Cannabinoid (CB1) receptor activation inhibits trigeminovascular neurons

Migraine is a common and disabling neurological disorder that involves activation or the perception of activation of the trigeminovascular system. Cannabinoid (CB) receptors are present in brain and have been suggested to be antinociceptive. Here we determined the effect of cannabinoid receptor activation on neurons with trigeminovascular nociceptive input in the rat. Neurons in the trigeminocervical complex (TCC) were studied using extracellular electrophysiological techniques. Responses to both dural electrical stimulation and cutaneous facial receptive field activation of the ophthalmic division of the trigeminal nerve and the effect of cannabinoid agonists and antagonists were studied. Nonselective CB receptor activation with R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2, 3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl) (WIN55,212; 1 mg kg(-1)) inhibited neuronal responses to A-(by 52%) and C-fiber (by 44%) afferents, an effect blocked by the CB(1) receptor antagonist SR141716 [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; 3 mg kg(-1)] but not the CB2 receptor antagonist AM630 (6-iodopravadoline; 3 mg kg(-1)). Anandamide (10 mg kg(-1)) was able to inhibit both A- and C-fiber-elicited TCC firing, only after transient receptor potential vanilloid 1 receptor inhibition. Activation of cannabinoid receptors had no effect on cutaneous receptive fields when recorded from TCC neurons. The data show that manipulation of CB1 receptors can affect the responses of trigeminal neurons with A- and C-fiber inputs from the dura mater. This may be a direct effect on neurons in the TCC itself or an effect in discrete areas of the brain that innervate these neurons. The data suggest that CB receptors may have therapeutic potential in migraine, cluster headache, or other primary headaches, although the potential hazards of psychoactive side effects that accompany cannabinoid treatments may be complex to overcome.     

Elucidation of the vasoactive intestinal peptide pharmacophore for VPAC(2) receptors in human and rat and comparison to the pharmacophore for VPAC(1) receptors

Vasoactive intestinal peptide (VIP) functions as a neurotransmitter involved in a number of physiological and pathological conditions. The actions of VIP are mediated through VPAC(1) and VPAC(2). In contrast to VPAC(1), which has been extensively studied, little is known about the pharmacology of VPAC(2). In this study we investigated the VIP pharmacophore for VPAC(2) by using alanine and D-amino acid scanning. We found significant species differences, and the human VPAC(2) (hVPAC(2)) expressed in Chinese hamster ovary (CHO) cells, which have been used in previous studies, differed significantly from the native hVPAC(2) in Sup T(1) cells and hVPAC(2) expressed in PANC1 cells. There was a close agreement between binding affinities and potencies for VPAC(2) activation. The amino acids whose backbone or side chain orientations were most important for high affinity potency are Asp(3), Phe(6), Thr(7), Tyr(10), Arg(12), Tyr(22), and Leu(23), whereas the side chains of Ser(2), Asp(8), Asn(9), Gln(16), Val(19), Lys(20), Lys(21), Asn(24), and Ser(25) are not essential. Comparison of the VIP pharmacophore between hVPAC(1) and hVPAC(2) demonstrated that the side chains of Thr(7), Tyr(10), Thr(11), and Tyr(22) were much more critical for high affinity for the hVPAC(2) than the hVPAC(1). In contrast, the orientation of the side chain of Asn(24) was more important for high affinity for the hVPAC(1). This study shows that in assessing the pharmacophore of VIP analogs for the VPAC(2), important species differences need to be considered as well as the expression system used. These results of our study should be useful for designing VPAC subtype-selective analogs, simplified analogs, and possibly metabolically stable analogs.     

Molecular basis for agonist selectivity and activation of the orphan bombesin receptor subtype 3 receptor

The persistent environmental contaminant and immunotoxicant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), markedly suppresses humoral immune responses. We recently reported impaired down-regulation of paired box 5 (Pax5), a repressor of B cell differentiation and concomitant suppression of the IgM response by TCDD in the murine CH12.LX B cell line. The objectives of the current study were to determine the impact of TCDD treatment on molecular outcomes characteristic of terminal B cell differentiation and to assess the role that Pax5 isoforms plays in the suppression of B cell differentiation by TCDD. In this study, we show that the highly abundant full-length Pax5 isoform, Pax5a, and at least two additional modestly expressed Pax5 isoforms were expressed in CH12.LX and splenic B cells. In lipopolysaccharide (LPS)-activated B cells, all of the identified Pax5 isoforms were synchronously down-regulated, and in the presence of TCDD cotreatment they were abnormally and synchronously elevated, suggesting a common mechanism of regulation. Furthermore, B cell differentiation markers X-box protein-1 and major histocompatibility complex class II showed that the levels to which Pax5 was derepressed by TCDD were sufficient to impair B cell differentiation and immunoglobulin gene expression. Confirming the involvement of Pax5, ectopic expression of Pax5a in the LPS-activated CH12.LX cells closely mimicked the suppression of the IgM response by TCDD. In summary, our results demonstrate that Pax5a has a critical role in both the TCDD-mediated impairment of B cell differentiation and the suppression of the humoral immune response.     

Novel pyrazole cannabinoids: insights into CB(1) receptor recognition and activation

Synthesis of an antagonist, SR141716A, that selectively binds to brain cannabinoid (CB(1)) receptors without producing cannabimimetic activity in vivo, suggests that recognition and activation of cannabinoid receptors are separable events. In the present study, a series of SR141716A analogs were synthesized and were tested for CB(1) binding affinity and in a battery of in vivo tests, including hypomobility, antinociception, and hypothermia in mice. These analogs retained the central pyrazole structure of SR141716A with replacement of the 1-, 3-, 4-, and/or 5-substituents by alkyl side chains or other substituents known to impart potent agonist activity in traditional tricyclic cannabinoid compounds. Although none of the analogs alone produced the profile of cannabimimetic effects seen with full agonists, several of the 3-substituent analogs with higher binding affinities showed partial agonism for one or more measures. Cannabimimetic activity was most noted when the 3-substituent of SR141716A was replaced with an alkyl amide or ketone group. None of the 3-substituted analogs produced antagonist effects when tested in combination with 3 mg/kg Delta(9)-tetrahydrocannabinol (Delta(9)-THC). In contrast, antagonism of Delta(9)-THC's effects without accompanying agonist or partial agonist effects was observed with substitutions at positions 1, 4, and 5. These results suggest that the structural properties of 1- and 5-substituents are primarily responsible for the antagonist activity of SR141716A.     

Compensatory insulin receptor (IR) activation on inhibition of insulin-like growth factor-1 receptor (IGF-1R): rationale for cotargeting IGF-1R and IR in cancer

Insulin-like growth factor-1 receptor (IGF-1R) is a receptor tyrosine kinase (RTK) and critical activator of the phosphatidylinositol 3-kinase-AKT pathway. IGF-1R is required for oncogenic transformation and tumorigenesis. These observations have spurred anticancer drug discovery and development efforts for both biological and small-molecule IGF-1R inhibitors. The ability for one RTK to compensate for another to maintain tumor cell viability is emerging as a common resistance mechanism to antitumor agents targeting individual RTKs. As IGF-1R is structurally and functionally related to the insulin receptor (IR), we asked whether IR is tumorigenic and whether IR-AKT signaling contributes to resistance to IGF-1R inhibition. Both IGF-1R and IR(A) are tumorigenic in a mouse mammary tumor model. In human tumor cells coexpressing IGF-1R and IR, bidirectional cross talk was observed following either knockdown of IR expression or treatment with a selective anti-IGF-1R antibody, MAB391. MAB391 treatment resulted in a compensatory increase in phospho-IR, which was associated with resistance to inhibition of IRS1 and AKT. In contrast, treatment with OSI-906, a small-molecule dual inhibitor of IGF-1R/IR, resulted in enhanced reduction in phospho-IRS1/phospho-AKT relative to MAB391. Insulin or IGF-2 activated the IR-AKT pathway and decreased sensitivity to MAB391 but not to OSI-906. In tumor cells with an autocrine IGF-2 loop, both OSI-906 and an anti-IGF-2 antibody reduced phospho-IR/phospho-AKT, whereas MAB391 was ineffective. Finally, OSI-906 showed superior efficacy compared with MAB391 in human tumor xenograft models in which both IGF-1R and IR were phosphorylated. Collectively, these data indicate that cotargeting IGF-1R and IR may provide superior antitumor efficacy compared with targeting IGF-1R alone.     

Evaluation of the permeability of pancreaticogastric anastomoses (PGA) with dynamic magnetic resonance pancreatography after secretin stimulation (secretin MRCP)

Background: Because some investigators have reported the systematic occurrence of exocrine pancreatic insufficiency after pancreaticoduodenectomy with pancreaticogastric anastomosis (PGA), we assessed PGA patency after pancreaticoduodenectomy. Methods: Nineteen patients underwent pancreaticoduodenectomy, and their PGAs were studied prospectively with secretin magnetic resonance cholangiopancreatography (MRCP). After administration of negative bowel contrast agent, single-shot fast spin-echo T2-weighted dynamic MR pancreatograms were obtained before and every minute for 12 min after secretin injection. Morphologic features of the pancreatic parenchymal and pancreatic duct were monitored (diameter and winding aspect of the pancreatic duct, pancreatic thickness, direct visualization of the anastomotic site). PGA permeability was classified into four grades, from 0 (obstruction) to 3 (good permeability). Pancreatic function was assessed by fecal-1 elastase concentration, fasting blood glucose, and fasting serum insulin level. Results: MRCP grades were 0 in two patients, 1 in four, 2 in five, and 3 in eight. The anastomotic site was visualized in 10 patients. Pancreatic parenchymal atrophy was discovered in four patients. There were statistically significant relations between secretin MRCP permeability grade and fecal-1 elastase concentration (p < 0.03) and between secretin MRCP permeability grade and pancreatic atrophy (p < 0.005). In contrast, fecal-1 elastase concentration was lower than the normal value in all but one case. There was no statistically significant relation between fecal-1 elastase concentration and other morphologic data. Conclusion: Secretin MRCP may indicate PGA stenosis or dysfunction, but it is not the only factor suggesting exocrine pancreatic insufficiency. Thus the major role of PGA may be the preservation of long-term endocrine function.     

Elucidation of vasoactive intestinal peptide pharmacophore for VPAC(1) receptors in human, rat, and guinea pig

Vasoactive intestinal peptide (VIP) is a neurotransmitter involved in a number of pathological and physiological processes. VIP is rapidly degraded and simplified stable analogs are needed. VIP's action was extensively studied in rat and guinea pig. However, it is largely unknown whether its pharmacophore in these species resembles human. To address this issue we investigated the VIP pharmacophore for VPAC(1) (the predominant receptor subtype in cancers and widely distributed in normal tissues) by using alanine and D-amino acid scanning. Interaction with rat, guinea pig, and human VPAC(1) was assessed using transfected Chinese hamster ovary (CHO) and PANC1 cells and cells possessing native VPAC(1). Important species differences existed in the VIP pharmacophore. The human VPAC(1) expressed in CHO cells, which were used almost exclusively in previous studies, differed markedly from the native VPAC(1) in T47D cells. The most important amino acids for determining affinity are His(1), Asp(3), Phe(6), Arg(12), Arg(14), and Leu(23). Ser(2), Asp(8), Asn(9), Thr(11), Val(19), Asn(24), Ser(25), Leu(27), and Asn(28) are not essential for high-affinity interaction/activation. [Ala(2,8,9,11,19,24,25,27,28)]VIP, which contained 11 alanines, was synthesized and it was equipotent to VIP at VPAC(1) receptors in all species and was metabolically stable. Our results show in any design of simplified VIP analogs for VPAC(1) it will be important to consider species differences and it is essential to use transfected systems that reflect the native receptor's pharmacophore. Last, with our results a simplified, metabolically stable VIP analog was identified that should be useful as a prototype for design of selective agonists/antagonists that could be useful therapeutically.     

Inhibition of alpha(1)-adrenergic-mediated responses in rat ventricular myocytes by adenosine A(1) receptor activation: role of the K(ATP) channel

Both beta- and alpha(1)-adrenoceptors mediate the myocardial effects of catecholamines. It is well known that adenosine inhibits beta-dependent effects; however, whether alpha(1)-dependent responses can be similarly modulated is unclear. Accordingly, rat ventricular myocytes were exposed for 25 min to the alpha(1) agonist phenylephrine (2 microM, in the presence of 1 microM propranolol) in the absence or presence of adenosine (100 microM) or the A(1) receptor-selective agonist N(6)-cyclopentyladenosine (CPA, 1 microM). We also investigated the effects of K(ATP) blockade with glibenclamide (1 microM), the protein kinase C inhibitor bisindolylmaleimide (20 nM), and pertussis toxin (300 ng/ml), which uncouples G(i) protein/receptor interaction, and assessed whether effects of adenosine were mimicked by K(ATP) activation with either pinacidil or cromakalim (5 microM). Phenylephrine significantly increased cell shortening by 190% and the Ca(2+) transient by 24%, which was abolished by either adenosine or CPA, but not in the presence of the A(1) receptor-selective antagonist 8-cyclopentyl-1, 3-dipropylxanthine (1 microM), and was abolished by pertussis toxin. The effect of adenosine or CPA was reversed by glibenclamide and mimicked by either cromakalim or pinacidil. Bisindolylmaleimide was without effect. The A(2) or A(3) receptor agonists 2-(4-(2-carboxyethyl)phenylethylamino)-5'-N-ethylcarboxamidoade nos ine and N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (1 microM each), respectively, were without effect. Neither CPA nor adenosine modulated the effect of endothelin-1 (5 nM), which also acts via the phosphoinositide hydrolysis pathway. We conclude that adenosine selectively inhibits alpha(1)-adrenergic-mediated effects in rat ventricular myocytes through a G(i) protein-dependent mechanism involving A(1) receptor and K(ATP) activation. Our study further suggests that endogenous adenosine may modulate alpha(1)-mediated effects of catecholamines.     

Functional selectivity and biased receptor signaling

With the emergence of information describing functional selectivity and biased agonists and antagonists has come a lack of confidence in "one size fits all" assays for detection of agonism. Seven-transmembrane receptors are pleiotropic with respect to the signaling protein to which they couple in a cell, and many conformations of the receptor can be formed; this leads to systems where ligands can stabilize unique conformations that go on to selectively activate signaling pathways. Thus, such "biased" ligands can produce cell-specific agonism that may require targeted assays to detect and quantify. It also predicts that ligands can have many different efficacies for the many behaviors that the receptor can exhibit (referred to as "pluridimensional efficacy"), leading to a breakdown in the common classifications of agonist and antagonist. This all poses unique challenges to the pharmacologic nomenclature of drugs, the detection and optimization of new drugs, and the association of phenotypic clinical profiles with pharmacological properties of drugs.     

Effects of spinal alpha(2)-adrenoceptor and I(1)-imidazoline receptor activation on hindlimb movement induction in spinal cord-injured mice

A partial recovery of locomotor functions has been shown in spinal cord-transected (Tx) cats after regular treadmill training and repeated administration of clonidine, an alpha(2)-adrenoreceptor agonist. However, clonidine has generally failed to show prolocomotor effects in other models (e.g., rat or mudpuppy in vitro-isolated spinal cord preparations). The reasons for this discrepancy remain unclear, but they may suggest condition- or species-specific effects induced by clonidine. This study is aimed at examining both the acute (at 6 or 41 days post-Tx) and chronic effects of repeated (once a week for one month) clonidine administration (0.25-5.0 mg/kg i.p.) on hindlimb movement generation in Tx mice (thoracic segment9/10). Locomotor-like (LM) and nonlocomotor movements (NLM) were assessed both in open-field and treadmill conditions. The results show that clonidine consistently failed, in both conditions, to induce LM and NLM at all time points even though control experiments revealed hindlimb movements steadily induced by 8-hydroxy-2-(di-N-propylamino)-tetralin (8-OH-DPAT), a serotonin receptor agonist. In turn, clonidine acutely suppressed (I(1)-imidazoline receptor-mediated) the frequency of spontaneously occurring LM and NLM but apparently increased spinal excitability over time, because the frequency of spontaneous LM and NLM was significantly greater in clonidine-treated (before an injection) than vehicle-treated animals after repeated administration for a few weeks. The results clearly show that clonidine can not acutely induce hindlimb movements in untrained and otherwise nonstimulated (e.g., no tail or perineal pinching) Tx mice, although repeated administration may progressively facilitate the expression of spontaneous hindlimb movements.     

Adenosine A1 receptor activation mediates suppression of (-) bicuculline methiodide-induced seizures in rat prepiriform cortex

The protective effects of a series of stable adenosine analogs against generalized seizures initiated by focal injection of bicuculline methiodide into the rat prepiriform cortex (PPC) were studied by microinjection of these compounds into this brain area. The adenosine agonists, 5'-N-(ethyl)carboxamido-adenosine (NECA), cyclohexyladenosine, cyclopentyadenosine, 2-chloroadenosine and R- and S-phenylisopropyladenosine (R- and S-PIA), protected animals against seizures in a dose-dependent, and extremely potent manner. NECA, the most potent compound evaluated, completely prevented seizures at doses greater than or equal to 6.8 pmol. In contrast, heroic doses of the A2 selective ligand, 2-phenylaminoadenosine, afforded no protection against seizures. The rank order of potency of these compounds in suppressing seizures is as follows: NECA greater than cyclohexyladenosine greater than cyclopentyladenosine greater than or equal to R-PIA greater than 2-chloroadenosine greater than S-PIA much greater than 2-phenylaminoadenosine. These data suggest that the antiseizure activity of these compounds in the PPC results from activation of A1 adenosine receptors. Quantitative autoradiographic analysis of the distribution of tritiated adenosine agonists 30 min after microinjection in the PPC reveals that [3H]NECA diffuses to a significantly greater extent than R-[3H]PIA, which may contribute to the relatively greater potency of the former compound in suppressing bicuculline methiodide-induced seizures. These results suggest that adenosine A1 receptors may participate in the normal inhibitory regulation of the PPC, a forebrain area which may play a significant role in the pathobiology of epilepsy.     

Ligation of protease-activated receptor 1 enhances alpha(v)beta6 integrin-dependent TGF-beta activation and promotes acute lung injury

Activation of latent TGF-beta by the alpha(v)beta6 integrin is a critical step in the development of acute lung injury. However, the mechanism by which alpha(v)beta6-mediated TGF-beta activation is regulated has not been identified. We show that thrombin, and other agonists of protease-activated receptor 1 (PAR1), activate TGF-beta in an alpha(v)beta6 integrin-specific manner. This effect is PAR1 specific and is mediated by RhoA and Rho kinase. Intratracheal instillation of the PAR1-specific peptide TFLLRN increases lung edema during high-tidal-volume ventilation, and this effect is completely inhibited by a blocking antibody against the alpha(v)beta6 integrin. Instillation of TFLLRN during high-tidal-volume ventilation is associated with increased pulmonary TGF-beta activation; however, this is not observed in Itgb6-/- mice. Furthermore, Itgb6-/- mice are also protected from ventilator-induced lung edema. We also demonstrate that pulmonary edema and TGF-beta activity are similarly reduced in Par1-/- mice following bleomycin-induced lung injury. These results suggest that PAR1-mediated enhancement of alpha(v)beta6-dependent TGF-beta activation could be one mechanism by which activation of the coagulation cascade contributes to the development of acute lung injury, and they identify PAR1 and the alpha(v)beta6 integrin as potential therapeutic targets in this condition.     

Ligand selectivity of dog coronary adenosine receptor resembles that of adenylate cyclase stimulatory (Ra) receptors

Adenosine receptors on the surface of coronary myocytes initiate coronary relaxation, but events subsequent to receptor occupancy are uncertain. We assayed the coronary vasoactivity of receptor-selective adenosine analogs in open chest dogs to test the hypothesis that the coronary adenosine receptor is an adenylate cyclase stimulatory (Ra) receptor. The potency order was: ethyl adenosine-5'-uronamide greater than cyclopropyl adenosine-5'-uronamide greater than 2-chloroadenosine greater than N6-[R(-)-1-phenyl-2-propyl] adenosine (R-PIA) greater than N6-cyclohexyladenosine greater than adenosine greater than S-PIA. Such a hierarchy of activity resembles that of the Ra receptors of thyroid and aortic myocyte adenylate cyclases and of tracheal smooth muscle relaxation. The potency order R-PIA greater than adenosine and the stereoselective coronary vasoactivity of R-PIA suggest that the coronary receptor may be a hybrid receptor which contains the "N6 site" typical of inhibitory (Ri) receptors in addition to the "5'-uronamide site' which expresses Ra activity.     

与人体肥胖相关的神经肽Y受体Y1基因的克隆及序列分析

目的 克隆与肥胖相关的人神经肽Y受体Y1(NPYlR)基因,并鉴定该克隆基因序列的正确性.方法 从人的脂肪组织中提取总RNA并进行反转录,以此为模板用PCR扩增NPY1R基因的cDNA,然后与pET 28a+载体重组,筛选阳性克隆和DNA序列分析鉴定,测序后与GeneBank中NPY1R基因进行同源性比较和序列分析.结果 PER扩增出一个1100 bp左右的DNA片段,与载体重组后DNA序列分析鉴定,DNA序列分析显示克隆的DNA片段是人NPY1R基因,且所克隆的基因共编码384个氨基酸,分子量为44 KD,与GeneBank中NPY1R基因序列同源性达100%.结论 克隆的人NPY1R基因与GeneBank中NPY1R序列完全一致,为进一步应用分子生物学技术深入研究NPY1R与人体肥胖发生、发展及转化等相关作用机制及应用该基因进行其基因蛋白表达奠定了基础.     

Opioid receptor selectivity of dynorphin gene products

In the guinea-pig ileum myenteric plexus-longitudinal muscle preparation, products from the dynorphin gene fell into three groups according to their potency. Dynorphin A was the most potent; dynorphin-32, dynorphin B, dynorphin B-29 and alpha-neo-endorphin were about equipotent and 10 to 20 times less potent than dynorphin A; dynorphin A-(1-8) and beta-neo-endorphin were about 200 times less potent than dynorphin A. Dynorphin A (a kappa agonist) was about 10 times less sensitive to antagonism by naloxone (as measured by naloxone Ke) than was normorphine (a mu agonist). Ke values for dynorphin-32, dynorphin B and alpha-neo-endorphin were the same as for dynorphin A, indicating that these peptides are also highly selective kappa agonists. Dynorphin A-(1-8), dynorphin B-29 and beta-neo-endorphin had Ke values intermediate between dynorphin A and normorphine, suggesting that they interact at both kappa and mu receptors. Addition of peptidase inhibitors to the bathing medium increased the potencies of dynorphin B, dynorphin B-29, alpha-neo-endorphin, dynorphin A-(1-8) and beta-neo-endorphin, but not of dynorphin A, dynorphin-32 or normorphine. The inhibitors did not change the naloxone Ke for dynorphin A or normorphine, or for dynorphin B-29, dynorphin A-(1-8) and beta-neo-endorphin, suggesting that the intermediate values were not caused by degradation to products with different receptor selectivities from the parent compounds. Ke for dynorphin-32, dynorphin B and alpha-neo-endorphin changed from being the same as dynorphin A in the absence of inhibitors to intermediate between dynorphin A and normorphine in the presence of inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

5-Hydroxytryptamine(1A) receptor activation enhances norepinephrine release from nerves in the rabbit saphenous vein.

Although serotonergic receptor agonists are known to modulate release of central serotonin, less is known about the ability of serotonin to alter neurotransmission in peripheral adrenergic nerves. The present study used field stimulation (40V, 0.7 ms duration, 1-16 Hz) to contract the rabbit saphenous vein, an effect that was abolished in the presence of tetrodotoxin and prazosin (10(-6) M), consistent with stimulation of neuronal norepinephrine release. Furthermore, the field-stimulated contraction was not altered by the 5-hydroxytryptamine (5-HT)(1B/1D) receptor antagonist GR127935 (10(-6) M), but was markedly inhibited by the 5-HT(1A) receptor antagonist WAY 100635 (10(-6) M). GR127935 (10(-8) M) inhibited contraction to sumatriptan, documenting that the concentration used was sufficient to block 5-HT(1B/1D-like) vascular receptors in this tissue. Likewise, WAY 100635 (10(-6) M) inhibited contraction to the 5-HT(1A) receptor agonists (+/-)-8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT) and LY238729, without altering contraction to norepinephrine or sumatriptan. Furthermore, both 8-OH-DPAT and LY228729 enhanced the contractile response to field stimulation (1. 0-8.0 Hz) and activated norepinephrine release in the absence of field stimulation. Contractile responses of the rabbit saphenous vein to both 5-HT(1A) receptor agonists were markedly inhibited by prazosin and dextrally shifted by WAY 100635, supporting the idea that the 5-HT(1A) receptor agonists were activating presynaptic 5-HT(1A) receptors to enhance norepinephrine release even in the absence of field stimulation. Thus, in the rabbit saphenous vein, 5-HT(1A) but not 5-HT(1B/1D) receptors enhanced neurotransmitter release from adrenergic nerves. These observations suggested that serotonergic nerves or other cell types in the saphenous vein are activated by field stimulation to release serotonin, which in turn activates presynaptic 5-HT(1A) receptors on adrenergic neurons to effect norepinephrine release. To support this hypothesis, serotonin levels were measured in the saphenous vein and were increased after pargyline pretreatment (30 mg/kg s.c.), decreased after dl-p-chlorophenylalanine methyl ester pretreatment (300 mg/kg s.c.), and unaltered after pretreatment with 6-hydroxydopamine hydrobromide (100 mg/kg s.c.). Thus, we provide strong evidence for the 1) presence of serotonin and its direct synthesis independent of adrenergic nerves and 2) a novel excitatory effect of presynaptic 5-HT(1A) receptor activation on adrenergic nerves in a peripheral blood vessel.     

Regulation of thromboxane receptor (TP) phosphorylation by protein phosphatase 1 (PP1) and PP2A

To investigate the protein phosphatases that dephosphorylate TP, human embryonic kidney cells (HEK293 cells) stably transfected with 12CA5-tagged TP were treated with TP agonist, washed, and then allowed to recover in the presence or absence of the cell-permeable PP1 and PP2A inhibitors calyculin or okadaic acid (OKA). After recovery, cells were rechallenged with TP agonist and TP responsiveness was assessed by measuring inositol trisphosphate generation. TP responsiveness recovered over a 20-min time period. Recovery of TP responsiveness was inhibited by calyculin and OKA and was associated with dephosphorylation of receptor proteins. To further identify the TP phosphatase, TP phosphorylated in the intact cell were isolated by immunoprecipitation and were used as substrate for protein phosphatases prepared from HEK293 cells. TP were dephosphorylated by whole-cell homogenates. Dephosphorylation of TP was completely inhibited by the PP1 and PP2A inhibitors calyculin and microcystin-LR, suggesting that the decrease in TP phosphorylation was not due to receptor degradation. TP phosphatase activity was partially blocked by 1) inhibitor 2, a specific protein inhibitor of PP1; and 2) OKA at concentrations (1 nM) that specifically inhibit PP2A. TP phosphatase activity did not have an absolute requirement for divalent cations and was found primarily in cytosolic fractions of the cell. These data suggest that PP1- and PP2A-like protein phosphatases dephosphorylate TP. By regulating the phosphorylation state of TP, protein phosphatases may modulate tissue responsiveness to thromboxane.