Randomized controlled studies on the efficacy of antiarthritic agents in inhibiting cartilage degeneration and pain associated with progression of osteoarthritis in the rat.

BACKGROUND: As an initial step in the development of a local therapeutic to treat osteoarthritis (OA), a number of agents were tested for their ability to block activation of inflammation through nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), subchondral bone changes through receptor activator of nuclear factor κB ligand (RANKL)-mediated osteoclastogenesis, and proteolytic degradation through matrix metalloproteinase (MMP)-13 activity. Candidates with low toxicity and predicted efficacy were further examined using either of two widely accepted models of OA joint degeneration in the rat: the monoiodoacetic acid (MIA) model or the medial meniscal tear/medial collateral ligament tear (MMT/MCLT) model. METHODS: Potential therapeutics were assessed for their effects on the activation of nuclear factor (NF)-κB, RANKL-mediated osteoclastogenesis, and MMP-13 activity in vitro using previously established assays. Toxicity was measured using HeLa cells, a synovial cell line, or primary human chondrocytes. Drugs predicted to perform well in vivo were tested either systemically or via intraarticular injection in the MIA or the MMT/MCLT model of OA. Pain behavior was measured by mechanical hyperalgesia using the digital Randall-Selitto test (dRS) or by incapacitance with weight bearing (WB). Joint degeneration was evaluated using micro computed tomography and a comprehensive semiquantitative scoring of cartilage, subchondral bone, and synovial histopathology. RESULTS: Several agents were effective both in vitro and in vivo. With regard to pain behavior, systemically delivered clonidine was superior in treating MIA-induced changes in WB or dRS, while systemic clonidine, curcumin, tacrolimus, and fluocinolone were all somewhat effective in modifying MMT/MCLT-induced changes in WB. Systemic tacrolimus was the most effective in slowing disease progression as measured by histopathology in the MMT/MCLT model. CONCLUSIONS: All of the agents that demonstrated highest benefit in vivo, excepting clonidine, were found to inhibit MMP-13, NF-κB, and bone matrix remodeling in vitro. The MIA and MMT/MCLT models of OA, previously shown to possess inflammatory characteristics and to display associated pain behavior, were affected to different degrees by the same drugs. Although no therapeutic was remarkable across all measures, the several which showed the most promise in either model merit continued study with alternative dosing and therapeutic strategies.

Gap junction assembly: roles for the formation plaque and regulation by the C-terminus of connexin43.

Using an established gap junction (GJ) assembly system with experimentally reaggregated cells, we analyzed "formation plaques" (FPs), apparent sites of GJ assembly. Employing freeze-fracture electron microscopy methods combined with filipin labeling of sterols and immunolabeling for connexin43 (Cx43), we demonstrated that FPs constitute distinct membrane "domains" and that their characteristic 10-nm particles contain connexin43, thus representing precursors (i.e., GJ hemichannels) engaged in assembly. Analysis of FPs in new systems-HeLa and N2A cells-resolved questions surrounding several key but poorly understood steps in assembly, including matching of FP membranes in apposed cells, reduction in the separation between FP membranes during assembly, and the process of particle aggregation. Findings also indicated that "docking" of GJ hemichannels occurs within FP domains and contributes to reduction of intermembrane separation between FPs. Other experiments demonstrated that FPs develop following a major C-terminal truncation of Cx43 (M257), although assembly was delayed. Particle aggregation also occurred at lower densities, and densities of particles within developing GJ aggregates failed to achieve full-length levels. With regard to regulation, inhibition of assembly following protein kinase C activation failed to occur in the M257 truncation mutants, as measured by intercellular dye transfer. However, several C-terminal serine mutations failed to disrupt inhibition.

Hysteresis in human HCN4 channels: a crucial feature potentially affecting sinoatrial node pacemaking.

The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels modulate and regulate cardiac rhythm and rate. It has been suggested that, unlike the HCN1 and HCN2 channels, the slower HCN4 channel may not exhibit voltage-dependent hysteresis. We studied the electrophysiological properties of human HCN4 (hHCN4) channels and its modulation by cAMP to determine whether hHCN4 exhibits hysteresis, by using single-cell patch-clamp in HEK293 cells stably transfected with hHCN4. Quantitative real-time RT-PCR was also used to determine levels of expression of HCNs in human cardiac tissue. Voltage-clamp analysis revealed that hHCN4 current (I(h)) activation shifted in the depolarizing direction with more hyperpolarized holding potentials. Triangular ramp and action potential clamp protocols also revealed hHCN4 hysteresis. cAMP enhanced I(h) and shifted activation in the depolarizing direction, thus modifying the intrinsic hHCN4 hysteresis behavior. Quantitative PCR analysis of human sinoatrial node (SAN) tissue showed that HCN4 accounts for 75% of the HCNs in human SAN while HCN1 (21%), HCN2 (3%), and HCN3 (0.7%) constitute the remainder. Our data suggest that HCN4 is the predominant HCN subtype in the human SAN and that I(h) exhibits voltage-dependent hysteresis behavior that can be modified by cAMP. Therefore, hHCN4 hysteresis potentially plays a crucial role in human SAN pacemaking activity.

Electrophysiological characterization of murine HL-5 atrial cardiomyocytes.

HL-5 cells are cultured murine atrial cardiomyocytes and have been used in studies to address important cellular and molecular questions. However, electrophysiological features of HL-5 cells have not been characterized. In this study, we examined such properties using whole cell patch-clamp techniques. Membrane capacitance of the HL-5 cells was from 8 to 62 pF. The resting membrane potential was -57.8 +/- 1.4 mV (n = 51). Intracellular injection of depolarizing currents evoked action potentials (APs) with variable morphologies in 71% of the patched cells. Interestingly, the incidence of successful, current-induced APs positively correlated with the hyperpolarizing degrees of resting membrane potentials (r = 0.99, P < 0.001). Only a few of the patched cells (4 of 51, 7.8%) exhibited spontaneous APs. The muscarinic agonist carbachol activated the acetylcholine-activated K(+) current and significantly shortened the duration of APs. Immunostaining confirmed the presence of the muscarinic receptor type 2 in HL-5 cells. The hyperpolarization-activated cation current (I(f)) was detected in 39% of the patched cells. The voltage to activate 50% of I(f) channels was -73.4 +/- 1.2 mV (n = 12). Voltage-gated Na(+), Ca(2+), and K(+) currents were observed in the HL-5 cells with variable incidences. Compared with the adult mouse cardiomyocytes, the HL-5 cells had prolonged APs and small outward K(+) currents. Our data indicate that HL-5 cells display significant electrophysiological heterogeneity of morphological appearance of APs and expression of functional ion channels. Compared with adult murine cardiomyocytes, HL-5 cells show an immature phenotype of cardiac AP morphology.

Endogenous channels in HEK cells and potential roles in HCN ionic current measurements.

A transformed line of human embryonic kidney epithelial cells (HEK 293) is commonly used as an expression system for exogenous ion channel genes. Previously, it has been shown that these cells contain mRNAs for a variety of ion channels. Expression of some of these genes has been confirmed at the protein level. Patch-clamp electrophysiology experiments confirm the presence of multiple ion channels and molecular data agree with pharmacological profiles of identified channels. In this work, we show that endogenous voltage-gated potassium channels in HEK cells are a significant source of outward current at positive potentials. We show that both non-transfected HEK cells and HEK cells transfected with hyperpolarization-activated cyclic-nucleotide gated (HCN) channels have a significant amount of voltage-gated potassium (K(V)) current when certain tail current voltage-clamp protocols are used to assay HCN current activation. Specifically, tail current protocols that use a depolarized holding potential of -40 mV followed by hyperpolarizing pulses (-80 to -140 mV) and then a tail pulse potential of +20 mV indicate K(V) channels undergo closed-state inactivation at the more depolarized holding potential of -40 mV, followed by recovery from inactivation (but no activation) at hyperpolarizing potentials and high amount of activation at the positive tail potential. Our results indicate that pulse protocols with positive tail pulses are inaccurate assays for HCN current in certain HEK cells. Surprisingly, HEK-293 cells were found to contain mRNA for HCN2 and HCN3 although we have not detected a significant and consistent endogenous I(f)-like current in these cells.

Connexin43 phosphorylation at S368 is acute during S and G2/M and in response to protein kinase C activation.

Phorbol esters such as 12-O-tetradeconylphorbol-13-acetate (TPA) activate protein kinase C, increase Connexin43 (Cx43) phosphorylation, and decrease cell-cell communication via gap junctions in many cell types. Previous work has implicated protein kinase C (PKC) in the direct phosphorylation of Cx43 at S368, which results in a change in single channel behavior that contributes to a decrease in intercellular communication. We have examined Cx43 phosphorylation in several cell lines with an antibody specific for phosphorylated S368. We show that this antibody detects Cx43 only when it is phosphorylated at S368 and, consistent with previous results, TPA treatment causes a dramatic increase in phosphorylation at S368. However, in some cell types, the increased phosphorylation at S368 did not cause a detectable shift in migration as compared with the nonphosphorylated Cx43. Immunofluorescence showed increased S368 immunolabeling in cytoplasmic and plasma membrane structures in response to TPA. Immunoblot analysis of synchronized cells showed increased phosphorylation at S368 during S and G2/M phases of the cell cycle. S-phase cells contained more total Cx43 but assembled fewer functional gap junctional channels than G0-phase cells. Since M-phase cells also communicate poorly and contain few assembled gap junctions, phosphorylation at S368 appears to be negatively correlated with gap junction assembly. Thus, both gap junctional communication and S368 phosphorylation change during S phase and G2/M, implying that phosphorylation at S368 might play a role in key cell-cycle events.