Kay-Pong Yip

Chronic hypoxia-induced upregulation of store-operated and receptor-operated Ca2+ channels in pulmonary arterial smooth muscle cells : A novel mechanism of hypoxic pulmonary hypertension

Chronic hypoxic pulmonary hypertension is associated with profound vascular remodeling and alterations in Ca2+ homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Recent studies show that transient receptor potential (TRPC) genes, which encode store-operated and receptor-operated cation channels, play important roles in Ca2+ regulation and cell proliferation. However, the influence of chronic hypoxia on TRPC channels has not been determined. Here we compared TRPC expression, and store- and receptor-operated Ca2+ entries in PASMCs of normoxic and chronic hypoxic rats. Reverse-transcription polymerase chain reaction (RT-PCR), Western blot, and immunostaining showed consistently that TRPC1, TRPC3, and TRPC6 were expressed in intralobar pulmonary arteries (PAs) and PASMCs. Application of 1-oleoyl-2-acetyl-sn-glycerol (OAG) to directly activate receptor-operated channels, or thapsigargin to deplete Ca2+ stores, caused dramatic increase in cation entry measured by Mn2+ quenching of fura-2 and by Ca2+ transients. OAG-induced responses were ≈700-fold more resistant to La3+ inhibition than thapsigargin-induced responses. siRNA knockdown of TRPC1 and TRPC6 specifically attenuated thapsigargin- and OAG-induced cation entries, respectively, indicating that TRPC1 mediates store-operated entry and TRPC6 mediates receptor-operated entry. In hypoxic PAs, there were 2- to 3-fold increases in TRPC1 and TRPC6 expression. They were accompanied by significant increases in basal, OAG-induced, and thapsigargin-induced cation entries in hypoxic PASMCs. Moreover, removal of Ca2+ or inhibition of store-operated Ca2+ entry with La3+ and SK&F-96365 reversed the elevated basal [Ca2+]i in PASMCs and vascular tone in PAs of chronic hypoxic animals, but nifedipine had minimal effects. Our results for the first time to our knowledge show that both store- and receptor-operated channels of PASMCs are upregulated by chronic hypoxia and contribute to the enhanced vascular tone in hypoxic pulmonary hypertension.

Regulation of Aquaporin-2 Trafficking by Vasopressin in the Renal Collecting Duct ROLES OF RYANODINE-SENSITIVE Ca2+ STORES AND CALMODULIN

In the renal collecting duct, vasopressin increases osmotic water permeability (P f ) by triggering trafficking of aquaporin-2 vesicles to the apical plasma membrane. We investigated the role of vasopressin-induced intracellular Ca2+ mobilization in this process. In isolated inner medullary collecting ducts (IMCDs), vasopressin (0.1 nm) and 8-(4-chlorophenylthio)-cAMP (0.1 mm) elicited marked increases in [Ca2+] i (fluo-4). Vasopressin-induced Ca2+ mobilization was completely blocked by preloading with the Ca2+ chelator BAPTA. In parallel experiments, BAPTA completely blocked the vasopressin-induced increase in P f without affecting adenosine 3′,5′-cyclic monophosphate (cAMP) production. Previously, we demonstrated the lack of activation of the phosphoinositide-signaling pathway by vasopressin in IMCD, suggesting an inositol 1,4,5-trisphosphate-independent mechanism of Ca2+ release. Evidence for expression of the type 1 ryanodine receptor (RyR1) in IMCD was obtained by immunofluorescence, immunoblotting, and reverse transcription-polymerase chain reaction. Ryanodine (100 μm), a ryanodine receptor antagonist, blocked the arginine vasopressin-mediated increase in P f and blocked vasopressin-stimulated redistribution of aquaporin-2 to the plasma membrane domain in primary cultures of IMCD cells, as assessed by immunofluorescence immunocytochemistry. Calmodulin inhibitors (W7 and trifluoperazine) blocked the P f response to vasopressin and the vasopressin-stimulated redistribution of aquaporin-2. The results suggest that Ca2+ release from ryanodine-sensitive stores plays an essential role in vasopressin-mediated aquaporin-2 trafficking via a calmodulin-dependent mechanism.

Coupling of vasopressin-induced intracellular Ca2+ mobilization and apical exocytosis in perfused rat kidney collecting duct.

Arginine vasopressin (AVP) regulates the osmotic water permeability of the kidney collecting duct by inducing exocytotic insertion of aquaporin‐2 into apical membrane. The coupling between AVP‐induced intracellular Ca2+ mobilization and apical exocytosis was investigated in isolated perfused rat inner medullary collecting duct (IMCD) segments using confocal fluorescence microscopy. Changes of [Ca2+]i in IMCD cells were measured with fluo‐4. A novel confocal imaging technique using a styryl dye, FM1‐43, was developed to monitor real‐time exocytosis induced by arginine vasopressin. AVP (0.1 nm) triggered a rapid increase of [Ca2+]i in IMCD cells, followed by sustained oscillations. Ratiometric measurement of [Ca2+]i confirmed that the observed [Ca2+]i oscillation was a primary event and was not secondary to changes in cell volume. The frequencies of [Ca2+]i oscillations in each IMCD cell were independent and time variant. 1‐Deamino‐8‐d‐arginine vasopressin (a V2 receptor agonist, 0.1 nm) simulated the effects of AVP by triggering [Ca2+]i oscillations. In the absence of extracellular Ca2+, ryanodine (0.1 mm) inhibited AVP‐induced Ca2+ mobilization. AVP (0.1 nm) triggered accumulative apical exocytosis in IMCD cells within 20 s after application. Pre‐incubating the IMCD with an intracellular Ca2+ chelator, BAPTA, prevented AVP‐induced intracellular Ca2+ mobilization, apical exocytosis, and increase of osmotic water permeability. These results indicate that AVP, via the V2 receptor, triggers a calcium signalling cascade observed as [Ca2+]i oscillations in the IMCD and that intracellular Ca2+ mobilization is required for exocytotic insertion of aquaporin‐2.

Redistribution of Na+/H+ exchanger isoform NHE3 in proximal tubules induced by acute and chronic hypertension

Redistribution of apical Na+/H+exchangers (NHE) in the proximal tubules as a plausible mechanism of pressure natriuresis was investigated with confocal immunofluorescence microscopy in Sprague-Dawley rats (SD), spontaneously hypertensive rats (SHR), and two-kidney, one-clip Goldblatt hypertensive rats (GH). NHE isoform NHE3 was localized in the brush border of proximal tubules in SD. Twenty minutes of induced acute hypertension (20-40 mmHg) resulted in a pronounced redistribution of isoform NHE3 from the brush border into the base of microvilli, where clathrin-coated pits were localized. Prehypertensive young SHR (5 wk old, mean blood pressure 105 ± 3 mmHg, n = 11) produced similar findings. However, NHE3 was found to concentrate in the base of microvilli in adult SHR (12 wk old, mean blood pressure 134 ± 6 mmHg, n = 12) and nonclipped kidneys of GH (mean blood pressure 131 ± 6 mmHg, n = 6). In clipped kidneys of GH, which were not exposed to the hypertension because of the arterial clips, NHE3 was localized on the brush border as in normal SD. No further redistribution of NHE3 was detected in adult SHR or GH when acute hypertension was induced. Since both acute and chronic increase of arterial pressure can provoke the redistribution of apical NHE in proximal tubules, the pressure-induced NHE redistribution could be a physiological response and an integral part of pressure natriuresis.

Synchronization phenomena in nephron–nephron interaction

Experimental data for tubular pressure oscillations in rat kidneys are analyzed in order to examine the different types of synchronization that can arise between neighboring functional units. For rats with normal blood pressure, the individual unit (the nephron) typically exhibits regular oscillations in its tubular pressure and flow variations. For such rats, both in-phase and antiphase synchronization can be demonstrated in the experimental data. For spontaneously hypertensive rats, where the pressure variations in the individual nephrons are highly irregular, signs of chaotic phase and frequency synchronization can be observed. Accounting for a hemodynamic as well as for a vascular coupling between nephrons that share a common interlobular artery, we develop a mathematical model of the pressure and flow regulation in a pair of adjacent nephrons. We show that this model, for appropriate values of the parameters, can reproduce the different types of experimentally observed synchronization. (c) 2001 American Institute of Physics.

Na+/H+ Exchanger Isoform 2 (NHE2) is Expressed in the Apical Membrane of the Medullary Thick Ascending Limb

Abstract. Apical Na+/H+ exchangers (NHE) in the proximal tubule and medullary thick ascending limb (MTAL) display similar functions and regulation, suggesting that similar NHE isoforms are present. In the rat proximal tubule, NHE2 and NHE3 are present in the apical membrane, however, in the MTAL, NHE3, but not NHE2, mRNA has been found. In this study, the expression and subcellular localization of NHE2 in both rat and mouse MTAL were studied. To detect NHE2 mRNA, reverse transcription-polymerase chain reaction (RT-PCR) was performed in microdissected MTAL tubules using primers specific for NHE2. Analysis of PCR products with and without digestion by restriction enzymes chosen from the published NHE2 sequence gave predicted sizes. Subcloning and sequencing of the PCR product from mouse MTAL revealed 91% and 75% identity to the published NHE2 nucleotide sequence of comparable regions in rat and rabbit, respectively. Thus, NHE2 mRNA is expressed in the MTAL of mouse and rat. The subcellular localization of NHE2 was determined by immunochemistry using a specific NHE2 antibody. Immunofluorescence staining was observed in the apical, but not basolateral, membrane of MTAL of both species. In addition, anti-NHE2 antibody recognized an 85 kD protein in plasma membranes prepared from mouse and rat renal outer medulla and a MTAL cell line by Western analysis, which further support that NHE2 protein is expressed in the MTAL of both species. We conclude that NHE2 is expressed in the apical membrane of MTAL in both mouse and rat.

Controlled release of plasmid DNA

Abstract Very large molecular weight reporter plasmids (pCMV- β gal, pSV- β gal) and lower molecular weight (herring sperm) DNA were encapsulated in polyethylene vinyl co-acetate (EVAc). In vitro release studies were performed to determine release rates and duration of delivery. The swelling behavior and morphological changes of these formulations were studied to elucidate the potential mechanism of DNA release. The bioactivity of the released plasmid DNA was assessed through analysis of conformation using agarose gel electrophoresis and in vitro transfection of C 2 C 12 myoblasts using liposome (Lipofectin™) complexation. Extraction of plasmid DNA from the delivery systems indicated that the fabrication conditions did not degrade the DNA. Depending on initial DNA loading, detectable levels were released for 1–6 months. Pore formation was accompanied by swelling which varied according to DNA loading as well as the type of DNA (herring sperm, plasmid). Conformational analysis of released plasmid DNA showed DNA was released without degradation and retained the ability to transfect cells in vitro. The results demonstrate that controlled release systems can be fabricated for the release of very large molecular weight plasmid DNA which may provide an alternative approach to plasmid-based gene transfer.

NBC3 expression in rabbit collecting duct: colocalization with vacuolar H+-ATPase

We have recently cloned and characterized a unique sodium bicarbonate cotransporter, NBC3, which unlike other members of the NBC family, is ethylisopropylamiloride (EIPA) inhibitable, DIDS insensitive, and electroneutral (A. Pushkin, N. Abuladze, I. Lee, D. Newman, J. Hwang, and I. Kurtz. J. Biol. Chem.274: 16569-16575, 1999). In the present study, a specific polyclonal antipeptide COOH-terminal antibody, NBC3-C1, was generated and used to determine the pattern of NBC3 protein expression in rabbit kidney. A major band of ∼200 kDa was detected on immunoblots of rabbit kidney. Immunocytochemistry of rabbit kidney frozen sections revealed specific staining of the apical membrane of intercalated cells in both the cortical and outer medullary collecting ducts. The pattern of NBC3 protein expression in the collecting duct was nearly identical to the same sections stained with an antibody against the vacuolar H+-ATPase 31-kDa subunit. In addition, the NBC3-C1 antibody coimmunoprecipitated the vacuolar H+-ATPase 31-kDa subunit. Functional studies in outer medullary collecting ducts (inner stripe) showed that type A intercalated cells have an apical Na+-dependent base transporter that is EIPA inhibitable and DIDS insensitive. The data suggest that NBC3 participates in H+/base transport in the collecting duct. The close association of NBC3 and the vacuolar H+-ATPase in type A intercalated cells suggests a potential structural/functional interaction between the two transporters.

An Arg-Gly-Asp peptide stimulates constriction in rat afferent arteriole.

The potential role of integrins in the myogenic mechanism was studied in the rat afferent arteriole (AA) by fluorescence immunolocalization and microperfusion of isolated AA. Confocal fluorescence images were acquired from frozen sections of rat kidney after indirect immunostaining for various integrin beta- and alpha-subunits. The beta 1-, beta 3-, alpha 3-, alpha 5-, and alpha V-integrins were found on the plasma membrane in smooth muscle of AA, providing the morphological basis for participation of integrins in mechanotransduction. With 1 mM nitro-L-arginine methyl ester (L-NAME) in the luminal perfusate to inhibit endogenous nitric oxide (NO) production from AA, the hexapeptide GRGDSP (10(-7)-10(-3)M) induced immediate vasoconstriction. The constriction was dose dependent and specific or peptides with arginine-glycine-aspartic acid (RGD) motifs, commonly found on the binding sites of extracellular matrix to integrins. In controls, the hexapeptide GRGESP induced no constriction. GRGDSP, 1 mM, induced a 21.6 +/- 2.6% decrease (P < 0.05, n = 6) in lumen diameter for 30 s and an 18.3 +/- 4.1% increase (P < 0.05, n = 6) in smooth muscle intracellular calcium concentration for 18 s, as measured by the emission ratio of Fluo-3/Fura Red. Binding of exogenous RGD motifs with exposed integrins on AA smooth muscle therefore triggers calcium-dependent vasoconstriction. However, the dose response to RGD was not sensitive to the myogenic tone of the vessel, which suggests that the integrin-mediated vasoconstriction is different from myogenic constriction.The potential role of integrins in the myogenic mechanism was studied in the rat afferent arteriole (AA) by fluorescence immunolocalization and microperfusion of isolated AA. Confocal fluorescence images were acquired from frozen sections of rat kidney after indirect immunostaining for various integrin β- and α-subunits. The β1-, β3-, α3-, α5-, and αV-integrins were found on the plasma membrane in smooth muscle of AA, providing the morphological basis for participation of integrins in mechanotransduction. With 1 mM nitro-l-arginine methyl ester (l-NAME) in the luminal perfusate to inhibit endogenous nitric oxide (NO) production from AA, the hexapeptide GRGDSP (10-7-10-3 M) induced immediate vasoconstriction. The constriction was dose dependent and specific for peptides with arginine-glycine-aspartic acid (RGD) motifs, commonly found on the binding sites of extracellular matrix to integrins. In controls, the hexapeptide GRGESP induced no constriction. GRGDSP, 1 mM, induced a 21.6 ± 2.6% decrease ( P < 0.05, n = 6) in lumen diameter for 30 s and an 18.3 ± 4.1% increase ( P < 0.05, n = 6) in smooth muscle intracellular calcium concentration for 18 s, as measured by the emission ratio of Fluo-3/Fura Red. Binding of exogenous RGD motifs with exposed integrins on AA smooth muscle therefore triggers calcium-dependent vasoconstriction. However, the dose response to RGD was not sensitive to the myogenic tone of the vessel, which suggests that the integrin-mediated vasoconstriction is different from myogenic constriction.

Time-varying properties of renal autoregulatory mechanisms

In order to assess the possible time-varying properties of renal autoregulation, time-frequency and time-scaling methods were applied to renal blood flow under broad-band forced arterial blood pressure fluctuations and single-nephron renal blood flow with spontaneous oscillations obtained from normotensive (Sprague-Dawley, Wistar, and Long-Evans) rats, and spontaneously hypertensive rats. Time-frequency analyses of normotensive and hypertensive blood flow data obtained from either the whole kidney or the single-nephron show that indeed both the myogenic and tubuloglomerular feedback (TGF) mechanisms have time-varying characteristics. Furthermore, we utilized the Renyi entropy to measure the complexity of blood-flow dynamics in the time-frequency plane in an effort to discern differences between normotensive and hypertensive recordings. We found a clear difference in Renyi entropy between normotensive and hypertensive blood flow recordings at the whole kidney level for both forced (p < 0.037) and spontaneous arterial pressure fluctuations (p < 0.033), and at the single-nephron level (p < 0.008). Especially at the single-nephron level, the mean Renyi entropy is significantly larger for hypertensive than normotensive rats, suggesting more complex dynamics in the hypertensive condition. To further evaluate whether or not the separation of dynamics between normotensive and hypertensive rats is found in the prescribed frequency ranges of the myogenic and TGF mechanisms, we employed multiresolution wavelet transform. Our analysis revealed that exclusively over scale ranges corresponding to the frequency intervals of the myogenic and TGF mechanisms, the widths of the blood flow wavelet coefficients fall into disjoint sets for normotensive and hypertensive rats. The separation of the scales at the myogenic and TGF frequency ranges is distinct and obtained with 100% accuracy. However, this observation remains valid only for the whole kidney blood pressure/flow data. The results suggest that understanding of the time-varying properties of the two mechanisms is required for a complete description of renal autoregulation.