Leptin is an adipokine produced by the adipose tissue regulating body weight through its appetite-suppressing effect and, as such, exerts a relevant action on the adipo-adrenal axis. Leptin has a dual action on adrenal mouse chromaffin cells both at rest and during stimulation. At rest, the adipokine inhibits the spontaneous firing of most cells by enhancing the probability of BK channel opening through the phosphoinositide 3-kinase signalling cascade. This inhibitory effect is absent in db(-) /db(-) mice deprived of Ob receptors. During sustained stimulation, leptin preserves cell excitability by generating well-adapted action potential (AP) trains of lower frequency and broader width and increases catecholamine secretion by increasing the size of the ready-releasable pool and the rate of vesicle release. In conclusion, leptin dampens AP firing at rest but preserves AP firing and enhances catecholamine release during sustained stimulation, highlighting the importance of the adipo-adrenal axis in the leptin-mediated increase of sympathetic tone and catecholamine release.

Mouse chromaffin cells (MCCs) of the adrenal medulla possess fast-inactivating Nav channels whose availability alters spontaneous action potential firing patterns and the Ca(2+)-dependent secretion of catecholamines. Here, we report MCCs expressing large densities of neuronal fast-inactivating Nav1.3 and Nav1.7 channels that carry little or no subthreshold pacemaker currents and can be slowly inactivated by 50% upon slight membrane depolarization. Reducing Nav1.3/Nav1.7 availability by tetrodotoxin or by sustained depolarization near rest leads to a switch from tonic to burst-firing patterns that give rise to elevated Ca(2+)-influx and increased catecholamine release. Spontaneous burst firing is also evident in a small percentage of control MCCs. Our results establish that burst firing comprises an intrinsic firing mode of MCCs that boosts their output. This occurs particularly when Nav channel availability is reduced by sustained splanchnic nerve stimulation or prolonged cell depolarizations induced by acidosis, hyperkalaemia and increased muscarine levels.

Using a Ca2+-imaging technique, we studied the action of ATP on the intracellular Ca2+ concentration ([Ca2+]i) of fura-2-loaded mixtures of type I and type II cells dissociated from rat carotid bodies. ATP (100 micro M) triggered a transient rise in [Ca2+]i in the spindle-shaped type II (sustentacular) cells, but not the ovoid type I (glomus) cells. When challenged with ionomycin (1 micro M), no amperometry signal could be detected from the ATP-responsive type II cells, suggesting that these cells lacked catecholamine-containing granules. In contrast, KCl depolarization triggered robust quantal catecholamine release from type I cells that were not responsive to ATP. In type II cells voltage clamped at -70 mV, the ATP-induced [Ca2+]i rise was not accompanied by any current change, suggesting that P2X receptors are not involved. The ATP-induced Ca2+ signal could be observed in the presence of Ni2+ (a blocker of voltage-gated Ca2+ channels) or in the absence of extracellular Ca2+, indicating that Ca2+ release from intracellular stores was the dominant mechanism. The order of purinoreceptor agonist potency in triggering the [Ca2+]i rise was UTP > ATP > 2-methylthioATP >> alpha,beta-methyleneATP, implicating the involvement of P2Y2 receptors. In carotid body sections, immunofluorescence revealed localization of P2Y2 receptors on spindle-shaped type II cells that partially enveloped ovoid type I cells. Since ATP is released from type I cells during hypoxia, we suggest that the ATP-induced Ca2+ signal in type II cells can mediate paracrine interactions within the carotid bodies.

The catechol metabolites of 17β-oestradiol (E2 β), 2-hydroxyoestradiol (2-OHE2 ) and 4-hydroxyoestradiol (4-OHE2 ), stimulate proliferation of pregnancy-derived ovine uterine artery endothelial cells (P-UAECs) through β-adrenoceptors (β-ARs) and independently of the classic oestrogen receptors (ERs). Herein we show that activation of ERK1/2, p38 and JNK mitogen activated protein kinases (MAPKs) is necessary for 2-OHE2 - and 4-OHE2 -induced P-UAEC proliferation, as well as proliferation induced by the parent hormone E2 β and other β-AR signalling hormones (i.e. catecholamines). Conversely, although 2-OHE2 and 4-OHE2 rapidly activate phosphatidylinositol 3-kinase (PI3K), its activation is not involved in catecholoestradiol-induced P-UAEC proliferation. We also show for the first time the signalling mechanisms involved in catecholoestradiol-induced P-UAEC proliferation; which converge at the level of MAPKs with the signalling mechanisms mediating E2 β- and catecholamine-induced proliferation. The present study advances our understanding of the complex signalling mechanisms involved in regulating uterine endothelial cell proliferation during pregnancy.

Before the preganglionic regulation of the adrenal medulla is established, hypoxia acts directly on the chromaffin cells to evoke the secretion of catecholamines. This direct action of hypoxia is suppressed by the gradual development of the preganglionic innervation and we have proposed that opioid peptides released from the adrenal splanchnic nerves may be responsible for this suppression. The effects of the specific opioid agonists DPDPE (delta-agonist), U-62066 (kappa-agonist) and DALDA (mu-agonist) on the hypoxia-evoked response were investigated in both a whole-gland preparation and in isolated adrenal chromaffin cells using amperometry, whole-cell patch clamping and measurement of cytosolic [Ca(2+)]. The combined application of mu- and kappa-type agonists abolished the hypoxia-evoked catecholamine secretion from whole perfused adrenal gland. In isolated chromaffin cells, mu- and kappa-opioid agonists reduced the rise in [Ca(2+)](i) that results from exposure to hypoxia. Both agonists decreased the voltage-dependent Ca(2+) current in these cells. The mu-agonist increased the conductance through SK-type K(+) channels and this action offset the decrease in K(+) conductance produced by exposure to hypoxia. The kappa-type agonist decreased the conductance through an action on BK-type K(+) channels, a class of channels that are not involved in initiating the direct response to hypoxia. These data suggest that opioids, through their action on SK channels and voltage-dependent Ca(2+) channels, may be responsible for the nerve-induced suppression of the hypoxic response of adrenal chromaffin cells and that these effects of endogenous opioids are mediated via mu- and kappa-type receptors.

The major source of serotonin (5-HT) in the body is the enterochromaffin (EC) cells lining the intestinal mucosa of the gastrointestinal tract. Despite the fact that EC cells synthesise ∼95% of total body 5-HT, and that this 5-HT has important paracrine and endocrine roles, no studies have investigated the mechanisms of 5-HT release from single primary EC cells. We have developed a rapid primary culture of guinea-pig and human EC cells, allowing analysis of single EC cell function using electrophysiology, electrochemistry, Ca(2+) imaging, immunocytochemistry and 3D modelling. Ca(2+) enters EC cells upon stimulation and triggers quantal 5-HT release via L-type Ca(2+) channels. Real time amperometric techniques reveal that EC cells release 5-HT at rest and this release increases upon stimulation. Surprisingly for an endocrine cell storing 5-HT in large dense core vesicles (LDCVs), EC cells release 70 times less 5-HT per fusion event than catecholamine released from similarly sized LDCVs in endocrine chromaffin cells, and the vesicle release kinetics instead resembles that observed in mammalian synapses. Furthermore, we measured EC cell density along the gastrointestinal tract to create three-dimensional (3D) simulations of 5-HT diffusion using the minimal number of variables required to understand the physiological relevance of single cell 5-HT release in the whole-tissue milieu. These models indicate that local 5-HT levels are likely to be maintained around the activation threshold for mucosal 5-HT receptors and that this is dependent upon stimulation and location within the gastrointestinal tract. This is the first study demonstrating single cell 5-HT release in primary EC cells. The mode of 5-HT release may represent a unique mode of exocytosis amongst endocrine cells and is functionally relevant to gastrointestinal sensory and motor function.