Amy Tse

Local Ca2+ release from internal stores controls exocytosis in pituitary gonadotrophs

Exocytosis and the cell-averaged cytosolic [Ca2+], [Ca2+]i, were tracked in single gonadotrophs. Cells released 100 granules/s at 1 microM = [Ca2+]i when gonadotropin-releasing hormone (GnRH) activated IP3-mediated Ca2+ release from internal stores, but only 1 granule/s when [Ca2+]i was raised uniformly to 1 microM by other means. Strong exocytosis was then seen only at higher [Ca2+]i (half-maximal at 16 microM). Parallel second messengers did not contribute to GnRH-induced exocytosis, because IP3 alone was as effective as GnRH, and because even GnRH failed to trigger rapid exocytosis when the [Ca2+]i rise was blunted by EGTA. When [Ca2+]i was released from stores, exocytosis depended on [Ca2+]i rising rapidly, as if governed by Ca2+ flux into the cytosol. We suggest that IP3 releases Ca2+ selectively from subsurface cisternae, raising [Ca2+] near exocytic sites 5-fold above the cell average.

Calcium homeostasis in identified rat gonadotrophs.

1. Whole‐cell voltage clamp was used in conjunction with the fluorescent Ca2+ indicator indo‐1 to measure extracellular Ca2+ entry and intracellular Ca2+ concentrations ([Ca2+]i) in rat gonadotrophs identified with the reverse haemolytic plaque assay. 2. Depolarizations to potentials more positive than ‐40 mV elicited inward Ca2+ current (ICa) and transient elevations of [Ca2+]i. 3. The relationship between [Ca2+]i elevations and Ca2+ entry with different Ca2+ buffer concentrations in the pipette showed that endogenous Ca2+ buffers normally bind approximately 99% of the Ca2+ entering the cell. 4. With [Ca2+]i elevations less than 500 nM, decay of [Ca2+]i could be approximated by an exponential whose time constant increased with the concentration of exogenous Ca2+ buffers. 5. Inhibitors of intracellular Ca(2+)‐ATPases, thapsigargin, cyclopiazonic acid (CPA) and 2,5‐di‐(tert‐butyl)‐1,4‐benzohydroquinone (BHQ), caused [Ca2+]i to rise. Application of BHQ during [Ca2+]i oscillations induced by gonadotrophin‐releasing hormone (GnRH) terminated the oscillation in a slowly decaying elevation. BHQ slowed the decay of depolarization‐induced [Ca2+]i elevations about 3‐fold. 6. Taking into account the Ca2+ buffering properties of the cytoplasm permitted estimation of the fluxes and rate constants for Ca2+ movements in gonadotrophs. The intracellular store is a major determinant of Ca2+ homeostasis in gonadotrophs.

Mechanism underlying corticotropin-releasing hormone (CRH) triggered cytosolic Ca2+ rise in identified rat corticotrophs.

1 The patch‐clamp technique was used in conjunction with the fluorescent Ca2+ indicator indo‐1 to measure simultaneously cytosolic Ca2+ concentration ([Ca2+]i) and membrane potential in single rat corticotrophs identified with the reverse haemolytic plaque assay. 2 Application of the adrenocorticotropin (ACTH) secretagogue, corticotropin‐releasing hormone (CRH), triggered a sustained [Ca2+]i elevation and membrane depolarization. 3 The CRH action was mediated via the cAMP‐dependent protein kinase cascade. Both the CRH‐induced depolarization and [Ca2+]i elevation could be mimicked by extracellular application of the adenylate cyclase activator forskolin or the membrane‐permeable cAMP analogue, 8‐(4‐chlorophenylthio)‐adenosine‐3’,5’‐cyclic monophosphate (8‐CPT‐cAMP). Intracellular adenosine cyclic 3’,5’‐(Rp)‐phosphothioate (Rp‐cAMPS), a protein kinase A inhibitor, abolished the CRH effects. 4 Voltage‐clamp studies suggest that the CRH‐triggered depolarization was due to the reduction of background K+ conductances. The CRH‐sensitive current was Ca2+ independent and was insensitive to the K+ channel blockers tetraethylammonium (TEA) or 4‐amino‐pyridine (4‐AP), but could be partially inhibited by Ba2+. 5 The CRH‐triggered steady‐state depolarization stimulated extracellular Ca2+ entry via voltage‐gated Ca2+ channels and raised [Ca2+]i. CRH failed to stimulate [Ca2+]i rise in cells that were voltage clamped at their resting potential. Removal of extracellular Ca2+ or inhibition of Ca2+ channels by Ni2+ abolished the [Ca2+]i rise. 6 Voltage‐clamp studies of voltage‐gated Ca2+ channels using Ba2+ as charge carrier show that ∼90% of the channels were available for activation at the resting potential. CRH did not enhance the voltage‐gated Ca2+ channels.

ATP triggers intracellular Ca2+ release in type II cells of the rat carotid body

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 μm) 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 μm), 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 ≫α,β‐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.

Signaling Mechanisms during the Response of Pituitary Gonadotropes to GnRH

Publisher Summary The pituitary gland is a versatile hormonal interface between the nervous system and the rest of the body, a warehouse of peptides waiting for nervous requisitions. In mammals, the anterior pituitary can secrete at least six major peptide hormones from at least five cell types. Each cell is under the control of specific releasing hormones and neurotransmitters secreted into the pituitary portal circulation by hypothalamic neurons. This chapter describes a study of G protein-mediated modulation of ion channels, which involved working with muscarinic activation of a K+ channel in cardiac trial cells and GnRH and muscarinic inhibition of a K+ channel in frog sympathetic ganglia, where GnRH was a neurotransmitter. The work with GnRH has explained the activities of gondaotrope, where a chain of events is triggered by GnRH. The primary secretory response to GnRH is unambiguously attributable to a steady rise of IP3that leads to an oscillatory release of Ca2+ from intracellular stores. Each elevation brings [Ca2+]i well above the 300 nM level that suffices to initiate exocytosis of numerous secretory granules. The secretory stimulus for gonadotropes does not initiate a major entry of extracellular Ca2+ as a primary signal for exocytosis. The electrophysiological responses of stimulated gonadotropes include a hyperpolarization during periods of [Ca2+]i rise instead of a depolarization.

ATP inhibits the hypoxia response in type I cells of rat carotid bodies

During hypoxia, ATP was released from type I (glomus) cells in the carotid bodies. We studied the action of ATP on the intracellular Ca2+ concentration ([Ca2+]i) of type I cells dissociated from rat carotid bodies using a Ca2+ imaging technique. ATP did not affect the resting [Ca2+]i but strongly suppressed the hypoxia‐induced [Ca2+]i elevations in type I cells. The order of purinoreceptor agonist potency in inhibiting the hypoxia response was 2‐methylthioATP > ATP > ADP ≫ α, β‐methylene ATP > UTP, implicating the involvement of P2Y1 receptors. Simultaneous measurements of membrane potential and [Ca2+]i show that ATP inhibited the hypoxia‐induced Ca2+ signal by reversing the hypoxia‐triggered depolarization. However, ATP did not oppose the hypoxia‐mediated inhibition of the oxygen‐sensitive TASK‐like K+ background current. Neither the inhibition of the large‐conductance Ca2+‐activated K+ (maxi‐K) channels nor the removal of extracellular Na+ could affect the inhibitory action of ATP. Under normoxic condition, ATP caused hyperpolarization and increase in cell input resistance. These results suggest that the inhibitory action of ATP is mediated via the closure of background conductance(s) other than the TASK‐like K+, maxi‐K or Na+ channels. In summary, ATP exerts strong negative feedback regulation on hypoxia signaling in rat carotid type I cells.

Regulation of exocytosis via release of Ca2 from intracellular stores

The release of Ca(2+) from intracellular stores is an important trigger for secretion in many cell types. Depending on the spatial relationship between the intracellular Ca(2+) stores and the site of exocytosis, the Ca(2+) signal can be very local or spread throughout the entire cell. Here, we review how the release of Ca(2+) from inositol trisphospate (IP(3))-sensitive stores contributes differently to the stimulus-secretion coupling in three types of secretory cells (acinar cells of the pancreas, gonadotrophs, and corticotrophs of the anterior pituitary gland). We propose that in both pancreatic acinar cells and pituitary gonadotrophs the IP(3)-sensitive stores may be in close proximity to the sites of exocytosis such that the concentration of Ca(2+) at these sites are transiently much higher than the average cytosolic Ca(2+) concentration. In contrast, the local Ca(2+) gradient is less prominent in pituitary corticotrophs. Finally, some recent technical developments that may contribute significantly to future investigations of local Ca(2+) signals are discussed.

Influence of Cholesterol on Catecholamine Release from the Fusion Pore of Large Dense Core Chromaffin Granules

Changes in cellular cholesterol can affect exocytosis, but the influence of cholesterol in fusion pore kinetics is unclear. Using carbon fiber amperometry, we monitored quantal catecholamine release from rat chromaffin cells. To bypass any possible effect of cholesterol perturbation on ion channels or the colocalization of voltage-gated Ca2+ channels with sites of exocytosis, exocytosis was stimulated via uniform elevation of cytosolic [Ca2+] (with whole-cell dialysis of a Ca2+-buffered solution). Under this condition, alterations of cellular cholesterol affected neither the mean number of amperometric events triggered per cell nor their quantal size and the kinetics of their main spike (which reflects the rapid release during and after rapid fusion pore dilation). In contrast, the reduction of cellular cholesterol shortened the “prespike foot” signals (which reflect the leakage of catecholamine via a semi-stable fusion pore) and reduced the proportion of “stand-alone foot” signals (which reflect the release via a flickering fusion pore that may close before it dilates significantly), whereas an oversupply of cholesterol had opposite effects. Acute extraction of cholesterol from the cytosol (via whole-cell dialysis of a cholesterol extractor) also shortened the prespike foot signals and reduced the proportion of stand-alone foot signals, but acute extracellular application of cholesterol extractor or “soluble” cholesterol had no effect. Our data raise the possibility that cholesterol molecules, particularly those in the cytoplasmic leaflet, helps to constrain the narrow waistline of a semi-stable fusion pore while it is flickering or before it starts to dilate rapidly.

α‐Adrenergic stimulation of cytosolic Ca2+ oscillations and exocytosis in identified rat corticotrophs

1 The patch clamp technique was used in conjunction with a fluorescent Ca2+ indicator (indo‐1, or indo‐1FF) to measure simultaneously cytosolic Ca2+ concentration ([Ca2+]i), ionic current and changes in membrane capacitance in single rat corticotrophs identified with the reverse haemolytic plaque assay. 2 Application of the adrenocorticotropin (ACTH) secretagogue noradrenaline (NA; norepinephrine), triggered [Ca2+]i oscillation in corticotrophs via α‐adrenergic receptors and the guanosine trisphosphate (GTP) binding protein‐coupled phosphoinositide pathway. 3 Simultaneous measurement of [Ca2+]i and capacitance shows that exocytosis was triggered during the first cycle of NA‐induced [Ca2+]i oscillation and the mean increase in cell membrane surface area was 1.4 ± 0.3 % (n= 6). 4 When Ca2+ was directly released from the inositol 1,4,5 trisphosphate (IP3)‐sensitive store via flash photolysis of caged IP3, the mean increase in cell surface area was 1.5 ± 0.5 % (n= 6). Thus, NA‐stimulated ACTH secretion in rat corticotrophs is closely coupled to intracellular Ca2+ release. 5 Large and rapid elevation of [Ca2+]i (>15 μm) via flash photolysis of caged Ca2+ triggered two phases of exocytosis: a rapid exocytic burst that was complete in ∼100 ms and a slow burst that continued for many seconds. 6 The rapid exocytic burst reflected the exhaustion of a pool of readily releasable granules and, on average, increased the cell surface by 2.8 ± 0.1 % (n= 14). 7 We suggest that the relatively weak exocytic response in corticotrophs during intracellular Ca2+ release may be partially attributed to a smaller pool of readily releasable granules.

Autocrine and paracrine actions of ATP in rat carotid body

Carotid bodies are peripheral chemoreceptors that detect lowering of arterial blood O(2) level. The carotid body comprises clusters of glomus (type I) cells surrounded by glial-like sustentacular (type II) cells. Hypoxia triggers depolarization and cytosolic [Ca(2+)] ([Ca(2+)](i)) elevation in glomus cells, resulting in the release of multiple transmitters, including ATP. While ATP has been shown to be an important excitatory transmitter in the stimulation of carotid sinus nerve, there is considerable evidence that ATP exerts autocrine and paracrine actions in carotid body. ATP acting via P2Y(1) receptors, causes hyperpolarization in glomus cells and inhibits the hypoxia-mediated [Ca(2+)](i) rise. In contrast, adenosine (an ATP metabolite) triggers depolarization and [Ca(2+)](i) rise in glomus cells via A(2A) receptors. We suggest that during prolonged hypoxia, the negative and positive feedback actions of ATP and adenosine may result in an oscillatory Ca(2+) signal in glomus cells. Such mechanisms may allow cyclic release of transmitters from glomus cells during prolonged hypoxia without causing cellular damage from a persistent [Ca(2+)](i) rise. ATP also stimulates intracellular Ca(2+) release in sustentacular cells via P2Y(2) receptors. The autocine and paracrine actions of ATP suggest that ATP has important roles in coordinating chemosensory transmission in the carotid body.