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Biomedical subjects

R P Millar

Publications and source records attributed to R P Millar.

At least 19 recordsLinked to original sources
PubMed1992

Activity of vertebrate gonadotropin-releasing hormones and analogs with variant amino acid residues in positions 5, 7 and 8 in the goldfish pituitary.

All non-mammalian vertebrates as well as marsupial mammals have two or more forms of gonadotropin-releasing hormone (GnRH) in the brain. Goldfish brain and pituitary contains two molecular forms of GnRH, salmon GnRH ([Trp7, Leu8]m-GnRH; s-GnRH) and chicken GnRH-II ([His5, Trp7, Tyr8]m-GnRH; cII-GnRH). Both sGnRH and cII-GnRH stimulate gonadotropin (GtH) as well as growth hormone (GH) release from the goldfish pituitary. The purpose of the present study was to study the activity of the five known forms of GnRHs as well as analogs of mammalian GnRH (m-GnRH) with variant amino acid residues in positions 5, 7 and 8 in terms of binding to GnRH receptors, and release of GTH and GH from the perifused fragments of goldfish pituitary in vitro. All five vertebrate GnRH peptides stimulated both GtH and GH release in a dose-dependent manner, although their potencies were very different. cII-GnRH was somewhat more active than s-GnRH in releasing GtH, whereas s-GnRH tended to have a greater potency than cII-GnRH in terms of GH release. Both chicken GnRH-I (cI-GnRH) and lamprey GnRH (l-GnRH) were significantly less potent than mGnRH, s-GnRH and cII-GnRH in releasing GtH and GH. cII-GnRH binds with higher affinity for the high affinity binding sites compared to all other native peptides. The activity of [Trp7]-GnRH was similar to both s-GnRH and cII-GnRH in releasing GtH and GH. Substitution of His5 resulted in a significant decrease in GtH releasing potencies compared to mGnRH, sGnRH and cII-GnRH. [His5]-GnRH also had lower GH releasing potency than mGnRH and sGnRH. Tyr8, His8 and Leu8 substitutions caused significant decreases in GtH releasing potencies compared to mGnRH, s-GnRH and cII-GnRH, but did not cause a significant change in GH releasing potency. The combination of [His5, Trp7]-GnRH had GtH and GH releasing activities similar to m-GnRH, s-GnRH and cII-GnRH.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1992

Regulation of corpus luteum function in the pouched mouse, Saccostomus campestris.

Regulation of corpus luteum function was studied in pouched mice, housed under controlled conditions (14L:10D), (22 +/- 2 degrees C). Prolactin in daily doses of 32 IU promoted luteal and uterine development and markedly increased plasma levels of progesterone in females exhibiting estrous cycles. Negligible levels of progesterone in prolactin-treated ovariectomized females ruled out the possibility of a significant nonovarian source of progesterone. Domperidone (a dopamine antagonist) induced pseudopregnancy in cycling females, with a marked increase of peripheral progesterone levels. Pseudopregnancy was also induced by artificial vaginal stimulation of cycling females according to a multiple intromission-multiple ejaculation pattern or a shorter, more concentrated stimulation pattern. The latter treatment, however, resulted in a much weaker histological response of the corpora lutea and uterine horns, although plasma levels of progesterone were similar in the two treatment groups. Recently mated females given domperidone daily did not have progesterone levels above those of mated females treated with vehicle only (both groups pregnant). Bromocriptine (a dopamine agonist) prevented newly mated females from becoming pregnant and interrupted pregnancy when given from Day 6 onward. The progesterone level in these females was lowered. A GnRH-antagonist given to ovariectomized females significantly suppressed LH levels, but intact females given artificial vaginal stimulation and treated with an GnRH antagonist became pseudopregnant. These findings indicate that, in the pouched mouse, as in many other rodent species, pseudopregnancy and progesterone production are dependent on prolactin and not on LH.

Animals↗
PubMed1992

Involvement of pertussis toxin-sensitive and -insensitive GTP-binding proteins in luteinizing hormone exocytosis distal to second messenger generation.

Inhibition of luteinizing hormone (LH) exocytosis by guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) in permeabilized pituitary cells has indicated the involvement of one or more GTP-binding proteins in the exocytotic mechanism distal to second messenger generation. We now report that two inhibitory sites of action of GTP gamma S can be distinguished by their dependence on GTP gamma S concentration and their sensitivity to pertussis toxin. Ca(2+)-stimulated exocytosis was half-maximally inhibited by 6.8 microM GTP gamma S, a six-fold higher concentration than that required for inhibition of exocytosis stimulated by phorbol ester plus cAMP. In addition, GTP gamma S inhibition of Ca(2+)-stimulated exocytosis was insensitive to pertussis toxin, in contrast to the inhibition of exocytosis stimulated by phorbol ester plus cAMP, which was abolished by pretreatment with pertussis toxin. These results indicate that at least two stimulus-specific GTP-binding proteins are involved in regulating LH exocytosis distal to second messenger generation.

Animals↗
PubMed1992

Prepubertal increases in gonadotropin-releasing hormone mRNA, gonadotropin-releasing hormone precursor, and subsequent maturation of precursor processing in male rats.

Changes in gonadotropins and gonadal steroids during sexual maturation in rats and humans are well documented but little is known about hypothalamic gonadotropin-releasing hormone (GnRH) gene expression in relation to these events. This study measured hypothalamic proGnRH mRNA, GnRH precursor, and fully processed GnRH from postnatal day 8 until day 62 in male rats. GnRH precursor increased on day 22, reached a peak on day 24, declined on day 25 and returned to infantile levels by day 28. A secondary rise in precursor occurred at about day 40 when testosterone levels increased. GnRH mRNA increased on day 22 and remained elevated over the study period to day 26. GnRH increased on day 24 and remained at this level until a secondary rise occurred coincident with the testosterone rise at about day 40. The ratio of GnRH precursor to GnRH was high until day 24 and was low from day 26 onwards, reflecting a maturation of the processing enzyme system between these 2 d. Thus, an abrupt increase in GnRH gene transcription (mRNA) occurs early in juvenile male rats (day 22), well before the onset of puberty. An increase in GnRH precursor accompanies these early changes and this is followed by the maturation of processing as evidenced by the rapid decline of precursor and increase in GnRH from day 24 onward.

Age Factors↗
PubMed1992

Cloning and functional expression of a mouse gonadotropin-releasing hormone receptor.

GnRH plays a pivotal role in the reproductive system, and GnRH analogs have wide therapeutic applications ranging from the treatment of prostatic cancer to infertility. Determination of the predicted structure of the GnRH receptor (GnRHR) would illuminate the mechanisms of receptor activation and regulation and allow directed design of improved GnRH analogs. We report the cloning of a cDNA representing the mouse GnRHR and confirm its identity using Xenopus oocyte expression. Injection of sense RNA transcript leads to the expression of a functional, high affinity GnRHR. Expression of the GnRHR using gonadotrope cell line RNA, however, is blocked by an antisense oligonucleotide. In situ hybridization in the rat anterior pituitary reveals a characteristic GnRHR distribution. The nucleotide sequence encodes a 327-amino acid protein which has the seven putative transmembrane domains characteristic of G protein-coupled receptors, but which lacks a typical intracellular C-terminus. The unusual structure and novel potential regulatory domain of the GnRHR may explain unique aspects of its signal transduction and regulation.

Amino Acid Sequence↗
PubMed1992

Arachidonic acid-induced LH release is ATP-independent and insensitive to N-ethyl maleimide.

The mechanism of arachidonic acid (AA)-induced LH release was characterized using sheep pituitary cells in primary culture permeabilized with Staphylococcal alpha-toxin. In intact cells, exogenous AA evoked release of LH in a manner which was partially dependent on extracellular Ca2+. At similar concentrations, AA also caused cell permeabilization as monitored by efflux of [3H]2-deoxyglucose metabolites. In alpha-toxin-permeabilized cells where cytosolic Ca2+ was clamped at resting levels, AA retained its ability to cause LH release. Unlike the stimulation of exocytosis produced by Ca2+, phorbol ester or cyclic AMP, AA-evoked release was independent of ATP and was not inhibited by pretreatment with N-ethyl maleimide. These findings indicated that exogenous AA does not cause LH release by Ca2+ influx or mobilization or by activating protein kinase C. The results suggest that LH release induced by exogenous AA is probably due to its detergent-like properties, and does not represent true exocytosis.

Adenosine Triphosphate↗
PubMed1991

Inhibition of luteinizing-hormone exocytosis by guanosine 5'-[gamma-thio]triphosphate reveals involvement of a GTP-binding protein distal to second-messenger generation.

Dual inhibitory and stimulatory actions of guanine nucleotides on luteinizing-hormone (LH) exocytosis were observed in primary sheep gonadotropes permeabilized with staphylococcal alpha-toxin. At resting cytosolic [Ca2+]free (pCa 7), 5'-[gamma-thio]triphosphate (GTP[S]) and guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) stimulated rapid LH exocytosis, which was maximal between 5 and 10 min. GTP[S] and p[NH]ppG had similar potencies (50% of maximum effect at 20-50 microM), but the effect of p[NH]ppG was more prolonged. Experiments carried out in the presence of saturating concentrations of phorbol 12-myristate 13-acetate (PMA), or in PMA-desensitized cells, suggested that stimulation by p[NH]ppG is mediated by a mechanism additional to protein kinase C (PKC) activation. Furthermore, p[NH]ppG stimulated LH exocytosis in the presence of saturating cyclic AMP (cAMP) concentrations, although its effect was less than additive. However, when both PMA and cAMP were present, p[NH]ppG did not stimulate a further increase in the rate of LH exocytosis. In contrast, pretreatment of cells with GTP[S] at low [Ca2+]free markedly inhibited subsequent responses to Ca2+, cAMP, PMA, and cAMP plus PMA. This inhibitory effect required lower GTP[S] concentrations than the stimulatory effect (50% inhibition at 1-10 microM), and was not observed with p[NH]ppG. A similar inhibition was observed with adenosine 5'-[gamma-thio]triphosphate, probably by its conversion into GTP[S]. These results suggest that the stimulatory actions of guanine nucleotides can be accounted for by the combined activation of PKC and generation of cAMP, resulting from activation of conventional signal-transducing GTP-binding proteins. The inhibitory effect of GTP[S] can be clearly distinguished and indicates the involvement of a distinct GTP-binding protein in exocytosis at a site distal to second-messenger generation.

1-Methyl-3-isobutylxanthine↗
PubMed1991

HLA class II induction by interferon-gamma in K562 variant cell line: inhibition by serum lipid.

The induction of class II antigen (Ag) by interferon-gamma (IFN-gamma) in variants of the K562 cell line has been examined in this study. Following incubation of K562A cells with IFN-gamma, surface expression of HLA-DR molecules was demonstrated by indirect immunofluorescence, confirmed by immunoblotting, and HLA-DR3 Ag specificity identified. HLA-DP and HLA-DRW52 Ag were co-expressed, but no HLA-DQ expression occurred. A variant of this line, designated K1A, spontaneously developed resistance to IFN-gamma induction of class II Ag, but continued to express class I Ag. In a third K562 variant, designated K562B, no class II gene products could be induced. Northern blot analysis indicated that mRNA levels correlated with surface class I and II Ag expression in all of the K562 cell lines. Resistance to IFN-gamma inducible class II Ag expression in K1A cells did not involve changes in IFN-gamma receptor affinity or number but was shown to be due to an inhibitory effect of serum lipid. These results indicate that cells derived from a common parental line may differ in susceptibility to a regulatory mechanism affecting IFN-gamma inducible class II Ag expression.

Blotting, Northern↗
PubMed1991

Patterns of Leydig cell and LH gonadotroph activity, and plasma testosterone concentrations in the seasonally reproducing Schreibers' long-fingered bat (Miniopterus schreibersii).

Spermatogenesis in Schreibers' long-fingered bat from approximately 33 degrees S in South Africa was seasonal, and occurred in the 3 months (February-April) preceding ovulation. The ultrastructure of the Leydig cells indicated a period of increased steroidogenesis at this time, and plasma testosterone concentrations were elevated from March to May (10.3 ng/ml). The reproductive accessory glands were secretorily active between March and May, and copulation occurred at the end of this period of activity. Changes in LH-beta immunoreactivity suggest that the LH gonadotrophs were secretorily active 1 month before the onset of spermatogenesis and that peak activity coincided with peak plasma testosterone concentrations, spermiogenesis and spermiation. During winter (May-August) there was no reproductive activity and the bats remained active, only entering prolonged periods of torpor during particularly cold spells. A secondary elevation in plasma testosterone concentration, during reproductive inactivity (October; 9.3 ng/ml), was not accompanied by any change in Leydig cell ultrastructure, and the biological significance of this peak is unknown. Such synchronous activity of the pituitary, Leydig cells, seminiferous epithelium and accessory glands is associated with the typical reproductive cycle of long-fingered bats in which copulation and fertilization are restricted to a brief period at the end of summer, and in which neither sperm storage nor a prolonged period of copulation occur.

Animals↗
PubMed1991

Plasma progesterone and luteinizing hormone concentrations and the role of the corpus luteum and LH gonadotrophs in the control of delayed implantation in Schreibers' long-fingered bat (Miniopterus schreibersii).

In Schreibers' long-fingered bat from South Africa (approximately 33 degrees S) copulation, ovulation and fertilization occurred in April and May, implantation was delayed until August, and parturition occurred in December. Delayed implantation coincided with winter, during which the bats remained active, only entering prolonged periods of torpor during particularly cold spells. Plasma progesterone concentration was low during non-pregnancy (1.54 ng/ml) and during delayed implantation (1.67 ng/ml), and thereafter increased to reach a peak mean of 64.82 ng/ml in late pregnancy. Changes in size and ultrastructure of the luteal cells indicated periods of steroidogenesis just after formation of the corpus luteum, and for about 2 months after implantation; reduced steroidogenic activity during delayed implantation; and luteolysis in the last 2 months of pregnancy. Plasma luteinizing hormone (LH) concentration and pituitary LH-beta immunoreactivity were highest during follicular development and peaked just before ovulation. During early delayed implantation, plasma LH concentration was low, and both plasma LH and pituitary LH-beta immunoreactivity increased from July, reaching peaks in late pregnancy. LH may be required to activate the corpus luteum and terminate delayed implantation, or, as in some small carnivores, it may be required for luteal maintenance.

Animals↗
PubMed1990

Cyclic AMP stimulates luteinizing-hormone (lutropin) exocytosis in permeabilized sheep anterior-pituitary cells. Synergism with protein kinase C and calcium.

Sheep anterior-pituitary cells permeabilized with Staphylococcus aureus alpha-toxin were used to investigate the role of cyclic AMP (cAMP) in exocytosis of luteinizing hormone (lutropin, LH) under conditions where the intracellular free Ca2+ concentration ([Ca2+]free) is clamped by Ca2+ buffers. At resting [Ca2+]free (pCa 7), cAMP rapidly stimulated LH exocytosis (within 5 min) and continued to stimulate exocytosis for at least 30 min. When cAMP breakdown was inhibited by 3-isobutyl-1-methylxanthine (IBMX), the concentration giving half-maximal response (EC50) for cAMP-stimulated exocytosis was 10 microM. cAMP-stimulated exocytosis required millimolar concentrations of MgATP, as has been found with Ca2(+)- and phorbol-ester-stimulated LH exocytosis. cAMP caused a modest enhancement of Ca2(+)-stimulated LH exocytosis by decreasing in the EC50 for Ca2+ from pCa 5.6 to pCa 5.9, but had little effect on the maximal LH response to Ca2+. Activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) dramatically enhanced cAMP-stimulated LH exocytosis by both increasing the maximal effect 5-7-fold and decreasing the EC50 for cAMP to 3 microM. This synergism between cAMP and PMA was further augmented by increasing the [Ca2+]free. Gonadotropin-releasing hormone (gonadoliberin, GnRH) stimulated cAMP production in intact pituitary cells. Since GnRH stimulation is reported to activate PKC and increase the intracellular [Ca2+]free, our results suggest that a synergistic interaction of the cAMP, PKC and Ca2+ second-messenger systems is of importance in the mechanism of GnRH-stimulated LH exocytosis.

1-Methyl-3-isobutylxanthine↗
PubMed1990

Staurosporine enhances gonadotrophin-releasing hormone-stimulated luteinizing hormone secretion.

In intact sheep gonadotropes, the protein kinase inhibitor, staurosporine, inhibited the stimulatory effect of phorbol 12-myristate 13-acetate (PMA) on luteinizing hormone (LH) secretion. Under the same conditions staurosporine enhanced gonadotrophin-releasing hormone (GnRH)-stimulated LH exocytosis without altering the EC50 of GnRH and without affecting basal LH exocytosis. These results suggest that PKC does not play a major role in mediating acute GnRH-stimulated LH exocytosis. Furthermore, they demonstrate that staurosporine enhances GnRH stimulus-secretion coupling. Both extracellular Ca2(+)-dependent and Ca2(+)-independent components of GnRH-stimulated LH secretion were enhanced by the drug. Staurosporine had no effect on GnRH stimulation of cAMP and inositol phosphate synthesis. In permeabilized cells staurosporine did not enhance Ca2(+)- and cAMP-stimulated LH exocytosis. Based on these results we hypothesize that staurosporine inhibits a protein kinase which is activated by GnRH and which negatively modulates GnRH stimulus-secretion coupling.

Alkaloids↗
PubMed1990

Calcium stimulates luteinizing-hormone (lutropin) exocytosis by a mechanism independent of protein kinase C.

Using permeabilized gonadotropes, we examined whether Ca2(+)-stimulated luteinizing-hormone (LH) exocytosis is mediated by the Ca2(+)-activated phospholipid-dependent protein kinase (protein kinase C). In the presence of high [Ca2+]free (pCa 5), alpha-toxin-permeabilized sheep gonadotropes secrete a burst of LH and then become refractory to maintained high [Ca2+]free. The protein kinase C activator phorbol myristate acetate (PMA) is able to stimulate further LH release from cells made refractory to high [Ca2+]free, suggesting that Ca2+ does not stimulate LH release by activating protein kinase C. Staurosporine, a protein kinase C inhibitor, inhibited PMA-stimulated (50% inhibition at 20 nM), but not Ca2(+)-stimulated, LH exocytosis. In cells desensitized to PMA by prolonged exposure to a high PMA concentration, Ca2(+)-stimulated LH exocytosis (when corrected for depletion of total cellular LH) was not inhibited. Ba2+ was able to stimulate LH exocytosis to a maximal extent similar to Ca2+, although higher Ba2+ concentrations were necessary. Ba2+ and Ca2+ stimulated LH exocytosis with a similar time course, and both were inhibitory at high concentrations. Furthermore, cells made refractory to Ca2+ were also refractory to Ba2+. These data strongly suggest that Ba2+ and Ca2+ act through the same mechanism. Since Ba2+ is a poor activator of protein kinase C, these findings are additional evidence against a major role for protein kinase C in mediating Ca2(+)-stimulated LH exocytosis.

Alkaloids↗
PubMed1990

Effects of GnRH-associated peptide and its component peptides on prolactin secretion from the tilapia pituitary in vitro.

The rostral pars distalis (RPD), containing mainly prolactin (PRL)-secreting cells, of the pituitary from immature and mature tilapia was incubated for 16 hr at 27 degrees in hypoosmotic medium (300 mOsm/kg) in the presence (10(-8) and 10(-11) M) or absence of the human GnRH-associated peptide (GAP) molecule, a potent PRL-inhibiting factor in mammals (Nikolics et al., Nature (London) 316, 511, 1985), and of a series of its component peptides. The release of the two forms of PRL in tilapia into the medium was measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by densitometry. The variability inherent in this method was normalized by calculating PRL release as the percentage of the total hormone present in both tissue and medium. Newly synthesized PRL was detected by incorporation of [35S]methionine, introduced into the culture medium, by the PRL molecules. In immature tilapia, GAP inhibited the release of total PRL while stimulating the release of newly synthesized large PRL. Among the GAP fragments tested, 28-36 was the fragment that most significantly affected PRL secretion. Both concentrations of fragment 28-36 stimulated the release of newly synthesized PRL from immature rostral pars distalis (RPDs). This stimulation appears to be dependent on the osmotic pressure of the medium since this fragment did not affect PRL secretion in hyperosmotic medium (340 mOsm/kg). Fragment 38-49 inhibited total PRL release from mature RPDs. Fragment 51-66 stimulated the release of total PRL from mature RPDs. Examination of tissue and medium values in densitometric units after incubation with fragments 28-36 and 51-66 indicated that while the tissue content of PRL was decreased, the medium content of PRL was not affected. This suggests that fragments 28-36 and 51-66, in opposition to the situation found when the data are expressed as percentage release of PRL, may not stimulate PRL release but may instead decrease the tissue content of PRL. These results suggest that the entire human GAP molecule, as well as some of its fragments, may have direct effects on the PRL cells in the tilapia pituitary.

Animals↗
PubMed1990

Selective FSH-releasing activity of [D-Trp9]GAP1-13: comparison with gonadotropin-releasing abilities of analogs of GAP and natural LHRHs.

We had previously shown that fragments of human gonadotropin-releasing hormone associated peptide (GAP) stimulated FSH and LH release in vivo. In particular, GAP1-13 had a preferential FSH-releasing activity. To decrease enzymatic degradation, analogs of GAP1-13 with D-amino acid substitutions were synthesized. The activities were tested in ovariectomized, estrogen-progesterone primed (OEP) rats and compared with those of GAP1-13, mammalian (m), chicken II (cII), and lamprey (1) LHRH. The peptides were injected (IV) into conscious, OEP rats and blood samples were obtained via the jugular catheter. [D-Trp9 )GAP1-13 selectively stimulated FSH release at a dose of 1 microgram. Multiple injections of this analog (10 micrograms every 30 min for 5 injections) induced a marked elevation of plasma FSH values which peaked (p less than 0.001) after the third injection. By contrast, [D-Trp9]GAP1-13 had no effect on LH and prolactin (PRL) release after either single or multiple injections. These doses of [D-Ala4]GAP1-13 had no effect on the release of FSH, LH or PRL. Both human GAP1-13 and its [D-Trp9] analog exerted a selective FSH-releasing effect at a dose of 10 micrograms, however, the [D-Trp9] analog was more potent than GAP1-13 on FSH release. The potency of [D-Trp9]GAP1-13 in releasing FSH was approximately 1/100th that of mLHRH. Chicken II LHRH had slightly selective FSH-releasing activity with a potency 1/10th that of mLHRH. Lamprey LHRH had a preferential LH-releasing activity and a potency 1000 times less than mLHRH. In conclusion. [D-Trp9]GAP1-13 is a selective FSH-releasing peptide of potential clinical value.

Animals↗
PubMed1990

Light and electron microscopic immunocytochemical analysis of antibodies directed against GnRH and its precursor in hypothalamic neurons.

A battery of antibodies directed against different portions of the precursor to gonadotropin-releasing hormone (GnRH), as well as to the mature decapeptide, were characterized immunocytochemically in two ways. Absorption experiments were used to determine the epitope recognized by each antiserum. Electron microscopic immunocytochemistry was then used to define the subcellular organelles that contained reaction product when tissue was incubated with these reagents. These latter observations helped to determine if the antibody recognized the epitope as part of the intact precursor or only after it had been cleaved from parent protein. Our results demonstrate that the GnRH precursor is routed from the rough endoplasmic reticulum through the Golgi apparatus to the secretory vesicles. Furthermore, we show that initial cleavage and processing of the GnRH precursor begin in the cell soma. These antibodies should be useful in the future in determining changes in processing of precursor in animals that differ in endocrine function.

Amino Acid Sequence↗
PubMed1990

A novel extracellular nucleotide receptor coupled to phosphoinositidase-C in pituitary cells.

In primary cultures of sheep pituitary cells extracellular nucleotides stimulated rapid increases in inositol tris- and bisphosphate, accompanied by intracellular Ca2+ mobilization. A similar stimulation of inositol phosphate production by extracellular nucleotides was observed in rat and baboon pituitary cells. The inositol phosphate response to nucleotides was greater than that elicited by any of the known hypothalamic releasing peptides. UTP, ATP, and ATP gamma S were the most potent agonists, with EC50 values for inositol phosphate production of 1.2, 2.6, and 2.7 microM. The relative potencies of a range of nucleotides indicates that the pharmacological specificity of the pituitary nucleotide receptor is different from that of the previously characterized P2X and P2Y purinoceptors present in other tissues. Increasing extracellular Mg2+ concentrations caused a shift to the right of the ATP dose-response curves, indicating that the predominantly active agonist species is not MgATP and may be ATP4-. In the absence of both Ca2+ and Mg2+ (1 mM EDTA) ATP stimulated inositol phosphate production with high potency (EC50 = 200 nM), indicating that an ectokinase or ecto-ATPase reaction is not involved in its mode of action. Phosphoinositidase-C activation by ATP was insensitive to pertussis toxin. The magnitude of the inositol phosphate and 45Ca2+ responses to extracellular nucleotides indicates that a substantial fraction of the cells in primary pituitary cultures bears nucleotide receptors. None of the major pituitary hormones appear to be released by extracellular nucleotides. The cell types in the pituitary that bear these nucleotide receptors are at present unidentified.

Adenosine Triphosphate↗