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CALCIUM SIGNALING AND
CALCIUM-CONTROLLED CELLULAR FUNCTIONS
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Stanko S. Stojilkovic, PhD, Head, Section on Cellular Signaling Melanija Tomić, PhD, Staff Scientist Silvana A. Andrić, PhD, Postdoctoral Fellow Arturo E.
Gonzales Iglesias, PhD, Postdoctoral Fellow Yonghua Jiang, PhD, Postdoctoral Fellow Karla Kretschmannová, PhD, Postdoctoral Fellow |
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We investigate the cellular signaling cascades
in endocrine and neuroendocrine cells and the interactions between plasma
membrane electrical events and receptor-controlled pathways. Our research
focuses on calcium signaling and cellular regulation. Our main objective is
to elucidate the receptors and channels involved in calcium signaling and the
role of calcium ions as messengers in controlling signaling, secretion, and
gene expression in anterior pituitary cells. Our approach calls for
characterizing calcium signaling from biophysical, physiological,
pharmacological, and molecular biology perspectives. Currently, we are
studying the dependence of calcium signaling function of purinergic receptor
channels on the channels’ structure, the pathways and roles of cyclic
nucleotides in control of calcium signaling, and the roles of G
protein– and tyrosine kinase–coupled receptors in controlling
calcium signaling, hormone secretion, and gene expression. Nucleotide-gated receptors and
ectonucleotidases He,a
Yan, Tomić, Gonzales Iglesias, Stojilkovic; in collaboration with
Koshimizu, Zemková Earlier published studies indicated that normal
and immortalized anterior pituitary cells express three subtypes of the G
protein–coupled purinergic P1 receptors (P1Rs): A1, A2A,
and A2B. Our work focuses on the expression and role of both
families of purinergic P2 receptors (P2Rs): the ligand-gated channels (P2XRs)
and the G protein–coupled receptors (P2YRs). The first P2R subtype
identified in anterior pituitary was P2Y2R. Recently, we
demonstrated transcripts for four additional members of this family of
receptors: P2Y1R, P2Y4R, P2Y6R, and P2Y12R
in anterior pituitary cells; we identified the P2Y1R subtype in
the lactotroph fraction of cells. Our studies also revealed the expression of
P2X2aR and its spliced form P2X2bR in somatotrophs and
gonadotrophs as well as the expression of P2X3R, P2X4R,
and P2X7R in other still unidentified pituitary cell types. In
lactotrophs, the P2X4R subtype provides a major pathway for
calcium influx–dependent signaling and prolactin (PRL) secretion.
Identification of P2Rs in other cell types is in progress. Our results
indicate that, of the 17 known nucleotide receptors, 12 are expressed in
anterior pituitary secretory cells. In that respect, only the brain expresses
more subtypes of these receptors, indicating the potential relevance of
nucleotide-receptor signaling pathways in pituitary cell functions. Nucleotides must be released by cells if they
are to act as extracellular messengers; they are then hydrolyzed by
ectonucleotidases, resulting in the formation of the respective nucleoside
and a free phosphate. The currently identified ectonucleotidases include
members of the ectonucleoside triphosphate diphosphohydrolase family of
enzymes (eNTPDase) and of several other subfamilies of enzymes. Experiments
in progress indicate that cultured normal and immortalized pituitary and
hypothalamic cells release ATP under resting conditions. RT-PCR analysis also
revealed the presence in these cells of transcripts for eNTPDase 1-3. The
enzymes were functional, as documented by degradation of endogenously
released and exogenously added ATP. Blocking the activity of eNTPDases by
ARL67156 led to an increase in ATP concentrations in pituitary perfusates and
inhibition of degradation of the extracellularly added ATP. On the other
hand, the addition of apyrase, a soluble ectonucleotidase, and the expression
of recombinant mouse eNTPDase-2 enhanced degradation of both endogenously
released and exogenously added ATP. The ATP released by resting hypothalamic
cells was sufficient to activate and desensitize high-affinity recombinant
P2X receptors, whereas facilitation of ATP metabolism by the addition of
apyrase protected their desensitization. The results indicate that
co-localization of ATP release sites and ectonucelotidase activity in
hypothalamic and pituitary cells provides an effective mechanism for the
operation of ATP as an extracellular signaling molecule. Our work with recombinant P2XRs focuses on the
dependence of channel activity on ectodomain structure and agonist potency.
We perform our work with human embryonic kidney 293 cells expressing rat P2X2aR,
P2X2bR, P2X3R,
and chimeras bearing the V60-R180 or V60-F301 ectodomain
sequences of P2X3R instead of the I66-H192 or I66-Y310 sequences
of P2X2aR and P2X2bR. Chimeric P2X2a/V60-F301X3R
and P2X2b/V60-F301X3R inherited the P2X3R ligand–selective
profile, whereas the potency of agonists for P2X2a/V60-R180X3R
was intermediate between those observed at parental receptors. Furthermore,
P2X2a/V60-F301X3R and P2X2a/V60-R180X3R
desensitized in a P2X2aR-specific manner, and P2X2b/V60-F301X3R
desensitized with rates comparable to those of P2X2bR. In striking
contrast to parental receptors, the rates of decay in P2X2a/V60-F301X3R
and P2X2b/V60-F301X3R currents after agonist withdrawal
were 15- to 200-fold slower. For these chimeras, the decays in currents were
not dependent on duration of stimuli and reflected both continuous
desensitization and deactivation of receptors. In addition, participation of
deactivation in closure of channels was inversely correlated with the potency
of agonists to activate receptors. The delay in deactivation was practically
abolished in P2X2a/V60-R180X3R–expressing cells.
However, the recovery from desensitization of P2X2a/V60-F301X3R
and P2X2a/V60-R180X3R was similar and was substantially
delayed compared with that of parental receptors. The results indicate that
both ectodomain halves participate in gating but that the C and N halves
influence the stability of open and desensitized conformation states,
respectively, which in turn reflects on rates of receptor deactivation and
resensitization. Our ongoing experiments are directed toward identification
of the P2XR domains responsible for ATP binding and gating. He
ML, Gonzalez-Iglesias, Stojilkovic SS. Role of nucleotide P2 receptors
in calcium signaling and prolactin release in pituitary lactotrophs. J
Biol Chem 2003;278:46270-46277. He ML, Zemková H, Koshimizu T, Tomić M,
Stojilkovic SS. Intracellular calcium measurements as a method in studies on
activity of purinergic P2X receptor-channels. Am J Physiol Cell Physiol
2003;285:C467-479. He ML, Zemková H, Stojilkovic SS. Dependence
of purinergic P2X receptor activity on ectodomain structure. J Biol Chem
2003;278:10182-10188. Stojilkovic SS. Ion channels and electrical
signaling. In: Conn PM, ed. Neuroscience in Medicine, Zemková H, He ML, Koshimizu T, Stojilkovic SS.
Identification of ectodomain regions contributing to gating, deactivation,
and resensitization of purinergic P2X receptors. J Neurosci
2004;24:6968-6978. Dependence of calcium signaling and secretion
on cyclic nucleotides Andrić,
Gonzalez Iglesias, Kostic,b Tomić, Jiang, Stojilkovic Our investigations focus on the
interrelationship between spontaneous and receptor-controlled electrical
activity and cyclic nucleotide signaling in pituitary lactotrophs and
immortalized GH cells. Our earlier results indicated that these cells fire
action potentials spontaneously and that the associated calcium transients
are sufficient to trigger PRL release. Experiments in progress showed that
addition of MDL-12330A, an adenylyl cyclase inhibitor, decreased basal cAMP
and cGMP production in a dose-dependent manner. This decreased production was
accompanied by a decrease in the frequency of spontaneous action potentials,
inhibition of calcium transients in single lactotrophs, and downregulation of
calcium-controlled basal (in the absence of agonist) PRL secretion. The
addition of either a calmodulin antagonist or a calmodulin kinase II
inhibitor also decreased both cAMP and cGMP levels and inhibited
voltage-gated calcium influx and basal PRL release. On the other hand,
forskolin, an activator of adenylyl cyclase, and IBMX, a nonselective
inhibitor of phosphodiesterase, increased cAMP and cGMP production as well as
PRL release in a time- and concentration-dependent manner. Forskolin also
initiated action potential firing and calcium transients in quiescent cells
and increased the frequency of firing in spontaneously active cells. The
stimulatory actions of forskolin on electrical activity, calcium signaling,
and PRL release were abolished in the presence of MDL-12330A. The results
indicate that an interplay between voltage-gated calcium influx and cyclic
nucleotide intracellular messenger systems underlies the excitability of
resting pituitary lactotrophs. Further studies should clarify which
intracellular messengers, cyclic nucleotides, or cyclic
nucleotide–dependent kinases and which plasma membrane channels are
responsible for adenylyl/gyanylyl cyclase–dependent modulation of
firing pattern in lactotrophs. The stimulatory action of adenylyl cyclase
activators on cGMP production prompted us to investigate the mechanism of
activation of soluble guanlylyl cyclase. In general, the stimulatory effects
of adenylyl cyclase activators on calcium signaling are in
accord with observations that intracellular calcium stimulates nitric oxide
(NO) synthase activity, leading to increase in cGMP production. Consistent
with these observations, we recently found that activation of adenylyl
cyclase by growth hormone–releasing hormone,
pituitary adenylate cyclase–activating polypeptide, vasoactive
intestinal peptide, and forskolin increased NO and cGMP levels and that
inhibition of NO synthase actvity abolished basal and stimulated cGMP production.
However, we found that activators of adenylyl cyclase enhanced the
NO-dependent cGMP production even when NO was held constant at basal levels
in cells bathed in calcium-deficient medium. Receptor-activated cGMP
production was mimicked by expression of a
constitutively active protein kinase A and was accompanied by phosphorylation
of native and recombinant alpha-1 soluble guanylyl cyclase subunit. Addition
of a protein kinase A inhibitor, overexpression of a dominant negative mutant
of regulatory protein kinase A subunit, and substitution of S107-S108
N-terminal residues of alpha-1 soluble guanylyl cyclase subunit with alanine
abolished adenylyl cyclase–dependent cGMP production without affecting
basal and NO-donor–stimulated cGMP production. The results indicate
that phosphorylation of alpha-1 soluble guanylyl cyclase subunit by protein
kinase A enhances the NO-dependent sGC activity, most likely by stabilizing
the NO/soluble guanylyl cyclase complex
(see Figure 4.3), which is probably the major pathway by which adenylyl
cyclase–coupled receptors stimulate cGMP production.
Andrić SA, Gonzalez-Iglesias AE,
Tomić M, Stojilkovic SS. Nitric oxide inhibits prolactin secretion in
pituitary cells downstream of voltage-gated calcium influx. Endocrinology
2003;144:2912-2921. Kostic TS, Andrić SA, Stojilkovic SS.
Receptor-controlled phosphorylation of alpha-1 soluble guanylyl cyclase
enhances nitric oxide-dependent cyclic guanosine 5'-monophosphate production
in pituitary cells. Mol Endocrinol 2004;18:458-470. Stojilkovic SS. Calcium signaling. In: Henry
HL, Receptor-controlled calcium signaling,
secretion, and gene expression Tomić,
Andrić, Kretschmannova, Stojilkovic; in collaboration with Wong, Zemková Several G protein–coupled receptors
expressed in pituitary cells stimulate or inhibit spontaneous voltage-gated
calcium influx and hormone secretion. Our ongoing work focuses on the control
of voltage-gated calcium influx by three calcium-mobilizing receptors:
gonadotropin-releasing hormone (GnRH), thyrotropin-releasing hormone (TRH),
and endothelin-A (ETA). GnRH receptors are expressed in
gonadotrophs, and extracellular calcium is not essential to initiate calcium
oscillations in these cells. We recently addressed the role of calcium influx
in sustained and repetitive calcium signaling as well as the mechanism of
calcium efflux and the kinetics of recovery of calcium signaling during
repetitive stimulation with short (10-30 s) GnRH pulses and variable
interpulse intervals; for this purpose we used neonatal gonadotrophs perfused
with calcium/sodium-containing, calcium-deficient/sodium-containing, and
calcium-containing/sodium-deficient media. In calcium/sodium-containing
medium, baseline calcium oscillations recovered without a refractory period
and with a time constant of about 20 seconds, whereas the recovery of the
spike response occurred after a 25- to 35-second refractory period and with a
time constant of about 30 seconds. During repetitive GnRH stimulation,
removal of calcium had only a minor effect on baseline oscillations but
abolished the spike response, whereas removal of sodium slightly extended
duration of baseline oscillations and considerably prolonged spike response.
The results indicate that two calcium-handling mechanisms are operative in
gonadotrophs: redistribution of calcium within InsP3-sensitive and
-insensitive pools and a sodium-dependent calcium efflux followed by calcium
influx. Redistribution of calcium within the cell leads to rapid recovery of
the Ins(1,4,5)-trisphosphate–dependent pool, whereas the
sodium-dependent calcium efflux pathway is activated by the spike response
and limits the time of exposure to elevated cytosolic calcium concentrations.
Two calcium-mobilizing receptors are expressed
in pituitary lactotrophs: ETA and TRH. Activation of both
receptors induced rapid calcium release from intracellular stores and PRL
secretion, but their actions differed during sustained stimulation; TRH
facilitates and ET-1 inhibits voltage-gated calcium influx and PRL secretion.
In pertussis toxin–treated cells, ET-1–induced inhibition of
voltage-gated calcium influx was abolished, and the pattern of calcium
signaling was comparable to that observed in TRH-stimulated cells. The
addition of cesium, a relatively specific blocker of inward rectifier
potassium channels, mimicked the effect of pertussis toxin on the pattern of
ET-1–induced sustained calcium signaling in about 50 percent of cells
and did not affect agonist-induced inhibition of PRL secretion. Extracellular
cesium was also ineffective in altering the TRH-induced facilitation of
voltage-gated calcium influx and PRL secretion. Furthermore, the agonist-specific
patterns of calcium signaling and PRL secretion were not affected by the
following agents: apamin and paxilline, specific blockers of
calcium-activated SK and BK type potassium channels, respectively; E-4031, a
blocker of the ether a-go-go potassium channel; and linopirdine, a
blocker of M-type potassium channels. The results suggest that ET-1 inhibits
voltage-gated calcium influx through activation of cesium-sensitive channels,
presumably the Gi/o-controlled inward rectifier K+ channels, and
that the agonist inhibits PRL release, but downstream of calcium influx.
Further studies are required to identify the mechanism of sustained
TRH-induced facilitation of voltage-gated calcium influx and PRL secretion. In collaboration with Anderson Wong, we
investigated the effects of growth hormone on the hormone’s secretion
at the pituitary level. Our initial experiments used grass carp pituitary
cells as a cell model. We identified GH receptors in somatotrophs and found
that their activation by exogenous GH increased steady-state GH mRNA levels
and GH production. Removal of endogenous GH by immunoneutralization with GH
antiserum inhibited basal as well as stimulated GH mRNA expression. Cytosolic
mature GH mRNA levels were elevated by GH treatment and reduced by GH
antiserum, whereas nuclear GH primary transcripts were almost undetectable
after GH immunoneutralization. Inhibition of Janus kinase-2, phosphoinositide
3-kinase, and MAPK also abolished GH-induced steady-state GH-mRNA levels. GH
immunoneutralization in pituitary cells pretreated with actinomycin D induced
a marked decrease in the half-life of GH mRNA, indicating that the clearance
of GH transcripts could be enhanced after removing endogenous GH. The results
provide evidence that GH can serve as an intrapituitary autocrine/paracrine
factor maintaining GH gene expression in somatotrophs and that such action is
mediated by Janus kinase-2/MAPK and Janus kinase-2/phosphoinositide 3-kinase
cascades coupled to GH receptors. Further experiments should clarify the
expression and role of GH receptors in the control of GH synthesis in
mammalian pituitary cells. Basta M, Van Goor F, Luccioli S, Billings EM,
Vortmeyer AO, Baranyi L, Szebeni J, Alving CR, Carroll MC, Berkower I,
Stojilkovic SS, Metcalfe DD. F(ab)’2-mediated neutralization
of C3a and C5a anaphylatoxins: a novel effector function of immunoglobulins. Nat
Med 2003;9:431-438. Tomić M, Andrić SA, Stojilkovic SS.
Dependence of prolactin release on coupling between Ca2+ mobilization
and voltage-gated Ca2+ influx pathways in rat lactotrophs. Endocrine
2003;20:45-52. Zemková H, Balik A, Kretschmannová K, Mazna P,
Stojilkovic SS. Recovery of Ins(1,4,5)-trisphosphate-dependent calcium
signaling in neonatal gonadotrophs. Cell Calcium 2004;36:89-97. Zemková H, Balik A, Stojilkovic SS. Expression
and signal transduction pathways of melatonin receptors in pituitary. In:
Pandi-Perumal SR, Cardinali D, eds. Melatonin: Biological Basis of Its
Function in Health and Disease. Zhou H, Ko WKW, Ho WKK, Stojilkovic SS, Wong
AOL. Novel aspects of growth hormone (GH) autoregulation: GH-induced GH gene
expression in grass carp pituitary cells through autocrine/paracrine
mechanisms. Endocrinology 2004;145:4615-4628. aMu-Lan
He, PhD, former Postdoctoral Fellow bTatjana
Kostic, PhD, former Postdoctoral Fellow COLLABORATORS Taka-aki Koshimizu, MD, PhD, National
Research Institute for Child Health and Development, Hana Zemková, PhD, For
further information, contact stojilks@mail.nih.gov |
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