Hiu-Yee Kwan

Store-operated Calcium Entry in Vascular Endothelial Cells Is Inhibited by cGMP via a Protein Kinase G-dependent Mechanism

Store-operated Ca2+ entry in vascular endothelial cells not only serves to refill the intracellular Ca2+ stores, but also acts to stimulate the synthesis of nitric oxide, a key vasodilatory factor. In this study, we examined the role of cGMP in regulating the store-operated Ca2+ entry in aortic endothelial cells. Cyclopiazonic acid (CPA) and thapsigargin, two selective inhibitors of endoplasmic reticulum Ca2+-ATPase, were used to induce store-operated Ca2+ entry. 8-Bromo-cGMP, an activator of protein kinase G, inhibited the CPA- or thapsigargin-induced Ca2+ entry in a concentration-dependent manner. An inhibitor of protein kinase G, KT5823 (1 μm) or H-8 (10 μm), abolished the inhibitory action of 8-bromo-cGMP and resumed Ca2+ entry. Addition ofS-nitroso-N-acetylpenicillamine (a nitric oxide donor) or dipyridamole (a cGMP phosphodiesterase inhibitor) during CPA treatment elevated cellular cGMP levels, stimulated protein kinase G activity, and at the same time reduced Ca2+ influx due to CPA. Patch clamp study confirmed the existence of a CPA-activated Ca2+-permeable channel sensitive to cGMP inhibition. These results suggest that cGMP via a protein kinase G-dependent mechanism may play a key role in the regulation of the store-operated Ca2+ entry in vascular endothelial cells.

TRPC1 Associates With BKCa Channel to Form a Signal Complex in Vascular Smooth Muscle Cells

TRPC1 (transient receptor potential canonical 1) is a Ca2+-permeable cation channel involved in diverse physiological function. TRPC1 may associate with other proteins to form a signaling complex, which is crucial for channel function. In the present study, we investigated the interaction between TRPC1 and large conductance Ca2+-sensitive K+ channel (BKCa). With the use of potentiometric fluorescence dye DiBAC4(3), we found that store-operated Ca2+ influx resulted in membrane hyperpolarization of vascular smooth muscle cells (VSMCs). The hyperpolarization was inhibited by an anti-TRPC1 blocking antibody T1E3 and 2 BKCa channel blockers, charybdotoxin and iberiotoxin. These data were confirmed by sharp microelectrode measurement of membrane potential in VSMCs of intact arteries. Furthermore, T1E3 treatment markedly enhanced the membrane depolarization and contraction of VSMCs in response to several contractile agonists including phenylephrine, endothelin-1, and U-46619. In coimmunoprecipitation experiments, an antibody against BKCa &agr;-subunit [BKCa(&agr;)] could pull down TRPC1, and moreover an anti-TRPC1 antibody could reciprocally pull down BKCa(&agr;). Double-labeling immunocytochemistry showed that TRPC1 and BKCa were colocalized in the same subcellular regions, mainly on the plasma membrane, in VSMCs. These data suggest that, TRPC1 physically associates with BKCa in VSMCs and that Ca2+ influx through TRPC1 activates BKCa to induce membrane hyperpolarization. The hyperpolarizing effect of TRPC1-BKCa coupling could serve to reduce agonist-induced membrane depolarization, thereby preventing excessive contraction of VSMCs to contractile agonists.

ACTIVITY OF VOLTAGE-GATED K+ CHANNELS IS ASSOCIATED WITH CELL PROLIFERATON AND Ca2+ INFLUX IN CARCINOMA CELLS OF COLON CANCER

Cell proliferation of carcinoma cells DLD-1 derived from colon cancer as measured by [3H] thymidine incorporation was drastically reduced in the presence of 4-aminopyridine, an inhibitors of voltage-gated K channel. A number of nonspecific K+ channel inhibitors including TPeA, TEA, verapamil and diltiazem also inhibited [3H] incorporation at the concentration reported to inhibit voltage-gated K+ channels. The presence of voltage-gated K+ channels was confirmed by reverse transcription-PCR and cDNA sequencing. Charybdotoxin and iberiotoxin, inhibitors for Ca2+-sensitive K+ channel, and glibenclamide, a specific inhibitor for ATP-sensitive K+ channel, did not have effect on cell proliferation. These experiments suggested a critical role of voltage-gated K+ channels in proliferation of colon cancer cells. Mechanism of action of K+ channel activity in cell proliferation was explored by studying the relationship between the K+ channel activity and Ca2+ entry. The results from experiments indicated that K+ channel inhibitors blocked [Ca2+]i influx. Therefore, it is likely that K+ channel activity may modulate Ca2+ influx into colon cancer cells, and subsequently modulate the proliferation of these cells.

Depletion of Intracellular Ca2+ Stores Sensitizes the Flow-Induced Ca2+ Influx in Rat Endothelial Cells

Abstract— Hemodynamic shear stress elicits a rise in endothelial [Ca2+]i, which may serve as a key second messenger to regulate many flow-associated physiological and biochemical processes. In the present study, we used Mn2+ quenching of fluorescent dye Fluo3 as an assay to investigate the Ca2+ influx of rat aortic endothelial cells in response to flow. We found that the Ca2+ signaling in response to flow could be greatly influenced by the status of intracellular Ca2+ stores. Depletion of intracellular Ca2+ stores by thapsigargin (4 &mgr;mol/L) or cyclopiazonic acid (10 &mgr;mol/L) drastically sensitized the Ca2+ influx in response to flow. Ca2+-mobilizing agonist bradykinin (100 nmol/L) or ATP (100 &mgr;mol/L) had similar sensitizing effect. The effect of bradykinin or ATP was blocked by Xestospongin C and U73122, suggesting that the sensitization was related to the IP3-mediated store depletion. On the other hand, the Mn2+ quenching in response to flow was greatly reduced by ochratoxin A (100 nmol/L), an agent that could increase the filling state of intracellular Ca2+ stores. In addition, we found that depletion-sensitized Ca2+ influx in response to flow was mediated by a PKG-inhibitable cation channel and that the influx was affected by membrane potential and K+ channel activity. In conclusion, the present study argues for a critical role of intracellular Ca2+ status in determining the Ca2+ signaling in response to flow and it provides a general mechanistic explanation for the stimulatory role of blood-borne agonists on flow-induced Ca2+ influx.

Protein kinase C can inhibit TRPC3 channels indirectly via stimulating protein kinase G

There are two known phosphorylation‐mediated inactivation mechanisms for TRPC3 channels. Protein kinase G (PKG) inactivates TRPC3 by direct phosphorylation on Thr‐11 and Ser‐263 of the TRPC3 proteins, and protein kinase C (PKC) inactivates TRPC3 by phosphorylation on Ser‐712. In the present study, we explored the relationship between these two inactivation mechanisms of TRPC3. HEK cells were first stably transfected with a PKG‐expressing construct and then transiently transfected with a TRPC3‐expressing construct. Addition of 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG), a membrane‐permeant analog of diacylglycerol (DAG), elicited a TRPC3‐mediated [Ca2+]i rise in these cells. This OAG‐induced rise in [Ca2+]i could be inhibited by phorbol 12‐myristate 13‐acetate (PMA), an agonist for PKC, in a dose‐dependent manner. Importantly, point mutations at two PKG phosphorylation sites (T11A‐S263Q) of TRPC3 markedly reduced the PMA inhibition. Furthermore, inhibition of PKG activity by KT5823 (1 µM) or H8 (10 µM) greatly reduced the PMA inhibition of TRPC3. These data strongly suggest that the inhibitory action of PKC on TRPC3 is partly mediated through PKG in these PKG‐overexpressing cells. The importance of this scheme was also tested in vascular endothelial cells, in which PKG plays a pivotal functional role. In these cells, OAG‐induced [Ca2+]i rise was inhibited by PMA, which activates PKC, and by 8‐BrcGMP and S‐nitroso‐N‐acetylpenicillamine (SNAP), both of which activate PKG. Importantly, the PMA inhibition on OAG‐induced [Ca2+]i rise was significantly reduced by PKG inhibitor KT5823 (1 µM) or DT‐3 (500 nM), suggesting an important role of PKG in the PMA‐induced inhibition of TRPC channels in native endothelial cells. J. Cell. Physiol. 207: 315–321, 2006.

Regulation of TRP Channels by Phosphorylation

The transient receptor potential (TRP) channels are a group of Ca2+-permeable cation channels (except TRPM4 and TRPM5) that function as cellular sensors of various internal and external stimuli. Most of these channels are expressed in the nervous system and they play a key role in sensory physiology. They may respond to temperature, pressure, inflammatory agents, pain, osmolarity, taste and many other stimuli. Recent development indicates that the activity of these channels is regulated by protein phosphorylation and dephosphorylation of serine, threonine, and tyrosine residues. In this review, we present a comprehensive summary of the literature regarding the TRP channel regulation by different protein kinases.

Mechanism of Non-Capacitative Ca2+ Influx in Response to Bradykinin in Vascular Endothelial Cells

Bradykinin is a potent vasoactive nonapeptide. It elicits a rise in cytosolic Ca2+ (Ca2+)i in endothelial cells, resulting in Ca2+-dependent synthesis and release of endothelial vasodilators. In the present study, we investigated the mechanism of bradykinin-induced Ca2+ influx in primary cultured rat aortic endothelial cells and in a mouse heart microvessel endothelial cell line (H5V). Bradykinin-induced Ca2+ influx was resolved into capacitative Ca2+ entry (CCE) and non-CCE. The non-CCE component was inhibited by a B2 receptor antagonist (HOE140; 1 µM) and a phospholipase C (PLC) inhibitor (U73122; 10 µM). The action of bradykinin could be mimicked by 1-oleoyl-2-acetyl-glycerol, an analogue of diacylglycerol (DAG), and by RHC80267, a DAG-lipase inhibitor. Immunoblots showed that TRPC6 was one of the main TRPC channels expressed in endothelial cells. Transfection of H5V cells with two siRNA constructs against TRPC6 both markedly reduced the TRPC6 protein level and, at the same time, decreased the percentage of cells displaying bradykinin-induced non-CCE. siRNA transfection also reduced the magnitude of non-CCE among the cells responding to bradykinin. Taken together, our data suggest that bradykinin-induced non-CCE is mediated via the B2-PLC pathway, and that DAG may be involved in this process. Further, TRPC6 is one of the important channels participating in bradykinin-induced non-CCE in endothelial cells.

Epinephrine-induced Ca2+ influx in vascular endothelial cells is mediated by CNGA2 channels

Epinephrine, through its action on beta-adrenoceptors, may induce endothelium-dependent vascular dilation, and this action is partly mediated by a cytosolic Ca(2+) ([Ca(2+)](i)) change in endothelial cells. In the present study, we explored the molecular identity of the channels that mediate epinephrine-induced endothelial Ca(2+) influx and subsequent vascular relaxation. Patch clamp recorded an epinephrine- and cAMP-activated cation current in the primary cultured bovine aortic endothelial cells (BAECs) and H5V endothelial cells. L-cis-diltiazem and LY-83583, two selective inhibitors for cyclic nucleotide-gated channels, diminished this cation current. Furthermore, this cation current was greatly reduced by a CNGA2-specific siRNA in H5V cells. With the use of fluorescent Ca(2+) dye, it was found that epinephrine and isoprenaline, a beta-adrenoceptor agonist, induced endothelial Ca(2+) influx in the presence of bradykinin. This Ca(2+) influx was inhibited by L-cis-diltiazem and LY-83583, and by a beta(2)-adrenoceptor antagonist ICI-118551. CNGA2-specific siRNA also diminished this Ca(2+) influx in H5V cells. Furthermore, L-cis-diltiazem and LY-83583 inhibited the endothelial Ca(2+) influx in isolated mouse aortic strips. L-cis-diltiazem also markedly reduced the endothelium-dependent vascular dilation to isoprenaline in isolated mouse aortic segments. In summary, CNG channels, CNGA2 in particular, mediate beta-adrenoceptor agonist-induced endothelial Ca(2+) influx and subsequent vascular dilation.

Rod-type cyclic nucleotide-gated cation channel is expressed in vascular endothelium and vascular smooth muscle cells

OBJECTIVES Ca(++)-permeable nonselective cation channels mediate the entry of extracellular Ca++ in vascular endothelium. They are also partly responsible for Ca++ entry in vascular smooth muscle cells (SMCs). The molecular identities of these channels have not been identified. The aim of this study is to examine whether rod-type nucleotide-gated nonselective cation (CNG1) channel, a channel which has been molecularly cloned, is related to the nonselective channels in vascular cells. METHODS We used RT-PCR, molecular cloning, northern Blot and in situ hybridization to examine the expression of CNG1 mRNA in a variety of guinea pig and rat blood vessels with different diameters and in cultured vascular endothelial cells and vascular smooth muscle cells. RESULTS We have cloned a 402-bp partial cDNA of CNG1 channel from guinea pig mesenteric arteries. RT-PCR and southern blot results indicate that the CNG1 mRNA is expressed in both cultured vascular endothelial and cultured vascular SMCs. Northern blot revealed the transcripts of approximately 3.2 kb, approximately 5.0 kb, and approximately 1.8 kb in cultured endothelial cells. In situ hybridization yielded strong labeling in endothelium layer of aorta, medium-sized mesenteric arteries, and small mesenteric arteries. CONCLUSION Our findings suggest a potential role of CNG protein for Ca++ entry in vascular endothelium and vascular smooth muscles. The high expression of CNG1 mRNA in the endothelium of medium-sized arteries and small-sized arteries implicates a possible involvement of CNG1 protein in the regulation of blood supply to different regions and in the regulation of arterial blood pressure.

CNGA2 Channels Mediate Adenosine-Induced Ca2+ Influx in Vascular Endothelial Cells

Objectives—Adenosine is a cAMP-elevating vasodilator that induces both endothelium-dependent and -independent vasorelaxation. An increase in cytosolic Ca2+ ([Ca2+]i) is a crucial early signal in the endothelium-dependent relaxation elicited by adenosine. This study explored the molecular identity of channels that mediate adenosine-induced Ca2+ influx in vascular endothelial cells. Methods and Results—Adenosine-induced Ca2+ influx was markedly reduced by L-cis-diltiazem and LY-83583, two selective inhibitors for cyclic nucleotide-gated (CNG) channels, in H5V endothelial cells and primary cultured bovine aortic endothelial cells (BAECs). The Ca2+ influx was also inhibited by 2 adenylyl cyclase inhibitors MDL-12330A and SQ-22536, and by 2 A2B receptor inhibitors MRS-1754 and 8-SPT, but not by an A2A receptor inhibitor SCH-58261 or a guanylyl cyclase inhibitor ODQ. Patch clamp experiments recorded an adenosine-induced current that could be inhibited by L-cis-diltiazem and LY-83583. A CNGA2-specific siRNA markedly decreased the Ca2+ influx and the cation current in H5V cells. Furthermore, L-cis-diltiazem inhibited the endothelial Ca2+ influx in mouse aortic strips, and it also reduced 5-N-ethylcarboxamidoadenosine (NECA, an A2 adenosine receptor agonist)-induced vasorelaxation. Conclusion—CNGA2 channels play a key role in adenosine-induced endothelial Ca2+ influx and vasorelaxation. It is likely that adenosine acts through A2B receptors and adenylyl cyclases to stimulate CNGA2.