Stimulation of atrial natriuretic peptide release by neurokinins in neonatal rat ventricular cardiomyocytes.

The effect of substance P (SP) on atrial natriuretic peptide (ANP) release was studied in neonatal rat ventricular cardiomyocytes. Incubation of cells with SP led to a marked increase in ANP secretion, a response accompanied by increases in alpha-type protein kinase C (PKC) in the membranous cell fraction and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) formation and a small increase in adenosine 3',5'-cyclic monophosphate (cAMP) production. A role for PKC in SP-induced 6-keto-PGF1 alpha formation and ANP release was apparent insofar as the responses were suppressed by PKC inhibitors and in PKC-downregulated cells. Furthermore, SP-induced 6-keto-PGF1 alpha production was strongly correlated with SP-induced ANP secretion (r = 0.91, P < 0.0001, n = 27), suggesting a role for prostaglandins in SP-mediated ANP release. Supporting this, indomethacin abolished SP-induced ANP release, whereas PGE2, PGF2 alpha, and prostacyclin (PGI2) promoted ANP secretion in this system. Both the profile of SP-induced cAMP production and results obtained with prostaglandin antagonists suggest that a prostanoid FP receptor is at the basis of this response. Finally, both neurokinins A and B induced similar ANP responses, whereas cultured cells were found to contain mRNA transcripts coding for both neurokinin NK1 and NK3 receptor subtypes. Overall, these results suggest that SP induces ANP secretion in neonatal ventricular cardiomyocytes through a PKC- and prostaglandin-dependent signaling pathway.

IL-8-induced signal transduction in T lymphocytes involves receptor-mediated activation of phospholipases C and D.

We have characterized the IL-8-induced signal transduction processes in T lymphocytes. A basal level of IL-8 receptor expression was shown on mixed PBL, as identified by using phycoerythrin (PE)-coupled IL-8, and this expression was increased following IL-2 stimulation. Scatchard analysis of T cells revealed competitive binding of IL-8 with a Kd of 0.55 nM, with approximately 1200 receptors per cell, on freshly isolated T cells. After 24 h in culture following purification, reverse transcriptase PCR (RT-PCR) analyses show the mRNA for only the type B IL-8R on these cultured T lymphocytes and the cell line MOLT-4. Stimulation of T lymphocytes or T cell clones with IL-8 led to generation of inositol trisphosphate and calcium flux. In addition, when T cells were prelabeled with [3H]oleic acid, IL-8 caused a long lasting, time- and dose-related increase in [3H]phosphatidylethanol (PtE), indicating activation of phospholipase D (PLD). By contrast, this IL-8-dependent PLD activity was undetectable in IL-8-stimulated neutrophils. PLD activation appeared to be downstream of protein kinase C, because several inhibitors abrogated the increase in [3H]PtE, whereas guanosine-5'-O-(3-thiotriphosphate (GTP(gamma)S) and inositol trisphosphorothioate (IP3S3) both increased the generation of [3H]PtE. Together, these results demonstrate that the IL-8RB receptor is sufficient to mediate phospholipase C (PLC) and PLD activation in T lymphocytes, but not in neutrophils, and indicate an important difference in receptor usage and signal transduction pathways between IL-8-stimulated lymphocytes and neutrophils.

Signal transduction mechanisms of recombinant bovine neurokinin-2 receptor stably expressed in baby hamster kidney cells.

The bovine neurokinin-2 (NK-2) receptor gene was stably transfected into Baby hamster kidney (BHK-21) fibroblasts and one recombinant clone expressing 17,700 high-affinity [125I]neurokinin A (NKA) binding sites/cell characterized further. [125I]NKA binding was displaced by unlabeled NKA with an IC50 of 8.26 +/- 2 nM (n = 5) and with the rank order of potency NKA > neurokinin B (NKB) > Substance P (SP) confirming pharmacological characteristics of an NK-2 receptor subtype. Stimulation with NKA resulted in a rapid and dose-dependent increase in inositol 1,4,5-trisphosphate (IP3) levels (EC50 = 32 +/- 10 nM; n = 7) which was paralleled by a transient biphasic rise in intracellular free calcium concentration [Ca2+]i (EC50 = 35 +/- 20 nM; n = 3). In addition to phosphoinositide (PI) hydrolysis and Ca2+ mobilization, NKA was found to stimulate both cyclic AMP formation (EC50 = 1.02 +/- 0.26 microM; n = 7) and [3H]arachidonic acid mobilization (EC50 = 0.65 +/- 0.45 microM; n = 4). Interestingly, cyclic AMP levels also rose after addition of an exogenous arachidonic acid metabolite, prostaglandin E2 (PGE2) (EC50 = 11.5 +/- 2 microM). Similar observations of NKA-induced IP3 production, Ca2+ mobilization, arachidonic acid liberation, and cAMP formation have been made previously following expression of the bovine NK-2 receptor in Chinese hamster ovary (CHO) epithelial cells. The present results suggest that activation of NK-2 receptors leads to characteristic and reproducible intracellular second messenger responses in a subclass of cell types which includes fibroblasts and epithelial cells irrespective of their genetic and phenotypic background.

Molecular characterisation, expression and localisation of human neurokinin-3 receptor.

The complete amino acid sequence of the human neurokinin-3 receptor was deduced by DNA sequence analysis of human genomic fragments. Comparison of the predicted primary structure with those for the human neurokinin receptors 1 and 2 shows a highly conserved pattern of seven hydrophobic regions with maximum divergence occurring at the amino- and carboxy-termini. The position of intron-exon junctions are identical to those in other reported neurokinin genes. Using a chimeric genomic-cDNA gene, the human NK-3 receptor was expressed in Xenopus laevis oocytes where it mediates membrane conductance changes in response to its agonist, neurokinin B. More significantly, expression of the gene in mammalian cells resulted in detection of receptor binding as well as neurokinin-stimulated calcium mobilization and arachidonic acid release, all displaying the pharmacological characteristics expected of a neurokinin-3 receptor. By using the polymerase chain reaction we have shown that mRNA for the human neurokinin-3 receptor is expressed predominantly in the central nervous system.

Recombinant bovine neurokinin-2 receptor stably expressed in Chinese hamster ovary cells couples to multiple signal transduction pathways.

Neurokinins are a family of neuropeptides with widespread distribution mediating a broad spectrum of physiological actions through three distinct receptor subtypes: NK-1, NK-2, and NK-3. We investigated some of the second messenger and cellular processes under control by the recombinant bovine NK-2 receptor stably expressed in Chinese hamster ovary cells. In this system the NK-2 receptor displays its expected pharmacological characteristics, and the physiological agonist neurokinin A stimulates several cellular responses. These include 1) transient inositol 1,4,5-trisphosphate (IP3) formation and Ca2+ mobilization, 2) increased out put of arachidonic acid and prostaglandin E2 (PGE2), 3) enhanced cyclic AMP (cAMP) generation, 4) increased de novo DNA synthesis, and 5) an induction of the "immediate early" genes c-fos and c-jun. Although NK-2 receptor-mediated IP3 formation involves activation of a pertussis toxin-insensitive G-protein, increased cAMP production is largely a secondary response and can be at least partially attributed to autocrine stimulation by endogenously generated eicosanoids, particularly PGE2. This is the first demonstration that a single recombinant neurokinin receptor subtype can regulate, either directly or indirectly, multiple signal transduction pathways and suggests several potential important mediators of neurokinin actions under physiological conditions.

Widespread distribution of Gq alpha/G11 alpha detected immunologically by an antipeptide antiserum directed against the predicted C-terminal decapeptide.

Antisera were raised to a synthetic peptide which represents the predicted C-terminal decapeptide of the alpha subunit of the G-proteins Gq and G11. Competitive ELISA indicated that antiserum CQ2 displayed strong reactivity against this peptide. Antiserum CQ2 identified an apparently single polypeptide of 42 kDa which was expressed widely. The mobility of this polypeptide in SDS-PAGE was not modified by pretreatment of cells with pertussis toxin, indicating that it was not a substrate for this toxin. Furthermore, the levels and mobility of this polypeptide were unaltered by treatment of cells with cholera toxin, defining that it was not related to Gs alpha.

Yeast alpha-mating factor receptor-linked G-protein signal transduction suppresses Ras-dependent activity.

Homologues of mammalian Ras conserved in Saccharomyces cerevisiae mediate glucose-stimulated cyclic AMP formation and we used this response to test for regulation of yeast Ras activity by the alpha-mating factor signal transduction pathway. alpha-Mating factor suppresses glucose-stimulated cyclic AMP formation by up to 57 +/- 12.6% (n = 5) and similar inhibition was observed in four different yeast strains (MATa cells). Moreover, this response is potent (IC50 = 0.14 +/- 0.19 microM (n = 4)), rapid (maximal within 1-2 min), and displays an absolute requirement for both the alpha-mating factor receptor (STE2) and associated G-protein beta-subunit (STE4). Inhibition appears independent of both phosphodiesterase activation and alpha-mating factor-stimulated cytoplasmic alkalinization. Also, basal cyclic AMP levels are unaffected by pheromone. This is the first demonstration that a cell-surface receptor linked to a heterotrimeric G-protein can suppress Ras-dependent activity and could provide important insight into mechanisms controlling p21ras in man. Inhibition of Ras-dependent cyclic AMP formation could also be a key event facilitating responses characteristic of yeast mating.

Influence of pregnancy on G-protein coupling to adenylate cyclase activation in guinea-pig myometrium.

The regulatory factors controlling uterine activity during pregnancy remain unclear in many species. Since myometrial relaxants raise intracellular cyclic AMP, modulation of signalling pathways coupling cell-surface receptors to adenylate cyclase activation could be an important site for control. To assess the functional activity of the stimulatory GTP-binding protein Gs we have measured adenylate cyclase activation by GTP, its non-hydrolysable analogue guanosine 5'-(beta-gamma-imido)triphosphate (Gpp(NH)p), fluoride, forskolin and manganese in a 50,000 g membrane fraction prepared from the myometrium of non-pregnant, mid-pregnant (30-32 days) and late-pregnant (62-66 days) guinea-pigs (full term 67 +/- 2 days). While forskolin- and manganese-dependent enzyme activation was unaltered by pregnancy, maximal stimulation by Gpp(NH)p and fluoride was enhanced by up to 200%. Recovery of adenylate cyclase activity in the 50,000 g fraction was essentially constant at 20-24% of the total activity throughout pregnancy, and thus cannot explain the increases observed. Since guanine nucleotides and fluoride stimulate adenylate cyclase through activating Gs, and forskolin and manganese act at the level of the catalytic unit, these data are consistent with a pregnancy-related increase in Gs functional coupling while adenylate cyclase activity is unaltered. These observations suggest a physiological regulation of myometrial Gs activity during pregnancy which could facilitate hormonal stimulation of adenylate cyclase and contribute to uterine quiescence by increasing uterine sensitivity to relaxants.

Pregnancy suppresses G protein coupling to phosphoinositide hydrolysis in guinea pig myometrium.

The regulatory factors controlling uterine contractile activity during pregnancy remain unclear, although pathways modulating intracellular Ca2+ and prostaglandin production play an important role. Because excitatory hormones raise myometrial Ca2+ levels and prostaglandin output through increasing phosphoinositide hydrolysis, regulation of G protein coupling to phospholipase C activation could be a key site for control. To measure the functional activity of this signaling pathway, we measured formation of [3H]inositol phosphates from prelabeled guinea pig myometrial membranes in response to G protein activation by guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) and fluoride. Although these agents stimulated a three- to fivefold increase in phosphoinositide phospholipase C activity in nonpregnant myometrium, at 46-47, 53-60, and 66-69 days of pregnancy (full term 67 +/- 2 days) this response fell by 43-83%. Moreover, the half-maximal effective dose (ED50) for GTP gamma S action was increased from 8.11 +/- 0.91 nM (n = 5) in the nonpregnant state to 307.4 +/- 142.3 (n = 9) and 209.7 +/- 155.1 nM (n = 8) at 53-60 and 66-69 days, respectively. Because phospholipase C levels displayed only a limited fall (28%) whether measured by direct Ca2+ activation or by immunoblotting, this study indicates a considerable suppression of G protein functional coupling to myometrial phosphoinositide hydrolysis throughout late gestation. Such a desensitization is likely to contribute to reports of diminished contractile sensitivity during pregnancy and to reflect an essential regulatory event in the processes maintaining uterine quiescence in pregnant guinea pig.

Alpha 2-adrenergic receptors in guinea pig myometrium in late pregnancy: evidence for a predominantly postjunctional location.

Uterine sympathetic nerves can exert an excitatory influence during pregnancy and parturition. Myometrial alpha 2-adrenergic receptor density falls sharply late in gestation, and diminished presynaptic neuromodulation could contribute to increased norepinephrine release reported at this time. Two studies were performed to investigate this potentially important mechanism. First, when alpha 2-adrenergic receptors were measured in well-innervated (tubal end and cervix), denervated (perifetal), and richly vascularized regions (placental) of late pregnant guinea pig uteri with the selective radioligand [3H]rauwolscine, high levels were detected in perifetal myometrium, particularly outside the site of placental implantation. Second, we report an unsuccessful attempt to demonstrate alpha 2-adrenergic receptor-mediated feedback suppression of electrically stimulated norepinephrine release from tubal and cervical regions of nonpregnant, midterm, and late-pregnant uteri. Together these results suggest that guinea pig myometrial alpha 2-adrenergic receptors are located mostly postjunctionally and are not associated with the vasculature. Thus an alpha 2-adrenergic feedback mechanism is unlikely to play an important role regulating uterine sympathetic neurotransmission.

Myometrial alpha 1-adrenoceptors in pregnant guinea pig: their distribution and increased number.

There is evidence for an excitatory influence in pregnant and parturient myometrium mediated by alpha 1-adrenoceptors. Despite this there are no reports of direct measurement of changes in the density of these receptors throughout pregnancy. Indirect evidence could suggest a fall early in gestation followed by a sharp rise near term. Hence, to follow pregnancy-related changes, [3H]prazosin was employed in this study to measure alpha 1-adrenoceptors in guinea pig myometrium. High-affinity [3H]prazosin binding [dissociation constant (Kd) = 0.51 +/- 0.05 nM (n = 29)] displayed the pharmacological characteristics of alpha 1-adrenoceptors and yielded linear Scatchard plots, which in nonpregnant myometrium indicated a maximum binding density (Bmax) of 48.6 +/- 6.1 fmol/mg protein (n = 5). At all times during pregnancy alpha 1-adrenoceptor density was approximately 40% higher than the nonpregnant value. This, together with uterine growth, resulted in a progressive increase in total alpha 1-adrenoceptor population of up to 34-fold with a mean value of 7.54 +/- 2.26 pmol/uterus (n = 6) at term (67 +/- 2 days). This slow rise in receptor number does not suggest a major role in the processes triggering labour in this species. The distribution of alpha 1-adrenoceptors in tubal, perifetal, and cervical regions was similar in the 60-day pregnant uterus. As much of the uterus is not innervated at this time, a considerable proportion of these receptors are probably extrasynaptic.

[3H]rauwolscine binding to myometrial alpha 2-adrenoceptors in pregnant guinea pig.

Uterine sympathetic nerves can exert an excitatory influence in late pregnancy and during parturition. Neuronal norepinephrine release is increased at these times and a diminished alpha 2-adrenoceptor-mediated prejunctional inhibition could account for this. To assess whether an altered receptor population may contribute, [3H]rauwolscine was used to measure alpha 2-adrenoceptors in myometrial membranes at time intervals throughout pregnancy. High affinity [3H]rauwolscine binding [Kd = 11.4 +/- 1.5 nM (n = 42)] yielded linear Scatchard plots that in nonpregnant myometrium indicated a maximum binding density (Bmax) of 217 +/- 42.4 fmol/mg protein. alpha 2-Adrenoceptor density was increased twofold at midpregnancy (31 days) and thereafter fell sharply by up to 90% toward term (67 +/- 2 days). When uterine growth is accounted for and data are expressed in terms of total myometrial population (uncorrected for plasma membrane recovery), alpha 2-adrenoceptor number was eightfold (midpregnancy) and fourfold (term) greater than the nonpregnant value of 804 +/- 322.4 fmol/uterus. alpha 2-Adrenoceptors were also found to bind dopamine with high affinity [Ki = 3.66 +/- 0.45 microM (n = 3)]. These observations could indicate a pregnancy-related change in uterine sympathetic autoinhibitory capacity and, since alpha 2-adrenoceptors appear also to be located postjunctionally, explain in part reports of altered myometrial responsiveness to norepinephrine infusion and also the uterotonic actions of dopamine.

Regional changes in catecholamine content of the pregnant uterus.

High-pressure liquid chromatography with electrochemical detection was used to identify and measure catecholamines in rat, rabbit, sheep, guinea-pig and human uteri and follow changes with pregnancy. Noradrenaline was consistently the major catecholamine and pregnancy caused a regionally specific fall in its concentration which, in rat, rabbit and guinea-pig, was associated with a decline in total content. Adrenaline was undetectable (less than 10 pmol/g myometrium) in all species and at all gestational ages studied. Dopamine and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) were detected at high concentrations in guinea-pig and particularly sheep uterus. In guinea-pig uterus the dopamine/DOPAC ratio fell dramatically with pregnancy, suggesting that increased quantities of dopamine were released and catabolized. The dopamine/noradrenaline ratios suggested that dopamine is stored with noradrenaline in adrenergic neurones in guinea-pig myometrium and within an additional neuronal or cellular store(s) in sheep uterus.