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K J Catt

Publications and source records attributed to K J Catt.

At least 91 records · Page 5Linked to original sources
PubMed1994

Inositol polyphosphates are not increased by overexpression of Ins(1,4,5)P3 3-kinase but show cell-cycle dependent changes in growth factor-stimulated fibroblasts.

NIH 3T3 fibroblasts were stably transfected with rat brain inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) 3-kinase to explore the relationship between increased production of Ins(1,3,4,5)P4 and the formation of InsP5 and InsP6. Mass measurements of InsP5 and InsP6 revealed no significant difference between kinase- and vector-transfected fibroblasts. However, such 3-kinase-transfected cells, when labeled with [3H]inositol for 48-72 h, showed lower levels of [3H]InsP5 and [3H]InsP6, as well as [3H]Ins(1,3,4,6)P4 and D/L[3H]Ins(1,4,5,6)P4, than their vector-transfected counterparts. Because Ins(1,4,5)P3 3-kinase-transfected cells grew less rapidly than vector-transfected controls, we determined whether the synthesis of InsP5 and InsP6 was related to a specific phase of the cell cycle. When NIH 3T3 cells prelabeled with [3H]inositol were synchronized by serum deprivation followed by stimulation with platelet-derived growth factor (PDGF), the amounts of labeled InsP5 and InsP6 began to increase only after 12 h of stimulation, when cells entered the S-phase as indicated by increased [3H]thymidine incorporation. The enhanced synthesis of these inositol polyphosphates was preceded by an early increase in Ins(1,4,5)P3 and its metabolites that was no longer evident by the fifth hour of PDGF action. There was also a prominent and biphasic increase in the level of D/L-Ins(1,4,5,6)P4 with an early peak at approximately 3 h and a second rise that paralleled the increases in InsP5 and InsP6. These results indicate that the formation of highly phosphorylated inositols is not tightly coupled to the receptor-mediated formation of Ins(1,4,5)P3 and its metabolites but is mainly determined by other factors that operate at specific points of the cell cycle.

3T3 Cells↗
PubMed1994

Modulatory action of progesterone and progesterone antagonists on hypothalamic-pituitary function.

The ability of ovarian steroids to sensitize and desensitize the pituitary gonadotroph to hypothalamic gonadotrophin-releasing hormone (GnRH) is essential for their modulatory actions on gonadotrophin secretion. The time-dependent actions of progesterone on GnRH-stimulated gonadotrophin secretion from cultured pituitary cells obtained from female rats were examined. Progesterone induced an acute stimulatory effect on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion in cell perifusion studies, from as early as 50 min after the onset of progesterone treatment. Long-term incubation (52 h) of pituitary cells in static culture reduced the responsiveness of the gonadotroph to GnRH. The antiprogestins RU486, ZK 98.299, and ZK 98.734 blocked both the acute facilitatory and the long-term inhibitory action of progesterone. In the absence of progesterone, the antiprogestins per se induced marked inhibitory and stimulatory effects on GnRH-stimulated LH secretion. In brief, short-term treatment of non-oestrogen-primed cells with antiprogestins was ineffective (ZK compounds) or reduced LH secretion (RU486), while long-term treatment was stimulatory. Oestrogen-primed cells exerted exclusively inhibitory effects on GnRH-induced LH secretion. In conclusion, antiprogestins are effective antagonists of progesterone actions in the gonadotroph. However, they exert diverse actions on gonadotrophin secretion in the absence of progesterone, which might interfere with their antagonistic properties.

Animals↗
PubMed1994

Glutamate modulates [Ca2+]i and gonadotropin-releasing hormone secretion in immortalized hypothalamic GT1-7 neurons.

Glutamate and its receptors are present in the hypothalamus and have been proposed to participate in neuroendocrine regulation, including the control of GnRH secretion. To address the mechanism of glutamate action, we measured [Ca2+]i, inositol phosphate, and secretory responses to glutamate receptor subtype agonists and antagonists in the immortalized GT1-7 cell line of GnRH-secreting hypothalamic neurons. Glutamate, N-methyl-D-aspartate (NMDA), kainate, and trans-(+/-)-1-amino-(1S,3R)-cyclopentanedicarboxylic acid increased GnRH secretion. In monolayer cultures of GT1-7 cells, L- but not D-glutamate induced a moderate, concentration-dependent rise in [Ca2+]i. The action of glutamate on [Ca2+]i was mimicked by NMDA, alpha-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid (AMPA), and kainate. Responses to NMDA were potentiated by the coagonist, glycine, and were inhibited by an antagonist of the glycine site on the NMDA receptor, 5,7-dichlorokynurenic acid (DCKA). NMDA-induced [Ca2+]i responses were also inhibited by Mg2+ and by the NMDA receptor antagonist, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,1 0-imine hydrogen maleate (MK-801), but not by the AMPA/kainate antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In contrast, responses to AMPA and kainate were inhibited by CNQX but not by Mg2+, DCKA, or MK-801. Responses to glutamate were more inhibited by MK-801 plus CNQX than by either antagonist alone. All [Ca2+]i responses were nearly abolished in Ca(2+)-free solution. None of the agonists stimulated inositol phosphate formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Regulation of angiotensin II receptors in rat brain during dietary sodium changes.

Activation of the renin-angiotensin system by sodium deficiency is associated with reciprocal changes in the expression of angiotensin II receptors in adrenal glomerulosa and vascular smooth muscle cells. The effects of dietary sodium changes on the expression of brain angiotensin receptor subtype 1 (AT1) mRNAs were examined in rats maintained on normal, low, and high sodium intake for 3 weeks. Plasma aldosterone and renin activity were elevated in rats maintained on a low salt diet compared with normal rats and were reduced in rats maintained on a high salt diet. These results are consistent with previous findings on the effects of altered dietary sodium on the renin-angiotensin system. The expression of AT1A and AT1B receptor subtype mRNAs was determined by quantitative reverse transcriptase-polymerase chain reaction during changes in sodium intake. The results revealed that sodium deprivation enhanced the expression of AT1B receptors in decorticated brains by 164% compared with high sodium intake. Conversely, high sodium diet increased the expression of AT1A receptors by 155% in the brain compared with low sodium intake. These data suggest that AT1A and AT1B receptors play reciprocal roles in central mechanisms for the control of fluid homeostasis. Further analysis of the molecular biology of angiotensin II receptor regulation in the brain may provide new insights into the interplay between the renin-angiotensin system and blood pressure regulation and also into the role of angiotensin II in the pathogenesis of essential hypertension.

Aldosterone↗
PubMed1994

Sequential activation of phospholipase-C and -D in agonist-stimulated gonadotrophs.

The contributions of phospholipase-C and -D to diacylglycerol (DG) formation during agonist-induced cell signaling were investigated in rat pituitary cells and alpha T3-1 gonadotrophs. In both cell types, GnRH caused a biphasic increase in DG formation, with an initial spike within 60 sec, followed by a larger and sustained rise to reach a second peak after 15 min of stimulation. Both phases of DG production were temporally correlated with inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] formation, consistent with the dependence of DG formation on phospholipase-C-mediated phosphoinositide hydrolysis. However, the ability of GnRH to stimulate phosphatidylethanol (PEt) in the presence of ethanol suggested that phospholipase-D may also participate in DG formation. Two inhibitors of phospholipase-C-dependent phosphoinositide hydrolysis, U73122 and neomycin sulfate, reduced the PEt as well as the Ins(1,4,5)P3 response to GnRH, indicating that phospholipase-D is activated during phospholipase-C-dependent signaling in pituitary gonadotrophs. The production of both DG and PEt was increased by treatment with the active phorbol ester phorbol 12-myristate 13-acetate (PMA), but not with inactive 4 alpha-phorbol 13-didecanoate, indicating that stimulation of protein kinase-C leads to activation of phospholipase-D. In accord with this, GnRH- and PMA-induced elevations of DG and PEt production were attenuated or abolished in protein kinase-C-depleted cells. In contrast, short and long term stimulation with PMA had no effect on basal inositol phosphate production. Also, GnRH-induced inositol phosphate production was not affected by protein kinase-C depletion. Finally, U73122 and neomycin sulfate did not inhibit PMA-induced PEt formation. These data indicate that GnRH activates a dual phospholipase pathway in a sequential and synchronized manner; phospholipase-C initiates the biphasic increase in Ins(1,4,5)P3 and DG formation, and protein kinase-C mediates the integration of phospholipase-D into the signaling response during the sustained phase of agonist stimulation.

Animals↗
PubMed1994

Coordinate actions of calcium and protein kinase-C in the expression of primary response genes in pituitary gonadotrophs.

Activation of GnRH receptors in cultured pituitary cells and alpha T3-1 gonadotrophs caused prominent, but transient, increases in messenger RNAs for primary response genes (PRGs) including c-fos, c-jun, and junB. GnRH-induced stimulation peaked at 30 min and was dose related, with similar EC50 values (approximately 1 nM) for all three PRGs and higher maximum responses for junB than for c-jun and c-fos. The agonist-induced expression of PRGs was mimicked by activation of protein kinase-C with the phorbol ester phorbol 12-myristate 13-acetate (PMA), which acted additively with GnRH at low concentrations of both stimuli. Depletion of cellular protein kinase-C by prior treatment with PMA reduced GnRH- and PMA-induced expression of PRGs. The protein kinase-C inhibitor staurosporine also attenuated agonist- and phorbol ester-induced PRG expression. Activation of Ca2+ entry by the calcium channel agonist BayK 8644 or high K(+)-induced depolarization caused a concentration-dependent rise in intracellular Ca2+ ([Ca2+]i) and a concentration-dependent and transient expression of PRGs, albeit of smaller amplitudes than those elicited by GnRH and PMA. Ca(2+)-dependent PRG expression was abolished by the calmodulin inhibitor W-7. Parallel measurements of [Ca2+]i and steady-state levels of PRG messenger RNAs indicated that intracellular Ca2+ exerted both additive and suppressive actions over its physiological concentration range on GnRH- and PMA-induced PRG expression. At lower intracellular calcium concentrations, calcium acted additively with low concentrations of GnRH and PMA. However, high calcium concentrations suppressed high agonist- and phorbol ester-induced PRG expression. In contrast, omission of Ca2+ from the extracellular medium significantly enhanced induction of PRGs. These findings indicate that GnRH-induced PRG expression in gonadotrophs is mediated by protein kinase-C and calcium, and that protein kinase-C-dependent induction of PRGs is modulated both positively and negatively by physiological changes in [Ca2+]i. Such coordinate actions of the two signaling molecules provide a mechanism for the control of PRG expression by preferential integration of low strength, and attenuation of high strength, extracellular signals.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1994

Homologous down-regulation of gonadotropin-releasing hormone receptor sites and messenger ribonucleic acid transcripts in alpha T3-1 cells.

GnRH is known to down-regulate its pituitary receptors by mechanisms that include endocytosis of the agonist-receptor complex. To evaluate the extent to which changes in receptor synthesis contribute to this process, the effects of GnRH and its analogs on GnRH receptor number and messenger RNA (mRNA) levels were analyzed in the alpha T3-1 gonadotroph cell line. Treatment with GnRH or its potent agonist analog, des-Gly10-[D-Ala6]GnRH N-ethylamide, reduced GnRH receptor number in a time- and dose-dependent manner, with a half-maximal decrease in response to 10(-6) M GnRH or agonist analog by 75 min. The maximum decrease in receptor number (to 31% of the control value) was sustained for up to 72 h. In alpha T3-1 cells incubated with 10(-8) M GnRH or agonist analog, the GnRH receptors fell by 28% and 46% after 2 h, respectively; no change in receptors occurred after treatment with 10(-8) M GnRH antagonist ([D-pGlu1,D-Phe2,D-Trp3,6]GnRH). Time- and dose-dependent reductions in the level of receptor mRNA were also observed after treatment of alpha T3-1 cells with GnRH and the agonist analog. However, the maximal reduction in mRNA levels (to 60-70% of the control value) was consistently less than the decline in receptor number. These results indicate that the mechanism of GnRH receptor down-regulation in alpha T3-1 gonadotrophs includes reduction of receptor synthesis secondary to decreases in receptor mRNA levels. The finding that reductions in mRNA levels were relatively less than the decreases in receptor number is consistent with the involvement of additional mechanisms, including endocytosis and degradation, in down-regulation of the GnRH receptor.

Animals↗
PubMed1994

Developmental and physiological aspects of Ca2+ signaling in agonist-stimulated pituitary gonadotrophs.

GnRH-induced Ca2+ signaling was analyzed in gonadotrophs from pituitary glands of neonatal, prepubertal, peripubertal, cycling, and ovariectomized female and adult male rats. In all cases, single cell studies showed three types of Ca2+ responses to increasing GnRH concentrations: subthreshold, baseline oscillatory, and biphasic (an early transient followed by sustained oscillations or a plateau). An increase in the frequency, but not the amplitude, of spiking, and the presence of both baseline oscillatory and biphasic Ca2+ responses were observed in single cells from all groups exposed to low and subsequently to higher GnRH concentrations. Thus, modulation of the frequency of Ca2+ spiking at low to medium GnRH concentrations and suppression of the oscillatory response at high agonist concentrations are characteristics of the gonadotroph that are not affected by age, sex, stage of the estrous cycle, or gonadectomy. Ca2+ influx through plasma membrane channels contributed to both baseline oscillatory and biphasic responses, and both types of Ca2+ signaling were associated with LH release. The sensitivity of gonadotrophs to GnRH stimulation was similar in cells from single and multigland preparations from the same group of donors, but varied according to age and physiological status. Thus, sensitivity was low in neonatal gonadotrophs and progressively increased in postnatal cells. In cycling females, gonadotrophs from proestrous animals were more sensitive than those from estrous and diestrous animals. Ca2+ signaling was more uniform in gonadotrophs from ovariectomized than in those from normal females. During prolonged culture there was an increase in the frequency of Ca2+ spiking in response to 1 nM GnRH over the first 3 days, followed by a gradual decrease over 7-10 days and loss of the response in older cultures. These changes in Ca2+ signaling were accompanied by similar changes in the GnRH-induced secretory responses. These data indicate that frequency-controlled spiking, with suppression of the oscillatory mechanism at high agonist concentrations, is a characteristic of Ca2+ signaling in rat gonadotrophs. In addition, developmental, physiological, and in vitro changes in pituitary sensitivity to GnRH are expressed through modulation of the Ca2+ signal and the consequent secretory response.

Aging↗
PubMed1994

Control of calcium spiking frequency in pituitary gonadotrophs by a single-pool cytoplasmic oscillator.

The mechanisms by which the generation and frequency of cytoplasmic Ca2+ oscillations are controlled were investigated in pituitary gonadotrophs. In these cells, two Ca(2+)-mobilizing receptors, the gonadotropin-releasing hormone and endothelin receptors, induce frequency-modulated Ca2+ spiking at the rate of up to 30 min-1. The cytoplasmic oscillator is also activated by discharge of luminal Ca2+ (initiated by ionomycin, thapsigargin, or thimerosal) but not by increased voltage-sensitive Ca2+ influx or treatment with caffeine. The basic difference between these two types of Ca2+ oscillations is related to their requirement for inositol-1,4,5-triphosphate (InsP3). Thapsigargin-, thimerosal-, and ionomycin-induced spiking occurs without the rise in InsP3 production that is essential for the generation of receptor-controlled oscillatory responses. The differential requirement for InsP3 in the two types of Ca2+ spiking is indicated by two lines of evidence. First, agonist-induced Ca2+ spiking of frequency similar to that of non-receptor-mediated oscillations was accompanied by a significant increase in InsP3, whereas none of the non-receptor-mediated oscillations was associated with measurable changes in inositol phosphate production. Second, agonist-induced InsP3 formation and Ca2+ spiking were abolished by treatment with the phospholipase C inhibitors U73122 and neomycin sulfate, whereas non-receptor-mediated Ca2+ spiking was not affected by these agents. When the oscillator was activated by agents that do not increase InsP3 formation, it operated only at the basal rate of approximately 5 min-1 and spiking frequency did not rise with increasing drug concentrations, in contrast to the situation in agonist-stimulated gonadotrophs. However, both types of oscillations were affected by depletion of luminal Ca2+ and by changes in the intracellular Ca2+ concentration ([Ca2+]i) but were not inhibited by ryanodine. These findings are consistent with the operation of a single-pool Ca2+ oscillator that is responsible for generation of both types of Ca2+ oscillations. The oscillator is controlled by the coagonist actions of InsP3 and Ca2+ on the InsP3 receptor channels and by the activation of Ca(2+)-ATPase by rising [Ca2+]i. It can be induced to operate at low frequency without an increase in InsP3 production by agents that reduce intraluminal [Ca2+]i, and it exhibits a dose-dependent increase in spiking frequency during agonist stimulation.

Animals↗
PubMed1993

Molecular cloning, sequencing and functional expression of an amphibian angiotensin II receptor.

Xenopus laevis and mammalian angiotensin AT1 receptors couple to the phospholipase C signal transduction pathway. However, amphibian AII receptors (xAT), unlike mammalian AT1 receptors, do not recognize the non-peptide antagonist Dup 753. To investigate the basis of this distinction, we have isolated a 3.0 kb Xenopus myocardial xAT cDNA that encodes a 41,039 MW protein containing 362 amino acids. The xAT receptor has 60% amino acid identity and 65% nucleotide homology with the coding regions of known mammalian AT1 receptors. xAT receptors expressed in Xenopus oocytes mediate AII-induced Ca2+ mobilization and are pharmacologically distinct from mammalian AT1 receptors. xAT transcripts are present in Xenopus lung, liver, kidney, spleen, and heart, but not in adrenal, intestine, and smooth muscle. Comparative analysis of xAT and AT1 receptors should facilitate elucidation of the structural basis of their binding and activation properties.

Amino Acid Sequence↗
PubMed1993

Expression of gonadotropin-releasing hormone receptors and autocrine regulation of neuropeptide release in immortalized hypothalamic neurons.

The hypothalamic control of gonadotropin secretion is mediated by episodic basal secretion and midcycle ovulatory surges of gonadotropin-releasing hormone (GnRH), which interacts with specific plasma membrane receptors in pituitary gonadotrophs. Similar GnRH receptors and their mRNA transcripts were found to be expressed in immortalized hypothalamic neurons, which release GnRH in a pulsatile manner in vitro. Activation of these neuronal GnRH receptors elicited dose-related intracellular Ca2+ concentration responses that were dependent on calcium mobilization and entry and were inhibited by GnRH antagonists. Exposure of perifused neurons to a GnRH agonist analog caused a transient elevation of GnRH release and subsequent suppression of the basal pulsatile secretion. This was followed by dose-dependent induction of less frequent but larger GnRH pulses and ultimately by single massive episodes of GnRH release. The ability of GnRH to exert autocrine actions on its secretory neurons, and to promote episodic release and synchronized discharge of the neuropeptide, could reflect the operation of the endogenous pulse generator and the genesis of the preovulatory GnRH surge in vivo.

Animals↗
PubMed1993

Mechanism of agonist-induced [Ca2+]i oscillations in pituitary gonadotrophs.

Gonadotropin-releasing hormone (GnRH) activates oscillatory Ca2+ signaling in pituitary gonadotrophs at a frequency (up to 25 min-1) that is dose-dependent and is determined by the degree of receptor-mediated inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) formation. Similar dose-dependent and frequency-modulated Ca2+ oscillations were elicited by intracellular administration of Ins(1,4,5)P3 and its nonhydrolyzable analogs, consistent with models in which Ins(1,4,5)P3 levels determine the frequency of Ca2+ oscillations but do not fluctuate in synchrony with [Ca2+]i. At constant agonist concentrations, Ca2+ spiking varied in amplitude, with a number of progressively larger transients before the onset of maximal oscillations, followed by a gradual decrease in spike amplitude that was accompanied by an increase in spiking frequency. The decline in the amplitude and increase in frequency of Ca2+ transients during stimulation by GnRH were not related to a decrease in the propagation of the Ca2+ signal within the cell but were associated with gradual depletion of the agonist-sensitive Ca2+ pool. Once initiated, the pattern of Ca2+ spiking was not altered by blockade of receptor occupancy, by inhibition of phospholipase C, or by reduction of extracellular [Ca2+]. Also, the endoplasmic reticulum (Ca2+)-ATPase blocker, thapsigargin, could substitute for Ins(1,4,5)P3 in initiating the oscillatory Ca2+ response. These findings indicate that although the Ins(1,4,5)P3 concentration determines the pattern of transients at the initiation of the oscillatory Ca2+ signal, maintenance of the signal does not require a sustained rise in Ins(1,4,5)P3. Since the frequency of Ca2+ oscillations is also influenced by depletion of luminal [Ca2+], it is possible that the Ins(1,4,5)P3-sensitive channels in the endoplasmic reticulum are tonically inhibited by high intraluminal Ca2+ levels and that Ins(1,4,5)P3 surmounts such inhibition by promoting Ca2+ discharge. When a critical level of Ca2+ discharge is attained, repetitive Ca2+ transients are generated by an autocatalytic mechanism in which a sustained rise in Ins(1,4,5)P3 is not an essential requirement.

Animals↗
PubMed1993

Differential activation of inositol 1,4,5-trisphosphate-sensitive calcium pools by muscarinic receptors in Xenopus laevis oocytes.

Muscarinic acetylcholine (ACh) receptors activate the phospholipase C signal transduction pathway to promote the formation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and the consequent elevation of cytoplasmic calcium (Ca2+). The inositol phosphate and Ca(2+)-mobilization responses to ACh were analyzed in Xenopus oocytes possessing endogenous receptors, and in oocytes expressing exogenous receptors from injected muscarinic RNA transcripts, to evaluate the patterns of signal transduction mediated by native and expressed receptors. Activation of native ACh receptors elicited dose- and time-dependent increases in Ins(1,4,5)P3 and inositol bisphosphate (InsP2) production. ACh-induced Ins(1,4,5)P3 production increased rapidly within the first 2 min and continued to rise over the next 20 min. ACh was a much more effective stimulus of inositol phosphate production at native (up to 35-fold) than at expressed receptors (less than 2-fold). In contrast, measurements of Ca(2+)-mobilization in oocytes injected with the Ca(2+)-specific photoprotein, aequorin, revealed that ACh stimulation of expressed receptors evoked up to 200-fold increase in light emission, whereas ACh stimulation of native receptors elicited less than a 2-fold response. These observations indicate that the oocyte possesses functionally distinct agonist-sensitive Ca2+ pools which differ markedly in their sensitivity to Ins(1,4,5)P3 production and suggest that these pools are mobilized by different effector mechanisms. The finding that the magnitude of the intra-oocyte Ca2+ response is not necessarily determined by the degree of Ins(1,4,5)P3 production, but rather by another aspect of the signal transduction pathway (e.g. the nature and/or location of the Ins(1,4,5)P3 releasable Ca2+ pool), reveals an additional level of complexity in the transduction mechanisms responsible for intracellular Ca2+ signaling.

Acetylcholine↗
PubMed1993

Atrial natriuretic factor activates cyclic adenosine 3',5'-monophosphate phosphodiesterase in Xenopus laevis oocytes and potentiates progesterone-induced maturation via cyclic guanosine 5'-monophosphate accumulation.

Xenopus oocytes were found to express atrial natriuretic factor (ANF) receptors that activate guanylate cyclase and stimulate cyclic guanosine 5'-monophosphate (cGMP) production in a dose- and time-dependent manner. A truncated fragment of ANF, known to bind to mammalian ANF receptors without stimulating cGMP accumulation, did not elicit a cGMP response in oocytes. In addition, preincubation with ANF increased the number of oocytes that underwent progesterone-induced maturation. The maximally effective dose of ANF (1 microM) elevated intracellular and extracellular cGMP accumulation from 50 to 200 and 5 to 800 fmol/oocyte, respectively, and increased the number of maturing oocytes by up to 3-fold. ANF's effects on progesterone-induced maturation were mimicked by nonhydrolyzable analogues of cGMP. ANF and 8-Br-cGMP had no effect on maturation in the absence of progesterone, indicating that elevation of cGMP alone is not sufficient to induce maturation. Dibutyryl-cGMP was as effective as 8-Br-cGMP, whereas 8-Br-guanosine monophosphate, 8-Br-guanosine, and 8-Br-cyclic inosine monophosphate did not potentiate ovum maturation. In Xenopus oocytes, an initial step in progesterone-induced maturation is the reduction of intracellular cAMP levels; both ANF and 8-Br-cGMP lowered cAMP levels and enhanced progesterone's ability to do so. This decrease in cAMP levels was attributable to increased cAMP-phosphodiesterase activity, which was enhanced by both ANF and 8-Br-cGMP. These findings, and the presence of functional ANF receptors on Xenopus oocytes, demonstrate that ANF can participate in ovum development by stimulation of cGMP accumulation and activation of cAMP phosphodiesterase, thereby potentiating progesterone's ability to decrease cAMP levels and promote ovum maturation.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1993

High-affinity uptake of noradrenaline in postsynaptic neurones.

1. Neurotransmitters released from nerve endings are inactivated by re-uptake into the presynaptic nerve terminals and possibly into neighbouring glial cells. While analysing the functional properties of alpha 1-adrenoceptors in the hypothalamus, we observed a high-affinity uptake process for noradrenaline in postsynaptic peptidergic neurones. 2. In primary hypothalamic cell cultures and in a hypothalamic neuronal cell line, [3H]-prazosin bound with high affinity and was displaced by unlabelled prazosin in concentrations of 10(-10) to 10(-7) M. However, at concentrations of unlabelled prazosin above 10(-7) M, there was a paradoxical increase in apparent [3H]-prazosin binding. 3. Methoxamine, an alpha 1-adrenoceptor ligand that is not subject to significant neuronal uptake, displaced [3H]-prazosin but did not cause the paradoxical increase in the apparent binding of [3H]-prazosin. Cooling the cells to 4 degrees C reduced the total amount of prazosin associated with the cells; under these conditions, methoxamine almost completely inhibited [3H]-prazosin binding to the cells. 4. In the presence of desipramine (DMI), unlabelled prazosin displaced [3H]-prazosin as before, but no paradoxical increase in apparent binding was seen above 10(-7) M. 5. The paradoxical increase of [3H]-prazosin binding was not observed in membrane preparations of hypothalamic neurones. These findings indicated that the paradoxical increase in apparent [3H]-prazosin binding was due to a cellular uptake process that becomes evident at high concentrations of the ligand. 6. DMI (10(-5) M) had no effect on the specific binding of [3H]-prazosin. The presence of alpha1-adrenoceptors was confirmed by binding of [125]-HEAT, but [3H]-idazoxan (an alpha2- ligand) did not bind to the cells.7. The uptake of prazosin obeyed the Michaelis-Menten model, with similar Km and Vmax values in both types of cultures.8. Noradrenaline was taken up with high affinity by both types of cultures. (+/-)-[3H]-noradrenaline uptake was reduced by DMI and by excluding sodium from the medium, indicating that this process has some of the properties of uptake 1. (+/-)-[3H]-noradrenaline uptake in the cell line was unaffected by testosterone.9. The measured uptake of (-)-noradrenaline in the cell line was considerably increased by blockade of catechol-omicron-methyl-transferase and monoamine oxidase, suggesting that (-)-noradrenaline is metabolized to lipophilic products that escape across the plasma membrane.10. Studies in rats, in which the noradrenaline isomer 6-hydroxydopamine was used, suggested that the post synaptic uptake process is operative in hypothalamic CRH and vasopressin neurones in vivo.11. The Km for (-)-noradrenaline was within the range for the high affinity uptake, process in noradrenergic neurones. Uptake takes place in concentrations at which noradrenaline activates alpha1-adrenoceptors.Removal of noradrenaline from the vicinity of the receptors may prevent desensitization,thus maintaining the responsiveness of postsynaptic neurones to the actions of the neurotransmitter.

Animals↗
PubMed1993

Ovarian steroids modulate endothelin-induced luteinizing hormone secretion from cultured rat pituitary cells.

It has been demonstrated that endothelins (ETs) induce LH secretory responses in cultured rat pituitary cells. Because estradiol and progesterone are known to be potent modulators of GnRH-induced gonadotropin secretion, we examined whether these steroids also influence the secretory responses of gonadotrophs to ETs. Cultured female rat pituitary cells were treated for 48 h with vehicle (0.2% ethanol), 1 nM estradiol alone, or a combination of 1 nM estradiol and 100 nM progesterone or for 48 h with 1 nM estradiol and a further 4 h with 100 nM progesterone and subsequently stimulated with 10 pM-100 nM ET-1 or ET-3. Forty-eight-hour estradiol treatment led to enhanced LH secretory responses to both ETs. This action was facilitated by short term progesterone treatment (3-fold vs. vehicle), while long term progesterone treatment was inhibitory. Perifusion experiments were performed to study the kinetics of individual and pulsatile LH secretory responses after steroid exposure of pituitary cells. ET-1 induced immediate biphasic LH responses that were augmented by long term estradiol treatment. Two-hour progesterone exposure led to marked increases in LH secretion, whereas 48-h progesterone treatment was inhibitory. Estradiol and progesterone were able to modulate both the initial spike and the secondary plateau phase of the secretory profile in response to ET-1, although these actions did not always reach statistical significance. The steroid treatment paradigms employed also induced inhibitory and stimulatory effects in cells that were stimulated with ET-3 in a pulsatile fashion. In these experiments it could be demonstrated that the facilitatory action of progesterone was present after 50 min of treatment and was maximum after 150 min (5-fold enhancement). The present data support the hypothesis that ETs are involved in the physiological regulation of gonadotropin secretion and demonstrate that ovarian steroids can act as potent modulators of ET-induced LH secretion.

Animals↗