The shark liver antimicrobial polyaminosterol squalamine is an angiogenesis inhibitor under clinical investigation as an anti-cancer agent and as a treatment for the choroidal neovascularization associated with macular degeneration of the retina. The related polyaminosterol MSI-1436 is an appetite suppressant that decreases systemic insulin resistance. However, the mechanisms of action of these polyaminosterols are unknown. We report effects of MSI-1436 on Xenopus oocytes consistent with the existence of a receptor for polyaminosterols. MSI-1436 activates bidirectional, trans-chloride-independent Cl- flux in Xenopus oocytes. At least part of this DIDS-sensitive Cl− flux is conductive, as measured using two-electrode voltage-clamp and on-cell patch-clamp techniques. MSI-1436 also elevates cytosolic Ca2+ concentration ([Ca2+]) and increases bidirectional 45Ca2+ flux. Activation of Cl− flux and elevation of cytosolic [Ca2+] by MSI-1436 both are accelerated by lowering bath Ca2+ and are not acutely inhibited by extracellular EGTA. Elevation of cytosolic [Ca2+] by MSI-1436 requires heparin-sensitive intracellular Ca2+ stores. Although injected EGTA abolishes the increased conductive Cl− flux, that Cl− flux is not dependent on heparin-sensitive stores. In low-bath Ca2+ conditions, several structurally related polyaminosterols act as strong agonists or weak agonists of conductive Cl− flux in oocytes. Weak agonist polyaminosterols antagonize the strong agonist, MSI-1436, but upon addition of the conductive Cl− transport inhibitor DIDS, they are converted into strong agonists. Together, these properties operationally define a polyaminosterol receptor at or near the surface of the Xenopus oocyte, provide an initial description of receptor signaling, and suggest routes toward further understanding of a novel class of appetite suppressants and angiogenesis inhibitors.
- calcium-dependent chloride conductance
- Xenopus oocyte
- 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid
animal epithelial tissues and other cell types synthesize and secrete several types of broad-spectrum antibiotic substances. Antibiotic peptides (63) include magainins (62), cryptdins (43), and defensins (52) and are found among many phyla of the animal kingdom. A more recently discovered nonpeptide class of broad-spectrum antibiotics is exemplified by squalamine (40) and its structural homologs (31), discovered in the dogfish Squalus acanthias. The polyaminosterol squalamine is a sulfated bile acid-like sterol conjugated to spermidine at the 3′ position of the sterol nucleus (Fig. 1). Multiple structural homologs of squalamine have been isolated, structurally characterized (45), and synthesized (50), but their physiological roles in the dogfish, as well as those of squalamine itself, remain unclear.
Squalamine has been shown to inhibit endothelial cell proliferation and angiogenesis (51). This property has been exploited to treat hyperoxia-induced retinopathy in a mouse model (23, 24), retinal choroidal revascularization in a rat laser injury model (11), and ocular neovascularization in a monkey laser injury model (15). Squalamine’s angiogenesis inhibitory properties also likely contribute to its anti-tumor actions (22, 37). In contrast, the structurally related polyaminosterol MSI-1436 suppresses mammalian appetite (2, 64) and decreases insulin resistance while normalizing hepatic steatosis in leptin-deficient mice (54). Both squalamine and MSI-1436 mediate slow-onset inhibition of mammalian Na+/H+ exchange mediated by NHE3 but not by NHE2 or NHE1 (3). However, the relationship between this inhibitory effect on pH regulatory ion transport and the above cellular and organ properties remains poorly understood.
In extending our studies on inhibition in Xenopus oocytes of SLC4/AE anion exchange (4), we noted that MSI-1436, unlike the polyaminosterols squalamine, MSI-1361, and MSI-1360, activated endogenous Cl− transport in Xenopus oocytes. Although this property of MSI-1436 prevented its further study in oocytes as a potential inhibitor of heterologous Cl−/anion exchange, the diversity of potential therapeutic effects of MSI-1436 prompted further investigation of its regulation of oocyte Cl− permeability.
Xenopus oocytes express endogenous Cl− channels of uncertain molecular identity (19, 60) regulated variously by volume or tonicity (1, 58), intracellular Ca2+ (36), and voltage (46). Xenopus oocytes also express putative plasmalemmal progesterone receptors that activate kinase cascades and mobilize intracellular Ca2+ signals triggering meiotic maturation and germinal vesicle breakdown (59). The Xenopus oocyte response to progesterone is a classic example of numerous reports of rapid cellular responses to steroids, proposed to be mediated by cell surface steroid receptors distinct in structure from nuclear steroid receptors (61). In addition to modulation of GABA and oxytocin receptors by progestins (7, 21), G protein-coupled receptors believed to mediate acute steroid effects have been cloned as cDNAs from fish eggs (67), followed by cDNA cloning of their mammalian homologs (66). A novel surface estrogen receptor, GPR30, also has been detected in a human breast cancer cell line devoid of nuclear estrogen receptor activity (55).
Because Xenopus oocyte Cl− currents remain widely studied readouts for ligand-activated and store-operated mechanisms of Ca2+ entry, we investigated further the actions of MSI-1436 and related polyaminosterols on endogenous Cl− transport in the Xenopus oocyte. We report that extracellular application of MSI-1436 activates bidirectional, trans-Cl−-independent Cl− transport that is, at least in part, conductive. MSI-1436 stimulation of Cl− transport is enhanced in magnitude and rate of onset by lowering bath [Ca2+]. Although MSI-1436 signals release of Ca2+ from heparin-sensitive intracellular stores, MSI-1436 activation of Cl− transport appears to require Ca2+ release from heparin-insensitive intracellular stores. Several additional naturally occurring polyaminosterols of shark origin are both weak agonists for oocyte Cl− transport and antagonists of MSI-1436. Some of these weak agonists alter the oocyte response to the anion transport inhibitor DIDS such that DIDS acts as a conditional Cl− transport agonist even while reducing polyaminosterol-elevated cytosolic [Ca2+].
The present study defines a new property of shark liver-derived polyaminosterols. The characteristics of polyaminosterol signaling in Xenopus oocytes reported in this study suggest a mechanism involving activation of one or more receptors for this novel class of ligands. The putative receptor(s) exhibit topographical specificity in their response only to ligand introduced to the extracellular medium, ligand selectivity among closely related structures acting as agonists or as weak agonist-antagonists, and reversibility of response to ligand. Although suggested by the data, saturability of the signaling response has yet to be proven, and development of a saturable ligand binding assay remains to be achieved. Despite these shortcomings, this signaling pathway is a new addition to previously defined rapid membrane responses to steroid ligands. It is the first report of rapid cellular actions of polyaminosterols, enhanced in its importance by the multiple clinical uses for which polyaminosterols are being investigated in intact animals and in humans.
The shark liver-derived polyaminosterols used in these experiments are shown in Fig. 1. Squalamine was synthesized as previously described (29). The additional structurally related polyaminosterols were isolated and their structures determined as previously described (45). Polyaminosterol stock solutions of 1–5 mg/ml in water were stored at −20°C and were stable at 0.1 mg/ml. DIDS, EGTA, BAPTA, and calcium green dextran (70 kDa) were obtained from Molecular Probes (Eugene, OR). Heparin, heparan sulfate, spermidine, progesterone, taurodeoxycholate, and taurocholate were obtained from Sigma (St. Louis, MO). Na36Cl was obtained from either Amersham-ICN (Costa Mesa, CA) or Dupont-NEN (Boston, MA). 45CaCl2 was obtained from Dupont-NEN. All other chemicals (Sigma or Fisher) were of reagent grade.
36Cl− influx and efflux measurements.
Female Xenopus laevis (NASCO, Madison, WI) were subjected to partial ovariectomy according to protocols approved by the Beth Israel Deaconess Medical Center Institutional Animal Care and Use Committee. Individual oocytes obtained from collagenase digestion of ovarian segments were manually defolliculated and then maintained in ND-96 medium at 19°C for 2–6 days until measurement of 36Cl− influx or 36Cl− efflux with analysis as previously described (9). ND-96 contained (in mM) 96 NaCl, 2 KCl, 1.8 MgCl2, 1 CaCl2, and 5 HEPES (Na), pH 7.40. In some experiments NaCl was replaced by Na-iodide, Na-gluconate, or N-methyl-d-glucamine (NMDG)-chloride. Thirty minutes before injection of 36Cl− in some experiments, either low-molecular-weight heparin [an inhibitor of the inositol 1,4,5-trisphosphate (IP3) receptor] or its inactive analog, heparan sulfate (Sigma), was injected to a final estimated concentration of 0.5 mg/ml. Lag times for activation of 36Cl− efflux by MSI-1436 were computed as follows: the time of MSI-1436 addition was subtracted from the time at which the tangent of the maximal MSI-1436-stimulated efflux rate intersected the tangent for the efflux rate before introduction of the drug. Oocytes studied for SLC4/AE anion exchanger activity were injected with 1–5 ng of cRNA, and ion transport was assayed 2–6 days later. Oocytes from at least two frogs were used for each experiment, although the exemplar 36Cl− efflux time courses presented represent oocytes from a single frog measured in a single representative experiment. Efflux rate constants were compared with Student’s paired two-tailed t-test.
45Ca2+ influx and efflux measurements.
For influx experiments, groups of oocytes were incubated for 1 h in ND-96 (150 μl) modified to contain 5 μCi 45Ca2+ (final [Ca2+] 0.38 mM), with or without 5 μg/ml MSI-1436. Oocytes were then washed in ND-96, lysed with 2% SDS, and counted in a scintillation counter.
For efflux experiments, oocytes were injected with 50 nl of Na-HEPES, pH 7.4, containing 100 μM 45CaCl2 (34,000 cpm) and allowed 2 h to recover and to equilibrate intracellular Ca2+. Oocytes were then incubated in successive 1-ml aliquots of efflux medium, from which 950 nl was removed for gamma counting at the end of each flux time period and replaced with fresh medium. 45Ca2+ efflux rate constants were calculated as for 36Cl− (9). Oocytes from two frogs were used.
Measurement of Xenopus oocyte cytosolic [Ca2+].
Oocytes preloaded for 45 min with fura-2 AM (5–10 μM) in ND-96 medium were mounted in a superfusion chamber on the stage of an Olympus IMT-2 epifluorescence microscope equipped with a DAGE image intensifier and a video camera. Fluorescence emission images were recorded at 510 nm from fura-2-labeled oocytes irradiated with alternating excitation wavelengths of 340 and 380 nm. Excitation ratio images were collected at 15- to 90-s intervals, recorded, and analyzed using an imaging board and software from Universal Imaging (West Chester, PA) as previously described (56, 57). Alternatively, oocytes injected with 35 nl of 5 mg/ml (∼70 μM) calcium green dextran (70 kDa) were excited at 490 nm and imaged at 530 nm. Intracellular concentration of calcium green dextran was between ∼5 μM (if dye space excluded yolk platelets only) and ∼15 μM (if dye space excluded all organelles). Intracellular Ca2+ concentration ([Ca2+]i) values were estimated for each method by in vitro solution calibration as previously described (56, 57). Estimates based on nonratiometric calcium green fluorescence assumed an initial (resting) [Ca2+] value of 80 nM (56). Oocytes from at least two frogs were used for each experiment.
Two-electrode voltage-clamp recordings.
Oocytes were placed in a 1-ml bath chamber (model RC-11; Warner Instruments, Hamden, CT) on the stage of a dissecting microscope and impaled under direct view with 2- to 3-MΩ microelectrodes pulled from borosilicate glass (Garner Glass, Claremont, CA) with a two-stage Narishige puller, sylgarded, fire-polished, and filled with 3 M KCl. After stabilization of membrane potential (Vm), currents were monitored under voltage clamp at room temperature with a Geneclamp 500 amplifier (Axon Instruments, Foster City, CA) interfaced to a personal computer via a TL-1 analog-to-digital board (Axon Instruments). The pCLAMP 6.0 program Clampex (Axon) generated current-voltage (I-V) routines, and the Fetchex program monitored holding current during time course studies. Bath resistance was minimized by using a 3 M KCl agar bridge, and a virtual ground head stage was used to clamp bath potential to zero. Bath addition of drugs was accomplished by gravity feed. Oocytes were injected with EGTA (50 nl of a 20 mM solution) via a Drummond microinjection pipette. Oocytes from at least two frogs were studied for each experiment. Experimental groups were compared using Student’s paired t-tests and t-tests for two means, or by repeated-measures ANOVA with Bonferroni posttest correction for multiple comparisons (InStat; GraphPad Software, San Diego, CA).
On-cell patch-clamp recording.
Defolliculated oocytes were manually devitellinized in ND-96 medium immediately before patch-clamp experiments. Cell-attached patches of 5–10 GΩ resistance were achieved to study single-channel I-V relationships with standard techniques (18). Bath solution was ND-96. Pipette solution contained (in mM) 96 NMDG-chloride, 1.8 CaCl2, 1 MgCl2, and 5 NMDG-HEPES pH 7.40. All voltages refer to the cell interior referenced to the patch pipette. Currents were measured with a 10-GΩ head stage, low-pass filtered at 100–500 Hz (Axopatch 1-D; Axon Instruments), digitized at 1–5 kHz, and stored to a personal computer hard drive. pCLAMP 6.0.3 software (Axon) controlled data acquisition via a Digidata 1200 interface, further filtering at 100 Hz (Fetchan subroutine) for calculation of open channel activity (NPo) from 30-s records as follows where T is the record time, n is the number of channels open, and tn is the time during which n channels are open. NPo values are presented as means ± SE. Oocytes from at least two frogs were studied.
MSI-1436 activates bidirectional Cl− fluxes in Xenopus oocytes.
In an earlier study (4), the naturally occurring polyaminosterols 1360A and 1361A differentially inhibited the SLC4A1/AE1 and SLC4A2/AE2 anion exchanger polypeptides expressed in Xenopus oocytes. Therefore, the additional polyaminosterols pictured in Fig. 1 were also tested as potentially selective inhibitors of SLC4A1 and SLC4A2 (see supplemental data for this article, which may be found at http://ajpregu.physiology.org/cgi/content/full/00098.2005/DC1). Although some of the compounds were indeed SLC4 inhibitors of modest potency [half-inhibitory concentration (ID50) ≤ 10 μM], MSI-1436 activated 36Cl− efflux from native or water-injected oocytes. This observation formed the basis of the current study.
As shown in Fig. 2A, acute exposure to 5 μg/ml MSI-1436 increased 36Cl− influx into native Xenopus oocytes 25-fold (range: 5- to 100-fold increase over 2 yr of experiments). This increased influx was accompanied by gradual acceleration of 36Cl− efflux (22.5 ± 3.0-fold stimulation). MSI-1436-stimulated 36Cl− efflux was sensitive to inhibition by 500 μM DIDS, which reduced stimulation (3.3 ± 0.6-fold stimulation, n = 23, mean ± SE, Fig. 2B), and to NS-3623 (100 μM), but not to 100 μM concentrations of niflumate, tenidap, or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) (Fig. 2D). The 36Cl− efflux activated by bath MSI-1436 was slowly reversible after the drug’s withdrawal (n = 6, not shown). MSI-1436 was inactive when injected into oocytes 30 min before initiation of the 36Cl− efflux assay (Fig. 2C) or when coinjected acutely with 36Cl− (data not shown). Injected MSI-1436 did not inhibit the stimulatory effect of bath MSI-1436 (n = 3, not shown). Addition of DIDS to oocytes in the absence of MSI-1436 had no effect on 36Cl− efflux (9, 27) (see also Fig. 7C). MSI-1436-activated 36Cl− efflux was unaffected by substitution of bath Cl− with cyclamate (Fig. 2, E and F) or with iodide (not shown), either of which maneuvers inhibit SLC4A2-mediated anion transport in Xenopus oocytes by >90% (27) (not shown). Thus extracellular but not injected MSI-1436 stimulates bidirectional Cl− transport characterized by lack of a trans-Cl− requirement, suggesting a mechanism other than Cl−/anion exchange.
MSI-1436 activates Cl− current in Xenopus oocytes.
Oocytes were studied using two-electrode voltage-clamp methods to test the hypothesis that MSI-1436-activated Cl− transport might be conductive. At a holding potential of −50 mV, bath-applied MSI-1436 increased inward current from −6 ± 3 to −210 ± 30 nA, and the current was rapidly and almost completely inhibited by DIDS in the continued presence of agonist (Fig. 3A, n = 4, P < 0.001).
Inhibition by DIDS of steady-state inward current at −100 mV was 80% (n = 3, not shown). The inward current was linear, with mild (variably observed) outward rectification, and was rapidly reversible (Fig. 3C). More marked outward rectification was noted at potentials more positive than +60 mV. The majority of the inward current likely represented conductive anion efflux rather than conductive cation influx, because bath Na+ substitution by NMDG (n = 6) only minimally changed current magnitude (Fig. 3B) and reversal potential (Erev), whereas bath Cl− substitution by gluconate shifted Erev +13 ± 7 mV toward positive potentials (Fig. 3D, n = 12). This small (sub-Nernstian) Erev shift likely reflects a coexisting component of activated nonspecific cation current, in addition to the anion:cation permselectivity of ≤10 often characteristic of anion conductances (19).
MSI-1436 injected to a final estimated intracellular concentration of 5 μg/ml increased inward current by only −12 ± 9 nA, but subsequent bath exposure of the same oocytes to 5 μg/ml MSI-1436 increased inward current by −151 ± 73 nA. This stimulated current was reduced by 62 ± 8 nA upon subsequent bath addition of 0.5 mM DIDS (n = 8, P < 0.01). Thus MSI-1436 is active only from outside the oocyte.
The pharmacological profile of MSI-1436-activated oocyte current was characterized further with additional compounds previously reported to block anion channels. At a holding potential of −100 mV, 1 mM Cd2+ inhibited MSI-1436-activated inward current by 69% (n = 6, P < 0.05). At a holding potential of −60 mV, 400 μM niflumate inhibited MSI-1436-stimulated inward current by 27% (n = 9, P < 0.05). Because MSI-1436 contains a spermine moiety and a bile acid-like moiety, representatives of these component moieties were tested separately in individual oocytes. Oocyte currents measured between −100 and +40 mV were unaltered by spermidine (2 μM, n = 4), taurocholate (2 or 750 μM, each n = 3), or taurodeoxycholate (2 μM, n = 3). Taurodeoxycholate at the much higher concentration of 750 μM modestly increased DIDS-sensitive oocyte current measured at −50 mV from −26 ± 7 to −82 ± 18 nA (n = 3). However, this current was unaccompanied by detectable stimulation of 36Cl− efflux from oocytes (n = 7, not shown), suggesting a limited effect restricted to cation current. Thus the shark polyaminosterol MSI-1436 elicits activities in Xenopus oocytes that are elicited by neither the polyamine representative spermidine nor the representative bile acids taurocholate and taurodeoxycholate.
MSI-1436 activates single Cl− channels in Xenopus oocytes.
Because MSI-1436 elicited currents measured using two-electrode voltage clamp with apparent contributions from both anion channels and nonspecific cation channels, the effect of MSI-1436 was monitored on channel activity in the on-cell patch-clamp conjuration. Figure 3E shows an on-cell patch current recorded before and several minutes after oocyte exposure to 5 μg/ml bath MSI-1436. Channel NPo increased from 0.02 ± 0.02 to 0.26 ± 0.09 after bath addition of MSI-1436 (n = 9, P < 0.05). On-cell patch current exhibited an inward unitary conductance of 12 pS between −25 and −50 mV and was mildly outwardly rectifying at positive potentials, with conductance of 50 pS between +25 and +50 mV (Fig. 3F). Given that the pipette solution contained (in mM) 96 NMDG-chloride, 1.8 CaCl2, 1 MgCl2, and 5 HEPES, pH 7.4, a substantial proportion of inward current likely represented conductive efflux of Cl− from the oocyte.1
MSI-1436-activated 36Cl− efflux from oocytes is regulated by bath Ca2+ and requires intracellular Ca2+.
Oocytes express endogenous Ca2+-activated Cl− current mediated by anion channels of unknown molecular identity (19, 36, 60). We therefore tested the effects of intracellular and bath [Ca2+] on the regulation of oocyte Cl− permeability by MSI-1436. Figure 4A shows that activation of 36Cl− efflux by MSI-1436 (18.5 ± 4.5-fold stimulation, n = 8) was not reduced in “nominally Ca2+-free solutions.” DIDS (500 μM) inhibited this degree of stimulation (less effectively than in the presence of bath Ca2+) to a level of 7.8 ± 1.7 (n = 8). Injected MSI-1436 did not alter these results (n = 3, not shown). The nominal absence of bath Ca2+ had no effect on basal 36Cl− efflux from oocytes unexposed to MSI-1436 (n = 4, not shown). Stimulation of 36Cl− efflux by bath MSI-1436 in the presence of 2 mM bath EGTA was undiminished in both magnitude (27.8 ± 5.3-fold stimulation, n = 4; results with 0.5 mM EGTA were similar, n = 5) and DIDS sensitivity (reduced to 10.9 ± 1.9-fold stimulation by subsequent addition of 500 μM DIDS; not shown). In addition, the apparent affinity of MSI-1436 for activation of 36Cl− efflux was higher in nominally Ca2+-free medium (Fig. 4B).2 Moreover, as evident from comparison of Fig. 4A with Fig. 4, C and E, reduction in bath [Ca2+] also reduced the lag period for activation of 36Cl− efflux by MSI-1436 from 7.8 ± 1.1 min in ND-96 to 1.6 ± 0.3 min in nominally Ca2+-free bath (n = 22).
[Ca2+]i was also important for MSI-1436 action. Prior injection of oocytes with an estimated final concentration of 5 mM EGTA (Fig. 4, C and D) or 0.5 mM BAPTA (Fig. 4, E and F) each reduced MSI-1436-induced efflux of 36Cl− by ∼70%. However, the action of intracellular Ca2+ on 36Cl− efflux was not inhibited by injection of the calmodulin inhibitor calmidazolium (10 μM final intracellular concentration; n = 3, not shown). Prior injection of EGTA into control oocytes (5 mM final concentration) was without effect on two-electrode voltage-clamp current in resting conditions (n = 4, P > 0.1). In contrast, injected EGTA abolished MSI-1436-induced current measured at −50 mV (−52 ± 35 nA before vs. −69 ± 40 nA after exposure to 5 μg/ml MSI-1436; n = 4, not shown). In oocytes in which MSI-1436 first activated inward current from −86 ± 11 to 162 ± 22 nA (measured at −100 mV), subsequent injection of EGTA (5 mM estimated final intracellular concentration) rapidly reduced current to 124 ± 11 nA (n = 6, not shown).
MSI-1436 increases oocyte [Ca2+]i while accelerating both influx and efflux of 45Ca2+.
Figure 5A shows that MSI-1436 addition to ND-96 bath increased [Ca2+]i sixfold, but only after a lag period of 5.8 ± 0.8 min (n = 9), approximating that observed for activation of 36Cl− efflux (see Fig. 2). As previously noted for 36Cl− efflux, this lag period was greatly shortened to 1.7 ± 0.5 min (n = 6, P < 0.001) without decrease in signal magnitude when MSI-1436 was added in nominally Ca2+-free bath (Fig. 5B). Injected EGTA (5 mM estimated final concentration) attenuated the increase in [Ca2+]i by ∼75% (n = 4), as shown in the representative oocyte in Fig. 5B, whereas bath addition of EGTA minimally reduced the elevation of [Ca2+]i by MSI-1436 (not shown). This agonist-induced elevation in [Ca2+]i was accompanied by fourfold-increased 45Ca2+ influx (Fig. 5C) and, in ND-96 (n = 4, not shown) or nominally Ca2+-free conditions (n = 6, Fig. 5D), by 8.8 ± 1.7-fold accelerated 45Ca2+ efflux, which exhibited modest DIDS sensitivity.
Oocytes express type I IP3 receptors and respond to IP3 injection with elevation of [Ca2+]i (20). Injection of heparin, an inhibitor of IP3-induced Ca2+ release (Fig. 6A) greatly inhibited elevation of [Ca2+]i (estimated Δ[Ca2+]i = 15 ± 4 nM, n = 11) compared with that in oocytes injected with the inactive heparan sulfate (estimated Δ[Ca2+]i = 80 ± 7 nM; n = 11, P < 0.02, fura-2). Inclusion of EGTA in the nominally Ca2+-free bath (Fig. 6B) did not reduce acute elevation of [Ca2+]i by MSI-1436 in oocytes injected with heparan sulfate (estimated Δ[Ca2+]i = 91 ± 17 nM, n = 3), and injected heparin still reduced this elevation (estimated Δ[Ca2+]i = 33 ± 10 nM, n = 3).3 In contrast, injected heparin did not attenuate MSI-induced 36Cl− efflux, whether measured in ND-96 bath (Fig. 6C) or in nominally Ca2+-free bath (Fig. 6D).
Inhibition of oocyte IP3 generation by bath addition of the phospholipase C (PLC) inhibitor U-73122 (20 μM with 1-h preincubation, Fig. 6, E and F; or 10 μM, not shown, n = 6) or by injection of U-73122 to an estimated final concentration of 20 μM (n = 6, not shown) failed to inhibit MSI-1436-stimulated 36Cl− efflux (Fig. 6, E and F). Indeed, the enhancement of 36Cl− efflux by U-73122 and its inhibition by the inactive analog U-73343 (20 μM with 1-h preincubation) suggests that PLC activity, if anything, might antagonize 36Cl− efflux. Thus activation of 36Cl− efflux, although dependent on intracellular Ca2+, appears not to require IP3-sensitive intracellular Ca2+ stores. Moreover, this released Ca2+ does not act via a (10 μM) calmidazolium-sensitive mechanism (n = 5, not shown).
MSI-1436 signaling in Xenopus oocytes is likely mediated by one or more polyaminosterol receptors in or near the plasma membrane.
The polyaminosterols in Fig. 1 were screened at 5 μg/ml as 36Cl− transport agonists in Xenopus oocytes (Table 1). In ND-96 bath, only MSI-1436 activated 36Cl− efflux. Because MSI-1436 differs from squalamine only with respect to the polyamine residue (spermine vs. spermidine) covalently linked to the sulfated sterol, activity in ND-96 is associated with the more cationic polyamine moiety. MSI-1521 is more cationic than MSI-1436 but lacks a sulfate on its C-24 hydroxyl residue and is inactive in ND-96, suggesting that the putative MSI-1436 “receptor” recognizes something more than a cationic steroid. Moreover, in nominally Ca2+-free bath, the spermidine-containing polyaminosterols MSI-1437 and MSI-1256 activated 36Cl− efflux at rates equivalent to that activated by MSI-1436. The presence of a CH2OH group on C-24 of the sterol side chain in MSI-1437 had minimal consequence, but the side chain alterations in MSI-1360 and -1361 were associated with reduced activity. The modest activity of MSI-1508 (MSI-1236 with its spermine moiety carrying a terminal aminopropyl group) was consistent with the importance of the polyamine’s net cationic charge and/or with spatial constraints on the polyamine. In Ca2+-free conditions, MSI-1521 (5 μg/ml) exhibited weak agonist activity but also strongly inhibited the 36Cl− efflux activated by 375 ng/ml MSI-1436 (Fig. 7A). These data suggest that structurally similar polyaminosterols act on Xenopus oocytes through binding to at least one receptor that discriminates among these ligands. This receptor appears to be in the plasma membrane or in a (functionally defined) subplasmalemmal membrane compartment.
MSI-1521 exhibited another interesting property in Xenopus oocytes. Whereas MSI-1436 (Fig. 2B) and most other polyaminosterols activated 36Cl− efflux sensitive to inhibition by DIDS (Table 1), oocytes previously exposed to MSI-1521 responded to subsequent addition of 0.5 mM DIDS (Fig. 7B) with an increase in relative rate of 36Cl− efflux from 1.7 ± 0.1 to 7.8 ± 0.5 times that of oocytes before MSI-1521 exposure (n = 12). This stimulatory effect of DIDS was not evident in oocytes treated with DIDS before MSI-1521 exposure (n = 4, Fig. 7C). Moreover, the related but noncovalently reactive stilbene DNDS (4,4′-dinitrostilbene-2,2′-disulfonic acid) failed to stimulate 36Cl− efflux from MSI-1521-treated oocytes (n = 8, Fig. 7D). MSI-1521 also elevated oocyte [Ca2+]i by 42 ± 4 nM (n = 6, Fig. 7E), but this Ca2+ signal did not mediate the stimulatory action of DIDS on 36Cl− efflux from MSI-1521-pretreated oocytes. Rather than potentiating the MSI-1521-stimulated increase in [Ca2+]i, subsequent addition of DIDS inhibited that increase by 91 ± 6% (n = 6, Fig. 7E). Exposure to DIDS alone left basal oocyte [Ca2+]i unaltered (n = 4; Fig. 7F).
Among the polyaminosterols tested in addition to MSI-1521, only MSI-1360A also conferred on oocytes the property of DIDS-stimulated 36Cl− efflux (Table 2), but at rates only 30% that of MSI-1521. MSI-1360A (5 μg/ml) also inhibited stimulation of 36Cl− efflux by 375 ng/ml MSI-1436. However, the presence of MSI-1436 together with either MSI-1521 or MSI-1360A did not prevent stimulation of 36Cl− efflux by subsequent addition of 0.5 mM DIDS (Table 2).
MSI-1521 confers on oocytes a DIDS-stimulated inward current.
MSI-1521 was tested in Xenopus oocytes in Ca2+-containing ND-96 bath to minimize the likelihood of eliciting Ca2+-inhibited Cl− current (60). Even in the presence of extracellular Ca2+, MSI-1521 produced a small increase in oocyte inward current that was substantially enhanced by subsequent application of DIDS (Fig. 8, A and B). Thus the response to DIDS of voltage-clamp current in MSI-1521-treated oocytes resembled the oocytes’ 36Cl− efflux response (Fig. 7B). Moreover, just as 36Cl− efflux from oocytes exposed to MSI-1436 was inhibited by MSI-1521 (Fig. 7A), the current activated in oocytes by MSI-1436 (1,478 ± 211 nA) was similarly reduced by MSI-1521 to a value of −789 ± 245 nA (Fig. 8C; P < 0.05).
Squalamine and related polyaminosterols were originally identified by screening shark tissues for antimicrobial activities (40, 45). Squalamine has since been characterized as an angiogenesis inhibitor and anti-tumor agent (11, 15, 22, 23, 24, 37, 51). The squalamine analog MSI-1436 has shown appetite suppressant activity and the ability to relieve insulin resistance and hepatic steatosis (2, 54, 64). Both squalamine and MSI-1436 mediate slow-onset inhibition of NHE3-mediated Na+/H+ exchange in transfected PS120 lung fibroblasts and in ileal brush-border membrane vesicles (3), but the signaling pathways leading to this inhibition are not known. Because NHE3 is not known to be expressed in endothelial cells, the role of this inhibition of ion transport in inhibition of angiogenesis remains unclear.
The current study presents several novel findings. These include the first reported data on rapid cellular actions of squalamine-related polyaminosterols, the first data suggesting polyaminosterol activity at or near the cell surface, and the first data suggesting that polyaminosterols trigger intracellular signaling by liganding specific receptors at or near the cell surface. When added to Xenopus oocytes, MSI-1436 activates bidirectional Cl− fluxes and currents sensitive to DIDS. This activation follows addition of MSI-1436 to the bath but not its injection into oocytes, suggesting interaction with a membrane-associated receptor. Enhanced whole cell anion conductance induced by MSI-1436 is accompanied by increased activity of unitary anion channels as detected by on-cell patch-clamp recordings. Activation of Cl− flux by MSI-1436 is accelerated and enhanced in potency by nominal removal of bath Ca2+ or bath addition of EGTA. In contrast, lowering [Ca2+]i with injected EGTA inhibits activation of Cl− flux and current by MSI-1436. The observed elevation of [Ca2+]i by MSI-1436 is sensitive to injected heparin, consistent with IP3 receptor-mediated Ca2+ release from intracellular stores. However, injected heparin fails to inhibit stimulation of 36Cl− flux by MSI-1436. Inhibition of PLC by U-73122 at a high concentration is similarly without effect. Thus the intracellular Ca2+ required for activation of Cl− transport by MSI-1436 may be derived from IP3-insensitive stores that contribute little to MSI-1436-induced elevation of total oocyte cytoplasmic [Ca2+]. In the presence of the weak agonist MSI-1521, DIDS addition activates, rather than inhibits, both Cl− flux and current.
MSI-1436 activates an undefined receptor at the Xenopus oocyte surface or in an intracellular compartment near the surface.
A previous report of Cl− secretion by Fischer rat thyroid cells and CFT-1 human tracheobronchial cells activated by the squalamine-related drug GL-172 postulated an ionophore effect to explain drug-induced secretion (30). However, ionophore-related effects of squalamine at the aminosterol concentrations used in the current investigation are minimal (48). Several findings support the existence of a specific receptor for polyaminosterols at or near the surface of Xenopus oocytes. First, MSI-1436 was active in the bath in a concentration-dependent manner but was inactive when injected into the oocyte (Fig. 2). This supports a surface membrane receptor without ruling out MSI-1436 entry into the cell by diffusion or via a less specific pathway (such as a low-density lipoprotein receptor-related protein class receptor) to act at a site inaccessible to injected MSI-1436 (perhaps due to binding by yolk platelets or other organelles). The receptor could be protein, glycan, or lipid. Second, the effects of MSI-1436 were not mimicked by representatives of each of the two moieties comprising MSI-1436, spermine as a polyamine and bile acids or progesterone as steroids. Third, the onset of MSI-1436 action in oocytes was potentiated and accelerated by reduction in bath [Ca2+] (Fig. 4), consistent with modulation of ligand binding by ionic strength and/or Ca2+. Fourth, the effects of MSI-1436 were reversible. Fifth, additional agonists (e.g., squalamine, MSI-1508) were found among the homologous polyaminosterols tested, and these weaker agonists also functioned as inhibitors of MSI-1436. Sixth, in the presence of the weak agonist MSI-1521, the Cl− transport blocker DIDS instead activated Cl− flux and conductance. However, a polyaminosterol binding assay is not yet available. Thus specific and saturable MSI-1436 binding to oocyte membranes remains to be demonstrated.
The potentiating effect of reducing bath Ca2+ may reflect direct enhancement of MSI-1436 binding or slowing of dissociation of bound MSI-1436 via enhanced interaction of the putative receptor(s) with the polyamine moiety of MSI-1436 or other polyaminosterols. Reduced bath Ca2+ might favor a cyclic form of polyaminosterol with internal pairing of the negatively charged sulfate with the positively charged terminal amine. Alternatively, reduced bath Ca2+ might unmask binding sites for other activating cations on the postulated polyaminosterol receptor, as has been reported for the interaction of α5β1-integrin with fibronectin (41). The relationship of the polyaminosterol receptor(s) of Xenopus oocytes to the oocyte progesterone receptor (59, 61) or to recently reported piscine and mammalian plasmalemmal steroid receptors of the G protein-coupled receptor superfamily (55, 66, 67) is unknown.
MSI-1436 binding to its putative receptor elevates oocyte [Ca2+]i, apparently by release from intracellular stores.
Liganding of the putative polyaminosterol receptor by MSI-1436 leads to both elevation of [Ca2+]i and increased bidirectional 45Ca2+ flux. The elevation of [Ca2+]i is greatly attenuated by injected heparin, suggesting that the bulk of the increased cytoplasmic [Ca2+] arises from the IP3-activated, IP3 receptor-mediated release from endoplasmic reticulum Ca2+ stores. This effect of heparin is consistent with persistence of MSI-1436-induced elevation of [Ca2+]i in the presence of bath EGTA. It also suggests likely generation of IP3 from phosphatidylinositol 4,5-bisphosphate (PIP2) by activation of PLC. This activation may follow MSI-1436 liganding of its receptor or, alternatively, may occur by interaction of MSI-1436 with an anionic phospholipid (48) such as PIP2, perhaps facilitating the interaction between PLC and its substrate.
Remarkably, however, injected heparin has no effect on MSI-1436-stimulated Cl− efflux, despite its effect on [Ca2+]i. Similarly, the PLC inhibitor U-73122 has no effect on ligand-stimulated Cl− efflux, despite its documented inhibition at these concentrations of multiple downstream targets of PLC in Xenopus oocytes (10, 32, 44). These discordant effects of heparin contrast with the coordinate inhibition by injected EGTA of increased 36Cl− efflux, Cl− current, and [Ca2+]i stimulated by MSI-1436. Thus, despite activation of heparin-sensitive Ca2+ release from (nominally) IP3-sensitive stores, MSI-1436 activates Ca2+-sensitive Cl− flux and current with Ca2+ released from heparin-insensitive (and nominally IP3 insensitive) stores. Xenopus oocytes do not express ryanodine receptors, and oocyte Ca2+ release by cADP-ribose, nicotinic acid adenine dinucleotide phosphate, or β-NAD has not been detected. Heparin-insensitive Ca2+ stores responsive to sphingosine-1-phosphate (28, 53), GTP, or cGMP (6) have not been reported in oocytes. Thus MSI-1436-sensitive Cl− channels might be activated exclusively by IP3 receptor-mediated Ca2+ release from a subplasmalemmal compartment either inaccessible to injected heparin or rapidly rendered insensitive to heparin (49).
Relationship between Cl− flux and current activated by MSI-1436.
MSI-1436-stimulated Cl− influx in ND-96 was ∼14 nmol/h per oocyte (Fig. 2A). Intracellular [Cl−] after 36Cl− injection in isotopic efflux experiments was ∼13 mM higher than the estimated resting value of ∼30 mM. Assuming this value of 43 mM, the mean MSI-1436-stimulated Cl− efflux rate measured in 28 oocytes from 6 different frogs was between 50 (median) and 70 nmol/h (mean). Thus estimated net flux was ∼36–56 nmol Cl− efflux per hour. Open-circuit Vm of control oocytes was −44 ± 2 mV (n = 8), and Vm estimated from the Erev of voltage-clamped oocytes ranged between −50 and −20 mV in these experiments. Figures 3C and 8C show MSI-1436-stimulated inward current at −40 mV between −150 and −400 nA, or 5.6 to 15 nmol of outward negative charge per hour. The difference between estimated net flux and measured current could represent parallel or secondary stimulation of cation current by MSI-1436, because bath ion substitution suggested a component of stimulated current carried by cations (Fig. 3, D and F). Alternatively, MSI-1436 might also stimulate electroneutral anion transport. In view of the assumptions required for these estimates, the bidirectional Cl− flux and apparent anion current stimulated by MSI-1436 are in reasonable agreement.
It is not clear why MSI-1436 added to ND-96 bath activated inward current with a short lag time (Fig. 3A), whereas 36Cl− efflux exhibited a longer lag time for activation (Fig. 2B). This difference might represent distinct pathways of chloride transport, such as acute activation in the membrane and slower recruitment of additional chloride channels to the membrane. Alternatively, a surface polyaminosterol receptor might rapidly activate Cl− current, with slower activation of a component of 36Cl− efflux by subsequent receptor internalization or by a distinct intracellular receptor.
Weak agonist polyaminosterols convert the anion transport inhibitor DIDS to an activator.
MSI-1436-stimulated 36Cl− efflux and Cl− conductance are both inhibited by DIDS. However, in the presence of the weak agonist MSI-1521, DIDS activates both 36Cl− efflux and the associated current. In contrast, the structurally related noncovalent inhibitor DNDS is without this effect on MSI-1521. DIDS might act directly on a Ca2+-activated Cl− channel conformationally altered by MSI-1521 binding to the putative polyaminosterol receptor or by MSI-1521 bound to the channel itself. The difference between DIDS and DNDS as agonists in the presence of MS-1521 might reflect different binding affinities to the receptor or a requirement for covalent interaction of DIDS with the putative receptor or the channel protein. In Xenopus oocytes, DIDS has been previously documented to increase 22Na efflux (16) and to activate two types of cation currents (13). Extracellular DIDS also activates the CD3-associated Ca2+ entry pathway in activated T lymphocytes (47), a biliary K+ secretion pathway (25), and, in a single report, transepithelial Cl− secretion by Calu-3 cell monolayers through apparent enhancement of basolateral K+ conductance (29).
In principle, DIDS might activate Cl− transport through elevation of [Ca2+]i, as has been observed in T84 human colon carcinoma cell monolayers (8). In intact rat pulmonary artery vascular smooth muscle cells, extracellular DIDS rapidly elevated resting [Ca2+]i without reduction in caffeine-releasable intracellular Ca2+ stores and in a manner unaltered by bath Cl− substitution (12). However, in Xenopus oocytes, DIDS did not itself elevate [Ca2+]i but instead reversed the elevation of [Ca2+]i produced by MSI-1521 even as it activated 36Cl− efflux (Fig. 7) and Cl− current (Fig. 8). These properties of DIDS and MS-1521 remain consistent with direct interaction of both ligands with the channel or with a channel-binding protein.
Why these effects of DIDS require MSI-1521 rather than MSI-1436 remains unclear. The isothiocyanate moiety of DIDS might selectively bind to or covalently react with a terminal amino group of MSI-1521. In any case, this unusual interaction between weak agonist and transport inhibitor should serve as a useful functional marker for future identification of the postulated surface receptor for polyaminosterols.
Role of Ca2+ signaling and chloride channel activation in the antiproliferative and appetite suppressant effects of MSI-1436.
The mechanisms of the appetite suppressant and metabolic effects of MSI-1436 remain unknown. The metabolic effects of MSI-1436 share some properties with leptin (2, 54). Leptin and orexin produce cell type-specific but consistently reciprocal effects on proopiomelanocortin-positive or neuropeptide Y-positive neurons of the arcuate nucleus or on glucose-responsive neurons in the ventromedial hypothalamus (40). MSI-1436 might have similarly cell type-specific properties. However, MSI-1436 appears to act either downstream from the leptin or melanocortin 4 (MC4) receptors or independently of their signaling pathways (64).
Leptin also activates ATP-sensitive K+ channels of pancreatic islet β-cells. The resulting hyperpolarization closes voltage-gated Ca2+ channels, lowers cytosolic [Ca2+], and inhibits Ca2+-dependent insulin release (33). The ability of MSI-1436 to correct the hyperinsulinemia, insulin resistance, and hyperglycemia of the ob/ob mouse (54) suggests that, like leptin, MSI-1436, might modulate β-cell membrane potential to control insulin secretion. However, activation of volume-sensitive (5) or Ca2+-sensitive chloride channels in the β-cell would hyperpolarize only in the context of depolarized membrane potential, perhaps contributing to shaping of oscillatory Ca2+ signaling (34). In contrast, neuronal cells that express high levels of the KCC2 K+-Cl− cotransporter often maintain sufficiently low intracellular [Cl−] as to render the opening of Cl− channels hyperpolarizing (26, 65). In this way, both leptin and MSI-1436 might decrease neuronal excitation. MSI-1436 might also hyperpolarize hypothalamic neurons by binding to the positive allosteric neurosteroid sites of GABAA receptors (7).
Further experiments on the cellular mechanisms of polyaminosterol action may identify a new class of receptors or assign new ligands to known receptors. In addition, these experiments may identify new intracellular Ca2+ stores or new subcellular localizations of known stores. The polyaminosterols will also be useful ligands for further study of the Cl− channels and cation channels activated by elevation of [Ca2+]i. Finally, such experiments will increase our knowledge of shark physiology and of shark tissues as a source of novel lead compounds for drug development.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-43495 and DK-51059 (to S. L. Alper), Harvard Digestive Diseases Center Grant DK-34854 (to S. L. Alper), and a sponsored research grant from Magainin Pharmaceuticals, Inc. (now Genaera Corporation).
Genaera Corporation was named Magainin Pharmaceuticals, Inc., at the time the experiments presented in this article were performed. M. A. Zasloff and J. I. Williams were employees of Magainin Pharmaceuticals at the time these studies were performed.
We thank Dr. Yoram Oron and Dr. Rexford Ahima for helpful discussions and Dr. Mike McLane for comments on the manuscript.
Present addresses: M. Zasloff, Georgetown University Medical Center, 4000 Reservoir Rd. NW, Washington DC 20057-2197; J. I. Williams, Exelixis Corporation, 170 Harbor Way, South San Francisco, CA 94083.
↵* M. N. Chernova and D. H. Vandorpe contributed equally to this work.
↵1 The −20 mV Erev of MSI-1436-induced oocytes current measured by two-electrode voltage clamp (Fig. 3, C and D) differed from that of about −5 mV measured by cell-attached patch clamp (Fig. 3E). This difference likely reflects at least two contributions. First, the patch membrane potential in the cell-attached mode, measured with reference to the bath, represents the difference between the cell membrane potential and the pipette potential. Second, the cell potential is not known and is not likely to remain constant as pipette potential is varied (18, 39). Thus Erev need not be identical in the two recording configurations.
↵2 ED50 values (concentrations producing half-maximal activation) for MSI-1436 could not be calculated in ND-96 or in nominally Ca2+-free bath for at least two reasons. First, 5 μg/ml was not confirmed to be a maximally effective concentration in all oocytes. Second, the lag time itself appeared to be concentration dependent. In the experiment shown, the apparent ED50 for MSI-1436 in the nominal absence of bath Ca2+ was 0.57 ± 0.28 μM, a minimal value based on the unproven assumption that maximal Cl− flux is evoked by 5 μg/ml (7.3 μM) MSI-1436.
↵3 After 30-min preincubation of oocytes in 0.5 mM EGTA, bath addition of MSI-1436 in the continued presence of EGTA produced an attenuated increase in [Ca2+]i of 15 ± 19 nM (n = 7), as compared with an estimated 63 ± 20 nM increase (n = 4) after MSI-1436 exposure in ND-96 bath (not shown).
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