Acute and chronic inflammatory responses to local administration of recombinant IL-1α, IL-β, TNFα, IL-2 and Ifnγ in mice

The contention that cytokines are important mediators of inflammation prompted the present studies which were designed to compare acute and chronic pathological effects of locally-administered recombinant (r) IL-1, IL-1, TNF, IL-2 and Ifn. Acute (6 hr), resolving (48 hr) inflammation was induced by the following, in order of potency: rIL-1>rIL-1>rTNF>rIfn=BSA (control) following a single sc. injection. However, only rIL-1 and rIL-2 initiated and maintained chronic granulomatous reactions when delivered locally from a sc. ethylene vinyl acetate (EVA) slow-release polymer. The predominance of macrophages in EVA-rIL-1 lesions contrasted with the proliferative lymphoid granulomata induced by EVA-rIL-2 implants. These in vivo observations reinforce, the roles of both IL-1 and IL-2 as potent mediators of chronic immunoinflammatory disease.     

Effects of recombinant human IL-Iβ on production of prostaglandin E2, leukotriene B4, NAG,and superoxide by human synovial cells and chondrocytes

The effects of recombinant human IL-1 on the production of prostaglandin E₂ (PGE₂), leukotriene B₄ (LTB₄),N-acetyl--D-glucosaminidase (NAG), and superoxide by synovial cells and chondrocytes derived from osteoarthritis patients were determined. IL-1 markedly enhanced PGE₂ production in chondrocytes and, to the lesser extent, in synovial cells. Synovial cells and chondrocytes spontaneously released LTB₄ into culture medium and IL-1 significantly inhibited LTB₄ production by these cells. IL-1 significantly suppressed the release of NAG and superoxide by synovial cells, whereas it significantly enhanced the production of NAG and superoxide by chondrocytes. Production of intracellular superoxide dismutase by synovial cells was significantly enhanced on incubation with IL-1, but that of chondrocytes was not altered. IL-6, unlike IL-1, significantly suppressed the production of NAG and superoxide by synovial cells and chondrocytes.These results suggest that IL-1 has differing effects on the release of mediators by synovial cells and chondrocytes and that these cells also vary in their responses to IL-1 and IL-6.     

A selective role for brain histamine in prolactin release induced by opiates

We studied the effects of histamine (HA) antagonists on the facilitatory action of morphine (M) and-endorphin (E) on prolactin (PRL) release and the effect of -fluoromethylhistidine (-FMH, inhibitor of HA synthesis) onE-induced PRL secretion. Male rats were injected intracerebroventricularly (i.c.v.) with mepyramine (MEP, H₁-antagonist, 0.8 mol/rat) or ranitidine (RAN, H₂-antagonist, 0.4 mol/rat) 10 min before M (6 mg/kg, intracarotid, i.a.) orE (0.25 g/rat, i.c.v.). -FMH (200 g/rat, i.c.v.) was administered 3 h beforeE. Plasma PRL levels were measured at various times before and after drug treatment. RAN but not MEP significantly reduced PRL release induced by M whereas neither HA-antagonists nor -FMH modifiedE-induced PRL release. The results obtained show that brain HA contributes through activation of H₂-receptors to the PRL facilitatory action of M but not ofE.     

Inhibition of interleukin-1 (IL-1) induced neutral proteases from rabbit articular chondrocytes by WY-46,135 and WY-48,989

The effects of potential anti-osteoarthritic compounds both on the direct inhibition of collagenase and neutral protease activities and on IL-1 induced release of neutral proteases from rabbit articular chondrocytes were investigated. WY-46,135 ((+)-N-[[[(5-chloro-2-benzothiazolyl)thio]phenyl]acetyl]-l-cysteine) directly inhibited collagenase activity (IC₅₀=15.4 M). This inhibition was reversible upon dialysis. WY-46,135 also directly inhibited neutral protease activity (IC₅₀=16.8 M) but did not significantly block bacterial collagenase activity at a concentration of 80 M. In contrast, WY-48,989 (4-[[2-(7-chloro-2-phenyl-2H-pyrazolo[4,3-c]quinolin-4-yl)ethyl]amino]benzonitrile) did not directly inhibit either collagenase (10M) or neutral protease (100 M) activity. Both WY-48,989 and WY-46,135 inhibited IL-1 stimulated release of neutral proteases (IC₅₀=3M). The activities of these compounds represents two potential approaches for the treatment of osteoarthritis. WY-46,135 combines direct metalloprotease inhibitory activity with the inhibition of IL-1 stimulated neutral protease release from articular chondrocytes while WY-48,989 selectively inhibits the IL-1 induced release of metalloproteases.     

Oral Administration of Gammalinolenic Acid, an Unsaturated Fatty Acid with Anti-inflammatory Properties, Modulates Interleukin-1β Production by Human Monocytes

Administration of gammalinolenic acid (GLA), an unsaturated fatty acid, reduces joint inflammation in patients with rheumatoid arthritis. Addition of GLA in vitro suppresses release of interleukin-1 (IL-1) from human monocytes stimulated with lipopolysaccharide (LPS). LPS-induced IL-1 release is followed by IL-1-induced IL-1 release, an amplification process termed autoinduction. We show here, using IL-1 stimulation to simulate autoinduction, that administration of GLA to healthy volunteers and to patients with inflammatory arthritis reduces LPS-induced IL-1 secretion mainly by reducing autoinduction of IL-1. GLA reduces LPS-induced pro-IL-1 mRNA modestly and IL-1-induced pro-IL-1 gene expression markedly. In addition to reducing amplification of IL-1, GLA increases the amount of IL-1 receptor antagonist (IL-1Ra) secreted from stimulated cells, thereby facilitating an increase in the secreted IL-1Ra/IL-1 ratio. IL-1 is important to host defense, but the amplification mechanism may be excessive in genetically predisposed individuals. Thus, reduction of IL-1 autoinduction may be protective in some patients with endotoxic shock and with diseases characterized by chronic inflammation.     

Synoviocytes in chronic synovitis in situ and cytokine stimulated synovial cells in vitro neo-express α1, α3 and α5 chains of β1 integrins

The expression of the 1 integrins was examined immunohistochemically in synoviocytes from normal synovial membrane and from chronic synovitis of different aetiology and intensity. Normal synoviocytes were 61-positive but lacked 1 through 5. In mild inflammation type A synoviocytes neo-expressed 1, 3, and 5 chains. In severe inflammation both type A and B synoviocytes expressed 3, 4, 5, and 6 chains. The effects of inflammatory cytokines, as single agents or in combination, on the 1 integrin expression in cultured normal synoviocytes was determined by immunocytochemistry and flow cytometry. The 1 chain, while absent in unstimulated synoviocytes, was induced by interleukin-1 (IL-1), tumour necrosis factor- (TNF-), and interferon- (INF-). This effect was enhanced by combining IL-1 and TNF-. Expression of the 3 chain was up-regulated by IL-1 and, more intensely, by IFN-. Transforming growth factor (TGF-) inhibited the up-regulating effect of IL-1 and antagonized the effect of IFN- on 3 chain expression. Expression of the 5 chain was up-regulated significantly by co-stimulation through IL-1 together with TGF- or TNF-. Thus, the 1 integrin profile of cytokine activated synoviocytes in vitro resembled that of synoviocytes in synovitis in situ. These data suggest that IL-1, TNF-, IFN-, and TGF- are likely to be among the effectors regulating 1 integrin expression in synoviocytes in vivo.     

Recombinant human interleukin-1α and recombinant human interleukin-1β stimulate cartilage matrix degradation and inhibit glycosaminoglycan synthesis

Recombinant human interleukin-1 (rhIL-1) and recombinant human interleukin 1 (rhIL-1) stimulated the time- and concentration-dependent release of glycosaminoglycan (GAG) from bovine nasal cartilage expiants. Maximum GAG release occurred during six to eight days of cartilage exposure to either species of rhIL-1; and rhIL-1 was consistently more potent than rhIL-1. In addition to inducing cartilage matrix resorption, rhIL-1 and rhIL-1 also inhibited the incorporation of [³⁵SO₄]sulfate into cartilage, which is a reflection of the suppression of GAG synthesis. IL-1 had no capacity to stimulate GAG relase from or inhibit GAG synthesis by dead cartilage. Cycloheximide, an inhibitor of protein synthesis, and 1, 10-phenanthroline, a metalloproteinase inhibitor, suppressed rhIL-1-stimulated cartilage matrix resorption. Polyclonal antisera to rhIL-1 and rhIL-1 specifically neutralized the respective cytokines.     

Interleukin-4 and Interferon-γ Inhibit Prostaglandin Production by Interleukin-1β-Stimulated Human Periodontal Ligament Fibroblasts

The purpose of the present study was to investigate the involvement of cyclooxygease-1 (COX-1) and cyclooxygenase-2 (COX-2) in prostaglandin (PG) production by human periodontal ligament (PDL) fibroblasts stimulated with a proinflammatory cytokine, inerleukin-1 (IL-1), and to examine the effect of interleukin-4 (IL-4), a Th2 cytokine, and interferon- (IFN-), a Th1 cytokine, on PG production by the cells. IL-1-stimulated PDL fibroblasts produced prostaglandin E₂ (PGE₂) in a time-dependent manner. Indomethacin, a non-selective COX-1/COX-2 inhibitor, and NS-398, a selective COX-2 inhibitor, completely inhibited PGE₂ production by IL-1-stimulated cells. Northern blot analysis showed that COX-2 mRNA was detected in IL-1-stimulated PDL cells, although not detected in unstimulated cells, while expression of COX-1 mRNA was in the same extent in both the cells. Dexamethasone inhibited COX-2 mRNA expression, COX activity and PGE₂ production in IL-1-stimulated cells. IL-4 and IFN- suppressed PGE₂ production by IL-1-stimulated PDL fibroblasts, but COX activity enhanced by IL-1 treatment was significantly inhibited by IL-4, not by IFN-. Northern blot analysis showed that IL-4 depressed COX-2 mRNA expression with no effect on COX-1 mRNA expression. On the other hand, IFN- had no effect on expression of COX-1 and -2 mRNA. These data suggest that COX-2 is primarily responsible for PGE₂ production by IL-1-stimulated human PDL fibroblasts and that IL-4 inhibited PGE₂ production by IL-1-stimulated PDL fibroblasts through down-regulation of COX-2 expression, while IFN- suppressed the PGE₂ production with no effect on COX-2 expression.     

Cytokine mRNA in the Joints and Draining Lymph Nodes of Rats with Adjuvant Arthritis and Effects of Cyclosporin A

TNF- and IL-1 promote leukocyte recruitment to arthritic joints and may contribute to cartilage degradation while regulatory cytokines such as IL-4 and IL-1RA may in part determine the course of arthritis. Here we report the pattern of TNF-, IL-1, IL-6, IFN-, IL-1RA, and IL-4 mRNA expression, detected by RT/PCR, in the talar joint and draining popliteal lymph node (PLN) of rats with adjuvant arthritis (AA). Levels of TNF- and IFN- mRNA were increased in the PLN before clinical signs of arthritis. This was followed by increases in IL-1 and IL-1RA mRNA at d9 and IL-6 mRNA at d12. PLN IL-1RA mRNA levels were positively correlated with those of IL-1 and TNF- throughout d5-d20. IL-4 mRNA levels were highest on days 7 and 20. In the synovium, a small increase in TNF-, IL-1, and IL-6 mRNA was detected on d5 then again on d12. Maximal synovial TNF- levels were reached on d20, while IL-1 peak expression was on d16 and IL-6 on d14. IL-4, IL-1RA, and IFN- mRNA was undetectable in the synovium. Cyclosporin treatment for 4 days, initiated at the height of arthritis, rapidly decreased clinical disease, and decreased migration of neutrophils and T lymphocytes into the joints. Yet no significant effect of CyA was observed on inflammatory cytokine expression, although the correlation between PLN IL-1RA and IL-1 or TNF- was lost in treated animals. Thus there is a variable pattern of cytokine gene expression in rat AA, the undetectable IL-4 and IFN- mRNA in synovium being analogous to human rheumatoid arthritis.     

Differential effects of interleukin-1α and β on the arachidonic acid cascade in human synovial cells and chondrocytes in culture

The effects of interleukin-1 and were tested on the [³H]-arachidonic acid release and the prostaglandin synthesis by human cultured synovial cells and chondrocytes. Both forms of interleukin-1 stimulated the arachidonic acid release but interleukin-1 was more potent than IL-1. Human synovial cells and chondrocytes synthesized three types of prostaglandins upon stimulation with interleukin-1 or : prostaglandin E₂, F₂ and 6-keto-prostaglandin F₁. Regarding the synthesis of these prostaglandins, IL-1 was again more potent than IL-1. A comparison between interleukin-1-stimulated synovial cells and chondrocytes revealed neither significant quantitative nor qualitative differences in both the arachidonic acid release and the prostaglandin synthesis.     

Glucocorticoids modulate TGF-beta production

TGF- is thought to play a central role in pulmonary fibrosis inducing fibroblast differentiation and extracellular matrix synthesis. In human lung fibroblasts, it is still unclear how various TGB- isoforms affect TGF- production and whether glucocorticoids, commonly used agents to treat fibrotic lung disease, modulate these processes. To this end, human fetal lung fibroblasts (HFL-1) were cultured with various concentrations of glucocorticoids (budesonide, dexamethasone or hydrocortisone) with and without TFG-1, -2, and -3. TGF- mRNA was assessed by real time RT-PCR. Smad 2, 3, and 4 and AP-1 complex (c-fos and c-Jun) cellular localization were evaluated by immunostaining. TGF-2 and -3 stimulated TGF-1 production significantly (p < 0.01 relative to control). TGF-1 stimulated TGF-2 production (p < 0.01 relative to control). TGF-3 was undetectable. Glucocorticoids significantly inhibited TGF-1 and -2 production and reduced expression of the upregulated TGF-1 and -2 mRNA induced by exogenous TGF-1, -2 or -3 (p < 0.01 for each) but had no effect on Smads. Although c-jun-related nuclear staining was not intensified in TGF--stimulated cells, it was reduced by glucocorticoids. Thus, TGF- isoforms may stimulate production of various TGF- isoforms in the lung. Glucocorticoids then may block TGF- production by modulating mRNA levels and c-Jun.     

Glucocorticoids modulate TGF-beta production by human fetal lung fibroblasts

TGF- is thought to play a central role in pulmonary fibrosis inducing fibroblast differentiation and extracellular matrix synthesis. In human lung fibroblasts, it is still unclear how various TGF- isoforms affect TGF- production and whether glucocorticoids, commonly used agents to treat fibrotic lung disease, modulate these processes. To this end, human fetal lung fibroblasts (HFLF) were cultured with various concentrations of glucocorticoids (budesonide, dexamethasone or hydrocortisone) with and without TGF-1, -2, or -3. Post-culture media were collected for ELISA assays of TGF-1, -2, and -3 . TGF- mRNA was assessed by real time RT-PCR. Smad 2, 3, and 4 and AP-1 complex (c-fos and c-Jun) cellular localization were evaluated by immunostaining. TFG-2 and -3 stimulated TGF-1 production significantly (p < 0.01 relative to control). TGF-1 stimulated TGF-2 production (p < 0.01 relative to control). TGF-3 was undetectable. Glucocorticoids significantly inhibited TGF-1 and TGF-2 production and reduced expression of the up-regulated TGF-1 and TGF-2 mRNA induced by exogenous TGF-1, -2, or -3 (p < 0.01 for each) but had no effect on Smads. Although c-jun-related nuclear staining was not intensified in TGF--stimulated cells, it was reduced by glucocorticoids. Thus, TGF- isoforms may stimulate production of various TGF- isoforms in the lung. Glucocorticoids then may block TGF- production by modulating mRNA levels and c-Jun.     

Isolation and bioactivities of three IL-1β N-terminal variants

Three distinct N-terminal variants of rhIL-1 can be generated by expression of the IL-1 gene in E. coli; the naturally occurring Ala¹ species, Met⁰-Ala¹ and des-Ala¹ proteins. Since most studies with rhIL-1 have used a mixture of two or more variants, we have evaluated their individual bioactivities. The variants were resolved by cation exchange HPLC. Bioactivity measurement on murine thymocytes gave a potency order of Ala¹ > des-Ala¹ > Met⁰-IL-1. Analysis using human T-cells co-stimulated with PMA showed a potency order of Ala¹ > des-Ala¹ > Met⁰-IL-1. Thus changes in the N-terminal amino acid of IL-1 changes the activity of the protein. Since murine and human T-cells respond similarly, the interactions between the N-terminus of rhIL-1 and their receptors probably occur through comparable mechanisms.     

Transforming growth factor-β (TGFβ) inhibition of epstein-barr virus (EBV)- and interleukin-4 (IL-4)-induced immunoglobulin production in human B lymphocytes

This study reports on the effects of TGF on the secretion of Ig isotypes by highly purified (>99% CD20-positive) human peripheral blood B cells. Stimulation of these B cell preparations with EBV resulted in the secretion of IgM, IgG, and IgA and the addition of IL-4 induced readily detectable levels (>100 ng/ml) of IgE between 10 and 25 days of culture. TGF1 and TGF2 showed similar dose-dependent suppression of IgM, IgG, and IgA, and the relative proportion of IgG and IgA remained unchanged in the presence of TGF. IgE production induced by EBV and IL-4 was significantly inhibited by TGF. TGF effects on Ig secretion were not related to inhibition of B cell proliferation by this cytokine. In contrast to these TGF effects on EBV activation of primary B cells, the constitutive Ig secretion by EBV-transformed B cells was resistant to TGF, while the increase in Ig secretion induced by IL-6 was inhibited by TGF. Thus, TGF inhibits the EBV-induced secretion of the major Ig isotypes in peripheral blood B cells and has differential effects on Ig secretion by transformed B cells.     

Bicarbonate permeability of epithelial chloride channels

To investigate the relation of arachidonate metabolism to the induction of fever by interleukin-1, indomethacin was administered in either an intracerebro-ventricular (icv) or a subcutaneous (sc) route in conscious rabbits. Fever induced by icv administration of recombinant human interleukin-1 (rhIL-1) was depressed by either icv or sc pretreatment with indomethacin. Fever induced by intravenous (iv) administration of rhIL-1 was significantly inhibited, though initial small increase in colonic temperature still remained, and was completely depressed by combination of icv and sc pretreatment with indomethacin. Intracerebroventricularly administered recombinant rabbit IL-1 (rrIL-1) induced dose-dependent increases in colonic temperature, which was depressed by sc pretreatment with indomethacin. There is little species specificity between human and rabbit IL-1, in terms of the pyrogenic potency and the inhibitory effect of sc indomethacin on fever induced by icv IL-1. Further, fever caused by icv administration of sodium arachidonate was significantly depressed by sc pretreatment with indomethacin. These results show that the inhibitory effect of indomethacin, administered either icv or sc, on IL-1-induced fever is similar to that of IL-1-induced fever reported previously [11]. This suggests that the site of arachidonate metabolism significantly involved in the mechanism of fever induction by IL-1 is easily accessible to the brain from the blood.     

Cartilage destruction and osteophytes in instability-induced murine osteoarthritis: Role of TGFβ in osteophyte formation?

Osteoarthritis is characterized by focal cartilage destruction and marked formation of osteophytes. We have investigated the possible relationship between site specific occurrence of cartilage damage and osteophytes in the collagenase induced murine osteoarthritis model. The degree of instability of the joint correlated with the amount of cartilage loss. Moreover, cartilage damage in the medial tibial plateau correlated only strongly with the osteophyte at the medial plateau, whereas a similar, site directed trend was noted for lateral damage and lateral osteophytes. A separate study with intraarticular injection of TGF1 in normal murine knee joints revealed that this factor can induce osteophytes at characteristic sites, suggesting a role of endogenous TGF in this phenomenon.Lecture presented at the Symposium on Models of Osteoarthritis during the Conference, Inflammation '93, Vienna, Austria, October 12, 1993.     

Effects of serine protease inhibitor,tame, on IL-1β in LPS-stimulated human monocytes: Relationship between synthesis and release of a 33-kDa precursor and the 17-kDa biologically active species

LPS stimulation of human monocytes in vitro induced release of the 17-kDa mature IL-1 (mIL-1) but did not result in release of precursor IL-1 (pIL-1). In contrast, the presence of a serine protease inhibitor, N-(p-toluene sulfonyl)-L-arginine methyl ester (TAME; 10 mM) for 6 or 18 h was associated with the LPS-stimulated release of the 33-kDa pIL-1 as well. These effects were initially discerned from observations that the fraction of the total IL-1 produced (as detected by ELISA) that was released from monocytes increased in the presence of TAME, and immunoblot assays confirmed that this fraction was predominantly 33-kDa IL-1. A global decrease in monocyte protein synthesis was also observed after prolonged (18-h) exposure to TAME and was associated with a decrease in IL-1 synthesis, predominantly affecting 31-kDa pIL-1, and a dose-dependent inhibition of TNF- production. Parallel examination of lactate dehydrogenase (LDH) release indicated thatpIL-1 release was unrelated to cell lysis. These results demonstrate that TAME-inhibitable serine proteases are probably involved in the production and eventual proteolysis of the 33-kDa pIL-1 in situ but are probably not mechanistically related to either maturation of the IL-1 molecule or signaling of IL-1 release. IL-1 release appears to be dependent on the amount of total IL-1 synthesized. Serine proteolysis may constitute a degradative pathway for excess precursor, which, if interfered with, could result in release of the higher-molecular-weight forms of IL-1.     

Characterization of interleukin-15 (IL-15) and the IL-15 receptor complex

IL-15 interacts with a heterotrimeric receptor that consists of the and subunits of the IL-2 receptor (IL-2R) as well as a specific, high-affinity IL-15-binding subunit, which is designated IL-15R. Since both the and the subunits of the IL-2R are required for signaling by either IL-2 or IL-15, it is not surprising that these cytokines share many activitiesin vitro. However, the differential expression of these cytokines and the chains of their receptors within various tissues and cell types suggests that IL-2 and IL-15 may perform at least partially distinct physiological functions. The production of IL-15 by macrophages, and possibly other cell types, in response to environmental stimuli and infectious agents suggests that IL-15 may play a role in protective immune responses, allograft rejection, and the pathogenesis of autoimmune diseases.     

Differential induction of human monocyte transforming growth factorβ1 production and its regulation by interleukin 4

We have previously shown that trauma patients' monocytes which arein vivo activated by multiple injury-induced mediators have elevated transforming growth factor-beta (TGF) bioactivity. Interleukin-4 (IL-4), a Th₂ and B lymphocyte stimulatory factor, has been shown to inhibit monocyte production of a number of mediators both after lipopolysaccharide stimulation and after trauma-induced stimulation. However, IL-4 inhibitory effects appears to vary, depending on the mixture of inducing stimuli. Here we describe thein vitro IL-4 inhibition of human monocyte TGF bioactivity using several stimulation induction protocols: muramyl dipeptide stimulation alone, or after FcRI (CD64) cross-linking induction, interferon-gamma (IFN) priming, or trauma-generatedin vivo mediator induction. IL-4 suppressed both muramyl dipeptide-induced TGF bioactivity and TGF mRNA in a dose-dependent fashion and was most effective when IL-4 was administered at initiation of normal monocyte stimulation. Muramyl dipeptide (MDP)-induced increases in trauma patients' monocyte TGF bioactivity were also inhibited by high doses of IL-4 (25 ng/ml). FcRI cross-linking increased MDP-induced normal monocyte TGF bioactivity, but this increase could be consistently inhibited only by very high IL-4 concentrations (50 ng/ml). IL-4 did not consistently downregulate MDP-induced TGF bioactivity in IFN-primed monocytes. IL-4 can suppress monocyte TGF production, as well as other monocyte mediators, but its efficiency depends on the stimuli combination present in the microenvironment.     

Modulation of Nav1.5 by β1- and β3-subunit co-expression in mammalian cells

Cardiac sodium channels (Na_v1.5) comprise a pore-forming -subunit and auxiliary -subunits that modulate channel function. In the heart, 1 is expressed throughout the atria and ventricles, whilst 3 is present only in the ventricles and Purkinje fibers. In view of this expression pattern, we determined the effects of 3 and 1 co-expression alone, and in combination, on Na_v1.5 stably expressed in Chinese hamster ovary cells. The current/voltage relationship was shifted –5 mV with either 1 or 3 co-expression alone and –10 mV with co-expression of both 1 and 3. In addition, 3 and 1/3 co-expression accelerated macroscopic current decay. There were significant hyperpolarizing shifts in equilibrium gating relationships with co-expression of 1 and 3 alone and in combination. Co-expression of 1/3 together resulted in a greater hyperpolarizing shift in channel availability, and an increase in the slopes of equilibrium gating relationships. Co-expression of 3 and 1/3, but not 1, slowed recovery from inactivation at –90 mV. Development of inactivation at –70 and –50 mV was accelerated by -subunit co-expression alone and in combination. -Subunit co-expression also reduced the late Na current measured at 200 ms. In conclusion, -subunits modulate Na_v1.5 gating with important differences between co-expression of 1 and 3 alone and 1/3 together.