Cross-talk regulation between cyclic AMP production and phosphoinositide hydrolysis induced by prostaglandin E2 in osteoblast-like cells.

In cloned osteoblast-like MC3T3-E1 cells, PGE2 stimulated both cAMP accumulation and the formation of inositol trisphosphate (IP3) dose dependently. The cAMP accumulation showed the peak value at 5 min and decreased thereafter, whereas the IP3 formation reached a plateau almost within 10 min and sustained it up to 30 min. The effect of PGE2 on cAMP accumulation (EC50 was 80 nM) was more potent than that on IP3 formation (EC50 was 0.8 microM). 12-O-Tetradecanoyl-phorbol-13-acetate (TPA), a protein kinase C (PKC)-activating phorbol ester, reduced the PGE2-induced cAMP accumulation, whereas 4 alpha-phorbol 12,13-didecanoate, a PKC-nonactivating phorbol ester, had little effect on the cAMP accumulation. 1-Oleoyl-2-acetyl-glycerol, a specific activator for PKC, inhibited PGE2-induced cAMP accumulation. TPA had little effect on cAMP accumulation induced by forskolin or NaF, a GTP-binding protein activator. So, the effect of TPA is presumed to be exerted at the point between the PGE2 receptor and Gs. On the other hand, forskolin and dibutyryl cAMP had little effect on the IP3 formation stimulated by PGE2. H-7, a PKC inhibitor, enhanced the PGE2-induced cAMP accumulation in comparison with HA1004, a control for H-7. Our data suggest that PGE2 regulates cAMP production through self-induced activation of PKC. These results strongly suggest that there is an autoregulatory mechanism in PGE2 signaling, and PGE2 modulates osteoblast functions through a cross-talk interaction between cAMP production and phosphoinositide hydrolysis in osteoblast-like cells.     

Selective agonists for receptors of substance P and related neurokinins

Neurokinins and their receptors are a complex system consisting of at least three endogenous agents--substance P (SP), neurokinin A (NKA), and neurokinin B (NKB)--and their corresponding receptor types, respectively, NK-1, NK-2, and NK-3. Investigations on receptors have been made using sensitive and fairly selective pharmacological preparations (the dog carotid artery for the NK-1, the rabbit pulmonary artery devoid of endothelium for the NK-2, and the rat portal vein for the NK-3 receptor), and some natural peptides of mammalian and nonmammalian origin. Because of the nonselectivity of the natural peptides, analogues of the neurokinins have been found that act on one receptor only and show therefore high selectivity. The selective agonists [Sar9,Met(O2)11]SP, [Nle10]NKA (4-10), and [MePhe7]-NKB have been used successfully for (a) characterizing the three neurokinin receptors, (b) identifying isolated organs whose responses to neurokinins depend on the activation of a single (monoreceptor systems) or of more than one (multireceptor systems) receptor, and (c) elucidating some of the physiological function of the three receptor types. It is suggested that NK-1 mediate peripheral vasodilatation and exocrine secretions, NK-2 stimulate bronchial muscles and facilitate the release of catecholamines, and NK-3 promote the release of acetylcholine in peripheral organs.     

PGE1 and PGE2 modify platelet function through different prostanoid receptors

There is evidence that the overall effects of prostaglandin E(2) (PGE(2)) on human platelet function are the consequence of a balance between promotory effects of PGE(2) acting at the EP3 receptor and inhibitory effects acting at the EP4 receptor, with no role for the IP receptor. Another prostaglandin that has been reported to affect platelet function is prostaglandin E(1) (PGE(1)), however the receptors that mediate its actions on platelet function have not been fully defined. Here we have used measurements of platelet aggregation and P-selectin expression induced by the thromboxane A(2) mimetic U46619 to compare the effects of PGE(1) and PGE(2) on platelet function. Their effects on vasodilator-stimulated phosphoprotein (VASP) phosphorylation, as a marker of cAMP, were also determined. We also investigated the ability of the selective prostanoid receptor antagonists CAY10441 (IP antagonist), DG-041 (EP3 antagonist) and ONO-AE3-208 (EP4 antagonist) to modify the effects of the prostaglandins on platelet function. The results obtained confirm that PGE(2) interacts with EP3 and EP4 receptors, but not IP receptors. In contrast PGE(1) interacts with EP3 and IP receptors, but not EP4 receptors. In both cases the overall effects on platelet function reflect the balance between promotory and inhibitory effects at receptors that have opposite effects on adenylate cyclase.     

Neurokinin receptors antagonists: old and new.

Four neurokinin antagonists of different size have been used to counteract the myotropic effects of substance P, neurokinin A and neurokinin B in isolated organs containing a single receptor type (monoreceptor systems). These are: the dog carotid artery, the rabbit jugular and cava veins and the guinea pig ileum (NK-1), the rabbit pulmonary artery (NK-2) and the rat portal vein (NK-3). Undeca and octapeptides containing 2 D-Trp residues in their sequences were slightly more active on the NK-1, than on the NK-2 and NK-3 receptors and showed little selectivity. In contrast, compound AcThr-D.Trp(For)-Phe.NMe Bz was found to be as good an antagonist as the larger compounds and showed some selectivity for the NK-1 receptors. When tested against kinins or angiotensin, all compounds were found to be inactive, suggesting that they are specific for neurokinins. The present results show that NK-1 receptor antagonism can be obtained with compounds of different size, including tripeptides and nonpeptides.     

Regulation of epithelial transport in the jejunal mucosa of the guinea pig by neurokinins

This study characterizes the actions of the neurokinins and calcitonin-gene related peptide (CGRP) on electrolyte transport across the mucosa of the guinea pig jejunum in vitro in a modified Ussing chamber. By following changes in short circuit current (Isc) induced by substance P (SP) and neurokinins A & B (NKA & NKB) in the presence and absence of tetrodotoxin (TTX) and atropine, it was established that two distinct neurokinin receptors are involved in the regulation of electrolyte transport. NKA preferentially activates a neuronal receptor since the actions of this neurokinin were inhibited by both TTX and atropine. SP, whose actions were reduced to a lesser extent by TTX and atropine, is considered to activate preferentially a receptor on the epithelial cells. The third neurokinin, NKB, appears to act non-selectively on both the neuronal and epithelial receptors. CGRP, which per se did not affect Isc, markedly potentiated the increases in Isc induced by SP and NKB, and thus acts synergistically with the epithelial neurokinin receptor. These results suggest that two distinct neurokinin receptors (the NK-1 and the NK-2) regulate epithelial transport in the jejunal mucosa of the guinea pig, and furthermore indicate that at least one of the peptides found in enteric nerves (i.e. CGRP) modulates the actions of neurokinins on epithelial cells.     

Endogenous Gs-coupled receptors in smooth muscle exhibit differential susceptibility to GRK2/3-mediated desensitization

Although G protein-coupled receptor (GPCR) kinases (GRKs) have been shown to mediate desensitization of numerous GPCRs in studies using cellular expression systems, their function under physiological conditions is less well understood. In the current study, we employed various strategies to assess the effect of inhibiting endogenous GRK2/3 on signaling and function of endogenously expressed G s-coupled receptors in human airway smooth muscle (ASM) cells. GRK2/3 inhibition by expression of a Gbetagamma sequestrant, a GRK2/3 dominant-negative mutant, or siRNA-mediated knockdown increased intracellular cAMP accumulation mediated via beta-agonist stimulation of the beta-2-adrenergic receptor (beta 2AR). Conversely, neither 5'-( N-ethylcarboxamido)-adenosine (NECA; activating the A2b adenosine receptor) nor prostaglandin E2 (PGE 2; activating EP2 or EP4 receptors)-stimulated cAMP was significantly increased by GRK2/3 inhibition. Selective knockdown using siRNA suggested the majority of PGE 2-stimulated cAMP in ASM was mediated by the EP2 receptor. Although a minor role for EP3 receptors in influencing PGE 2-mediated cAMP was determined, the GRK2/3-resistant nature of EP2 receptor signaling in ASM was confirmed using the EP2-selective agonist butaprost. Somewhat surprisingly, GRK2/3 inhibition did not augment the inhibitory effect of the beta-agonist on mitogen-stimulated increases in ASM growth. These findings demonstrate that with respect to G s-coupled receptors in ASM, GRK2/3 selectively attenuates beta 2AR signaling, yet relief of GRK2/3-dependent beta 2AR desensitization does not influence at least one important physiological function of the receptor.     

Pharmacology of neurokinin receptors.

The neurokinins are a group of naturally occurring peptides with the common C-terminal sequence Phe-X-Gly-Leu-Met.NH2. They include substance P (SP), neurokinin A (NKA), and neurokinin B (NKB). SP and NKA are coded on the same gene, the PPT-A, while NKB is coded on a separate gene, the PPT-B. Neurokinins are present in the central nervous system and in peripheral organs where they exert various actions. They act on three receptors--NK-1, NK-2, and NK-3--characterized through pharmacological, biochemical, and histochemical studies. Selective agonists for each neurokinin receptor were developed and evaluated on isolated smooth muscle preparations containing only one neurokinin receptor type. All three neurokinin receptors were cloned and expressed in Xenopus oocytes. Relative affinities of those receptors to neurokinins are the same as in their respective smooth muscle preparation. Finally, the mechanism of action of SP on histamine release from rat peritoneal mast cell has been studied and a direct activation of G proteins by peptides with basic amino acids is proposed as a working hypothesis.     

Heterogeneity of NK-2 tachykinin receptors in hamster and rabbit smooth muscles.

The possible existence of NK-2 receptor subtypes in peripheral smooth muscle preparations from rabbit and hamster was investigated by studying the effect of neurokinin A, the selective NK-2 receptor agonist [beta Ala8] neurokinin A (4-10), the selective NK-2 tachykinin receptor antagonists, MEN 10,376, L 659,877 and R 396, and the pseudopeptide derivative of neurokinin A (4-10), MDL 28,564. All experiments were performed in the presence of peptidase inhibitors (captopril, bestatin and thiorphan, 1 microM each). Both neurokinin A and [beta Ala8] neurokinin A (4-10) produced concentration-dependent contractions of the rabbit isolated bronchus and hamster isolated stomach and colon, as well as enhancement of the nerve-mediated twitches of rabbit isolated vas deferens (pars prostatica). MEN 10,376, L 659,877 and R 396 antagonized the effect of the NK-2 receptor selective agonist in all four tissues under study, although marked differences in antagonist potency were evident for the three antagonists. Thus MEN 10,376 was distinctly more potent (about 100 times) in rabbit than in hamster preparations while L 659,877 and R 396 were more potent in hamster than rabbit preparations. MDL 28,564 showed a distinct agonist character in rabbit preparations while it was virtually inactive in hamster preparations, where it antagonized the effect of the NK-2 receptor selective agonist.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two Possibly Distinct Prostaglandin E1 Receptors in N1E-115 Clone: One Mediating Inositol Trisphosphate Formation, Cyclic GMP Formation, and Intracellular Calcium Mobilization and the Other Mediating Cyclic AMP Formation

Prostaglandin E1 (PGE1)-mediated transmembrane signal control systems were investigated in intact murine neuroblastoma cells (clone N1E-115). PGE1 increased intracellular levels of total inositol phosphates (IP), cyclic GMP, cyclic AMP, and calcium ([Ca2+]i). PGE1 transiently increased inositol 1,4,5-trisphosphate formation, peaking at 20 s. There was more than a 10-fold difference between the ED50 for PGE1 at cyclic AMP formation (70 nM) and its ED50 values at IP accumulation (1 microM), cyclic GMP formation (2 microM), and [Ca2+]i increase (5 microM). PGE1-mediated IP accumulation, cyclic GMP formation, and [Ca2+]i increase depended on both the concentration of PGE1 and extracellular calcium ions. PGE1 had more potent intrinsic activity in cyclic AMP formation, IP accumulation, and cyclic GMP formation than did PGE2, PGF2 alpha, or PGD2. A protein kinase C activator, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, had opposite effects on PGE1-mediated IP release and cyclic GMP formation (inhibitory) and cyclic AMP formation (stimulatory). These data suggest that there may be subtypes of the PGE1 receptor in this clone: a high-affinity receptor mediating cyclic AMP formation, and a low-affinity receptor mediating IP accumulation, cyclic GMP formation, and intracellular calcium mobilization.     

Tachykinin receptors and the airways.

The tachykinins, substance P, neurokinin A and neurokinin B, belong to a structural family of peptides. In mammalian airways, substance P and neurokinin A are colocalized to afferent C-fibres. Substance P-containing fibres are close to bronchial epithelium, smooth muscle, mucus glands and blood vessels. Sensory neuropeptides may be released locally, possibly as a result of a local reflex, and produce bronchial obstruction through activation of specific receptors on these various tissues. Three types of tachykinin receptors, namely NK-1, NK-2 and NK-3 receptors, have been characterized by preferential activation by substance P, neurokinin A and neurokinin B respectively. NK-1 and NK-2 receptors were recently cloned. The determination of receptor types involved in the effects of tachykinins in the airways has been done with synthetic agonists and antagonists binding specifically to NK-1, NK-2 and NK-3 receptors. Although the existence of species differences, the conclusion that bronchial smooth muscle contraction is mainly related to activation of NK-2 receptors on bronchial smooth muscle cell has been drawn. The hypothesis of a NK-2 receptor subclassification has been proposed with NK-2A receptor subtype in the guinea-pig airways. Other effects in the airways are related to stimulation of NK-1 receptors on mucus cells, vessels, epithelium and inflammatory cells. A non-receptor-mediated mechanism is also involved in the effect of substance P on inflammatory cells and mast cells.     

Conformation and orientation of regulatory peptides on lipid membranes. Key to the molecular mechanism of receptor selection

The reaction of regulatory peptides with their membrane-bound receptors often occurs via a membrane-associated state of the peptide. From infrared studies on thin lipid films, we have shown that several ligands of the opioid kappa receptor and the neurokinin NK-1 receptor insert their message segments as an alpha-helix, more or less perpendicularly, into the membrane. The binding parameters for these membrane-associated states were determined from the capacitance minimization potential of lipid bilayers. A theory has been developed to account for the observed binding constants and the preferred conformation and orientation of these peptides. In contrast to the kappa and NK-1 receptors, ligands of the opioid mu and delta, and the neurokinin NK-2 and NK-3 receptors, are predicted not to form the inserted alpha-helical structure. A selection between the mu and delta (or NK-2 and NK-3) receptors appears to be made on the basis of an electrostatic gradient near the membrane surface. The molecular mechanism of receptor selection thus appears to be based to a large extent on the membrane-induced compartmentalization of ligands for the different receptors.     

The cellular actions of vasopressin on corticotrophs of the anterior pituitary: resistance to glucocorticoid action

The cellular actions of vasopressin (AVP) in the anterior pituitary were investigated. HPLC analysis of [3H]inositol-labeled cells indicated that AVP stimulated a rapid increase in inositol-1,4,5 trisphosphate (IP3), inositol-1,4 bisphosphate, and inositol-4 monophosphate levels. While CRF had no effect on basal IP3 levels, it blocked their stimulation by AVP. CRF-stimulated ACTH secretion and cAMP accumulation were potentiated by AVP. AFter dexamethasone (DEX) treatment (20 nM, 18 h), CRF-dependent ACTH secretion and cAMP accumulation were attenuated but AVP was still able to potentiate both of these actions of CRF suggesting that cellular actions of AVP may be resistant to DEX effects. Therefore, [3H]AVP binding was determined in control and DEX-treated cells. Pretreatment with DEX had no effect on either AVP receptor affinity or on the number of available binding sites. Consistently, stimulation of IP3 production by AVP in DEX-treated cells was comparable to that of control cells. Protein kinase C activators such as 12-O-tetradecanoyl-phorbol-13-acetate and dioctanoylglycerol were either near additive with CRF or also potentiated the action of CRF on ACTH secretion, respectively, even after DEX pretreatment. These results indicate that, in the anterior pituitary, distinct intracellular signaling pathways mediate the actions of CRF and AVP; cAMP mediates CRF actions and IP3/protein kinase C mediate the effects of AVP. Neuromodulation of ACTH secretion by dual effector mechanisms which exhibit a complex mode of interaction and only one of which is negatively influenced by glucocorticoids, provides these cells a mechanisms by which appropriate responses can be elicited under various physiological states.     

Prostanoid EP4 receptor is involved in suppression of 3T3-L1 adipocyte differentiation

Prostaglandins (PGs) have been shown to play various roles in adipogenesis. In this study, we investigated on which PGE receptor subtypes are involved in the inhibition of 3T3-L1 preadipocyte differentiation. The triglyceride content of cells, used as an index of differentiation, was decreased when PGE(2), the FP-agonist fluprostenol or dibutyryl cAMP, was exogenously added to differentiation cocktails. 3T3-L1 preadipocyte cells express mRNAs for the prostanoid EP4, FP, and IP receptors. PGE(2) and the EP4 agonist AE1-329 increased cAMP levels in preadipocytes in a dose-dependent manner. AE1-329 suppressed the expression induction of differentiation marker genes such as resistin and peroxisome proliferator-activated receptor-gamma. The inhibitory effect of PGE(2) but not that of fluprostenol was reversed by the addition of the EP4 antagonist AE3-208. AE3-208 mimicked the differentiation-promoting effects of indomethacin. These results suggest that the EP4 receptor mediates the suppressive action of PGE(2) in 3T3-L1 adipocyte differentiation.     

Dimerization of the human receptors for prostacyclin and thromboxane facilitates thromboxane receptor-mediated cAMP generation

Prostacyclin (PGI(2)) and thromboxane (TxA(2)) are biological opposites; PGI(2), a vasodilator and inhibitor of platelet aggregation, limits the deleterious actions of TxA(2), a vasoconstrictor and platelet activator. The molecular mechanisms involved in the counterregulation of PGI(2)/TxA(2) signaling are unclear. We examined the interaction of the receptors for PGI(2) (IP) and TxA(2) (TPalpha). IP-induced cAMP and TP-induced inositol phosphate generation were unaltered when the receptors were co-expressed in HEK 293 cells (IP/TPalpha-HEK). TP-cAMP generation, in response to TP agonists or a TP-dependent isoprostane, iPE(2)III, was evident in IP/TPalpha-HEK and in aortic smooth muscle cells, but not in cells expressing either receptor alone, or in IP-deficient aortic smooth muscle cells. Augmentation of TP-induced cAMP generation, with the IP agonist cicaprost, was ablated in IP-deficient cells and was independent of direct IP signaling. IP/TPalpha heterodimers were formed constitutively when the receptors were co-expressed, with no overt changes in ligand binding to the individual receptor sites. However, despite inefficient binding of iPE(2)III to either the IP or TPalpha, expressed alone or in combination, robust cAMP generation was evident in IP/TPalpha-HEK, suggesting the formation of an alternative receptor site. Thus, IP/TPalpha dimerization was coincident with TP-cAMP generation, promoting a "PGI(2)-like" cellular response to TP activation. This represents a previously unknown mechanism by which IP may limit the cellular effects of TP.     

Attenuation of reflex pressor and ventilatory responses to static contraction by an NK-1 receptor antagonist.

The chemical messengers released onto second-order dorsal horn neurons from the spinal terminals of contraction-activated group III and IV muscle afferents have not been identified. One candidate is the tachykinin substance P. Related to substance P are two other tachykinins, neurokinin A (NKA) and neurokinin B (NKB), which, like substance P, have been isolated in the dorsal horn of the spinal cord and have receptors there. Whether NKA or NKB plays a transmitter/modulator role in the spinal processing of the exercise pressor reflex is unknown. Therefore, we tested the following hypotheses. After the intrathecal injection of a highly selective NK-1 (substance P) receptor antagonist onto the lumbosacral spinal cord, the reflex pressor and ventilatory responses to static muscular contraction will be attenuated. Likewise, after the intrathecal injection either of an NK-2 (NKA) receptor antagonist or an NK-3 (NKB) receptor antagonist onto the lumbrosacral spinal cord, the reflex pressor and ventilatory responses to static contraction will be attenuated. We found that, 10 min after the intrathecal injection of 100 micrograms of the NK-1 receptor antagonist, the pressor and ventilatory responses to contraction were significantly (P < 0.05) attenuated. Mean arterial pressure was attenuated by 13 +/- 3 mmHg (48%) and minute volume of ventilation by 120 +/- 38 ml/min (34%). The cardiovascular and ventilatory responses to contraction before either 100 micrograms of the NK-2 receptor antagonist or 100 micrograms of the NK-3 receptor antagonist were not different (P > 0.05) from those after the NK-2 or the NK-3 receptor antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of differentiation in v-Ha-ras-transformed MDCK cells by prostaglandin E2 and 8-bromo-cyclic AMP is associated with a decrease in steady-state level of inositol 1,4,5-trisphosphate.

We used Ha-ras-transformed Madin-Darby canine kidney (MDCK) cells as a model to study possible signal transduction mechanisms underlying the induction of glucagon responsiveness by the differentiation inducers prostaglandin E2 (PGE2) and 8-bromo-cyclic (8-Br-cAMP) AMP and the inhibition of induction by phorbol ester or a serum factor. The steady-state level of inositol 1,4,5-trisphosphate (IP3) was higher in Ha-ras-transformed MDCK cells than in parental MDCK cells. In contrast, the steady-state level of intracellular cAMP of transformed cells was similar to that of normal cells. PGE2 and 8-Br-cAMP increased cAMP content but decreased IP3 levels in a concentration-dependent fashion after 5 days of treatment. We examined the time course for effects of PGE2 and 8-Br-cAMP and found that there was a lag period of 8 to 16 h between elevation of cAMP after the addition of 8-Br-cAMP or PGE2 and the decrease of IP3 levels. Another lag period of 2 days existed before the induction of differentiation. Both the reduction of IP3 levels and the induction of glucagon responsiveness were blocked by phorbol-12-myristate-13-acetate or serum, suggesting that a decrease in the IP3 level might be causally involved in induction of differentiation in transformed MDCK cells. However, induction of differentiation was not due to changes in the expression or guanine nucleotide-binding properties of p21 protein. It is likely that cAMP has a direct regulatory effect on the phospholipid signaling pathway. We conclude that perturbation of the inositol phosphate signaling pathway may be responsible for the induction of differentiation by PGE2 and 8-Br-cAMP in transformed MDCK cells.     

Involvement of substance P and the NK-1 receptor in cancer progression

Many data suggest the deep involvement of the substance P (SP)/neurokinin (NK)-1 receptor system in cancer: (1) Tumor cells express SP, NK-1 receptors and mRNA for the tachykinin NK-1 receptor; (2) Several isoforms of the NK-1 receptor are expressed in tumor cells; (3) the NK-1 receptor is involved in the viability of tumor cells; (4) NK-1 receptors are overexpressed in tumor cells in comparison with normal ones and malignant tissues express more NK-1 receptors than benign tissues; (5) Tumor cells expressing the most malignant phenotypes show an increased percentage of NK-1 receptor expression; (6) The expression of preprotachykinin A is increased in tumor cells in comparison with the levels found in normal cells; (7) SP induces the proliferation and migration of tumor cells and stimulates angiogenesis by increasing the proliferation of endothelial cells; (8) NK-1 receptor antagonists elicit the inhibition of tumor cell growth; (9) The specific antitumor action of NK-1 receptor antagonists on tumor cells occurs through the NK-1 receptor; (10) Tumor cell death is due to apoptosis; (11) NK-1 receptor antagonists inhibit the migration of tumor cells and neoangiogenesis. The NK-1 receptor is a therapeutic target in cancer and NK-1 receptor antagonists could be considered as broad-spectrum antitumor drugs for the treatment of cancer. It seems that a common mechanism for cancer cell proliferation mediated by SP and the NK-1 receptor is triggered, as well as a common mechanism exerted by NK-1 receptor antagonists on tumor cells, i.e. apoptosis.     

Substance P stimulates cyclooxygenase-2 and prostaglandin E2 expression through JAK-STAT activation in human colonic epithelial cells

Substance P (SP) via its neurokinin-1 receptor (NK-1R) regulates several gastrointestinal functions. We previously reported that NK-1R-mediated chloride secretion in the colon involves formation of PG. PGE2 biosynthesis is controlled by cyclooxygenase-1 (COX-1) and COX-2, whose induction involves the STATs. In this study, we examined whether SP stimulates PGE2 production and COX-2 expression in human nontransformed NCM460 colonocytes stably transfected with the human NK-1R (NCM460-NK-1R cells) and identified the pathways involved in this response. SP exposure time and dose dependently induced an early (1-min) phosphorylation of JAK2, STAT3, and STAT5, followed by COX-2 expression and PGE2 production by 2 h. Pharmacologic experiments showed that PGE2 production is dependent on newly synthesized COX-2, but COX-1 protein. Inhibition of protein kinase Ctheta (PKCtheta), but not PKCepsilon and PKCdelta, significantly reduced SP-induced COX-2 up-regulation, and JAK2, STAT3, and STAT5 phosphorylation. Pharmacological blockade of JAK inhibited SP-induced JAK2, STAT3, and STAT5 phosphorylation; COX-2 expression; and PGE2 production. Transient transfection with JAK2 short-interferring RNA reduced COX-2 promoter activity and JAK2 phosphorylation, while RNA interference of STAT isoforms showed that STAT5 predominantly mediates SP-induced COX-2 promoter activity. Site-directed mutation of STAT binding sites on the COX-2 promoter completely abolished COX-2 promoter activity. Lastly, COX-2 expression was elevated in colon of mice during experimental colitis, and this effect was normalized by administration of the NK-1R antagonist CJ-12,255. Our results demonstrate that SP stimulates COX-2 expression and PGE2 production in human colonocytes via activation of the JAK2-STAT3/5 pathway.     

Membrane-assisted molecular mechanism of neurokinin receptor subtype selection.

Based on the observed membrane structures of substance P, physalaemin, and eledoisin, preferred conformations, orientations and accumulations of 13 mammalian neurokinins and non-mammalian tachykinins were estimated and compared with pharmacologic and selective binding data taken from the literature. Principal site affinities and relative affinities supported the view that neurokinins bind to three principal mammalian sites: the NK-1 (preferring substance P), the NK-2 (preferring neurokinin A), and the NK-3 site (preferring neurokinin B). Strong hydrophobic membrane interaction of the C-terminal message segment as a perpendicularly oriented alpha-helical domain correlated with NK-1 selection. Electrostatic accumulation of the peptide at the anionic fixed charge layer of the membrane without hydrophobic interaction through a helix correlated with NK-2 preference. Electrostatic repulsion by the anionic fixed charge layer correlated with NK-3 selection. Thus, neurokinin receptor selection is guided by the same principles as opioid receptor selection. Membrane catalysis of specific agonist--receptor interactions may prove to be a quite general phenomenon, and the membrane structure of a peptide more important for its structure--activity relationship than its crystal structure or its mixture of conformers in solution or in vacuo.