Biomedical subjects

C Kordon

Publications and source records attributed to C Kordon.

At least 19 recordsLinked to original sources

PubMed1992

Precursor and deaminated forms of both luteinizing hormone-releasing hormone (LHRH) and (hydroxyproline9)LHRH are present in the rat hypothalamus.

A naturally occurring analog of the decapeptide luteinizing hormone-releasing hormone ([Hyp9]LHRH) has been described previously in the hypothalamus of several mammals. It derives from post-translational hydroxylation of the LHRH proline9 residue. In the present work, intermediate LHRH precursors exhibiting both Pro9 or Hyp9 residues in the LHRH sequence were characterized in the rat hypothalamus. Hydroxylation of the Pro9 residue can thus be assumed to occur at an early stage of post-translational maturation. Deaminated, free acid forms of both native decapeptides were also detected. They correspond most likely to catabolites from incompletely processed precursors.

Amino Acid Sequence↗

PubMed1992

Effect of an essential fatty acid deficiency on the phospholipid composition in anterior pituitary membranes.

The effects of an essential fatty acid deficient diet were investigated on the phospholipid fatty acids of several membrane fractions of the rat anterior pituitary, the secretion of which is known to be partly dependent on the membrane phospholipidic constituents. In standard dietary conditions, arachidonic acid (20:4n-6) and its elongation product, adrenic acid (22:4n-6), were the two main polyunsaturated fatty acids in all fractions studied. In rats deprived of EFA for 6 weeks after weaning, the levels of both 20:4n-6 and 22:4n-6 were not changed in microsomal + plasma membrane and nuclear fractions, whereas they were decreased in heavy mitochondrial and light mitochondrial fractions. The present data suggest a mechanism of compensation between membrane fractions which may preferentially preserve 20:4n-6 and 22:4n-6 in discrete membrane fractions.

Animals↗

PubMed1992

A Ca2+ calmodulin dependent kinase rather than protein kinase C is involved in up-regulation of the LHRH receptor.

Stimulation of protein kinase C (PKC) by phorbol ester (PMA) was reported previously to increase total binding of the peptide in whole rat pituitary cells. The effect could be obtained in cells from intact, not from spayed animals, suggesting a different level of spontaneous phosphorylation in both conditions. In the present work, endogenous PKC was desensitized in pituitary cells sampled from intact or 3 weeks castrated male rats and maintained in primary culture. Desensitization was induced by overnight incubation with 1 microM PMA. The maximum number of plasma membrane LHRH receptors (Bmax) present on cells from in intact animals was higher (+ 98 +/- 9%) when binding was performed at 0.5 degrees C instead of 21 degrees C as already observed in non PKC-desensitized cells. PMA (100 nM) was ineffective to increase Bmax, suggesting effectiveness of enzyme desensitization. In contrast, ionomycin 1 microM increased Bmax (53 +/- 10%). This increment was inhibited by W7, a calmodulin inhibitor, with an IC50 = 1 +/- 0.35 10(-6) M. No temperature dependency of the Bmax was observed in cells from castrated rats as already shown in the absence of PKC desensitization. Under these conditions, a Bmax decrease of 34 +/- 6% and 36.5 +/- 7.5% respectively was observed in the presence of H7, a PKC inhibitor, or of W7 (IC50 = 1 +/- 0.5 10(-5) M and IC50 = 0.8 +/- 0.2 10(-6) M). We conclude that a Ca2+ calmodulin dependent protein kinase rather than PKC itself is responsible for unmasking LHRH receptors.

Animals↗

PubMed1992

Regulation of somatostatin synthesis by GABAA receptor stimulation in mouse brain.

Neuroanatomical data have documented the existence of synaptic contacts between gamma-aminobutyric acid (GABA) terminals and periventricular hypothalamic somatostatin (SRIF) neurons. In other brain regions, like the cortex or hippocampus, GABA and SRIF are colocalized in short interneurons. These observations suggest that GABA modulates SRIF neuronal activity. In order to test this hypothesis, we studied the effects of the in vivo stimulation of the GABAA receptor (muscimol, 0.75 mg/kg + diazepam, 2.5 mg/kg) on SRIF content and preproSRIF mRNA levels, in mouse brain. Chronic (7 days), but not acute, treatment induced a 38% decrease in hypothalamic SRIF content (as estimated by RIA), a 20% decrease in cortex and no effect in the striatum. The decrease in hypothalamic and cortical SRIF levels lasted until 24 h after cessation of the treatment. In the hypothalamus, prosomatostatin mRNA levels were estimated by Northern blot analysis using a 32P-labeled 45-mere oligoprobe. ProSR1F mRNA hypothalamic levels were equally (48%) decreased by the acute and chronic treatments and remained lower than controls 48 h after the last injection. Quantitative in situ hybridization was used to examine the regional distribution of GABA-induced acute inhibition of proSR1F mRNA densities, using the same oligomere labeled with 35S. ProSR1F mRNA levels were decreased by 35% in the periventricular hypothalamic nucleus. In contrast, no significant modification was observed in cortex, striatum and hilus of the dentate gyrus of the dorsal hippocampus. The present data demonstrate a regionally selective inhibitory action of GABA, mediated by GABAA receptors stimulation, on the biosynthetic mechanisms of the long projecting neuroendocrine SRIF neurons of the anterior periventricular nucleus of the hypothalamus.

Animals↗

PubMed1992

A second endogenous molecular form of mammalian hypothalamic luteinizing hormone-releasing hormone (LHRH), (hydroxyproline9)LHRH, releases luteinizing hormone and follicle-stimulating hormone in vitro and in vivo.

In vitro and in vivo release of pituitary hormones were studied in the presence of (hydroxyproline9)LHRH ((Hyp)LHRH), a newly characterized endogenous molecular form of LHRH. Results were compared to those obtained with LHRH itself. (Hyp)LHRH, as LHRH, stimulated both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release in a homothetic manner. The hydroxylated compound was, however, 24 times (in vitro) and 5 times (in vivo) less potent than LHRH. The lower activity of (Hyp)LHRH than of LHRH in the in vitro assay correlated well with a 28-fold lesser potency in a binding test using pituitary membrane preparations. The higher relative potency and the prolonged effect of (Hyp)LHRH in the in vivo test were related to a lesser susceptibility of the hydroxylated form to proteolytic degradation. Effects of LHRH and of (Hyp)LHRH were not additive, both peptides were equally able to desensitize gonadotrophs to a subsequent challenge by the other. Taken together, these observations suggest that both forms of LHRH act at the same receptor site. The lesser affinity of the hydroxylated compound is compensated to a certain extent by its higher resistance to enzymatic degradation. It is concluded that in spite of its lesser potency, (Hyp)LHRH may participate in the regulation of gonadotropins.

Animals↗

PubMed1992

Alkaloids from Psychotria oleoides with activity on growth hormone release.

Bioactivity-guided purification of a crude alkaloid extract of Psychotria oleoides has afforded a new alkaloid, psycholeine [1], together with quadrigemine C [2], a tetrameric pyrrolidinoindoline compound of unknown stereochemistry. A comparison study of nmr and cd spectra of quadrigemine C and hodgkinsine [3], a trimeric pyrrolidinoindoline substance, led us to suggest the stereochemistry of quadrigemine C. The structure and configuration of psycholeine was determined by spectroscopic means and chemical correlation with quadrigemine C. Psycholeine interacts with somatostatin receptors and exhibits a somatostatin antagonistic activity on GH secretion by pituitary cells in primary culture.

Alkaloids↗

PubMed1992

Growth hormone-releasing hormone-synthesizing neurons are a subpopulation of somatostatin receptor-labelled cells in the rat arcuate nucleus: a combined in situ hybridization and receptor light-microscopic radioautographic study.

Distribution of growth-hormone-releasing hormone (GHRH) cell bodies and somatostatin binding sites were compared in the mediobasal hypothalamus of the rat. GHRH-synthesizing neurons were visualized by in situ hybridization, using as 35S-labelled synthetic oligonucleotide (45 mere), and 125I-Tyr0-DTrp8-somatostatin (125I-SRIH) binding sites by light-microscopic radioautography on adjacent 20-microns-thick frozen mirror sections. GHRH mRNA hybridizing cells were detected mostly in the ventrolateral portion of the arcuate nucleus (ARC) and around the perimeter of the ventromedial nucleus (VMN). Comparison with the distribution of pericellular 125I-SRIH binding sites allowed to differentiate three types of cells: (1) GHRH perikarya not associated with pericellular 125I-SRIH binding sites around the perimeter of the VMN, (2) 125I-SRIH-labelled cells, not associated with GHRH perikarya in the periventricular zone along the dorsal part of the third ventricle, and (3) in the ventrolateral portion of the ARC, GHRH mRNA-labelled neurons had the same distribution as 125I-SRIH-labelled cells. Furthermore, on adjacent sections, the number of both labelled cells were correlated (r = 0.68; p less than 0.001). In this last population, the extent of colocalization of 125I-SRIH binding sites on GHRH mRNA-labelled neurons was further investigated in adjacent 5-microns-thick sections. The proportions of cells GHRH mRNA and 125I-SRIH allowed to differentiate three subdivisions of the arcuate: the periventricular (PV), ventrobasal (VB) and lateral portions. In the PV-ARC, 27% of GHRH-synthesizing cells were coidentified as 125I-labelled while only 6% of 125I-labelled cells contained GHRH mRNA. In the VB-ARC the proportion of double-labelled cells was equivalent (31 and 26%, respectively for GHRH mRNA and 125I-SRIH).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Estradiol regulation of somatostatin receptors in the arcuate nucleus of the female rat.

Somatostatin receptors on lactotroph cells of the anterior pituitary are positively regulated by estradiol. In the present work, we investigated whether estradiol regulation of somatostatin receptors also occurred in the female rat brain. 125I-Tyr0-DTrp8-somatostatin (125I-SRIF: 780 Ci/mM) was used as a ligand. Female adult rats were ovariectomized and treated or not with estradiol benzoate (20 micrograms/day for 1 or 8 days). In female brains, 125I-SRIF binding, as assessed by film radioautography, was high in the basolateral amygdala, CA1 field and dentate gyrus of the hippocampus and locus coeruleus, moderate in the median habenula and deep layers all through the cortex. Castration or estradiol treatment did not modify 125I-SRIF binding in these regions. By light-microscopic radioautography, a subpopulation of 125I-SRIF-labeled cells was localized in the ventrolateral portion of the arcuate nucleus. Ovariectomy alone did not significantly affect the number and binding density of 125I-SRIF-labeled cells in the arcuate nucleus. However, estradiol treatment in ovariectomized animals significantly increased both parameters. Along the estrus cycle, the number of 125I-SRIF-labeled cells was not significantly modified but 125I-SRIF binding density was significantly higher in proestrus as compared to diestrus I, diestrus II and estrus. These results demonstrate that brain 125I-SRIF binding sites are positively regulated by estradiol only in the arcuate nucleus of the hypothalamus.

Animals↗

PubMed1992

Gonadotropin regulation, oestrogens and the immune system.

Several transmitters and neuropeptides participate in the regulation of gonadotropins. At the hypothalamic level, control of gonadotropin releasing hormone (GnRH) involves noradrenaline, GABA, glutamate, angiotensin II, neuropeptide Y, neurotensin, and 5-hydroxytryptamine as well as interleukins 1 and 2. Ovarian steroids can interfere with gonadotropin regulation by either direct effects on GnRH release or by increasing the sensitivity of anterior pituitary cells to GnRH, thus potentiating the release of pituitary hormones. These interactions involve discrete actions on second messenger systems; similar processes also account for brain and endocrine effects on immune cells.

Animals↗

PubMed1992

Resistance to somatostatin (SRIH) analog therapy in acromegaly. Re-evaluation of the correlation between the SRIH receptor status of the pituitary tumor and the in vivo inhibition of GH secretion in response to SRIH analog.

The development of a long-acting somatostatin (SRIH) analog (octreotide, Sandoz) has been a major breakthrough in the treatment of acromegaly. However, in 20-30% of the patients, growth hormone (GH) plasma levels remain elevated (> 10 micrograms/l) despite treatment with octreotide. This raised the concept of resistance to SRIH analog therapy in acromegaly. Indeed, in vivo response to SRIH analogs varies greatly among acromegalic patients. According to the reviews in the literature and our own autoradiographic data, no direct correlation can be established between the GH response to octreotide and the number or affinity of the SRIH receptors located on the tumor. In our series a greater density of SRIH receptors is present on tumors from patients very sensitive to the SRIH agonist. A subset of patients resistant to octreotide could result from a very low density of SRIH receptor although this type of GH-secreting tumor constitutes certainly a rare case. A subset of GH-secreting pituitary tumors can be characterized by a mutation on the alpha subunit of the guanine nucleotide-dependent protein coupled to the stimulation of adenylate cyclase (G alpha s). This mutation results in a high basal adenylate cyclase activity and a low GHRH-stimulated activity. However, when the adenomas are separated according to their basal adenylate cyclase activity, SRIH is able to decrease cAMP levels in both types of tumor. In addition, in our series no direct correlation is observed between the SRIH inhibition of adenylate cyclase and the amount of SRIH-binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Acromegaly↗

PubMed1992

Luteinizing hormone-releasing hormone (LHRH)- and (hydroxyproline9)LHRH-stimulated human chorionic gonadotropin secretion from perifused first trimester placental cells.

(Hydroxyproline9)LHRH [(Hyp9)LHRH] has been isolated from human, sheep, rodent, and frog hypothalamus. (Hyp9)LHRH is the major LHRH moiety in fetal rat hypothalamus. This study compared 1) synthetic LHRH-stimulated hCG secretion from term trophoblast vs. first trimester placental cells and 2) the ability and specificity with which synthetic LHRH and (Hyp9)LHRH could stimulate hCG secretion from 8- to 12-week gestation placenta. Physically dissociated cells from multiple placentae were pooled, plated on microcarrier beads, and perifused in 1.5-ml chambers (1.5 x 10(6) cells/chamber). Effluent fractions were analyzed for hCG. Each chamber was its own control. Basal hCG secretion did not depend upon exogenous LHRH stimulation. The amplitude of LHRH-stimulated hCG pulses was greater from first trimester placental than term trophoblast cells (mean +/- SEM, 6.99 +/- 1.47 vs. 0.50 +/- 0.05 mIU/ml perifusate; peak minus basal; n = 4 chambers; P less than 0.01). LHRH and (Hyp9)LHRH (10(-9) M) increased hCG secretion from first trimester placental cells (5.02 +/- 1.29 vs. 8.64 +/- 1.61 and 4.36 +/- 0.58 vs. 7.44 +/- 1.01 mIU/ml; n = 15 and 9, respectively; P less than 0.01). At the concentrations used, LHRH and (Hyp9)LHRH seemed to stimulate hCG secretion equipotently (P greater than 0.05). Simultaneous perifusion with an LHRH antagonist, (Nal-Glu)LHRH blocked the hCG secretory response to LHRH or (Hyp9)LHRH (equimolar 10(-9) M concentrations; n = 5; P less than 0.05). (Nal-Glu)LHRH alone (10(-9) M) did not affect hCG secretion (n = 5; P greater than 0.05). The results suggested that first trimester placental cells are more responsive to LHRH than are term trophoblast cells. (Hyp9)LHRH is a potential physiological secretagogue of hCG.

Chorionic Gonadotropin↗

PubMed1992

[New data on a second endogenous molecular form of mammalian hypothalamic LHRH, (hydroxyproline 9) LHRH].

An endogenous hydroxylated form of LHRH, (Hyp) LHRH, is able to displace LHRH bound to pituitary membrane preparations. In parallel, it stimulates release of both LH and FSH from pituitary cells in primary culture. The potency ratio of (Hyp)LHRH is approximately 1:20 and 1:5 with respect to the native decapeptide when peptidasic degradation is or is not inhibited. This correlates with a greater resistance of (Hyp) LHRH towards enzymatic degradation; in contrast to LHRH, the C-terminal (residues 6 to 10) end of (Hyp) LHRH is not degraded and generates C-terminal fragments which account for 64% of the LHRH immunoreactivity in extrahypothalamic areas as the hippocampus. Besides its weak gonadotropin releasing activity and its action or its localization in peripheral organs (placenta, gonads), a major role of the hydroxylated decapeptide may thus be to serve as a precursor of smaller active fragments on targets other than pituitary receptors.

Animals↗

PubMed1991

Activation of the CD3/T cell receptor (TcR) complex or of protein kinase C potentiate adenylyl cyclase stimulation in a tumoral T cell line: involvement of two distinct intracellular pathways.

A monoclonal antibody (OKT3) directed against the T cell receptor (TcR)/CD3 molecular complex, as well as a protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate, PMA) were added to a culture of tumoral Jurkat T cells, in order to precise the sequence of intracellular signals leading to T cell activation. The experiments were performed in the presence or in absence of various stimulators of adenylate cyclase (AC) such as forskolin (FK), cholera toxin (CT) or prostaglandin E2 (PGE2). OKT3 increased inositol phosphate (IP) production; in parallel, it induced a slight accumulation of cAMP. The effect was markedly potentiated in presence of FK or CT, and to a lesser extent in the presence of PGE2. FK stimulated adenylate cyclase of Jurkat cell membranes, but the effect was not potentiated by OKT3, suggesting that potentiation of cAMP accumulation requires intact cells and is not mediated by direct receptor coupling. On the other hand, elevated cAMP accumulation induced a negative feedback on IP production. The effect of OKT3 on cAMP was mimicked by A23187, a Ca2+ ionophore, and abolished in the absence of extracellular Ca2+. PMA had the same effect as OKT3 on basal or FK- and CT-induced accumulation of cAMP. In contrast, it inhibited the PGE2 effect on the cyclic nucleotide. After desensitization of PKC by pretreatment with a high concentration of PMA, the phorbol ester was no longer effective. Under those conditions, facilitation by OKT3 of FK-induced accumulation of cAMP was preserved, whereas potentiation by the monoclonal antibody of the PGE2 stimulation of AC was even enhanced. The data indicate that cAMP accumulation indirectly elicited by phospholipase C activation is, at least partly, mediated by IP-dependent Ca2+ mobilization, while PKC is preferentially effective as an inhibitor of PGE2 stimulation.

Adenylyl Cyclases↗

PubMed1991

Characterization of pericellular [125I]Tyr0 DTrp8 somatostatin binding sites in the rat arcuate nucleus by a newly developed method: quantitative high-resolution light microscopic radioautography.

In the present work we characterized the kinetic properties of [125I]somatostatin pericellular binding sites in the arcuate nucleus of the hypothalamus of the rat by quantitative high-resolution light microscopic radioautography. In order to determine whether these pericellular binding sites corresponded to functional receptors, their properties were compared with those of previously well-characterized [125I]somatostatin binding sites present on neuronal processes on the same sections in the stratum radiatum of the CA1 of the hippocampus. Radiolabelled sections were analysed by densitometry using a Biocom image analysis system coupled with a Leitz orthoplan microscope. The linear relationship between optical densities and radioactive standards allowed us to quantitate [125I]somatostatin-specific binding. Binding was time- and temperature-dependent, and saturable and specific in the arcuate nucleus as in the CA1 of the hippocampus. Saturation experiments indicated a single receptor population of binding sites with KD values of 0.2 +/- 0.1 nM in the arcuate nucleus and 0.6 +/- 0.4 nM in the CA1. In both structures, displacement curves obtained with somatostatin 14 and somatostatin 28 were monophasic, but shallow, while the somatostatin analogue SMS 201-995 induced a biphasic displacement, suggesting two populations of binding sites. In both regions binding was GTP-dependent. Desaturation procedures (in vivo by cysteamine and in vitro by preincubating with GTP) resulted in an increase in the number of measurable binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Alpha 1-adrenergic receptor coupling with phospholipase-C is negatively regulated by protein kinase-C in primary cultures of hypothalamic neurons and glial cells.

In the present work we evaluated the interactions of adrenergic receptors with phospholipase-C (PLC) and protein kinase-C (PKC), using an in vitro system of hypothalamic neurons and astroglial cells in primary cultures. The study was performed on immature neurons after 7 days in vitro (7 Div), that is before synaptogenesis, as well as on mature cells (14 Div). Comparisons were made between neurons and glial cells at the corresponding developmental stages. Norepinephrine (NE) increased inositol phosphates (IPs) formation in a dose- and time-dependent manner. The NE effect was mediated by alpha 1-receptor (alpha 1R) and was observed in young cells before synaptogenesis as well as in mature neuronal cultures; its amplitude was enhanced during the latter stage of the neuronal development. The coupling of alpha 1R with PLC was partially sensitive to pertussis toxin treatment and did not implicate the activation of calcium voltage-dependent channels. Activation of PKC by 12-O-Tetradecanoylphorbol 13-acetate (TPA) inhibited in a time-dependent manner the NE-stimulated production of IPs in young and mature hypothalamic neurons; however, in PKC depleted cells NE-induced IPs formation remained unchanged. In hypothalamic astroglial cell cultures the adrenergic stimulus of IPs generation was also mediated by alpha 1R. The effect was observed at both developmental stages, with a greater response in 14 Div cultures, and was insensitive to pertussis toxin treatment. As in neurons, activation of PKC resulted in inhibition of NE-induced IPs formation. These data indicate that functional interrelation between alpha 1R, PLC, and PKC is already present in immature neurons and glial cells and progressively develops in culture.

Animals↗

PubMed1991

Involvement of protein kinase-C in the effect of angiotensin-II on adenosine 3',5'-monophosphate production in lactotroph cells.

When applied to rat anterior pituitary cells, angiotensin-II (AII) exerted two opposite effects on adenylate cyclase (AC) activity: a pertussis toxin (PTX)-sensitive inhibition of the enzyme with a maximal effect of -42 +/- 2% in crude cell membrane preparations, and, in contrast, a non-PTX-sensitive stimulation of cAMP production (maximal effect = 38 +/- 3%) in intact cells. The apparent affinity of both effects was equal to 1.8 nM. The stimulation of cAMP formation parallels the stimulation of PRL release. Under the same conditions, dopamine (DA) inhibited both membrane AC activity and cAMP formation in intact cells by a PTX-sensitive mechanism. After separation of pituitary cell types by sedimentation at unit gravity, the effects of AII and DA on intracellular cAMP and membrane AC activity coincided in the same fractions (those enriched in PRL cells). The stimulatory effect of AII on cAMP formation was about 5 times weaker than that of peptides positively coupled to AC as vasoactive intestinal peptide in total as well as in PRL-enriched cells. Since the AII receptor is also coupled to phospholipase-C (PLC) in a non-PTX-sensitive manner, we investigated whether protein kinase-C (PKC) could indirectly account for the positive effect of AII on cAMP formation. 12-O-Tetradecanolylphorbol 13-acetate (TPA), a stimulator of PKC was indeed able to increase intracellular cAMP; this effect was not additive with that of AII. conversely, application of the PKC inhibitors H7 [1-(5-isoquinolylsulfonyl)2-methyl-piperazine] and staurosporine or desensitization of PKC by long exposure of the cells to TPA abolished the cAMP response to TPA as well as that to AII. In addition, thyreoliberin, another activator of the PLC pathway, was able to stimulate cAMP formation in a PKC-dependent manner. DA inhibition of intracellular cAMP was not affected by any PKC inhibition. We conclude that in lactotroph cells, 1) the AII inhibitory coupling to AC observed in membrane preparations does not exist in intact cells, at least under basal conditions; and 2) the AII intracellular cAMP stimulation observed is not accounted for by a direct coupling with AC; it is due to a cross-talk of the PLC pathway mediated by PKC, an effect that might be shared by other PLC-stimulating mediators and may participate in the regulation of PRL release.

Adenylyl Cyclases↗

PubMed1990

Functional maturation of somatostatin neurons and somatostatin receptors during development of mouse hypothalamus in vivo and in vitro.

Ontogenesis of somatostatin (SRIF) neurons and receptors was studied in fetal hypothalamic cell cultures kept in serum-free medium, and compared to the in vivo developmental pattern. Initial rise in neuronal content of SRIF occurred later in vitro than in vivo. In vitro, K(+)-induced SRIF release was only present after synaptogenesis. SRIF binding sites were measurable as early as 1 day after birth and at an equivalent time in culture, after 6 days in vitro (DIV); their affinity was in the nanomolar range. In cultured cells, binding reached a maximum at two weeks in vitro and decreased sharply thereafter as a consequence of binding site occupancy by the endogenous ligand. Indeed, pretreatment with cysteamine decreased SRIF concentration in the neuronal cultures and twice as many binding sites as in control cultures of 21 DIV were measured. Competition kinetics using unlabelled SMS 201-995 to displace [125I]SRIF revealed two distinct binding sites in the neuronal preparations (IC50 = 11 +/- 3 pM and 4.5 +/- 0.8 nM). In contrast, only the lower affinity site was present on glial cell preparations (1.7 +/- 0.4 nM). SRIF inhibited adenylate cyclase activity in glia and neurons, and the onset of SRIF coupling to the second messenger occurred earlier in vitro than in vivo. Pertussis toxin pretreatment was equally effective in neuronal and glial cell preparations to decrease SRIF binding and to inhibit adenylate cyclase activity.

Animals↗

PubMed1990

Dual mechanisms of inhibition by dopamine of basal and thyrotropin-releasing hormone-stimulated inositol phosphate production in anterior pituitary cells. Evidence for an inhibition not mediated by voltage-dependent Ca2+ channels.

In primary cultures of anterior pituitary cells, dopamine inhibited basal and thyrotropin-releasing hormone (TRH)-stimulated inositol monophosphate, bisphosphate, and trisphosphate production. This inhibition by dopamine can be resolved into two distinct components. One of the components was rapid and already present after 10 s. The other was slower, starting after 1 min, and was mimicked by nimodipine, a dihydropyridine calcium channel antagonist. The effects of dopamine and nimodipine were not additive on both basal and TRH-stimulated inositol phosphate production. Furthermore, the dopamine inhibition in the presence of TRH was much higher than the inhibition induced by nimodipine. It is thus likely that calcium entry through voltage-dependent calcium channels triggers a positive feedback on TRH stimulation of phospholipase C. However, depolarizing concentrations of K+ or BAY-K-8644, a voltage-dependent calcium channel agonist, had no effect on inositol monophosphate and bisphosphate accumulation. Ionomycin, even at a very high concentration (10 microM), had only a slight and transient effect on inositol phosphate formation. In addition, these agents did not affect the TRH dose-dependent stimulation of inositol phosphate production. These results suggest that the intracellular calcium concentrations that we measured under basal and TRH-stimulated conditions are sufficient to allow the maximal activity of phospholipase C which can be obtained under these two experimental conditions. In contrast, any decrease in the intracellular calcium concentration by a dihydropyridine antagonist, suppression of extracellular calcium, or inactivation of a voltage-dependent calcium channel by long term depolarization with K+ decreased the phospholipase C activities measured under basal and TRH-stimulated conditions. From these data it can be concluded that dopamine inhibits inositol phosphate production by two distinct mechanisms. The slow dopamine-induced inhibition of TRH-stimulated inositol phosphate production which is mimicked by nimodipine is likely because of an inhibition of a voltage-dependent calcium channel. This is substantiated further by the fact that ionomycin (10 microM) was able to reverse the nimodipine inhibitions as well as this slow component of dopamine inhibition. The nature of the rapid inhibition of TRH-stimulated inositol phosphate production induced by dopamine, but not by nimodipine, remains to be determined. It is suppressed in the absence of extracellular Ca2+. This may suggest that this inhibition is related to blockade of non-dihydropyridine-sensitive Ca2+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)

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