Maturation Alters the Contribution of Potassium Channels to Resting and 5HT-induced Tone in Small Cerebral Arteries of the Sheep

Brain Research. Developmental Brain Research. Feb, 2002  |  Pubmed ID: 11882339

To address the hypothesis that maturation alters the contribution of K-channels to resting and agonist-induced tone in small cerebral arteries, second branch middle cerebral arteries (approximately 200 microm) were taken from term fetal (139-141 days gestation) and adult sheep, denuded of endothelium, and mounted in myographs. After determination of length-tension relations, the arteries were stretched to 55, 100, and 145% of optimum length. At each level of stretch, contractile responses to 5 mM 4-aminopyridine (4-AP, voltage-sensitive K-channel blocker), 100 nM iberiotoxin (calcium-sensitive K-channel blocker), 10 microM glibenclamide (ATP-sensitive K-channel blocker), or 10 microM Ba(2+) (inward rectifier K-channel blocker) were recorded. In separate experiments, concentration--response relations were determined for 5-HT in the presence and absence of each of the four K-channel blockers at the same concentrations. Both 4-AP and iberiotoxin produced stretch-dependent contractions of greater magnitude in adult (37% for 4-AP and 43% for iberiotoxin at 100% optimum) than in fetal (5% for 4-AP and 7% for iberiotoxin at 100% optimum) arteries. 4-AP also enhanced the pD(2) for 5-HT in adult (from 7.15 to 7.49), but not in fetal, arteries. Conversely, glibenclamide attenuated the pD(2) for 5-HT in fetal (from 7.02 to 6.71), but not in adult, arteries. Iberiotoxin enhanced the pD(2) for 5-HT in both fetal (from 7.05 to 7.51) and adult (from 7.15 to 7.75) arteries. In addition, iberiotoxin enhanced maximum responses to 5-HT (from 59 to 82%) in adult but not fetal arteries. Finally, 4-AP enhanced the maximum responses to 5-HT in both fetal (from 67 to 85%) and adult (from 59 to 79%) arteries. These results indicate that maturation modulates the contribution of K(V), K(Ca), and K(ATP), but not K(IR) channels to basal and/or 5HT-induced cerebrovascular tone, and demonstrate that K(V) and K(Ca) channels are coupled to stretch-sensitive receptors, and that K(V) and K(Ca) limit contractile responses to 5-HT. To the extent that changes in pD(2) values reflect changes in agonist--ligand interactions, the data also suggest that K(V), K(Ca), and K(ATP) channels may possibly influence ligand--receptor binding for 5-HT.

Cardiovascular Responses and Neurotransmission in the Ventrolateral Medulla During Skeletal Muscle Contraction Following Transient Middle Cerebral Artery Occlusion and Reperfusion

Brain Research. Oct, 2002  |  Pubmed ID: 12376178

We hypothesized that static skeletal muscle contraction-induced systemic cardiovascular responses, and central glutamate/GABA release in rostral (RVLM) and caudal ventrolateral medulla (CVLM), would be modulated by cerebral ischemia. In sham-operated rats, a 2-min tibial nerve stimulation induced static contraction of the triceps surae, evoked pressor responses, increased glutamate in both the RVLM and CVLM, decreased GABA in the CVLM, and increased GABA in the RVLM. In rats with a temporary 90-min left middle cerebral artery occlusion (MCAO) followed by 24 h reperfusion, pressor responses during muscle contractions were attenuated, as were glutamate within the left RVLM and left CVLM. Glutamate within the right RVLM and right CVLM were unaltered and similar to those in sham rats. In contrast, GABA increases during muscle contractions were enhanced in the left RVLM and CVLM but changes within the right CVLM and RVLM were similar to those in sham rats. These results indicate that unilateral ischemia increases ipsilateral GABA/glutamate ratios during muscle contraction in the RVLM. In contrast, opposite changes in ipsilateral glutamate and GABA release within the RVLM and CVLM were observed following a 90-min right-sided MCAO followed by 24 h reperfusion. However, cardiovascular responses during muscle contraction were depressed following such an ischemic brain injury. These data suggest that transient ischemic brain injury attenuates cardiovascular responses to static exercise via modulating neurotransmission within the ventrolateral medulla.

Polycystins 1 and 2 Mediate Mechanosensation in the Primary Cilium of Kidney Cells

Nature Genetics. Feb, 2003  |  Pubmed ID: 12514735

Several proteins implicated in the pathogenesis of polycystic kidney disease (PKD) localize to cilia. Furthermore, cilia are malformed in mice with PKD with mutations in TgN737Rpw (encoding polaris). It is not known, however, whether ciliary dysfunction occurs or is relevant to cyst formation in PKD. Here, we show that polycystin-1 (PC1) and polycystin-2 (PC2), proteins respectively encoded by Pkd1 and Pkd2, mouse orthologs of genes mutated in human autosomal dominant PKD, co-distribute in the primary cilia of kidney epithelium. Cells isolated from transgenic mice that lack functional PC1 formed cilia but did not increase Ca(2+) influx in response to physiological fluid flow. Blocking antibodies directed against PC2 similarly abolished the flow response in wild-type cells as did inhibitors of the ryanodine receptor, whereas inhibitors of G-proteins, phospholipase C and InsP(3) receptors had no effect. These data suggest that PC1 and PC2 contribute to fluid-flow sensation by the primary cilium in renal epithelium and that they both function in the same mechanotransduction pathway. Loss or dysfunction of PC1 or PC2 may therefore lead to PKD owing to the inability of cells to sense mechanical cues that normally regulate tissue morphogenesis.

Maturation Alters Cyclic Nucleotide and Relaxation Responses to Nitric Oxide Donors in Ovine Cerebral Arteries

Biology of the Neonate. , 2003  |  Pubmed ID: 12576757

To examine the hypothesis that maturation modulates nitric oxide (NO)-induced relaxation in cerebral arteries, we quantified concentration-relaxation relations and the corresponding dynamic responses of guanosine 3':5'-cyclic monophosphate (cGMP) and adenosine 3':5'-cyclic monophosphate (cAMP) levels following administration of nitroglycerin and S-nitroso-N-acetyl-penicilamine (SNAP), an NO donor, in posterior communicating and middle cerebral arteries from newborn (3-7 days) and adult sheep. The results offer 5 main observations: (1) the efficacy and potency of NO donors were generally greater in newborn than in adult cerebral arteries; (2) rates of relaxation, and presumably rates of NO release, were faster for equimolar concentrations of SNAP than for nitroglycerin in both newborn and adult arteries; (3) basal concentrations were greater for cAMP than for cGMP, and both were greater in newborn than adult cerebral arteries; (4) in adult cerebral arteries, NO-induced increases in cGMP occurred faster but relaxation developed more slowly than in newborn cerebral arteries, and (5) responses to NO donors involved significant cross-reactivity between cGMP and cAMP, the characteristics of which were age, artery, and agent specific. From these results, we conclude that postnatal changes in reactivity to NO reflect corresponding changes in soluble guanylate cyclase activity and possible decreases in NO half-life. We also conclude that maturation slows the mechanisms mediating NO-induced relaxation, and that this effect is more pronounced in distal than in proximal cerebral arteries. The data also suggest that the rate-limiting step governing rates of response to NO is probably downstream from cGMP synthesis. From the basal cyclic nucleotide levels, we conclude that basal ratios of synthesis to hydrolysis were greater in fetal than adult arteries. Because NO increased both cGMP and cAMP, we speculate that Type III phosphodiesterase has a possible influence upon cerebrovascular responses to NO, and that this influence varies with postnatal age and artery type. Together, these findings emphasize that the cerebrovascular effects of NO are highly age dependent and artery specific, and should be carefully considered when administering NO therapeutically in the neonate.

Immortalized Epithelial Cells from Human Autosomal Dominant Polycystic Kidney Cysts

American Journal of Physiology. Renal Physiology. Sep, 2003  |  Pubmed ID: 12734101

Autosomal dominant polycystic kidney disease (ADPKD) is the result of mutations in one allele of the PKD1 or PKD2 genes, followed by "second hit" somatic mutations of the other allele in renal tubule cells. Continued proliferation of clonal cells originating from different nephron segments leads to cyst formation. In vitro studies of the mechanisms of cyst formation have been hampered by the scarcity of nephrectomy specimens and the limited life span of cyst-derived cells in primary culture. We describe the development of a series of immortalized epithelial cell lines from over 30 individual renal cysts obtained from 11 patients with ADPKD. The cells were immortalized with either wild-type (WT) or temperature-sensitive (TS) recombinant adeno-simian virus (SV)40 viruses. SV40 DNA integration into the cell genome was verified by PCR analysis. The cells have been passaged over 50 times with no apparent phenotypic change. By light microscopy, the cells appear pleomorphic but mostly polygonal and resemble the primary cultures. Transmission electron microscopy shows polarized epithelia with tight junctions. The SV40 large T antigen was detected by immunocytochemistry and by Western blot analysis at 37 degrees C in the WT cell lines and at 33 degrees C in the TS cell lines. It disappeared in TS cells 72 h following transfer to 39 degrees C. The majority (29) of the cell lines show binding of Dolichos biflorus lectin, suggesting distal tubule origin. Three cell lines show binding of Lotus tetragonolobus lectin or express aminopeptidase N, suggesting proximal tubule origin. Three cell lines were derived from a mixture of cysts and express features of both tubules. The PKD1 and PKD2 mRNA and protein were detected in all cells by RT-PCR and by immunocytochemistry. The majority of the cells tested also express the epidermal growth factor receptor, cystic fibrosis transmembrane conductance regulator, epithelial sodium channel, and renin. These new series of cyst-derived cell lines represent useful and readily available in vitro models for studying the cellular and molecular biology of ADPKD.

Cardiovascular Responses and Neurotransmitter Changes Following Blockade of NNOS Within the Ventrolateral Medulla During Static Muscle Contraction

Brain Research. Jul, 2003  |  Pubmed ID: 12788516

Nitric oxide (NO) is synthesized from L-arginine through the activity of the synthetic enzyme, NO synthase (NOS). Previous studies have demonstrated the roles of the three isoforms of NOS, namely endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS) in cardiovascular regulation. However, no investigation has been done to study their individual role in modulating cardiovascular responses during static skeletal muscle contraction. In this study, we determined the effects of microdialyzing a specific nNOS antagonist into the rostral (RVLM) and caudal ventrolateral medulla (CVLM) on cardiovascular responses and glutamatergic/GABAergic neurotransmission during the exercise pressor reflex using rats. We hypothesized that the NO modulation of the exercise pressor reflex was largely influenced by specific nNOS activity within the ventrolateral medulla. Bilateral microdialysis of a selective nNOS antagonist, 1-(2-trifluoromethylphenyl)-imidazole (1.0 microM), for 30 or 60 min into the RVLM potentiated cardiovascular responses and glutamate release during a static muscle contraction. Levels of GABA within the RVLM were decreased. The cardiovascular responses and neurochemical changes to muscle contraction recovered following discontinuation of the drug. In contrast, bilateral application of the nNOS antagonist into CVLM attenuated cardiovascular responses and glutamate release during a static muscle contraction, but augmented GABA release. These results demonstrate that nNOS in the ventrolateral medulla plays an important role in modulating glutamatergic/GABAergic neurotransmission that regulates the exercise pressor reflex, and contributes to the sympathoexcitatory and sympathoinhibitory actions of NO within the RVLM and CVLM, respectively.

Gating of the Polycystin Ion Channel Signaling Complex in Neurons and Kidney Cells

FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Apr, 2004  |  Pubmed ID: 14766803

Mutations in either polycystin-2 (PC2) or polycystin-1 (PC1) proteins cause severe, potentially lethal, kidney disorders and multiple extrarenal (including brain) disease phenotypes. PC2, a member of the transient receptor potential channel superfamily, and PC1, an orphan membrane receptor of largely unknown function, are thought to be part of a common signaling pathway. Here, we show that in rat sympathetic neurons and kidney cells, coassembly of full-length PC1 with PC2 forms a plasmalemmal ion channel signaling complex in which PC1 stimulation simultaneously activates PC2 ion channels and Gi/o-proteins. PC2 activation occurs through a structural rearrangement of PC1, independent of G-protein activation. Thus, PC1 acts as a prototypical membrane receptor that concordantly regulates PC2 channels and G-proteins, a bimodal mechanism that may account for the multifunctional roles of polycystin proteins in fundamental cellular processes of various cell types.

Polycystins and Mechanosensation in Renal and Nodal Cilia

BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology. Aug, 2004  |  Pubmed ID: 15273987

The external surfaces of the human body, as well as its internal organs, constantly experience different kinds of mechanical stimulations. For example, tubular epithelial cells of the kidney are continuously exposed to a variety of mechanical forces, such as fluid flow shear stress within the lumen of th nephron. The majority of epithelial cells along the nephron, except intercalated cells, possess a primary cilium, an organelle projecting from the cell's apical surface into the luminal space. Despite its discovery over 100 years ago, the primary cilium's function continued to elude researchers for many decades. However, recent studies indicate that renal cilia have a sensory function. Studies on polycystic kidney disease (PKD) have identified many of the molecular players, which should help solve the mystery of how the renal cilium senses fluid flow. In this review, we will summarize the recent breakthroughs in PKD research and discuss the role(s) of the polycystin signaling complex in mediating mechanosensory function by the primary cilium of renal epithelium as well as of the embryonic node.

Global Cytoskeletal Control of Mechanotransduction in Kidney Epithelial Cells

Experimental Cell Research. Nov, 2004  |  Pubmed ID: 15501441

Studies of mechanotransduction mediated by stress-sensitive ion channels generally focus on the site of force application to the cell. Here we show that global, cell-wide changes in cytoskeletal structure and mechanics can regulate mechanotransduction previously shown to be triggered by activation of the mechanosensitive calcium channel, polycystin-2, in the apical primary cilium of renal epithelial cells [S.M. Nauli, F.J. Alenghat, Y. Luo, E. Williams, P. Vassilev, X. Li, A.E. Elia, W. Lu, E.M. Brown, S.J. Quinn, D.E. Ingber, J. Zhou, Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells. Nat. Genet. 33 (2003) 129-37]. Disrupting cytoplasmic microfilaments or microtubules in these cells eliminated fluid shear stress-induced increase of intracellular calcium. Altering the cytoskeletal force balance by inhibiting actomyosin-based tension generation (using 2,3-butanedione monoxime), interfering with microtubule polymerization (using nocodazole, cochicine, or taxol), or disrupting basal integrin-dependent extracellular matrix adhesions (using soluble GRGDSP peptide or anti-beta1 integrin antibody), also inhibited the calcium spike in response to fluid stress. These data indicate that although fluid stress-induced displacement of the primary cilium may be transduced into a calcium spike through activation of polycystin-2 and associated calcium-induced calcium release from intracellular stores, this mechanotransduction response is governed by global mechanical cues, including isometric tension (prestress) within the entire cytoskeleton and intact adhesions to extracellular matrix.

Involvement of Extracellular Ca2+ Influx Through Voltage-independent Ca2+ Channels in Endothelin-1 Function

Cellular Signalling. Aug, 2005  |  Pubmed ID: 15894164

This article reviews the types and roles of voltage-independent Ca(2+) channels involved in the endothelin-1 (ET-1)-induced functional responses such as vascular contraction, cell proliferation, and intracellular Ca(2+)-dependent signaling pathways and discusses the molecular mechanisms for the activation of voltage-independent Ca(2+) channels by ET-1. ET-1 activates some types of voltage-independent Ca(2+) channels, such as Ca(2+)-permeable nonselective cation channels (NSCCs) and store-operated Ca(2+) channels (SOCC). Extracellular Ca(2+) influx through these voltage-independent Ca(2+) channels plays essential roles in ET-1-induced vascular contraction, cell proliferation, activation of epidermal growth factor receptor tyrosine kinase, regulation of proline-rich tyrosine kinase, and release of arachidonic acid. The experiments using various constructs of endothelin receptors reveal the importance of G(q) and G(12) families in activation of these Ca(2+) channels by ET-1. These findings provide a potential therapeutic mechanism of a functional interrelationship between G(q)/G(12) proteins and voltage-independent Ca(2+) channels in the pathophysiology of ET-1, such as in chronic heart failure, hypertension, and cerebral vasospasm.

Chronic Hypoxia Modulates Relations Among Calcium, Myosin Light Chain Phosphorylation, and Force Differently in Fetal and Adult Ovine Basilar Arteries

Journal of Applied Physiology (Bethesda, Md. : 1985). Jul, 2005  |  Pubmed ID: 16036903

The present study tests the hypothesis that age-related differences in contractility of cerebral arteries from hypoxic animals involve changes in myofilament Ca2+ sensitivity. Basilar arteries from term fetal and nonpregnant adult sheep maintained 110 days at 3,820 m were used in measurements of cytosolic calcium concentration ([Ca2+]i), myosin light chain phosphorylation, and contractile tensions induced by graded concentrations of K+ or serotonin (5-HT). Slopes relating [Ca2+]i to tension were similar in fetal (0.83 +/- 0.07) and adult (1.02 +/- 0.08) arteries for K+ contractions but were significantly greater for fetal (3.77 +/- 0.64) than adult (2.00 +/- 0.13) arteries for 5-HT contractions. For both K+ and 5-HT contractions, these relations were left shifted in fetal compared with adult arteries, indicating greater Ca2+ sensitivity in fetal arteries. In contrast, slopes relating [Ca2+]i and %myosin phosphorylation for K+ contractions were less in fetal (0.37 +/- 0.08) than adult (0.81 +/- 0.07) arteries, and the fetal curves were right shifted. For 5-HT contractions, the slope of the Ca2+-phosphorylation relation was similar in fetal (0.33 +/- 0.09) and adult (0.33 +/- 0.23) arteries, indicating that 5-HT depressed Ca2+-induced myosin phosphorylation in adult arteries. For slopes relating %myosin phosphorylation and tension, fetal values (K+: 1.52 +/- 0.22, 5-HT: 7.66 +/- 1.70) were less than adult values (K+: 2.13 +/- 0.30, 5-HT: 8.29 +/- 2.40) for both K+- and 5-HT-induced contractions, although again fetal curves were left shifted relative to the adult. Thus, in hypoxia-acclimatized basilar arteries, a downregulated ability of Ca2+ to promote myosin phosphorylation is offset by an upregulated ability of phosphorylated myosin to produce force yielding an increased fetal myofilament Ca2+ sensitivity. Postnatal maturation reprioritizes the mechanisms regulating hypoxic contractility through changes in the source of activator Ca2+, the pathways governing myosin light chain phosphorylation, and its interaction with actin.

Molecular Changes in NNOS Protein Expression Within the Ventrolateral Medulla Following Transient Focal Ischemia Affect Cardiovascular Functions

Brain Research. Sep, 2005  |  Pubmed ID: 16084499

The majority of human strokes involve an occlusion of the middle cerebral artery and subsequent damage to the brain tissues it perfuses. We have previously reported that reflex cardiovascular changes during a static muscle contraction are attenuated following transient middle cerebral artery occlusion (MCAO) and reperfusion [A. Ally, S.M. Nauli, T.J. Maher, Cardiovascular responses and neurotransmission in the ventrolateral medulla during skeletal muscle contraction following transient middle cerebral artery occlusion and reperfusion, Brain Res. 952 (2002) 176-187]. We hypothesized that the attenuation is a result of altered expression of neuronal nitric oxide synthase (nNOS) within the rostral (RVLM) and caudal ventrolateral medulla (CVLM). In this study, we have compared cardiovascular responses and nNOS protein expression within the four quadrants, i.e., left and right sides of both RVLM and CVLM in sham-operated rats (n = 10) and in rats with a temporary 90-min left-sided MCAO followed by 24 h reperfusion (n = 10). Increases in mean arterial pressure during a static muscle contraction were significantly attenuated in MCAO rats when compared to sham rats. The transient ischemia reduced nNOS expression within the ipsilateral RVLM quadrant compared to the contralateral RVLM or RVLM quadrants of control rats. In contrast, compared to sham rats and the right CVLM quadrant of MCAO rats, nNOS expression was significantly augmented in the ipsilateral CVLM in left-sided MCAO rats. These data suggest that the attenuation of cardiovascular responses during static muscle contraction in MCAO rats is partly due to a reduction in nNOS expression within the ipsilateral RVLM and an overexpression of nNOS abundance within the ipsilateral CVLM. Results demonstrate that nNOS expression within the medulla plays a significant role in mediating cardiovascular responses during static exercise in intact and pathophysiological conditions.

Loss of Polycystin-1 in Human Cyst-lining Epithelia Leads to Ciliary Dysfunction

Journal of the American Society of Nephrology : JASN. Apr, 2006  |  Pubmed ID: 16565258

A "two-hit" hypothesis predicts a second somatic hit, in addition to the germline mutation, as a prerequisite to cystogenesis and has been proposed to explain the focal nature for renal cyst formation in autosomal dominant polycystic kidney disease (ADPKD). It was reported previously that Pkd1(null/null) mouse kidney epithelial cells are unresponsive to flow stimulation. This report shows that Pkd1(+/null) cells are capable of responding to mechanical flow stimulation by changing their intracellular calcium concentration in a manner similar to that of wild-type cells. This paper reports that human renal epithelia require a higher level of shear stress to evoke a cytosolic calcium increase than do mouse renal epithelia. Both immortalized and primary cultured renal epithelial cells that originate from normal and nondilated ADPKD human kidney tubules display normal ciliary expression of the polycystins and respond to fluid-flow shear stress with the typical change in cytosolic calcium. In contrast, immortalized and primary cultured cyst-lining epithelial cells from ADPKD patients with mutations in PKD1 or with abnormal ciliary expression of polycystin-1 or -2 were not responsive to fluid shear stress. These data support a two-hit hypothesis as a mechanism of cystogenesis. This report proposes that calcium response to fluid-flow shear stress can be used as a readout of polycystin function and that loss of mechanosensation in the renal tubular epithelia is a feature of PKD cysts.

Fibrocystin/polyductin, Found in the Same Protein Complex with Polycystin-2, Regulates Calcium Responses in Kidney Epithelia

Molecular and Cellular Biology. Apr, 2007  |  Pubmed ID: 17283055

Recent evidence suggests that fibrocystin/polyductin (FPC), polycystin-1 (PC1), and polycystin-2 (PC2) are all localized at the plasma membrane and the primary cilium, where PC1 and PC2 contribute to fluid flow sensation and may function in the same mechanotransduction pathways. To further define the exact subcellular localization of FPC, the protein product encoded by the PKHD1 gene responsible for autosomal recessive polycystic kidney disease (PKD) in humans, and whether FPC has direct and/or indirect cross talk with PC2, which, in turn, is pivotal for the pathogenesis of autosomal dominant PKD, we performed double immunostaining and coimmunoprecipitation as well as a microfluorimetry study of kidney tubular epithelial cells. FPC and PC2 are found to completely or partially colocalize at the plasma membrane and the primary cilium and can be reciprocally coimmunoprecipitated. Although incomplete removal of FPC by small interfering RNA and antibody 803 to intracellular epitopes of FPC did not abolish flow-induced intracellular calcium responses, antibody 804 to extracellular epitopes of FPC blocked cellular calcium responses to flow stimulation. These findings suggest that FPC and polycystins share, at least in part, a common mechanotransduction pathway.

Endothelial Cilia Are Fluid Shear Sensors That Regulate Calcium Signaling and Nitric Oxide Production Through Polycystin-1

Circulation. Mar, 2008  |  Pubmed ID: 18285569

When challenged with extracellular fluid shear stress, vascular endothelial cells are known to release nitric oxide, an important vasodilator. Here, we show that the ability of cultured endothelial cells to sense a low range of fluid shear depends on apical membrane organelles, called cilia, and that cilia are compartments required for proper localization and function of the mechanosensitive polycystin-1 molecule.

Ciliary Dysfunction in Polycystic Kidney Disease: an Emerging Model with Polarizing Potential

Frontiers in Bioscience : a Journal and Virtual Library. , 2008  |  Pubmed ID: 18508522

The majority of different cell types in the human body have a cilium, a thin rod-like structure of uniquely arranged microtubules that are encapsulated by the surface plasma membrane. The cilium originates from a basal body, a mature centriole that has migrated and docked to the cell surface. The non-motile cilia are microtubule-based organelles that are generally considered sensory structures. The purpose of this review is to discuss the practicality of the ciliary hypothesis as a unifying concept for polycystic kidney disease and to review current literature in the field of cilium biology, as it relates to mechanosensation and planar cell polarity. The polycystins and fibrocystin localization at the cilium and other subcellular localizations are discussed, followed by a hypothetical model for the cilium's role in mechanosensing, planar cell polarity, and cystogenesis.

Ciliary Polycystin-2 is a Mechanosensitive Calcium Channel Involved in Nitric Oxide Signaling Cascades

Circulation Research. Apr, 2009  |  Pubmed ID: 19265036

Cardiovascular complications such as hypertension are a continuous concern in patients with autosomal dominant polycystic kidney disease (ADPKD). The PKD2 encoding for polycystin-2 is mutated in approximately 15% of ADPKD patients. Here, we show that polycystin-2 is localized to the cilia of mouse and human vascular endothelial cells. We demonstrate that the normal expression level and localization of polycystin-2 to cilia is required for the endothelial cilia to sense fluid shear stress through a complex biochemical cascade, involving calcium, calmodulin, Akt/PKB, and protein kinase C. In response to fluid shear stress, mouse endothelial cells with knockdown or knockout of Pkd2 lose the ability to generate nitric oxide (NO). Consistent with mouse data, endothelial cells generated from ADPKD patients do not show polycystin-2 in the cilia and are unable to sense fluid flow. In the isolated artery, we further show that ciliary polycystin-2 responds specifically to shear stress and not to mechanical stretch, a pressurized biomechanical force that involves purinergic receptor activation. We propose a new role for polycystin-2 in transmitting extracellular shear stress to intracellular NO biosynthesis. Thus, aberrant expression or localization of polycystin-2 to cilia could promote high blood pressure because of inability to synthesize NO in response to an increase in shear stress (blood flow).

Failure to Ubiquitinate C-Met Leads to Hyperactivation of MTOR Signaling in a Mouse Model of Autosomal Dominant Polycystic Kidney Disease

The Journal of Clinical Investigation. Oct, 2010  |  Pubmed ID: 20852388

Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disorder that is caused by mutations at two loci, polycystin 1 (PKD1) and polycystin 2 (PKD2). It is characterized by the formation of multiple cysts in the kidneys that can lead to chronic renal failure. Previous studies have suggested a role for hyperactivation of mammalian target of rapamycin (mTOR) in cystogenesis, but the etiology of mTOR hyperactivation has not been fully elucidated. In this report we have shown that mTOR is hyperactivated in Pkd1-null mouse cells due to failure of the HGF receptor c-Met to be properly ubiquitinated and subsequently degraded after stimulation by HGF. In Pkd1-null cells, Casitas B-lineage lymphoma (c-Cbl), an E3-ubiquitin ligase for c-Met, was sequestered in the Golgi apparatus with α₃β₁ integrin, resulting in the inability to ubiquitinate c-Met. Treatment of mouse Pkd1-null cystic kidneys in organ culture with a c-Met pharmacological inhibitor resulted in inhibition of mTOR activity and blocked cystogenesis in this mouse model of ADPKD. We therefore suggest that blockade of c-Met is a potential novel therapeutic approach to the treatment of ADPKD.

A Comparative Study of Embedded and Anesthetized Zebrafish in Vivo on Myocardiac Calcium Oscillation and Heart Muscle Contraction

Frontiers in Pharmacology. , 2010  |  Pubmed ID: 21833178

The zebrafish (Danio rerio) has been used as a model for studying vertebrate development in the cardiovascular system. In order to monitor heart contraction and cytosolic calcium oscillations, fish were either embedded in methylcellulose or anesthetized with tricaine. Using high-resolution differential interference contrast and calcium imaging microscopy, we here show that dopamine and verapamil alter calcium signaling and muscle contraction in anesthetized zebrafish, but not in embedded zebrafish. In anesthetized fish, dopamine increases the amplitude of cytosolic calcium oscillation with a subsequent increase in heart contraction, whereas verapamil decreases the frequency of calcium oscillation and heart rate. Interestingly, verapamil also increases myocardial contraction. Our data further indicate that verapamil can increase myocardial calcium sensitivity in anesthetized fish. Taken together, our data reinforce in vivo cardiac responses to dopamine and verapamil. Furthermore, effects of dopamine and verapamil on myocardial calcium and contraction are greater in anesthetized than embedded fish. We suggest that while the zebrafish is an excellent model for a cardiovascular imaging study, the cardio-pharmacological profiles are very different between anesthetized and embedded fish.

Dopamine Receptor Type 5 in the Primary Cilia Has Dual Chemo- and Mechano-sensory Roles

Hypertension. Aug, 2011  |  Pubmed ID: 21709211

Polycystic kidney disease is characterized by cardiovascular irregularities, including hypertension. Dopamine, a circulating hormone, is implicated in essential hypertension in humans and animal models. Vascular endothelial primary cilia are known to function as mechano-sensory organelles. Although both primary cilia and dopamine receptors play important roles in vascular hypertension, their relationship has never been explored. To determine the roles of the dopaminergic system and mechano-sensory cilia, we studied the effects of dopamine on ciliary length and function in wild-type and mechano-insensitive polycystic mutant cells (Pkd1(-/)(-) and Tg737(orpk/orpk)). We show for the first time that mouse vascular endothelia exhibit dopamine receptor-type 5 (DR5), which colocalizes to primary cilia in cultured cells and mouse arteries in vivo. DR5 activation increases cilia length in arteries and endothelial cells through cofilin and actin polymerization. DR5 activation also restores cilia function in the mutant cells. In addition, silencing DR5 completely abolishes mechano-ciliary function in WT cells. We found that DR5 plays very important roles in ciliary length and function. Furthermore, the chemo-sensory function of cilia can alter the mechano-sensory function through changes in sensitivity to fluid-shear stress. We propose that ciliary DR5 has functional chemo- and mechano-sensory roles in endothelial cells.

The Mechanosensory Role of Primary Cilia in Vascular Hypertension

International Journal of Vascular Medicine. , 2011  |  Pubmed ID: 21748021

Local regulation of vascular tone plays an important role in cardiovascular control of blood pressure. Aside from chemical or hormonal regulations, this local homeostasis is highly regulated by fluid-shear stress. It was previously unclear how vascular endothelial cells were able to sense fluid-shear stress. The cellular functions of mechanosensory cilia within vascular system have emerged recently. In particular, hypertension is insidious and remains a continuous problem that evolves during the course of polycystic kidney disease (PKD). The basic and clinical perspectives on primary cilia are discussed with regard to the pathogenesis of hypertension in PKD.

Calcium-mediated Mechanisms of Cystic Expansion

Biochimica Et Biophysica Acta. Oct, 2011  |  Pubmed ID: 20932898

In this review, we will discuss several well-accepted signaling pathways toward calcium-mediated mechanisms of cystic expansion. The second messenger calcium ion has contributed to a vast diversity of signal transduction pathways. We will dissect calcium signaling as a possible mechanism that contributes to renal cyst formation. Because cytosolic calcium also regulates an array of signaling pathways, we will first discuss cilia-induced calcium fluxes, followed by Wnt signaling that has attributed to much-discussed planar cell polarity. We will then look at the relationship between cytosolic calcium and cAMP as one of the most important aspects of cyst progression. The signaling of cAMP on MAPK and mTOR will also be discussed. We infer that while cilia-induced calcium fluxes may be the initial signaling messenger for various cellular pathways, no single signaling mediator or pathway is implicated exclusively in the progression of the cystic expansion. This article is part of a Special Issue entitled: Polycystic Kidney Disease.

Endothelial Cells from Humans and Mice with Polycystic Kidney Disease Are Characterized by Polyploidy and Chromosome Segregation Defects Through Survivin Down-regulation

Human Molecular Genetics. Jan, 2011  |  Pubmed ID: 21041232

Autosomal-dominant polycystic kidney disease (ADPKD) is the most common hereditary and systemic disorder associated with various cardiovascular complications. It has been implicated with dysfunction in primary cilia. We and others have shown that the immediate function of endothelial cilia is to sense extracellular signal. The long-term function of cilia is hypothesized to regulate cell cycle. Here, we show that ciliary function (polycystins) and structure (polaris) are required for proper cellular division. Cilia mutant cells undergo abnormal cell division with apparent defects in mitotic spindle formation, cellular spindle assembly checkpoint and centrosome amplification. Down-regulation of the chromosomal passenger survivin contributes to these abnormalities, which further result in cell polyploidy. Re-expression of survivin restores a competent spindle assembly checkpoint and reduces polyploidy. Aged animals show a more severe phenotype in cellular division, consistent with progression of cardiovascular complications seen in older ADPKD patients. For the first time, we show that structure and function of mechanosensory cilia are crucial in maintaining proper cellular proliferation. Furthermore, developmental aging plays a crucial role in the progression of these abnormal cellular phenotypes. We propose that abnormal function or structure of primary cilia not only causes failure to transmit extracellular signals, but also is associated with cytokinesis defects in both mice and humans with polycystic kidney disease.

Multiple Protein Kinases Determine the Phosphorylated State of the Small Heat Shock Protein, HSP27, in SH-SY5Y Neuroblastoma Cells

Neuropharmacology. Jul-Aug, 2011  |  Pubmed ID: 21338617

In SH-SY5Y human neuroblastoma cells, the cholinergic agonist, carbachol, stimulates phosphorylation of the small heat shock protein 27 (HSP27). Carbachol increases phosphorylation of both Ser-82 and Ser-78 while the phorbol ester, phorbol-12, 13-dibutyrate (PDB) affects only Ser-82. Muscarinic receptor activation by carbachol was confirmed by sensitivity of Ser-82 phosphorylation to hyoscyamine with no effect of nicotine or bradykinin. This response to carbachol is partially reduced by inhibition of protein kinase C (PKC) with GF 109203X and p38 mitogen-activated protein kinase (MAPK) with SB 203580. In contrast, phosphorylation produced by PDB is completely reversed by GF 109203X or CID 755673, an inhibitor of PKD. Inhibition of phosphatidylinositol 3-kinase or Akt with LY 294002 or Akti-1/2 stimulates HSP27 phosphorylation while rapamycin, which inhibits mTORC1, does not. The stimulatory effect of Akti-1/2 is reversed by SB 203580 and correlates with increased p38 MAPK phosphorylation. SH-SY5Y cells differentiated with a low concentration of PDB and basic fibroblast growth factor to a more neuronal phenotype retain carbachol-, PDB- and Akti-1/2-responsive HSP27 phosphorylation. Immunofluorescence microscopy confirms increased HSP27 phosphorylation in response to carbachol or PDB. At cell margins, PDB causes f-actin to reorganize forming lamellipodial structures from which phospho-HSP27 is segregated. The resultant phenotypic change in cell morphology is dependent upon PKC, but not PKD, activity. The major conclusion from this study is that the phosphorylated state of HSP27 in SH-SY5Y cells results from integrated signaling involving PKC, p38 MAPK and Akt.

Lack of Primary Cilia Primes Shear-induced Endothelial-to-mesenchymal Transition

Circulation Research. Apr, 2011  |  Pubmed ID: 21393577

Primary cilia are cellular protrusions that serve as mechanosensors for fluid flow. In endothelial cells (ECs), they function by transducing local blood flow information into functional responses, such as nitric oxide production and initiation of gene expression. Cilia are present on ECs in areas of low or disturbed flow and absent in areas of high flow. In the embryonic heart, high-flow regime applies to the endocardial cushion area, and the absence of cilia here coincides with the process of endothelial-to-mesenchymal transition (EndoMT).

Multimodal Spectral Imaging of Cells Using a Transmission Diffraction Grating on a Light Microscope

Applied Spectroscopy. Jun, 2011  |  Pubmed ID: 21639978

A multimodal methodology for spectral imaging of cells is presented. The spectral imaging setup uses a transmission diffraction grating on a light microscope to concurrently record spectral images of cells and cellular organelles by fluorescence, darkfield, brightfield, and differential interference contrast (DIC) spectral microscopy. Initially, the setup was applied for fluorescence spectral imaging of yeast and mammalian cells labeled with multiple fluorophores. Fluorescence signals originating from fluorescently labeled biomolecules in cells were collected through triple or single filter cubes, separated by the grating, and imaged using a charge-coupled device (CCD) camera. Cellular components such as nuclei, cytoskeleton, and mitochondria were spatially separated by the fluorescence spectra of the fluorophores present in them, providing detailed multi-colored spectral images of cells. Additionally, the grating-based spectral microscope enabled measurement of scattering and absorption spectra of unlabeled cells and stained tissue sections using darkfield and brightfield or DIC spectral microscopy, respectively. The presented spectral imaging methodology provides a readily affordable approach for multimodal spectral characterization of biological cells and other specimens.

An ACE Inhibitor Improves Vascular Outcomes in a PKD Model

American Journal of Physiology. Renal Physiology. Nov, 2011  |  Pubmed ID: 21880832

Polycystic Diseases in Visceral Organs

Obstetrics and Gynecology International. , 2011  |  Pubmed ID: 22242024

Primary cilia are nonmotile, microtubule-based, antenna-like organelles projecting from the apical surface of most mammalian cells. Elegant studies have established the importance of ciliary structure and function in signal transduction and the sensory roles of cilia in maintaining healthy cellular state. In particular, dysfunctional cilia have been implicated in a large number of diseases mainly characterized by the presence of fluid-filled cysts in various organs. Aside from polycystic kidney disease (PKD), however, the roles of cilia in polycystic liver disease (PLD), polycystic pancreas disease (PPD), and polycystic ovarian syndrome (PCOS) are still very vague. In addition, although gender and sex hormones are known to regulate cyst formation, their roles in regulating physiological functions of cilia need to be further explored.

Endothelin

Cellular and Molecular Life Sciences : CMLS. Jan, 2011  |  Pubmed ID: 20848158

Endothelin-1 is the most potent vasoconstrictor agent currently identified, and it was originally isolated and characterized from the culture media of aortic endothelial cells. Two other isoforms, termed endothelin-2 and endothelin-3, were subsequently identified, along with structural homologues isolated from the venom of Actractapis engaddensis known as the sarafotoxins. In this review, we will discuss the basic science of endothelins, endothelin-converting enzymes, and endothelin receptors. Only concise background information pertinent to clinical physician is provided. Next we will describe the pathophysiological roles of endothelin-1 in pulmonary arterial hypertension, heart failure, systemic hypertension, and female malignancies, with emphasis on ovarian cancer. The potential intervention with pharmacological therapeutics will be succinctly summarized to highlight the exciting pre-clinical and clinical studies within the endothelin field. Of note is the rapid development of selective endothelin receptor antagonists, which has led to an explosion of research in the field.

Mechanisms Regulating Cilia Growth and Cilia Function in Endothelial Cells

Cellular and Molecular Life Sciences : CMLS. Jan, 2012  |  Pubmed ID: 21671118

The primary cilium is an important sensory organelle present in most mammalian cells. Our current studies aim at examining intracellular molecules that regulate cilia length and/or cilia function in vitro and ex vivo. For the first time, we show that intracellular cAMP and cAMP-dependent protein kinase (PKA) regulate both cilia length and function in vascular endothelial cells. Although calcium-dependent protein kinase modulates cilia length, it does not play a significant role in cilia function. Cilia length regulation also involves mitogen-activated protein kinase (MAPK), protein phosphatase-1 (PP-1), and cofilin. Furthermore, cofilin regulates cilia length through actin rearrangement. Overall, our study suggests that the molecular interactions between cilia function and length can be independent of one another. Although PKA regulates both cilia length and function, changes in cilia length by MAPK, PP-1, or cofilin do not have a direct correlation to changes in cilia function. We propose that cilia length and function are regulated by distinct, yet complex intertwined signaling pathways.

Primary Cilia Regulates the Directional Migration and Barrier Integrity of Endothelial Cells Through the Modulation of Hsp27 Dependent Actin Cytoskeletal Organization

Journal of Cellular Physiology. Jan, 2012  |  Pubmed ID: 21837772

Cilia are mechanosensing organelles that communicate extracellular signals into intracellular responses. Altered functions of primary cilia play a key role in the development of various diseases including polycystic kidney disease. Here, we show that endothelial cells from the oak ridge polycystic kidney (Tg737(orpk/orpk) ) mouse, with impaired cilia assembly, exhibit a reduction in the actin stress fibers and focal adhesions compared to wild-type (WT). In contrast, endothelial cells from polycystin-1 deficient mice (pkd1(null/null) ), with impaired cilia function, display robust stress fibers, and focal adhesion assembly. We found that the Tg737(orpk/orpk) cells exhibit impaired directional migration and endothelial cell monolayer permeability compared to the WT and pkd1(null/null) cells. Finally, we found that the expression of heat shock protein 27 (hsp27) and the phosphorylation of focal adhesion kinase (FAK) are downregulated in the Tg737(orpk/orpk) cells and overexpression of hsp27 restored both FAK phosphorylation and cell migration. Taken together, these results demonstrate that disruption of the primary cilia structure or function compromises the endothelium through the suppression of hsp27 dependent actin organization and focal adhesion formation, which may contribute to the vascular dysfunction in ciliopathies.