Ceramide is responsible for the failure of compensatory nerve sprouting in apolipoprotein E knock-out mice.

Apolipoprotein E (apoE) is a key transporter of the cholesterol and phospholipids required for membrane synthesis and nerve growth. We now report a virtual absence in apoE knock-out (KO) mice of normal nerve growth factor (NGF)-driven compensatory sprouting of undamaged cutaneous nociceptive nerves. In contrast, NGF-independent regeneration of crushed axons was unaffected. Essentially similar results came from aged wild-type mice. In apoE KO mice, the endogenous sprouting stimulus was suspect, because NGF administration induced normal sprouting; nevertheless, NGF increased normally in denervated skin, transported normally in the axons, and led to phosphorylation of trkA, erk1, and erk2. However, sprouting was restored in apoE KO mice (although not in aged mice) by fumonisin B1, an inhibitor of ceramide synthesis. A shotgun analysis revealed a wide array of changes in individual ceramide species in DRG neurons of apoE KO mice, and the changes for ceramide species OH_N15:0 made it a candidate inhibitor of sprouting (increased in apoE KO mice and normalized by fumonisin B1). Nevertheless, the unknown effects of individual ceramide species on sprouting, as well as the variability of their changed levels in apoE KO mice and how these were affected by fumonisin B1, support a different conclusion. We suggest that absence of apoE expression alters the balance among ceramide species to one that collectively inhibits compensatory sprouting, whereas fumonisin B1 establishes a new balance that allows sprouting. Nontoxic ceramide modulators might usefully promote sprouting and circuitry repair in neurodegenerative disorders in which ceramide species are perturbed, adding to the benefits of reducing ceramide-induced neuronal apoptosis.

Isoprostanes constrict human radial artery by stimulation of thromboxane receptors, Ca2+ release, and RhoA activation.

OBJECTIVES: Radial artery vasospasm remains a potential cause of early graft failure after coronary bypass graft surgery, despite pretreatment with alpha-adrenergic or calcium channel blockers. We examined the roles of isoprostanes and prostanoid receptors selective for thromboxane A2 in the vasoconstriction of human radial arteries. METHODS: Human radial arterial segments were pretreated intraoperatively with verapamil/papaverine or nitroglycerine/phenoxybenzamine, or not treated. In the laboratory, we measured isometric contractions in ring segments, vasoconstriction in pressurized segments, and changes in [Ca2+] and K+ currents in single cells. RESULTS: Although phenoxybenzamine eliminated adrenergic responses, the isoprostane 15-F(2t)-IsoP and 2 closely related E-ring molecules (15-E(1t)-IsoP and 15-E(2t)-IsoP) still evoked powerful contractions; 15-E(2t)-IsoP was approximately 10-fold more potent than the other 2 agents. Responses were mediated through thromboxane receptors because they were sensitive to ICI-192605. Furthermore, they were sensitive to the Rho-kinase inhibitors Y-27632 or H-1152 (both 10(-5) mol/L) or to cyclopiazonic acid (which depletes the internal Ca2+ pool), but not to nifedipine. In single cells, 15-E(2t)-IsoP elevated [Ca2+]i and suppressed K+ current. CONCLUSIONS: Isoprostanes accumulate after coronary artery bypass graft surgery, yet none of the currently available antispasm treatments for radial artery grafts is effective against isoprostane-induced vasoconstriction. It is imperative that more specific treatment strategies be developed. We found that isoprostane responses in radial arteries are mediated by prostanoid receptors selective for thromboxane A2 with activation of Rho-kinase and release of Ca2+. Pretreatment of radial artery grafts with Rho-associated kinase inhibitors may potentially reduce postoperative graft spasm. Clinical studies to test this are indicated.

The reverse mode of the Na(+)/Ca(2+) exchanger provides a source of Ca(2+) for store refilling following agonist-induced Ca(2+) mobilization.

Agonist-induced contraction of airway smooth muscle (ASM) can be triggered by an elevation in the intracellular Ca(2+) concentration, primarily through the release of Ca(2+) from the sarcoplasmic reticulum (SR). The refilling of the SR is integral for subsequent contractions. It has been suggested that Ca(2+) entry via store-operated cation (SOC) and receptor-operated cation channels may facilitate refilling of the SR. Indeed, depletion of the SR activates substantial inward SOC currents in ASM that are composed of both Ca(2+) and Na(+). Accumulation of Na(+) within the cell may regulate Ca(2+) handling in ASM by forcing the Na(+)/Ca(2+) exchanger (NCX) into the reverse mode, leading to the influx of Ca(2+) from the extracellular domain. Since depletion of the SR activates substantial inward Na(+) current, it is conceivable that the reverse mode of the NCX may contribute to the intracellular Ca(2+) pool from which the SR is refilled. Indeed, successive contractions of bovine ASM, evoked by various agonists (ACh, histamine, 5-HT, caffeine) were significantly reduced upon removal of extracellular Na(+); whereas contractions evoked by KCl were unchanged by Na(+) depletion. Ouabain, a selective inhibitor of the Na(+)/K(+) pump, had no effect on the reductions observed under normal and zero-Na(+) conditions. KB-R7943, a selective inhibitor of the reverse mode of the NCX, significantly reduced successive contractions induced by all agonists without altering KCl responses. Furthermore, KB-R7943 abolished successive caffeine-induced Ca(2+) transients in single ASM cells. Together, these data suggest a role for the reverse mode of the NCX in refilling the SR in ASM following Ca(2+) mobilization.

Intracellular Cl- fluxes play a novel role in Ca2+ handling in airway smooth muscle.

Intracellular Ca(2+) is actively sequestered into the sarcoplasmic reticulum (SR), whereas the release of Ca(2+) from the SR can be triggered by activation of the inositol 1,4,5-trisphosphate and ryanodine receptors. Uptake and release of Ca(2+) across the SR membrane are electrogenic processes; accumulation of positive or negative charge across the SR membrane could electrostatically hinder the movement of Ca(2+) into or out of the SR, respectively. We hypothesized that the movement of intracellular Cl(-) (Cl(i)(-)) across the SR membrane neutralizes the accumulation of charge that accompanies uptake and release of Ca(2+). Thus inhibition of SR Cl(-) fluxes will reduce Ca(2+) sequestration and agonist-induced release. The Cl(-) channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 10(-4) M), previously shown to inhibit SR Cl(-) channels, significantly reduced the magnitude of successive acetylcholine-induced contractions of airway smooth muscle (ASM), suggesting a "run down" of sequestered Ca(2+) within the SR. Niflumic acid (10(-4) M), a structurally different Cl(-) channel blocker, had no such effect. Furthermore, NPPB significantly reduced caffeine-induced contraction and increases in intracellular Ca(2+) concentration ([Ca(2+)](i)). Depletion of Cl(i)(-), accomplished by bathing ASM strips in Cl(-)-free buffer, significantly reduced the magnitude of successive acetylcholine-induced contractions. In addition, Cl(-) depletion significantly reduced caffeine-induced increases in [Ca(2+)](i). Together these data suggest a novel role for Cl(i)(-) fluxes in Ca(2+) handling in smooth muscle. Because the release of sequestered Ca(2+) is the predominate source of Ca(2+) for contraction of ASM, targeting Cl(i)(-) fluxes may prove useful in the control of ASM hyperresponsiveness associated with asthma.

8-isoprostaglandin E(2) activates Ca(2+)-dependent K(+) current via cyclic AMP signaling pathway in murine renal artery.

The inhibitory pathway of 8-isoprostaglandin E(2) was investigated in murine renal arterial smooth muscle. K(+) current was augmented in a concentration-dependent fashion, with an average increase of 123+/-28% (n=6) following application of 10(-5) M 8-iso PGE(2). This augmentation was observed in the presence of 4-aminopyridine (4-AP, 10(-3) M) but not that of charybdotoxin (Ch Tx, 10(-7) M). Fluorimetric recordings showed marked concentration-dependent increase of cytosolic Ca(2+) levels by 8-iso PGE(2), while an enzyme-linked immunosorbent assay (ELISA)-based cyclic AMP assay showed increased cAMP levels by 10(-7) M 8-iso PGE(2) challenge. The isoprostane-induced augmentation was prevented by the ryanodine receptor blocker ruthenium red (10(-5) M) or the adenylate cyclase blocker SQ 22536 (10(-4) M). The protein kinase A (PKA) inhibitor H 89 (10(-5) M) inhibited resting K(+) currents (78+/-5%, n=5) but did not prevent 8-iso PGE(2) from augmenting the remaining K(+) current. We conclude that 8-iso PGE(2) enhances Ca(2+)-dependent K(+) currents in murine renal artery through a cAMP-dependent pathway which may involve internally sequestered Ca(2+).

Cyclopiazonic acid activates a Ca2+-permeable, nonselective cation conductance in porcine and bovine tracheal smooth muscle.

Capacitative Ca2+ entry has been examined in several tissues and, in some, appears to be mediated by nonselective cation channels collectively referred to as "store-operated" cation channels; however, relatively little is known about the electrophysiological properties of these channels in airway smooth muscle. Consequently we examined the electrophysiological characteristics and changes in intracellular Ca2+ concentration associated with a cyclopiazonic acid (CPA)-evoked current in porcine and bovine airway smooth muscle using patch-clamp and Ca2+-fluorescence techniques. In bovine tracheal myocytes, CPA induced an elevation of intracellular Ca2+ that was dependent on extracellular Ca2+ and was insensitive to nifedipine (an L-type voltage-gated Ca2+ channel inhibitor). Using patch-clamp techniques and conditions that block both K+ and Cl- currents, we found that CPA rapidly activated a membrane conductance (I(CPA)) in porcine and bovine tracheal myocytes that exhibits a linear current-voltage relationship with a reversal potential around 0 mV. Replacement of extracellular Na+ resulted in a marked reduction of I(CPA) at physiological membrane potentials (i.e., -60 mV) that was accompanied by a shift in the reversal potential for I(CPA) toward more negative membrane potentials. In addition, I(CPA) was markedly inhibited by 10 microM Gd3+ and La3+ but was largely insensitive to 1 microM nifedipine. We conclude that CPA induces capacitative Ca2+ entry in porcine and bovine tracheal smooth muscle via a Gd3+- and La3+-sensitive, nonselective cation conductance.

Regulation of Rho/ROCK signaling in airway smooth muscle by membrane potential and [Ca2+]i.

Recently, we have shown that Rho and Rho-activated kinase (ROCK) may become activated by high-millimolar KCl, which had previously been widely assumed to act solely through opening of voltage-dependent Ca(2+) channels. In this study, we explored in more detail the relationship between membrane depolarization, Ca(2+) currents, and activation of Rho/ROCK in bovine tracheal smooth muscle. Ca(2+) currents began to activate at membrane voltages more positive than -40 mV and were maximally activated above 0 mV; at the same time, these underwent time- and voltage-dependent inactivation. Depolarizing intact tissues by KCl challenge evoked contractions that were blocked equally, and in a nonadditive fashion, by nifedipine or by the ROCK inhibitor Y-27632. Other agents that elevate intracellular calcium concentration ([Ca(2+)](i)) by pathways independent of G protein-coupled receptors, namely the SERCA-pump inhibitor cyclopiazonic acid and the Ca(2+) ionophore A-23187, evoked contractions that were also largely reduced by Y-27632. KCl directly increased Rho and ROCK activities in a concentration-dependent fashion that paralleled closely the effect of KCl on tone and [Ca(2+)](i), as well as the voltage-dependent Ca(2+) currents that were measured over the voltage ranges that are evoked by 0-120 mM KCl. Through the use of various pharmacological inhibitors, we ruled out roles for Ca(2+)/calmodulin-dependent CaM kinase II, protein kinase C, and protein kinase A in mediating the KCl-stimulated changes in tone and Rho/ROCK activities. In conclusion, Rho is activated by elevation of [Ca(2+)](i) (although the signal transduction pathway underlying this Ca(2+) dependence is still unclear) and possibly also by membrane depolarization per se.

KCl evokes contraction of airway smooth muscle via activation of RhoA and Rho-kinase.

Airway smooth muscle (ASM) cells express voltage-dependent Ca2+ channels, primarily of the L-subtype. These may play a role in excitation-contraction coupling of ASM, although other signaling pathways may also contribute: one of these includes Rho and its downstream effector molecule Rho-associated kinase (ROCK). Although voltage-dependent Ca2+ influx and Rho/ROCK signaling have traditionally been viewed as entirely separate pathways, recent evidence in vascular smooth muscle suggest differently. In this study, we monitored contractile activity (muscle baths) in bronchial and/or tracheal preparations from the pig, cow, and human, and further examined Rho and ROCK activities (Western blots and kinase assays) and cytosolic levels of Ca2+ (fluo 4-based fluorimetry) in porcine tracheal myocytes. KCl evoked substantial contractions that were suppressed in tracheal preparations by removal of external Ca2+ or using the selective L-type Ca2+ channel blocker nifedipine; porcine bronchial preparations were much less sensitive, and bovine bronchi were essentially unaffected by 1 microM nifedipine. Surprisingly, KCl-evoked contractions were also highly sensitive to two structurally different ROCK inhibitors: Y-27632 and HA-1077. Furthermore, the inhibitory effects of nifedipine and of the ROCK inhibitors were not additive. KCl also caused marked stimulation of Rho and ROCK activities, and both these changes were suppressed by nifedipine or by removal of external Ca2+. KCl-induced elevation of [Ca2+]i was not affected by Y-27632 but was reversed by NiCl2 or by BAPTA-AM. We conclude that KCl acts in part through stimulation of Rho and ROCK, possibly secondary to voltage-dependent Ca2+ influx.

Neotrofin, a novel purine that induces NGF-dependent nociceptive nerve sprouting but not hyperalgesia in adult rat skin.

We report peripheral actions in rats of Neotrofin, a purine derivative of therapeutic interest. Systemic injections mimicked NGF in eliciting sprouting of nociceptive nerves without affecting their regeneration. The sprouting was prevented by anti-NGF treatment, implicating endogenous NGF. We detected no Neotrofin-induced increases in cutaneous NGF levels or in retrograde NGF transport. In contrast, both NGF and phosphorylation of trkA increased significantly in DRGs, with a marginal appearance of phosphorylated trkA in axons. We conclude that the DRG effects of Neotrofin are responsible for its induction of sprouting. Neotrofin also induced a striking phosphorylation of axonal erk 1 and 2, which was, however, unaffected by anti-NGF treatment. We suggest that this NGF-independent MAP kinase activation is involved in nonsprouting functions of Neotrofin such as neuroprotection. Unlike injected NGF, Neotrofin did not induce hyperalgesia, supporting its candidacy as a treatment for peripheral neuropathies like those induced by diabetes and anticancer chemotherapy.