The key findings reported here relate to the functions of primary cilia in intrahepatic bile ducts. We demonstrated for the first time that: (i) luminal fluid flow induces in cholangiocytes of microperfused IBDUs [Ca2+]i and cAMP signaling responses through the bending of primary cilia; (ii) the flow-induced increase in [Ca2+]i reflects both extracellular and intracellular Ca2+ stores; (iii) PC-1 and PC-2 are localized to cholangiocyte cilia and account for the flow-induced increase in [Ca2+]i; (iv) the flow-induced decrease in cAMP is secondary to an increase in [Ca2+]i and is dependent on PC-2 and extracellular Ca2+; and (v) Ca2+-inhibitable AC6 is localized to cholangiocyte cilia accounting for the flow-induced decrease in cAMP. Collectively, these data support the concept that cholangiocyte cilia may function as sensory organelles that transmit luminal fluid flow stimuli into integrated [Ca2+]i and cAMP signaling.
In experiments described in this study, cholangiocytes were shown to respond to luminal flow by an increase in [Ca2+
and a decrease in cAMP when a flow rate of 3 μl/sec was applied. Since bile flow measured in the rat common bile duct is about of 0.3 μl/sec, a question regarding the physiologic relevance of our observations may be justifiably raised. Regarding this issue, at least three points are worth making. First, we believe that our results are proof of concept that flow-induced mechanical stimuli can affect cholangiocyte function via ciliary-associated mechanisms. Second, the in vivo
bile flow rate measured at the tip of the common bile duct almost certainly does not accurately reflect bile flow rates in the intrahepatic biliary system; indeed, current technology does not permit direct measurements of bile flow rates along the intrahepatic biliary system. Third, it has been previously shown in experimental models that mechanical forces greater than biomechanical forces of a physiological range disassemble cilia.27
Our data showing that luminal fluid flow bends cholangiocyte cilia but do not cause any damage in these organelles suggest that we developed a suitable model for addressing the mechanosensory function of cholangiocyte cilia.
Hypothetically, an increase in [Ca2+
and a decrease in cAMP observed in microperfused IBDUs may be induced by several mechanical forces. Cholangiocytes, like endothelial cells and renal tubular epithelial cells, may experience at least three types of mechanical forces that originate from variations in fluid (bile) flow (i.e., hydrostatic pressure, circumferential stretch, and fluid flow-induced drag forces), and potentially may exhibit [Ca2+
transient if exposed to any of them.20, 21, 28, 29
However, in intrahepatic bile ducts in vivo
and in in vitro
microperfused IBDU, in which the distal end is open, hydrostatic pressure and circumferential stretch are minimal. Thus, the primary mechanical forces to which the luminal surface of intrahepatic bile ducts and cholangiocyte cilia are exposed are bile (fluid)-flow induced drag forces that physically can affect cilia.20, 21
In our model, such forces bent cilia through an angle of approximately 70° in the middle portion of the axoneme; the findings are consistent with observations of others demonstrating that chondrocyte primary cilia are bent by mechanical forces in situ in the middle part of an axoneme through an angle of approximately 90°.30
The mechanosensory function of cholangiocyte cilia depends on PC-1, PC-2 and both extracellular and intracellular Ca2+
sources. The initial increase in [Ca2+
originates from the entry of extracellular Ca2+
via PC-2 which in turn stimulates Ca2+
-dependent release of Ca2+
from intracellular stores. In epithelial cells, the mechanisms of Ca2+
release from intracellular stores in response to fluid flow remains uncertain. In MDCK cells, this mechanism involves activation of IP3
whereas in cultured mouse renal epithelial cells, it involves activation of ryanodine receptors.18
Cholangiocytes do not express ryanodine receptors, but express three isoforms of IP3
receptors that are responsible for [Ca2+
Since in thapsigargin-treated cholangiocytes, the only partial suppression of the flow-induced increase in [Ca2+
was observed, mechanisms other than activation of IP3
receptors may contribute to the flow-induced ciliary-mediated release of Ca2+
from intracellular stores. However, currently, such mechanisms remain unknown.
The mechanosensory functions of cholangiocyte cilia are not limited to transmitting luminal mechanical flow stimuli into [Ca2+]i signaling response but also occur through cAMP signaling. To our knowledge, this study is the first demonstration of the expression of AC in primary cilia and of the involvement of the cAMP-signaling pathway in ciliary flow sensing in any epithelial cell type. Since the flow-induced decrease in cAMP was dependent on extracellular Ca2+ and the presence of PC-2 and AC6 in cholangiocytes and cilia, it suggested to us that at a molecular level, this may occur through the Ca2+-dependent inhibition of AC6.
AC6 is one of six membrane-bound ACs (i.e., AC4-AC9) expressed in rat cholangiocytes.26
In different cell types, AC6 is activated by hormones through G-protein-coupled receptors and is inhibited by extracellular Ca2+
in micromollar concentrations.32
Recent developments show that AC6 is co-localized with Ca2+
entry channels in discrete domains of the plasma membrane (i.e., “lipid rafts”) where its activity is inhibited by extracellular Ca2+
Our data suggest that an analogous mechanism may exist in cholangiocyte cilia where extracellular Ca2+
entering into the cell via PC-2 may inhibit AC6 activity.
The physiological and pathophysiological implications of the flow-induced ciliary-mediated increase in [Ca2+]i and decrease in cAMP remain unclear. Our findings that PC-1, PC-2 and AC6 are localized to cholangiocyte cilia suggest the existence on the apical plasma membrane of a previously unknown regulatory mechanism that may provide a molecular explanation for the initiation and cessation of ductal bile secretion. In a working model depicted on , we present the concept that cholangiocyte cilia function as sensory organelles located on the apical plasma membrane that monitor changes in bile flow within intrahepatic bile ducts and adjust cholangiocyte functional response (i.e., ductal bile secretion) to such changes.
Figure 10 Working model of coordinated regulation of cholangiocyte secretion. Cholangiocytes possess numerous transporters, exchangers and channels necessary for ductal bile formation. The functions of these proteins on the apical plasma membrane are regulated (more ...)
Physiologically, both canalicular and ductal bile secretion increase in response to a meal resulting in an increase in bile flow in the biliary tree. In the digestive phase, an increase in ductal bile secretion is stimulated by secretin, which is released into the portal circulation from endocrine S cells in the duodenum and jejunum. We and others have previously demonstrated that secretin stimulates ductal bile secretion via the cAMP-signaling pathway and its action may be potentiated or terminated by other regulatory molecules (i.e., hormones, regulatory peptides, neurotransmitters, etc.) via both cAMP and [Ca2+
Hormonal regulation of ductal bile secretion occurs primarily on the cholangiocyte basolateral plasma membrane by activation of specific receptors. However, it is now evident that ductal bile secretion is also controlled via regulatory mechanisms on the apical cholangiocyte plasma membrane. The regulatory molecules that may affect cholangiocyte function on their apical side include but are not limited to bile acids,41
etc. In the model that we propose, ductal bile secretion may also be regulated by luminal bile flow via apically-located ciliary-associated mechanisms. Physiologically, bile flow in intrahepatic bile ducts is pulsatile and its changes may alter the mechanical forces to which cholangiocyte cilia are exposed. Given that cholangiocyte cilia contain a mechanoreceptor, PC-1, and Ca2+
channel, PC-2, proteins thought to form a functional complex when a primary cilium is bent,18, 19
luminal bile flow may bend cholangiocyte cilia resulting in an increase in [Ca2+
which in turn suppresses cAMP signaling via ciliary-associated AC6. Functionally, luminal bile flow via activation of [Ca2+
signaling may terminate both global and local cAMP signaling (i.e., turn off regulation) initially activated by secretin and other regulatory molecules (i.e., turn on regulation), thus, providing a coordinated regulation of ductal bile secretion and fast functional adjustments of intrahepatic ductal system to physiological needs.
Thus, our study demonstrates that cholangiocyte cilia may function as fluid (bile) flow sensors in intrahepatic bile ducts. The precise mechanisms of a mechanosensory function of cholangiocyte cilia and the physiological and pathophysiological implications of this phenomenon remain to be fully characterized; however, our initial experiments show that cholangiocyte cilia may have a significant role in coordinated regulation of ductal bile formation.