Presence of oxytocinergic neuronal-like cells in the bovine pineal gland: an immunocytochemical and in situ hybridization study.

In the last decade, there is more and more evidence showing the role of the central innervation of the pineal gland, but there are controversies around the intra or extrapineal origin of oxytocin found within the pineal tissue. In order to check the amount and the site of synthesis of oxytocin in the bovine pineal gland, we performed a morphological and chromatographic study. The anatomical distribution of the pineal oxytocin was explored by immunohistochemistry and in situ hybridization for the corresponding mRNA. The results confirm the presence of oxytocinergic fibres in the bovine pineal, some of them endowed with big varicosities. Immunohistochemistry also displayed neuronal-like cells in the pineal body. The in situ hybridization for the mRNA encoding pre-pro/oxytocin-NFZ I used a mixture of three oligonucleotide probes labelled with (35)S. This allowed identification of positive cells in the bovine pineal. The content in oxytocin was evaluated by radioimmunoassay during 5 months, from July to November, and the peptidic extract revealed an increase of pineal oxytocin immunoreactivity in September as compared with July or November. The significance of intrinsic oxytocin innervation of the bovine pineal gland, as well as the threefold increase of the oxytocin content in the pineal in September, remains to be elucidated.

Desensitization of beta2-adrenoceptor function in non-pregnant rat myometrium is modulated by sex steroids.

The effects of in vivo treatment with estrogen and progesterone on isoproterenol-induced uterine relaxation and beta(2)-adrenoceptor (beta(2)AR) mRNA production in non-pregnant rat myometrium were investigated. Whether homologous myometrial desensitization of beta(2)AR function was dependent on or modulated by the two steroids was also examined. Estrogen treatment alone or in combination with progesterone reduced maximal relaxation (E(max)) of isolated uterine strips subsequently challenged with isoproterenol whereas progesterone alone had no effect on this parameter. The reduction was accompanied by an enhanced beta(2)AR mRNA concentration. The concentration of isoproterenol giving half-maximal relaxing response (EC(50)) increased following estrogen treatment and this effect was curbed by progesterone. Isoproterenol had no effect on beta(2)AR transcription irrespective of the steroid regimes employed. E(max) of isolated uterine strips was reduced following prolonged in vivo treatment with isoproterenol but the effect was found only when estrogen alone was administered concomitantly. Finally, in vivo treatment with isoproterenol increased EC(50) of uterine strips subsequently stimulated with isoproterenol in vitro. This effect was independent of steroid treatment. We conclude that homologous desensitization of beta(2)AR function in non-pregnant rat myometrium in terms of sensitivity (EC(50)) is independent of sex steroids but in terms of maximal response (E(max)) occurs only in the presence of estrogen. We speculate whether progesterone withdrawal in connection with the well-known estrogen dominance at rat parturition may strengthen the desensitization induced by beta(2)AR activation and thus contribute to the transformation of the uterus from a quiescent to a highly contractile organ.

Effect of oxytocin receptor blockade on rat myometrial responsiveness to prostaglandin f(2)(alpha).

In the present study we have shown that the genetic expression of prostaglandin (PG)F(2alpha) receptor (R) and cyclooxygenase (COX)-2 increases in laboring rat myometrium. This finding was associated with a relatively weak contractile in vitro response (E:(max)) of isolated uterine strips when challenged with PGF(2alpha). Five days postpartum PGF(2alpha)-R mRNA values exceeded those during labor while COX-2 mRNA was reduced to preparturient values. Maximal contractility of isolated strips stimulated with PGF(2alpha) at this time was enhanced and E:C(50) decreased. Oxytocin treatment of estrogen-primed nonpregnant rats down-regulated uterine contractile responsiveness to PGF(2alpha), leaving mRNA values for this receptor unchanged, whereas oxytocin receptor blockade with atosiban (an oxytocin receptor antagonist) left E:(max) unaltered. In contrast, atosiban treatment of pregnant rats resulted in a 2.5-fold increase in E:(max) and a considerably reduced EC(50) during labor when compared to untreated delivering rats. The increased contractile ability was associated with a threefold increase in PGF(2alpha)-R mRNA production, indicating that the regulation by atosiban of the PGF(2alpha)-induced response is exerted at the genetic level. Based on the present data we suggest that 1) PGF(2alpha)-R stimulation may not primarily exert a contracting role in the normally delivering myometrium, and 2) the presence of the PGF(2alpha)-R system in rat myometrium may explain the apparent functional redundancy of the oxytocinergic system during the process of birth in animals lacking oxytocin or where the oxytocin receptor is blocked. In this context PGF(2alpha) receptor stimulation may, in the absence of oxytocin receptor stimulation, exert the contractile forces needed for proper propulsion of the fetus.

Up-regulation of oxytocin receptors in non-pregnant rat myometrium by isoproterenol: effects of steroids.

The objective of the present study was to further elucidate our previous observation that beta2-adrenoceptor activation induces oxytocin receptor (OTR) expression in rat myometrium. We wanted to investigate whether the mechanism behind this effect was under the influence of gonadal steroids. Ovariectomized non-pregnant rats were treated with estrogen, progesterone or a combination of both for 3 days. Some rats were concomitantly treated with isoproterenol. Estrogen treatment increased both OTR mRNA production and maximal binding of [3H]-oxytocin to isolated myometrial plasma membranes, but it did not affect contractility of isolated uterine strips challenged with oxytocin. When the estrogen regimen was combined with isoproterenol treatment, an augmented maximal contractile response (Emax) to oxytocin was observed although no further increase in OTR mRNA and binding was seen. Progesterone treatment did not in itself alter OTR mRNA, OTR binding or Emax. However, OTRs were induced at the level of gene expression when progesterone was supplemented with isoproterenol infusion. Finally, progesterone suppressed the effect of estrogen on OTR mRNA production and binding when the two compounds were administered together. However, when isoproterenol treatment was added this effect was abolished and Emax was enhanced more than that seen following treatment with estrogen alone. These data suggest that beta2-adrenoceptor activation represents an important regulator of OTR expression/function in estrogen- and progesterone-dominated rat myometrium.

Effect of oxytocin receptor and beta2-adrenoceptor blockade on myometrial oxytocin receptors in parturient rats.

It has been proposed that the rise in myometrial oxytocin receptor (OTR) concentrations at term triggers parturition. In the present study, we have shown that in vivo infusion of the beta2-adrenoceptor (beta2AR) antagonist ICI-118.551 in late pregnant rats prevents the rise in myometrial OTR binding normally seen during delivery. A reduced contractile responsiveness of uterine strips isolated from rats in labor when challenged with oxytocin (OT) and a slight shortening of gestation accompanied this effect. OTR mRNA levels were, however, unaltered after the treatment, suggesting that the effect of beta2AR blockade on myometrial OTR was posttranscriptional or due to influences on extra-myometrial tissue. Infusion of the OTR antagonist atosiban down-regulated OTR binding sites in the parturient myometrium and resulted in an impaired contractile response to OT without affecting gestational length. OTR gene expression did not change, as seen from unchanged OTR mRNA values. Neither atosiban nor ICI-118.551 infusions alone changed fetal mortality. A significant increase in the incidence of fetal deaths was found, however, when rats were treated with a combination of atosiban and ICI-118.551. This treatment also down-regulated myometrial OTR and weakened the contractile response to OT, but it did not change gestational length. We conclude that the timing and onset of a normal parturition as well as a favorable outcome seem to be independent of a rise in OTR. This fact cannot exclude the possibility that an increase in OTR is of importance in the genesis of preterm labor. We suggest that beta2 stimulation up-regulates OTR during delivery. This effect may partly be responsible for the tachyphylaxis seen after the use of beta2 agonists to control preterm labor. We further suggest that OTR stimulation up-regulates OTR during labor. The OTR down-regulation seen after atosiban treatment adds to the direct relaxing effect of atosiban on the myometrium. In view of this, atosiban may prove to be a more useful tocolytic than the traditionally used beta2 agonists.

Oxytocin receptor binding and uterotonic activity of carbetocin and its metabolites following enzymatic degradation.

Metabolites of the analogue 1-deamino-1-carba-2-tyrosine(O-methyl)-oxytocin (carbetocin) following incubation with a rat kidney homogenate were isolated and their pharmacodynamic properties investigated. Apart from the parent compound two metabolites were identified namely desGlyNH2-carbetocin (carbetocin metabolite I) and desLeuGlyNH2-carbetocin (carbetocin metabolite II). Both carbetocin, carbetocin metabolite I and carbetocin metabolite II displayed binding affinities to the myometrial oxytocin receptor of a similar magnitude as oxytocin. Carbetocin was found to have agonistic properties on isolated myometrial strips and it was found to exert this effect through generation of inositol phosphates, as is the case for oxytocin. However, maximal contractile effect of carbetocin was approximately 50% lower than that of oxytocin (2.70 +/- 0.12 g compared to 5.22 +/- 0.26 g) and EC50 was approximately ten times higher (48.0 +/- 8.20 nM compared to 5.62 +/- 1.22 nM). Neither carbetocin metabolite I nor carbetocin metabolite II were able to contract isolated myometrial tissue. All three compounds displayed antagonistic properties against oxytocin in vitro, with carbetocin being the strongest inhibitor (pA2 = 8.21) and carbetocin metabolite II (pA2 = 8.01) being stronger than carbetocin metabolite I (pA2 = 7.81). These results indicate that carbetocin is a partial agonist/antagonist to the oxytocin receptor while the two metabolites carbetocin metabolite I and carbetocin metabolite II are pure antagonists. All three analogues bound to the myometrial vasopressin V1 receptor, albeit with much lower affinities than to the oxytocin receptor. Carbetocin metabolite II showed the weakest binding affinity of 33.7 +/- 7.34 nM compared to 7.24 +/- 0.29 nM for carbetocin and 9.89 + 2.80 nM for carbetocin metabolite I. Only carbetocin bound to the renal vasopressin V2 receptor though the binding affinity was very low (61.3 +/- 14.6 nM).

Beta2-adrenoceptor desensitization in non-pregnant estrogen-primed rat myometrium involves modulation of oxytocin receptor gene expression.

The nona-peptide oxytocin (OT) induces contraction of the myometrium by interaction with specific plasma membrane associated OT receptors (OTR), whereas stimulation of beta2-adrenoceptors (beta2AR) causes relaxation. Homologous desensitization of the myometrium to both hormones has been described. However, a possible interaction between the two systems has not been investigated. In the present study, long-term in vivo treatment of non-pregnant estrogen-primed rats with isoproterenol decreased maximal relaxation of isolated uterine strips challenged with isoproterenol. Increased EC50 values of similarly treated animals suggest that the coupling between receptor occupancy and contractile response was impaired. Since beta2AR mRNA levels were left unchanged, we conclude that the homologous desensitization to beta2 stimulation is not due to changes in beta2AR gene expression. OT infusion did not alter beta2AR mRNA levels or isoproterenol-induced relaxation of isolated uterine strips. Treatment with OT had no effect on the amount of myometrial OTR mRNA. We have previously found that OT down-regulates OTR in the non-pregnant rat myometrium, but this therefore does not appear to take place at the level of mRNA production. Isoproterenol treatment resulted in a three-fold increase in OTR mRNA. This was accompanied by a 91% rise in OTR binding and an augmented contractile response of isolated uterine strips to OT, suggesting that the increased production of mRNA reflects formation of active receptors. Neither OTR affinity nor EC50 of in vitro strips was affected by isoproterenol treatment. We conclude that stimulation of beta2AR causes heterologous up-regulation of OTR in the non-pregnant estrogen-primed rat myometrium.

Interaction of chymotrypsin with carbetocin ([1-deamino-1-monocarba-2-O-methyltyrosine]-oxytocin).

The results of the present study describe the course of reaction and the products following chymotrypsin treatment of the oxytocin analogue carbetocin ([1-deamino-1-monocarba-2-O-methyltyrosine]-oxytocin). The metabolites were analyzed and identified through TLC, HPLC and mass spectrometry. The main product emerging after treatment of carbetocin with chymotrypsin is 9-desglycineamide carbetocin indicating preferential hydrolysis of the peptide bond between leucine at position 8 and carboxyterminal glycineamide. At the same time the stability of the bond between tyrosine at position 2 and isoleucine at position 3 appears significantly enhanced through the alkylation of the hydroxyl group of tyrosine.

Cross talk between substance P and melittin-activated cellular signaling pathways in rat lactotroph-enriched cell cultures.

We have investigated the possible interaction (cross talk) between the phospholipase A2 (PLA2) and inositol 1,4,5-trisphosphate/protein kinase C (PKC) signaling pathways in rat lactotroph-enriched cell cultures. Melittin, a bee venom peptide, stimulated release of [3H]arachidonic acid ([3H]AA) from [3H]AA-labeled enriched lactotrophs in a dose-dependent manner. Moreover, melittin and exogenous AA induced a redistribution of PKC catalytic activity and PKC alpha and beta immunoreactivity from the soluble to the particulate fraction in resting and substance P (SP)-stimulated cells. Melittin had no effect on phospholipase C (PLC) activity. Pretreatment of cell cultures with the PLA2 inhibitors quinacrine and aristolochic acid resulted in a dose-dependent inhibition of melittin-stimulated PKC isozyme translocation as did the inhibitor of lipoxygenase, nordihydroguaiaretic acid, whereas the cyclooxygenase inhibitor indomethacin had no effect. SP and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) dose-dependently increased levels of [3H]AA released from cells. Pretreatment of cell cultures with quinacrine reduced the effect of SP on [3H]AA formation. After long-term treatment (24 h) of cells with TPA, the effect of TPA on [3H]AA production was not different from control, whereas SP still displayed [3H]AA-releasing abilities although not at full scale. Pretreatment of cells with thapsigargin, U 73122, methoxyverapamil, and RHC 80267, an inhibitor of diacylglycerol lipase, all resulted in reduced SP-stimulated [3H]AA liberation. Treatment of cell cultures with pertussis toxin (PTX) reduced the release of [3H]AA induced by SP, whereas PTX had no effect on SP-stimulated generation of 3H-inositol phosphates. On the basis of these results, it is concluded that (1) the PLA2 pathways interfere with the phosphoinositide-PLC signaling system at the level of PKC isozymes alpha and beta, the product responsible for this interaction being either AA or a metabolite produced by the action of lipoxygenase; (2) SP and TPA are able to activate the PLA2 pathway at a level at or beyond PLA2, and this effect is mediated, in part, through PKC alpha and beta species and (for SP) intracellular Ca2+ recruited from internal stores as well as from external sources; and (3) SP also activates PLA2 through a PTX-sensitive pathway distinct from the one coupled to phosphoinositide-PLC, which is PTX insensitive.

Effect of pregnancy on rat myometrial beta 2-adrenoceptor mRNA and isoproterenol-induced relaxation of isolated uterine strips.

The altered myometrial contractile activity near term of pregnancy is partly due to changes in the responsiveness to catecholamines. Previous experiments have basically been concerned with uterine adrenoceptor binding characteristics. In the present study we have evaluated total myometrial DNA, beta 2-adrenoceptor mRNA and isoproterenol-induced relaxation of rat isolated uterine strips pre-contracted with potassium on days 0, 7, 14 and 21 of pregnancy and on day 5 post-partum. Total myometrial DNA expressed per milligram wet tissue peaked at day 14 of pregnancy followed by a decrease at the end of gestation. This suggests that hyperplasia predominates in the growth of the uterus in early gestation, whereas hypertrophy may be more marked in late pregnancy. The concentration of beta 2-adrenoceptor mRNA decreased linearly throughout the gestational period (0.73 +/- 0.20 amol/mg wet tissue on day 0 vs 0.34 +/- 0.09 amol/mg wet tissue on day 21, P < 0.05). Five days after parturition, at which time the uterus had returned to its pre-pregnant weight, beta 2-adrenoceptor mRNA was found to have increased 8-fold (2.79 +/- 0.14 amol/mg wet tissue, P < 0.05) as compared with day 21. The maximal effect of isoproterenol on pre-contracted uterine strips in which alpha-receptors were blocked by phentolamine showed a similar decrease which on day 21 reached 67% of the day 0 level (P < 0.001). EC50 values were unchanged in all groups except day 21 pregnant rats in which an increase was observed. One-way ANOVA with Bonferroni's t-test showed statistically significant differences only between the day 21 group and either the day 5 post-partum group or the day 14 pregnant group (P < 0.05). The observed alteration in EC50 prior to the end of gestation indicates that the system becomes less sensitive to beta 2-adrenergic stimulation at this time. We conclude that a reduction of de novo synthesis of beta 2-adrenoceptors may play a role in contributing to the increased myometrial activity at term. We further suggest that the dramatic up-regulation of beta 2-adrenoceptor mRNA postpartum may protect the fully involuted uterus against excessive contractions induced by oxytocin secreted during lactation.

Cross-talk between cellular signaling pathways activated by substance P and vasoactive intestinal peptide in rat lactotroph-enriched pituitary cell cultures.

We have investigated cross-talk between the cAMP/protein kinase A (PKA) and protein kinase C (PKC)/inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) messenger systems probed by vasoactive intestinal peptide (VIP) and substance P (SP), respectively, in rat pituitary cell cultures enriched in lactotrophs. VIP and forskolin had no effect on the basal distribution pattern of the four PKC isozymes (alpha, beta, delta, and zeta) detectable in lactotroph-enriched cell cultures derived from peripubertal male rats, whereas both compounds significantly increased translocation of PKC alpha and beta from the cytosol to the plasma membrane induced by SP. The delta and zeta subspecies were not affected by VIP and forskolin. Moreover, VIP and forskolin also stimulated SP-induced formation of Ins(1,4,5)P3 while having no effect on basal inositol phosphate turnover. The effects of VIP and forskolin on PKC isozyme distribution could be blocked by pretreating cells with the PKA inhibitor rp-cAMP. On the other hand, SP potentiated the effect of VIP and forskolin on cAMP formation while having no effect on the cAMP pathway when it was not triggered by an appropriate agonist. Down-regulation of PKC activity by long term 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment (24 h) diminished, but did not abolish, the effect of SP on VIP-stimulated cAMP production. Staurosporine and dopamine inhibited the potentiating effect of SP on cAMP accumulation. TPA, which translocates PKC alpha, beta, and delta in lactotrophs, had a synergistic effect on cAMP formation induced by VIP, but did also, unlike SP, display cAMP rising abilities when cells were not exposed to VIP and forskolin. Discharging intracellular Ca2+ by thapsigargin pretreatment had no effect on the basal cAMP concentration or the VIP-induced cAMP response, whereas exposure of cells to SP, thapsigargin, and VIP resulted in a decrease of the cAMP response compared with SP + VIP. The potentiating effect of SP on the VIP response could also be inhibited, but not blocked, by staurosporine. On the basis of these results, it is concluded that there exists substantial cross-talk between the cAMP/PKA and PKC/Ins(1,4,5)P3 messenger systems in lactotroph-enriched cell cultures. Key effectors seem to be PKA, one or more of PKC alpha, beta, deleta and Ins(1,4,5)P3-sensitive Ca2+ stores.

Substance P increases intracellular Ca2+ in individual rat pituitary lactotrophs, somatotrophs, and gonadotrophs.

The present study has investigated transients in the intracellular calcium concentration [Ca2+]i in response to substance P (SP) in single pituitary cells. SP raised [Ca2+]i in three subtypes of pituitary cells: lactotrophs, somatotrophs, and gonadotrophs. In all three cell subtypes the [Ca2+]i response to SP was amplitude-modulated and a concentration of 100 nM was necessary to elicit well pronounced two phased [Ca2+]i transients. The first phase was associated with increased generation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) in all three cell types. In lactotrophs, the second phase, but not the first, was blunted by depletion of extracellular Ca2+ (Ca2+ free EGTA incubation buffer) and by addition of dopamine (1 microM). In somatotrophs, the second phase of the SP-induced [Ca2+]i response was inhibited by depletion of extracellular Ca2+ and by addition of somatostatin (100 nM), while the first phase was unaffected by this treatment. In gonadotrophs, the second phase, but not the first, was inhibited by the Ca2+ channel blocker methoxyverapamil and depletion of extracellular Ca2+. SP was compared with other agonists having an action on lactotrophs, somatotrophs or gonadotrophs. These experiments demonstrated that SP was a weaker agonist in terms of maximal [Ca2+]i response than thyrotropin-releasing hormone (TRH) (in lactotrophs), growth hormone-releasing hexapeptide (in somatotrophs) and GnRH (in gonadotrophs). On the basis of these results it is concluded that SP exerts direct Ca2+ mobilizing effects in single lactotrophs, somatotrophs, and gonadotrophs derived from male peripubertal rats. The first phase in SP-induced [Ca2+]i transients is likely to be brought about by inositol 1,4,5-trisphosphate-mediated Ca2+ release from internal stores while the second phase reflects an influx of calcium through voltage-gated calcium channels.

Characterization of the rat myometrial contractile response to neuropeptide Y.

The in vitro contractile effect of neuropeptide Y (NPY) on rat myometrial strips was for the first time demonstrated and characterised, and the EC50 value estimated to be 267 +/- 87 nM. This effect is presumably mediated by the NPY1 receptor being responsible for postsynaptic effects throughout the peripherial nervous system, thus indicating a direct uterotonic effect of NPY. Further, the effect was demonstrated to the dependent on extracellular Ca2+. Short-term exposures to NPY markedly desensitized the tissue affecting subsequent responses to NPY as well as to oxytocin (OT). This desensitization was time and concentration-dependent, but lasted less than three hours. However, long-term infusions of NPY for 5 days increased to response to both NPY and OT. Long-term infusions of OT caused a marked decrease of the NPY response, and it is concluded that common pathways for up and down regulation of the myometrial responsiveness to several peptide hormones may exist.

Translocation of protein kinase C isozymes in rat pituitary lactotroph-enriched cell cultures by substance P: effects of sex and age.

It is generally accepted that the phospholipid and calcium-dependent enzyme protein kinase C (PKC) plays a significant role in secretion of hormones from anterior pituitary cells. The present study was undertaken to study age and sex-related changes in 1. levels of immunoreactivity of PKC isozymes and 2. distribution of immunoreactivity of PKC isozymes after stimulation with substance P (SP) in rat lactotroph-enriched cell cultures. The alpha, beta, delta and zeta isozymes were present in both sexes and at all ages. There was a sex-specific differential regulation of the different PKC isozymes as a function of sexual maturation. In male rats there was an up-regulation of the alpha isozyme throughout the sexual development, while the beta subtype showed a small, but significant decrease in immunoreactivity with increasing age. In female rats, on the other hand, the beta species was up-regulated with increasing age while the other subtypes remained constant. The concentration of the delta and zeta isozymes was unaffected of sex and age. Stimulation of lactotroph-enriched cell cultures with substance P (SP) resulted in translocation of the alpha and beta isozymes from the soluble to the particulate fraction while the delta and zeta species were left unchanged independently of age and sex. However, a decrease in responsiveness was observed in adult male rats, although a significant degree of translocation of alpha and beta species was still detected. On the basis of these results it is suggested that in lactotroph-enriched cell cultures basal levels of PKC subtype immunoreactivity and distribution of immunoreactivity of PKC isozymes after SP challenge might be regulated as a function of sex and age.

Effects of the non-peptide inhibitor OPC-21268 on oxytocin and vasopressin stimulation of rat and human myometrium.

OPC-21268 (1-[-1-[4-(3-acetylaminopropoxy)benzoyl]-piperidyl]-3,4- dihydro-2(1H)-quinolinone), a non-peptide vasopressin V1 receptor antagonist, inhibited oxytocin- and vasopressin-induced contractions of myometrial strips from rats and from full-term pregnant women. Administered intravenously in rats the drug also inhibited uterine contractions caused by infusion of oxytocin. When incubated with purified plasma membranes from rat or human myometrial tissue, OPC-21268 inhibited the specific receptor binding of tritiated oxytocin and vasopressin in a dose-dependent and reversible way.

Growth hormone releasing hexapeptide (GHRP-6) activates the inositol (1,4,5)-trisphosphate/diacylglycerol pathway in rat anterior pituitary cells.

Growth hormone-releasing hexapeptide (GHRP-6) is known to stimulate secretion of growth hormone (GH) in vivo and in vitro in a variety of species. However, the cellular effects of GHRP-6 remain largely unknown. We have tested the influence of GHRP-6 on the inositol phospholipid second messenger system in cultured anterior pituitary cells. Cultured pituitary cells responded upon challenge with GHRP-6 with a dose-dependent release of GH. Moreover, incubation of GHRP-6 with pituitary cell cultures labelled with myo-[3H]inositol resulted in a dose-dependent rise in [3H]inositol phosphates. Brief stimulation of pituitary cells with GHRP-6 increased phosphorylation of MBP4-14, a specific protein kinase C substrate, when incubated with the cytosol- or plasma membrane fraction from the stimulated cells. Furthermore, introduction of MBP4-14 into the cytosol in digitonin permeabilized pituitary cells caused increased phosphorylation of this substrate. GHRP-6 induced a rise in intracellular Ca2+ in individual somatotrophs loaded with the Ca2+ indicator, Fura-2. Preincubation (3 min) with somatostatin (SRIF) diminished the Ca2+ spike elicited by GHRP-6, while no effect of SRIF was observed when added simultaneously with GHRP-6. These results indicate that GHRP-6-stimulated GH-secretion involves the diacylglycerol/inositol(1,4,5)trisphosphate pathway with a resulting rise in cytosolic Ca2+.

Tachykinins induce secretion of prolactin from perifused rat anterior pituitary cells by interactions with two different binding sites.

Substance P and the two other mammalian tachykinins, neurokinin A and B, are accepted to have direct regulating effects at the anterior pituitary level. We have examined the effects of substance P (SP) and neurokinin B (NKB), alone and in combination, on prolactin release from cultured anterior pituitary cells grown on collagen-coated micro beads and placed in a perfusion system. Prolactin (Prl) secretion was observed within 25 s after exposure to either secretagogue and reached a maximum within 60-80 s. Furthermore, the prolactin response induced by SP and NKB was dose-dependent. Prl secretion remained constant for up to 4 h when SP or NKB were perifused and then fell gradually towards basal levels. Simultaneous addition of submaximal concentrations of SP and NKB resulted in an additive response compared with the responses of either secretagogue alone. Continuous (8 h) perifusion with SP did not prevent a normal prolactin response by NKB or TRH. These results indicate that the tachykinins, substance P and neurokinin B, release Prl from perifused female rat anterior pituitary cells by interaction with two different receptors, possibly the NK1 and NK3 tachykinin receptor subtypes.

Mastoparan, a wasp venom peptide, stimulates release of prolactin from cultured rat anterior pituitary cells.

Studies have shown that mastoparan and other amphiphilic peptides induce exocytosis of hormones from anterior pituitary cells. We have studied the effect of mastoparan on the secretion of prolactin from cultured rat anterior pituitary cells and on the concomitant functional status of signal-transducing pathways in lactotroph-enriched cell cultures. Mastoparan stimulation of prolactin secretion was dose-dependent, time-dependent, reversible and required the presence of calcium. Pretreatment of pituitary cell cultures with cholera and pertussis toxin had no effect on the secretory response, whereas encapsulation of guanosine 5-[beta-thio]diphosphate (GDP-beta-S) by reversible electropermeabilization inhibited mastoparan-stimulated secretion. Incubation of mastoparan with myo-[3H]inositol-labelled lactotroph-enriched anterior pituitary cell cultures resulted in increased formation of inositol phosphates compared with control cells, and encapsulation of GDP-beta-S blocked mastoparan-induced inositol lipid hydrolysis. Mastoparan caused translocation of protein kinase C activity from a soluble to a membrane-attached form. Mastoparan was able to increase the intracellular Ca2+ concentration in Fura-2-loaded individual lactotrophs. Omission of Ca2+ from the extracellular medium did not change the Ca2+ response in lactotrophs when stimulated with mastoparan. On the basis of these results it is concluded that mastoparan-induced release of prolactin is preceded by activation of the inositol(1,4,5)trisphosphate/diacylglycerol pathway with resulting translocation of protein kinase activity and increment in intracellular Ca2+. However, other signal-transducing pathways may be involved in the secretory process.

Translocation of protein kinase C isozymes in lactotroph-enriched rat anterior pituitary cell cultures: differential effects of substance P and phorbol ester.

Translocation of protein kinase C (PKC) from the cytosol to the plasma membranes is believed to reflect activation of the enzyme. We have studied translocation of PKC in lactotroph-enriched anterior pituitary cell cultures by measuring the incorporation of gamma-32P from [gamma-32P]ATP into a synthetic peptide substrate, MBP4-14, and by immunoblotting of PKC isozymes. Using cells permeabilized with digitonin the effects of PKC cofactors on the distribution of the enzyme were studied. Ca2+ (50 nM) and dioctanoyl-sn-glycerol had no effect when tested alone, but in combination they caused a redistribution of PKC from the soluble to the particulate fraction. Arachidonic acid needed Ca2+ to induce translocation of PKC, while being ineffective under Ca(2+)-free conditions. Western blot analysis of partly purified PKC from lactotroph-enriched pituitary cells revealed the presence of the alpha, beta, delta and zeta isozymes. 12-O-Tetradecanoylphorbol 13-acetate (TPA) and substance P displayed different patterns of redistribution of PKC isozyme immunoreactivity from soluble to membrane-attached forms. Thus, TPA induced time- and dose-dependent (mean effective concentration (EC50) = 1 nM) translocation of the alpha, beta and delta species, while substance P stimulated time- and dose-dependent (EC50 = 1 nM) redistribution of the alpha and beta isozymes. zeta subtype immunoreactivity could not be translocated by either agonist; neither could the immunoreactivity of zeta be down-regulated by long-term treatment (24 h) with TPA. The results indicate that simultaneous activation of phospholipases C and A2 induces a synergistic activation of PKC. Finally it is suggested that substance P may exert some of its effects in lactotrophs by translocation of PKC isozymes alpha and beta.