Thomas H. Steinberg

Hypertension and prolonged vasoconstrictor signaling in RGS2-deficient mice

Signaling by hormones and neurotransmitters that activate G protein-coupled receptors (GPCRs) maintains blood pressure within the normal range despite large changes in cardiac output that can occur within seconds. This implies that blood pressure regulation requires precise kinetic control of GPCR signaling. To test this hypothesis, we analyzed mice deficient in RGS2, a GTPase-activating protein that greatly accelerates the deactivation rate of heterotrimeric G proteins in vitro. Both rgs2+/- and rgs2-/- mice exhibited a strong hypertensive phenotype, renovascular abnormalities, persistent constriction of the resistance vasculature, and prolonged response of the vasculature to vasoconstrictors in vivo. Analysis of P2Y receptor-mediated Ca2+ signaling in vascular smooth muscle cells in vitro indicated that loss of RGS2 increased agonist potency and efficacy and slowed the kinetics of signal termination. These results establish that abnormally prolonged signaling by G protein-coupled vasoconstrictor receptors can contribute to the onset of hypertension, and they suggest that genetic defects affecting the function or expression of RGS2 may be novel risk factors for development of hypertension in humans.

Inhibition of Fura-2 sequestration and secretion with organic anion transport blockers.

Fura-2 is widely used to measure the concentration of cytosolic free calcium, but in many cells the dye does not remain localized within the cytoplasmic matrix. In these cells, Fura-2 is sequestered within intracellular organelles, secreted into the extracellular medium, or both. We have found that, in mouse peritoneal macrophages, J774 cells, PC12 cells, and N2A cells, Fura-2 sequestration and secretion are mediated by organic anion transport systems and are blocked by the inhibitors probenecid and sulfinpyrazone. Under appropriate conditions these agents have little affect on calcium transients, and may facilitate the use of Fura-2 in a variety of cell types.

Connexin43 mediates direct intercellular communication in human osteoblastic cell networks.

We have examined cell coupling and expression of gap junction proteins in monolayer cultures of cells derived from human bone marrow stromal cells (BMC) and trabecular bone osteoblasts (HOB), and in the human osteogenic sarcoma cell line, SaOS-2. Both HOB and BMC cells were functionally coupled, since microinjection of Lucifer yellow resulted in dye transfer to neighboring cells, with averages of 3.4 +/- 2.8 (n = 131) and 8.1 +/- 9.3 (n = 51) coupled cells per injection, respectively. In contrast, little diffusion of Lucifer yellow was observed in SaOS-2 monolayers (1.4 +/- 1.8 coupled cells per injection, n = 100). Dye diffusion was inhibited by octanol (3.8 mM), an inhibitor of gap junctional communication. All of the osteoblastic cells expressed mRNA for connexin43 and connexin45, but not for connexins 26, 32, 37, 40, or 46. Whereas all of the osteoblastic cells expressed similar quantities of mRNA for connexin43, the poorly coupled SaOS-2 cells produced significantly less Cx43 protein than either HOB or BMC, as assessed by immunofluorescence and immunoprecipitation. Conversely, more Cx45 mRNA was expressed by SaOS-2 cells than by HOB or BMC. Thus, intercellular coupling in normal and transformed human osteoblastic cells correlates with the level of expression of Cx43, which appears to mediate intercellular communication in these cells. Gap junctional communication may serve as a means by which osteoblasts can work in synchrony and propagate locally generated signals throughout the skeletal tissue.

Cyclic Stretch Enhances Gap Junctional Communication Between Osteoblastic Cells

Mechanical loading is essential to maintain skeletal integrity. Because gap junctions in bone are affected by mechanical factors, we studied whether stretch, an anabolic stimulus for osteoblasts, modulates direct intercellular communication in these cells. Gap junctional communication during stretch was assessed using a newly developed method, the “parachute assay,” which allows monitoring of dye diffusion without disruption of the plasma membrane. Application of cyclic stretch for 2 or 24 h to well‐coupled ROS 17/2.8 cells resulted in a 56.5% and 30.4% increase in dye coupling, respectively, compared with resting conditions. Stretch increased dye diffusion less dramatically (12.4% compared with unstimulated cells) in the poorly coupled UMR 106‐01 cells. The stretch‐induced increase of cell coupling was abolished in the presence of the gap junctional inhibitor, heptanol. Steady‐state mRNA levels of connexin43 (Cx43), the gap junction protein that mediates cell‐to‐cell diffusion of negatively charged dyes between osteoblasts, were not different between control and stretched ROS 17/2.8 or UMR 106‐01 cultures after various periods of cyclic stretch. However, phosphorylated forms of Cx43 protein were more abundant in stretched ROS 17/2.8 than in controls. This was associated with increased punctate Cx43‐specific immunostain at appositional membranes of stretched cells. Thus, cyclic stretch increases gap junctional communication between osteoblastic cells by modulating intracellular localization of Cx43.

ZO-1 alters the plasma membrane localization and function of Cx43 in osteoblastic cells

ZO-1 is the major connexin-interacting protein in ROS 17/2.8 (ROS) osteoblastic cells. We examined the role of ZO-1 in Cx43-mediated gap junction formation and function in ROS cells that expressed the connexin-interacting fragment of ZO-1 (ROS/ZO-1dn) cells. Expression of this ZO-17-444 fusion protein in ROS cells disrupted the Cx43/ZO-1 interaction and decreased dye transfer by 85%, although Cx43 was retained on the plasma membrane as assessed by surface biotinylation. Fractionation of lysates derived from ROS/ZO-1dn cells on a 5-30% sucrose flotation gradient showed that 40% of the Cx43 floated into these sucrose gradients, whereas none of the Cx43 in ROS cell lysates entered the gradients, suggesting that more Cx43 is associated with lipid rafts in the transfected ROS cells than in lysates derived from untransfected ROS cells. In contrast to the ROS/ZO-1dn cells, ROS cells that over-expressed ZO-1 protein (ROS/ZO-1myc cells) exhibited increased gap junctional permeability and appositional membrane staining for Cx43. These data demonstrate that ZO-1 regulates Cx43-mediated gap junctional communication in osteoblastic cells and alters the membrane localization of Cx43. They suggest that ZO-1-mediated delivery of Cx43 from a lipid raft domain to gap junctional plaques may be an important regulatory step in gap junction formation.

P2Y Purinoceptors Are Responsible for Oscillatory Fluid Flow-induced Intracellular Calcium Mobilization in Osteoblastic Cells

We previously found that oscillatory fluid flow activated MC3T3-E1 osteoblastic cell Ca2+ i mobilization via the inositol 1,4,5-trisphosphate pathway in the presence of 2% fetal bovine serum (FBS). However, the molecular mechanism of fluid flow-induced Ca2+ i mobilization is unknown. In this study, we first demonstrated that oscillatory fluid flow in the absence of FBS failed to increase [Ca2+] i in MC3T3-E1 cells. Apyrase (10 units/ml), which rapidly hydrolyzes 5′ nucleotide triphosphates to monosphophates, prevented the fluid flow induced increases in [Ca2+] i in the presence of FBS. Adding ATP or UTP to flow medium without FBS restored the ability of fluid flow to increase [Ca2+] i , suggesting that ATP or UTP may mediate the effect of fluid flow on [Ca2+] i . Furthermore, adenosine, ADP, UDP, or adenosine 5′-O-(3-thiotriphosphate) did not induce Ca2+ i mobilization under oscillatory fluid flow without FBS. Pyridoxal phosphate 6-azophenyl-2,4′-disulfonic acid, an antagonist of P2X purinoceptors, did not alter the effect of fluid flow on the Ca2+ i response, whereas pertussis toxin, a Gi/o-protein inhibitor, inhibited fluid flow-induced increases in [Ca2+] i in the presence of 2% FBS. Thus, by the process of elimination, our data suggest that P2Y purinoceptors (P2Y2 or P2Y4) are involved in the Ca2+ i response to fluid flow. Finally, a decreased percentage of MC3T3-E1 osteoblastic cells treated with P2Y2 antisense oligodeoxynucleotides responded to fluid flow with an increase in [Ca2+] i , and an increased percentage of ROS 17/2.8 cells, which do not normally express P2Y2 purinoceptors, transfected with P2Y2 purinoceptors responded to fluid flow in the presence of 2% FBS, confirming that P2Y2 purinoceptors are responsible for oscillatory fluid flow-induced Ca2+ i mobilization. Our findings shed new light of the molecular mechanisms responsible for oscillatory fluid flow-induced Ca2+ i mobilization in osteoblastic cells.

Activation of L-type calcium channels is required for gap junction-mediated intercellular calcium signaling in osteoblastic cells

The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in “fast” ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting in “slow” ICW. Human osteoblastic cells transmit intercellular calcium signals by both of these mechanisms. In the current studies we have examined the mechanism of slow gap junction-dependent ICW in osteoblastic cells. In ROS rat osteoblastic cells, gap junction-dependent ICW were inhibited by removal of extracellular calcium, plasma membrane depolarization by high extracellular potassium, and the L-type voltage-operated calcium channel inhibitor, nifedipine. In contrast, all these treatments enhanced the spread of P2 receptor-mediated ICW in UMR rat osteoblastic cells. Using UMR cells transfected to express Cx43 (UMR/Cx43) we confirmed that nifedipine sensitivity of ICW required Cx43 expression. In human osteoblastic cells, gap junction-dependent ICW also required activation of L-type calcium channels and influx of extracellular calcium.

Cell-mediated cytotoxicity: ATP as an effector and the role of target cells

Cell-mediated cytotoxicity involves a number of distinct mechanisms as well as the active participation of the target cell. Recently, several investigators have demonstrated that extracellular ATP can act as a cytotoxic effector.

Redistribution of connexin45 in gap junctions of connexin43-deficient hearts.

OBJECTIVE Adult ventricular myocytes express two gap junction channel proteins: connexin43 (Cx43) and connexin45 (Cx45). Cx43-deficient mice exhibit slow ventricular epicardial conduction, suggesting that Cx43 plays an important role in intercellular coupling in the ventricle. Cx45 is much less abundant than Cx43 in working ventricular myocytes. Its role in ventricular conduction has not been defined, nor is it known whether expression or distribution of Cx45 is altered in Cx43-deficient mice. The present study was undertaken to determine (1) whether expression of Cx45 is upregulated and (2) whether gap junction structure and distribution are altered in Cx43-deficient mice. METHODS Ventricular tissue from neonatal Cx43(+/+), Cx43(+/-) and Cx43(-/-) and adult Cx43(+/+) and Cx43(+/-) mice was analyzed by immunoblotting and confocal immunofluorescence microscopy. RESULTS Total Cx45 protein abundance measured by immunoblotting was not different in Cx43-deficient or null hearts compared to wild-type control hearts. However, the amount and distribution of Cx45 immunoreactive signal measured by quantitative confocal analysis were markedly reduced in both Cx43(+/-) and Cx43(-/-) hearts. CONCLUSION Although the total content of Cx45 is not upregulated in Cx43-deficient hearts, the localization of Cx45 to cardiac gap junctions depends on the expression level of Cx43 and is dramatically altered in mice that express no Cx43.

Organic-anion transport inhibitors to facilitate measurement of cytosolic free Ca2+ with fura-2

Publisher Summary The fluorescent reporter dyes quin2 and fura-2 are used by many investigators to quantitate the cytosolic free-calcium concentration [Ca 2+ ] i in different types of cells. For the dyes to measure accurately [Ca 2+ ] i , they must be selectively and uniformly distributed within the cytoplasmic matrix of the cells. However, in a number of cell types fura-2 does not remain within the cytoplasmic matrix; it accumulates within intracellular compartments, is secreted from the cells entirely, or both. This chapter reviews the evidence for fura-2 sequestration and secretion by cells and describes the use of organic-anion transport inhibitors to ameliorate problems caused by these processes. Mouse macrophages possess organic-anion transporters that remove fluorescent dyes, including fura-2, from the cytoplasmic matrix of these cells. The dyes are sequestered within cytoplasmic vacuoles and secreted into the extracellular medium. The transporters that promote dye sequestration and secretion are inhibited by the drugs probenecid and sulfinpyrazone that block transport of organic anions in polarized epithelial cells.