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The release of hormones and neurotransmitters by regulated exocytosis requires the delivery of secretory vesicles to the plasma membrane, where they dock and become primed for fusion with the plasma membrane. Upon stimulation a fusion pore is formed through which cargo molecules diffuse out of the vesicle lumen into the extracellular space. After the cargo release the fusion pore either closes (kiss-and-run, transient exocytosis), fluctuates between an open and a closed state (for short times, fusion pore flickering, or for rather longer periods, ‘pulsing pore’) or expands irreversibly (full fusion exocytosis). In almost all secretory cells spontaneous secretion of vesicle cargo can be detected in the absence of stimulation. Spontaneous and stimulated exocytosis were thought to exhibit similar properties at elementary level, differing only in the probability of occurrence. However, recent studies indicate that spontaneous exocytosis differs from the stimulated one in many respects, therefore opening questions about the physiological role of spontaneous exocytosis. In this report we address the elementary properties of spontaneous and stimulated peptidergic vesicle discharge which appears to be modulated by fusion pore conductance (diameter) and fusion pore gating.

We have prepared fresh pituitary gland slices from adult and, for the first time, from newborn mice to assess modulation of secretory activity via voltage-activated Ca2+ channels (VACCs). Currents through VACCs and membrane capacitance have been measured with the whole-cell patch-clamp technique. Melanotrophs in newborns were significantly larger than in adults. In both newborn and adult melanotrophs activation of VACCs triggered exocytosis. All pharmacologically isolated VACC types contributed equally to the secretory activity. However, the relative proportion of VACCs differed between newborns and adults. In newborn cells L-type channels dominated and, in addition, an exclusive expression of a toxin-resistant R-type-like current was found. The expression of L-type VACCs was up-regulated by the increased oestrogen levels observed in females, and was even more emphasized in the cells of pregnant females and oestrogen-treated adult male mice. We suggest a general mechanism modulating endocrine secretion in the presence of oestrogen and particularly higher sensitivity to treatments with L-type channel blockers during high oestrogen physiological states.

1. We measured membrane capacitance (Cm) in cultured rat melanotrophs pretreated with Clostridium spiroforme toxin (CST), which specifically depolymerizes cortical filamentous actin (F-actin). Phalloidin staining confirmed that CST treatment depolymerised the F-actin. 2. In control cells, cytosol dialysis with 1 microM Ca2+i increased Cm by 23 +/- 4 % (n = 11) relative to the resting Cm 400 s after the start of patch rupture. In CST-treated cells the increase in Cm was 32 +/- 5 % (n = 15), not significantly different from controls. The rate of Cm increase was affected transiently by CST treatment, peaking at 1 min after patch rupture. The maximal rate of Cm increase was 4.27 +/- 0.85 fF s-1 (n = 12; measured 200 s after the start of patch rupture) in controls and 8.0 +/- 1.35 fF s-1 (n = 23; measured 75 s after the start of patch rupture) in CST-treated cells (P0.01). 3. In control cells cytosol dialysis with 0 microM Ca2+i decreased Cm by 9 +/- 3 % (n = 7), in CST-treated cells Cm increased by 11 +/- 3 % (n = 7) relative to resting Cm 400 s after the start of cytosol dialysis. The rate of change in Cm remained constant (controls: -1 to -2 fF s-1; CST treatment: 1-2 fF s-1). 4. Transient and sustained effects of CST treatment on changes in Cm at high or low [Ca2+]i, respectively, suggest a distinct role of cytoskeleton in Ca2+-dependent and Ca2+-independent changes in Cm. Transient enhancement of the rate of Cm by CST is consistent with a barrier role of cytoskeleton in regulated exocytosis. The sustained effect of CST on Ca2+-independent changes in Cm suggests cytoskeletal involvement in endocytosis.

1. Using the patch-clamp technique, we have monitored the secretory activity of single rat melanotrophs. Changes in membrane capacitance (Cm) were measured to detect small discrete femtofarad steps. These are believed to be due to interactions between single secretory organelles (granules) and plasmalemma. 2. A new approach was introduced to measure the amplitude of discrete steps in Cm. Records of Cm were converted into time derivatives, where discrete steps appeared as transients. A transient due to a 2 fF discrete step in Cm was easily distinguished from random noise, since the probability of such a transient being due to random noise was less than 0.01. To distinguish apparent steps from noise the computer-based analysis employed a threshold of 3 times the standard deviation of the noise time derivative (dCm/dt). A phase diagram was created by plotting dCm/dt versus Cm, from which the magnitude and direction of transients were determined. Transients due to 2 fF steps (equivalent to a signal-to-noise ratio of 1) were detected with a reliability of 100%, whereas steps of 1 fF were detected with a reliability of more than 60%. The amplitude of false steps detected by the program was less than 1 fF, and the frequency of false detections of 0.075 S-1 was equal for exocytotic and endocytotic events. 3. Electron microscopy was used to measure secretory organelle size and an immunogold technique was used to label the electron micrographs with an anti-adrenocorticotrophin (ACTH) antibody. Secretory organelles in cultured and non-cultured cells were of similar diameter. All sizes of secretory granules appear to contain ACTH, since secretory organelles of similar diameter stained positively with the anti-ACTH antibodies. 4. Small discrete steps in Cm, recorded with the whole-cell configuration and loosely buffered cytosolic calcium, were similar to the estimated Cm of secretory organelles from morphological data. Thus, measured discrete steps in Cm reflect interactions between single organelle size and plasma membrane. Exocytotic and endocytotic steps were found to be of similar size. 5. To separate exocytosis from endocytosis in Cm records, we assumed that the rates of exocytosis and endocytosis were related to the respective frequencies of discrete steps in Cm. A relationship between the frequency of exocytotic, but not endocytotic events, and the rate of change in Cm was observed. Thus, under our experimental conditions, an increase in Cm could be explained by an increased rate of exocytosis in rat melanotrophs.

We have prepared fresh pituitary gland slices from adult and, for the first time, from newborn mice to assess modulation of secretory activity via voltage-activated Ca(2+) channels (VACCs). Currents through VACCs and membrane capacitance have been measured with the whole-cell patch-clamp technique. Melanotrophs in newborns were significantly larger than in adults. In both newborn and adult melanotrophs activation of VACCs triggered exocytosis. All pharmacologically isolated VACC types contributed equally to the secretory activity. However, the relative proportion of VACCs differed between newborns and adults. In newborn cells L-type channels dominated and, in addition, an exclusive expression of a toxin-resistant R-type-like current was found. The expression of L-type VACCs was up-regulated by the increased oestrogen levels observed in females, and was even more emphasized in the cells of pregnant females and oestrogen-treated adult male mice. We suggest a general mechanism modulating endocrine secretion in the presence of oestrogen and particularly higher sensitivity to treatments with L-type channel blockers during high oestrogen physiological states.

The pituitary, sometimes called the master gland due to its governance over other glands, consists of three distinct anatomical and functional parts: posterior, intermediate (pars intermedia) and anterior lobes (Fig. 1). While the posterior pituitary consists of axon terminals originating from neurons with somata in the hypothalamus, cells in the pars intermedia release peptidergic hormones under the control of neurotransmitters released into the synaptic cleft from the innervating neurons. On the other hand, the secretory activity of anterior pituitary cells, which control important bodily functions including growth, development, reproduction and responses to stress, release hormones in response to blood-borne hypothalamic factors. Following their interaction with specific surface membrane receptors and subsequent activation of intracellular signalling mechanisms they control exocytosis and thus the secretory output from anterior pituitary cells. Figure 1 Diagram depicting hypothalamus pituitary gland and an anterior pituitary cell with the GABAA receptor, Na+-K−-2Cl− co-transporter (NKCCi), the K+-Cl− co-transporter (KCC2), voltage-dependent calcium channel (VDCC). The paper by Zemkova et al. (2008) in this issue of The Journal of Physiology, brings a new insight into the regulation of anterior pituitary cells. It seems that a novel regulating factor, γ-aminobutyric acid (GABA), will have to be firmly positioned into the list of factors stimulating hormone release from these cells. Although it has been known previously that anterior pituitary cells express ionotropic and metabotropic GABA receptors which mediate the secretory hormonal output from these cells (Anderson & Mitchell, 1986; Nakayama et al. 2006), it was unclear how the ionotropic GABA receptors, which gate chloride-specific channels, function in the stimulus–secretion coupling. Specifically, it was controversial whether GABA was stimulatory or inhibitory for the secretory output of anterior pituitary cells. To address these issues Zemkova et al. (2008) first used molecular biology techniques to study the expression of molecular subunits of GABAA receptor channels in cultured anterior pituitary cells from postpubertal female rats and immortalized αT3-1 and GH3 clonal pituitary cells. Interestingly, mRNAs for all GABAA receptor subunits were found to be expressed in pituitary cells and α1/β1 subunit proteins are present in all secretory cells. Second, by using electrophysiology to voltage clamp gramicidin-perforated cells to monitor GABA-induced currents, they have shown that GABA-induced dose-dependent increases in current amplitude were inhibited by bicuculline and picrotoxin, inhibitors of GABAA receptor channels, and facilitated by diazepam and zolpidem in a concentration-dependent manner. Third, they used fluorescence methods to test whether the application of GABA and the GABAA receptor agonist muscimol affected cytosolic calcium activity, an important cytosolic stimulus of anterior pituitary cell secretory activity. Since both GABA and muscimol caused a rapid and transient increase in intracellular calcium, whereas the GABAB receptor agonist baclofen was ineffective, their data are consistent with the view that chloride-mediated depolarization activates voltage-gated calcium channels. Furthermore, the GABAA channel reversal potential for chloride ions was positive to the resting membrane potential in most cells and the activation of ion channels by GABA resulted in depolarization of cells. This prompted Zemkova et al. (2008) to consider the mechanism of chloride homeostasis, which in most brain cells is controlled by NKCC1 and KCC2, two electrically neutral cation/chloride cotransporters. The ubiquitously expressed NKCC1 derives energy from the electrochemical gradient for Na+ to take up Cl−, promoting cytosolic accumulation of Cl−, whereas KCC2 facilitates Cl− extrusion coupled to the K+ gradient (Fiumelli & Woodin, 2007). The experiments have shown that the mRNA expression of NKCC1 greatly exceeded that of KCC2, which is consistent with the relatively positive reversal potential for chloride currents. While the results of Zemkova et al. (2008) establish the excitatory role of GABA in anterior pituitary cells, a number of questions have now been opened. For example, we will have to consider that GABA is now listed as a regulatory factor of anterior pituitary cells. However, it is unclear if it is delivered from the hypothalamus via the portal vascular system. While GABA is the major inhibitory neurotransmitter in the central nervous system and is released from nerve terminals innervating pars intermedia cells (Schneggenburger & Lopez-Barneo, 1992), the mode of delivery of GABA to the cells in the anterior pituitary is probably different. Is the source of GABA the portal vasculature capturing GABA released from neurons in the median eminence? It is known that radioactive GABA readily accumulates in the pituitary (Kuroda et al. 2000; Duvilanski et al. 2000). Is it possible that GABA is released from anterior pituitary cells and thus can be considered an autocrine regulator? These questions will have to be addressed in the future.

1. Caesium ions (Cs+), applied extracellularly, caused a decrease in the input resistance of cultured spinal cord (s.c.) neurones and depolarized the neurones when they contained 140 mM-CsCl. 2. The reversal potential for Cs+-activated currents shifted 56 mV on average for a 10-fold reduction in the intracellular chloride ion (Cl-) activity, indicating that the Cs+-activated currents were specific to Cl-. 3. The activation of Cl- currents by Cs+ was not due to the depolarization-evoked release of neurotransmitter from presynaptic terminals. We therefore suggest that Cs+ were acting directly on the extracellular surface of the s.c. neurones to activate Cl- currents. 4. Cs+-activated currents showed desensitization in the presence of 140 mM-Cs+. 5. The log-log plot of the dose-response data could be fitted with a straight line with a slope of 1.7 +/- 0.4 (n = 6), indicating that at least 2 Cs+ were needed to activate a single Cl- channel. The KD of the Cs+-induced response was greater than 69 mM. 6. In outside-out patches Cs+ activated single Cl- channels. These channels were not activated by sodium or potassium ions. 7. The Cs+-activated channels displayed a total of five distinct conductance states which had mean conductances of 20, 30, 43, 66 and 92 pS. The 30 and 43 pS states were the most frequently occurring states. 8. The conductance states of the Cs+-activated channel have the same conductances as those reported for gamma-aminobutyric acid (GABA)- and glycine-activated channels in rat s.c. neurones. We therefore conclude that Cs+ activate the same type of Cl- channel as GABA and glycine through an unidentified receptor.