Expression of sphingosine 1-phosphate receptors in the rat dorsal root ganglia and defined single isolated sensory neurons.

Previously, we demonstrated that sphingosine 1-phosphate (S1P) increased the excitability of small-diameter sensory neurons, in part, through activation of S1P receptor 1 (S1PR(1)), suggesting that other S1PRs can modulate neuronal excitability. Therefore, studies were undertaken to establish the expression profiles of S1PRs in the intact dorsal root ganglion (DRG) and in defined single isolated sensory neurons. To determine mRNA expression of S1PRs in the DRG, SYBR green quantitative PCR (qPCR) was used. To determine the expression of S1PR mRNAs in single neurons of defined diameters, a preamplification protocol utilizing Taqman primer and probes was used to enhance the sensitivity of detection. The preamplification protocol also permitted detection of mRNA for two hallmark neuronal receptor/ion channels, TRPV1 and P(2)X(3). Expression profiles of S1PR mRNA isolated from lung and brain were used as positive control tissues. In the intact DRG, the order of expression of S1PRs was S1PR(3)>>R(1)≈R(2)>R(5)≈R(4). In the single neurons, the expression of S1PRs was quite variable with some neurons expressing all five subtypes, whereas some expressing only one subtype. In contrast to the DRG, S1PR(1) was the highest expressing subtype in 10 of the 18 small-, medium-, and large-diameter sensory neurons. S1PR(1) was the second highest expressor in -50% of those remaining neurons. Overall, in the single neurons, the order of expression was S1PR(1)>>R(3)≈R(5)>R(4)>R(2). The results obtained from the single defined neurons are consistent with our previous findings wherein S1PR(1) plays a prominent but not exclusive role in the enhancement of neuronal excitability.

Nerve growth factor enhances the excitability of rat sensory neurons through activation of the atypical protein kinase C isoform, PKMζ.

Our previous work showed that nerve growth factor (NGF) increased the excitability of small-diameter capsaicin-sensitive sensory neurons by activating the p75 neurotrophin receptor and releasing sphingolipid-derived second messengers. Whole cell patch-clamp recordings were used to establish the signaling pathways whereby NGF augments action potential (AP) firing (i.e., sensitization). Inhibition of MEK1/2 (PD-98059), PLC (U-73122, neomycin), or conventional/novel isoforms of PKC (bisindolylmaleimide I) had no effect on the sensitization produced by NGF. Pretreatment with a membrane-permeable, myristoylated pseudosubstrate inhibitor of atypical PKCs (aPKCs: PKMζ, PKCζ, and PKCλ/ι) blocked the NGF-induced increase in AP firing. Inhibitors of phosphatidylinositol 3-kinase (PI3K) also blocked the sensitization produced by NGF. Isolated sensory neurons were also treated with small interfering RNA (siRNA) targeted to PKCζ. Both Western blots and quantitative real-time PCR established that PKMζ, but neither full-length PKCζ nor PKCλ/ι, was significantly reduced after siRNA exposure. Treatment with these labeled siRNA prevented the NGF-induced enhancement of excitability. Furthermore, consistent with the high degree of catalytic homology for aPKCs, internal perfusion with active recombinant PKCζ or PKCι augmented excitability, recapitulating the sensitization produced by NGF. Internal perfusion with recombinant PKCζ suppressed the total potassium current and enhanced the tetrodotoxin-resistant sodium current. Pretreatment with the myristoylated pseudosubstrate inhibitor blocked the increased excitability produced by ceramide or internal perfusion with recombinant PKCζ. These results demonstrate that NGF leads to the activation of PKMζ that ultimately enhances the capacity of small-diameter capsaicin-sensitive sensory neurons to fire APs through a PI3K-dependent signaling cascade.

Pharmacological characterization of a new class of nonpeptide neurokinin A antagonists that demonstrate species selectivity.

We examined the pharmacology of ZM253,270 and two representative examples of the pyrrolopyrimidines, a new class of nonpeptide, NK-2 receptor (NK-2R) antagonists. ZM253,270 competitively inhibited [3H]NKA binding to native or cloned NK-2R from hamster urinary bladder (Ki = 2 nM), but was a weaker (48-fold) inhibitor of [3H]NKA binding to cloned human NK-2R. A similar species selectivity was observed with less potent analogs of ZM253,270. The pyrrolopyrimidines demonstrated only marginal inhibition of [3H]SP binding to NK-1R in guinea pig lung membranes (Ki > 2 microM). In hamster trachea, ZM253,270 competitively antagonized the contractile response evoked by neurokinin A (NKA, -logKB = 7.5). In human bronchus, ZM253,270 was about 90-fold less potent as a competitive antagonist of NKA. The data from ligand binding assays in cloned receptors combined with functional receptor assays in airway smooth muscles, demonstrate that the nonpeptide antagonist ZM253,270 is selective for the NK2 receptor species that are prevalent in hamster, compared with those found in human tissues.

Pharmacological examination of receptors mediating contractile responses to tachykinins in airways isolated from human, guinea pig and hamster.

The abilities of agonists selective for neurokinin (NK)-1 (Ac-[Arg6,Sar9,Met(O2)11]-SP6-11, ASMSP), NK-2 ([beta-Ala8]-NKA4-10) and NK-3 ([Asp5,6,MePhe8]-SP5-11, senktide analog) receptors to contract human bronchus and guinea pig and hamster trachea were studied. The antagonism of these responses by selective antagonists was also examined. In the human bronchus and hamster trachea, [beta-Ala8]-NKA4-10 was the most potent agonist, whereas ASMSP and senktide analog failed to elicit contractions greater than 50% of the maximum response even at concentrations reaching 1 to 3 x 10(-4) M. By contrast, both ASMSP and [beta-Ala8]-NKA4-10 were potent contractile agonists in guinea pig trachea. In all tissues, the selective NK-1 receptor antagonist (2S,3S)-cis-2-(diphenylmethyl)-N-[(2-methoxyphenyl)-methyl]-1-a zab icyclo- [2.2.2]octan-3-amine (CP 96,345) was without effect on contractile responses to [beta-Ala8]-NKA4-10. Blockade by CP 96,345 of responses to ASMSP was, however, observed in the guinea pig trachea, but not in human bronchus or hamster trachea. Responses to ASMSP in human bronchus and hamster trachea were inhibited by NK-2 antagonists, whereas these compounds had little effect on responses to ASMSP in guinea pig trachea. In all tissue types, responses to senktide analog were inhibited by NK-2 antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)