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Introduction
An increase in cytosolic calcium concentration
([Ca2+]c) is among the earliest events that occur after stimulation of many
different types of cells by endogenous signaling molecules such as hormones, neurotransmitters, and reactive
oxygen species (ROS) including singlet
oxygen[1,2]. The
[Ca2+]c increase is frequently in the form of oscillations. Oscillations can, by default, be
encoded by at least three independent modes of modulation: amplitude modulation (AM), frequency modulation (FM), and
individual calcium spike-shape modulation (SM). Thus the simplest divalent cation, by temporal oscillation, encodes numerous,
diverse and specific signals to modulate various vital body functions such as oocyte
fertilization[3], cell
secretion[4_6], muscle contrac-
tion[7], neuronal migration and neurite
growth[8,9], development[10], and
apoptosis[11]. As such, calcium oscillation is a
multi-functional and universal signal.
Typical FM-encoded signaling and the corresponding specific activation of cellular function or protein functional
modulation are exemplified by the calcium/calmodulin dependent protein kinases and phosphatases. The multifunctional
calcium/calmodulin-dependent protein kinase II (CaM Kinase II) has been extensively investigated because of its involvement in
many cellular functions such as neuronal plasticity and memory
formation[12]. Detailed studies have delimited the specific
calcium oscillating frequency that this enzyme is sensitive to. It was found that this enzyme is maximally activated by calcium
oscillations in the frequency range of a few
Hz[13]. This is in sharp contrast with the frequency modulation of
calcium/calmodulin-dependent protein phosphatase IIB or calcineurin (CN). This enzyme is a vital component in the modulation of
transcription factor NFAT and is mainly modulated by calcium increases in the
cytosol[14,15]. Calcineurin is optimally
activated by calcium oscillations in the frequency range of about 10
mHz[16,17]. A comparison of the specific activation frequency
of CaM
Kinase II and calcineurin is illustrated in Figure 1.
The above examples are only the beginning of probably a very long list of such frequency-encoding cases. This list is
likely to be growing at a fast rate in the next few years as more detailed works are carried out to investigate the involvement
of calcium/calmodulin-dependent cytosolic proteins in specific cellular functions, which are modulated by calcium oscillating
frequencies. It may be possible sometime in the future to group these proteins to provide a modulation spectrum, based on
frequencies, similar to the light spectrum.
Other than frequency modulation, it would also be interesting to see protein-specific amplitude modulations. Although
most of the cell types after stimulation regularly oscillate at the level of 600_700 nmol/L such as pancreatic acinar
cells[18], brown
adipocytes[19], the magnitude of calcium oscillation spikes increases with increasing concentrations of the
stimuli[18]. It would also be interesting to analyze the on- and off-rates of each individual calcium spike in different cells and under
different circumstances, which determine the particular shape of the individual spike. Relative densities of
IP3R/calcium channels, and SERCA/PMCA isoforms and their activity status, such as phosphorylation status will shape the overall profile
for each spike. The off rate, for example, may determine the speed at which a particular enzyme returns to its de-activated
state.
During the course of our studies investigating the mechanisms of pace-making activities for calcium oscillations in
non-excitable cells, we noted that some isolated exocrine cells
in vitro did not typically respond to hormone or neurotransmitter
stimulation by oscillatory increases in
[Ca2+]c. The rodent submandibular gland acinar cells are noted to increase
[Ca2+]c not in the form of oscillations, especially at low stimulating concentrations, but rather in the form of graded plateaus. The rat
submandibular acinar cells responded to noradrenaline or acetylcholine stimulation by plateau increase in
[Ca2+]c, even at the minimal or threshold stimulating concentrations
used[20]. All of these works have been carried out on the isolated
submandibular acini bathed in a KrebĄ¯s-like buffered solution. But, in fact, these acinar cells and other components of the secretory
axis in submandibular gland in vivo may actually be bathed in extracellular medium with very different composition.
Submandibular gland: microanatomy, potas-sium and osmolarity gradients along the secre-tory
axis
The rat submandibular gland structure is illustrated in Figure 2. The secretory axis starts with acinus, a few acinar cells
form a spherical acinus structure. The luminal side of the acinus is connected to the rather narrow intercalated duct.
Intercalated duct cells are the smallest cells in the secretory axis. The intercalated duct is connected to the convoluted
granular tubules (GCT), striated duct, excretory duct, and ends with the main excretory duct in the mouth. Acinar cells, cells
in GCT and striated ducts are 20_30 µm in diameter, the intercalated duct is only about 10
µm[21].
The salivary gland cells have a large portion of their surface bathed in the saliva, which is different from ordinary
extracellular fluid. The usual inorganic compositions of extracellular fluids are rather similar to that of serum, with potassium
at approximately 5 mmol/L and total osmolarity at 310 mOsm. Most of the cells in the body are bathed in such extracellular
fluids. But the salivary cells are different in that at least one side of the cells is bathed in saliva. In fact in the submandibular
gland, some authors reported that the acinar cells are nearly encircled by the extended acinar lumen
system[22].
Saliva formation is divided into two steps. Primary saliva is formed at the site of acini/intercalated ducts, with a
composition similar to that of serum and extracellular fluid but with obviously enhanced potassium:
Na+ 126_136 mmol/L, K+
8.4_11.9 mmol/L, osmolarity 310
mOsm[21]. The potassium concentration increases and total osmolarity decreases progressively
along the secretory axis. The osmolarity drops to 307 mOsm at the intercalated duct, 229 mOsm at the main excretory duct, and
89 mOsm at the main excretory duct orifice, which opens to the oral
cavity[23]. At the main excretory duct,
K+ increases to 130
mmol/L[21,23]. To take into consideration of this peculiarity, some early researchers raised the potassium concentration in the
KrebĄ¯s buffer to 15 mmol/L; these authors noted that such a raised
potassium concentration was needed to maintain acinar
structure [24]. But the effects of the significant changes in inorganic components of the saliva along the secretory axis
(K+ from 8.4 to 30_130 mmol/L, osmolarity from 310 to 220
mOsm)
on the functions of the acinar and different ductal cells, and the
effects of any disturbances of such ordered gradients on both the physiology and pathology, have never been
systematically investigated at the cellular and molecular levels. The fact that large amounts of potassium ion are secreted after
stimulation of the salivary glands has long been noted, but the physiological significance of the secreted potassium has
never been properly addressed[20,25_27].
Calcium oscillations along the secretory axis in submandibular gland
The combination of AM, FM and SM of calcium oscillations as mentioned above ensures both complexity and diversity,
and probably also specificity in the encoded calcium signals. In rat submandibular gland acinar cells, both ACh and
noradrenaline induced plateau increases in cytosolic calcium concentrations, rather than oscillatory increases (Figure 3). To
trace the possible source of this unique property, we looked for typical differences in salivary acinar cells and other exocrine
acinar cells and found significant differences in the extracellular fluid that bathe the cells and the gradual changes in saliva
composition. Yoshida et al[26] made the initial discovery that when the potassium concentration in KrebĄ¯s buffer was raised
(to 30 mmol/L), ACh-induced calcium increases changed dramatically from plateau increases to oscillatory increases. This
discovery was confirmed in our laboratory (Figure 3). In addition, we found that this transformation was applicable to
cholinergic stimulation, but not to adrenergic
stimulation[20]. This provides a cellular basis for the differences in sympathetic
and parasympathetic stimulation of saliva secretion in the salivary glands (Figure 3).
The intercalated ducts are rather small. The GCT or granulated duct cells are the largest in the submandibular secretory
axis and may be up to 30 µm in diameter (Figure 2). Granulated ductal cells are typical exocrine cells, but surprisingly very little
is known about these cells, especially about their calcium signal-encoding and molecular mechanisms of exocytosis.
Submandibular granulated duct cells are known to contain multiple secretory products, such as tissue
kallikrein[28], growth factors epidermal growth factor (EGF) and nerve growth factor
(NGF)[29], carbonic
anhydrase[30], insulin-like
proteins[31],
angiotensin[32], and
chromogranins[33]. Fine-tuning may be required for specific release of desired secretory products after
nerve stimulation of the submandibular gland. But works on isolated mouse GCT so far observed only graded plateau
increases after cholinergic and adrenergic stimulations (JIA & CUI,unpublished). This is in contrast with works reported in
a human submandibular gland ductal cell line (HSG) where carbachol induced regular calcium
oscillations[34]. To clarify whether oscillatory calcium increases occur in rodent GCT
cell in vivo, in situ imaging or sophisticated
in vitro maneuverings in terms of the extracellular microenvironments are required.
Potassium modulation of other neurotransmitter stimula-tions, such as purinergic stimulation, is not known at the moment.
Neither is it known whether such potassium modulation applies to the ductal cells in the intercalated duct, granulated duct,
and striated duct. The effects of hypo-osmolarity remain to be determined also. These works may have important
implications for salivary diseases such as cystic fibrosis, and SjogrenĄ¯s syndrome.
Preliminary data indicated that high potassium-modulation of calcium signals in the submandibular gland may be related
to sodium/calcium exchanging activity (MA and CUI, unpublished). NCX1 has been found to exist in
submandibular gland[27,35_37], possibly working in the reverse
mode[27,38], although detailed distribution along the secretory axis is not known. Future
works involving primary cultures of granulated and striated duct cells, and iRNA downregulation of NCX1 will more
definitively elucidate the involvement of NCX1 in high potassium- and low osmolarity-induced transformations of calcium increases.
In conclusion, the submandibular gland is unique in that it has all the secretory components in the secretory axis: acinus,
intercalated duct, granular convoluted tubules, striated duct, and excretory duct. The potassium and osmolarity gradients in
saliva may play a feedback role in regulating neurotransmitter-induced calcium increases. High potassium transformation of
cholinergically-induced plateau increase into oscillatory increase serves as a typical example of such feedback regulation.
Additional works will be needed to reveal whether this is a general rule along the secretory axis, especially in the intro- and
inter-lobular ducts.
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