronary circulation; potassium channel; perfused coronary; membrane currents THE FUNCTIONAL CHARACTERISTICS of the coronary circulation may be described in terms of the relationship between steadystate perfusion pressure and the amount of blood flow. There are functional hemodynamic differences among coronary vascular beds that are linked to pressure differences along the vascular bed. These differences in compressive forces result in almost continuous flow in the right coronary bed, whereas flow in the anterior left descending bed occurs mainly during diastole (24, 30). Furthermore, when aortic pressure rises and the ventricle is loaded, the right coronary bed exhibits reflex increase of blood flow, whereas the anterior left descending coronary artery vasodilates poorly (29). Such differences in the hemodynamic profile between the left (LCA) and right coronary arteries (RCA) may underline differences in the regulation of basal tone between these two vascular beds.The coronary vascular tone is modulated by several factors, ...

The type 1 ryanodine receptor (RyR1) is expressed widely in the brain, with high levels in the cerebellum, hippocampus, and hypothalamus.

Recently, highly localized Ca 2ϩ release events, similar to Ca 2ϩ sparks in muscle, have been observed in neuronal preparations. Specifically, in murine neurohypophysial terminals (NHT), these events, termed Ca 2ϩ syntillas, emanate from a ryanodine-sensitive intracellular Ca 2ϩ pool and increase in frequency with depolarization in the absence of Ca 2ϩ influx. Despite such knowledge of the nature of these Ca 2ϩ release events, their physiological role in this system has yet to be defined. Such localized Ca 2ϩ 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 2ϩ imaging has enabled us to establish that spontaneous neuropeptide release and Ca 2ϩ 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.

The mechanism of action of halothane is not fully understood in pulmonary circulation and especially in chronic hypertension models. As the 5-hydroxytryptamine (5-HT) pulmonary vasoconstrictor response increases in chronic hypoxic rat, halothane could differentially attenuate this vasoconstriction response on normoxic and chronic hypoxic rats. The effect of halothane on 5-HT-induced contractions on pulmonary arteries isolated from normoxic and chronic hypoxic rats was compared. Rings dissected from proximal pulmonary artery without endothelium were attached to a force transducer to record tone and placed in an organ chamber gassed either by air or air + halothane (1-5%). Contractions induced by (10(-4) M) 5-HT were used to test the effect of halothane on rings isolated from normoxic and chronic hypoxic rats. 5-Hydroxytryptamine-mediated contractions were more sensitive to external calcium in normoxic than chronic hypoxic rings. In calcium-free solution, with verapamil or cadmium the amplitude of remaining 5-HT-induced contractions were greater in chronic hypoxic rings. Halothane (1-5%) decreased 5-HT-mediated contractions in normoxic and chronic hypoxic rings. The effect occurred with no change of pD2 for 5-HT and was more pronounced in normoxic rings. The effect of halothane on both rings was abolished in the absence of external calcium or in the presence of verapamil. In the presence of cadmium, 5% halothane had no effect on normoxic rings but still decreased the remaining 5-HT contraction on chronic hypoxic rings. The findings suggested that halothane decreased sarcolemmal calcium entry in pulmonary artery rings by a cadmium-sensitive pathway in normoxic rats and by a cadmium-insensitive pathway in chronic hypoxic rats.

In isolated coronary arteries, hypoxia induces an increase in tone by releasing an unidentified endothelium-derived contracting factor (EDCF). Isometric force was measured in an isolated rabbit coronary artery ring at 37 degrees C in control and high K+ (40 mM) pre-contracted conditions. Hypoxia (15 mmHg pO2) induced by equilibrating the perfusate with nitrogen. Hypoxia did not affect the resting tone but induced an endothelium-dependent contraction on pre-contracted rings. Inhibitors of nitric oxide (NO) were tested, L-NAME (10(-4) M) totally and L-NMMA (10(-4) M) partially convert the hypoxic contraction to an hypoxic relaxation. The addition of L-arginine (10(-4) or 10(-3) M) did not restore the response. Methylene blue (10( -5) M) and ODQ (1 H-[1,2,4] oxadiazolo-[4,3-a] quinoxalin-1-one, 10(-5) M), both inhibitors of guanylate cyclase, also changed the hypoxic contraction into a hypoxic relaxation. Catalase (1200 U/ml), which decomposes hydrogen peroxide (H2O2), and superoxide dismutase (150 U/ml, SOD), a free radical scavenger, did not change the hypoxic response but quinacrine (50 microM), an inhibitor of phospholipase A2, significantly decreased it. Inhibitors of arachidonic acid metabolism (indomethacin, diethylcarbamazine, miconazole) however did not affect the hypoxic response. We conclude that in K+ pre-contracted rabbit coronary artery rings, hypoxia induces a contraction which is nitric oxide and arachidonic acid dependent.