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M Wallis

Publications and source records attributed to M Wallis.

At least 55 records · Page 3Linked to original sources
PubMed1989

Effects of pretreatment with tetradecanoyl phorbol acetate on regulation of growth hormone and prolactin secretion from ovine anterior pituitary cells.

Tetradecanoyl phorbol acetate (TPA) stimulates growth hormone (GH) and prolactin secretion from ovine anterior pituitary cells. Pretreatment of the cells with TPA abolishes this effect, presumably due to down-regulation of protein kinase C. Such pretreatment did not alter effects of thyrotropin-releasing hormone or dopamine on prolactin secretion, suggesting no involvement of protein kinase C. Pretreatment with TPA attenuated actions of GH-releasing hormone on GH release (but not actions on cyclic AMP levels), possibly due to depletion of cellular stores of GH. Such pretreatment also attenuated inhibition of GH release by somatostatin, possibly due to phosphorylation of receptors or associated proteins by protein kinase C.

Animals↗
PubMed1989

Cloning, sequence and expression in Escherichia coli of cDNA for ovine pregrowth hormone.

cDNA prepared from mRNA from ovine anterior pituitary glands was cloned in Escherichia coli and the sequence of a clone encoding the full coding sequence of ovine pregrowth hormone (preGH) determined. The predicted sequence for ovine GH agrees with that determined previously on the protein, except that residue 99 is asparagine rather than aspartic acid. The cDNA sequence also accords with one of the two genomic sequences for the ovine GH gene that have been reported. Expression plasmids using trp and lac promoters were constructed which allowed expression at low levels of ovine preGH in E. coli, as detected by immunoblotting and immunoassay.

Amino Acid Sequence↗
PubMed1989

Production of plasmids giving high expression of recombinant DNA-derived ovine growth hormone variants in Escherichia coli.

A method for the production of plasmids giving different levels of expression of ovine growth hormone (oGH) variants in E. coli is described. The cDNA sequence coding for mature oGH was inserted into the multiple cloning site of plasmid pUC8 and random deletions were then introduced 3' to the initiation codon. Clones producing GH (with varying N-terminal extensions) were identified by immunological screening. Levels of expression of GH-related protein, measured by immunoassay or on SDS-polyacrylamide gels, varied from over 20% to less than 0.05% of total cell protein. The coding sequence of plasmid pOGHe101, giving very high expression of variant oGH1, was determined.

Amino Acid Sequence↗
PubMed1989

Human growth hormone stimulates somatomedin C/insulin-like growth factor I production by the human lymphoid cell line, IM-9.

The human lymphoid cell line, IM-9, is known to possess receptors for human growth hormone (hGH), but the only biological response that has been shown to follow binding of this hormone to the cells is receptor down-regulation. We have studied the actions of hGH on production of insulin-like growth factor I (IGF-I) by IM-9 cells. In order to demonstrate effects cells had to be transferred to a serum-free medium in which cell multiplication almost ceased, and cell viability fell to 50-60%. hGH stimulated IGF-I production by up to 400%. The effect was dose-related, but the dose-response curve was bimodal, with peaks of activity at approximately 15 ng/ml and 1000 ng/ml hGH. The effect of hGH was of slow onset, becoming significant only after about 24 h, and approaching a maximum after 2-5 days of treatment. hGH had a much greater stimulatory effect than non-primate growth hormones. The physiological significance of the effect observed is not yet clear, but it is apparent that the IM-9 line is a potentially useful model for study of the actions of growth hormone.

Cell Line↗
PubMed1988

A comparison of lactogenic receptors from rat liver and Nb2 rat lymphoma cells by using cross-linking techniques.

Lactogenic receptors were analysed with the use of the cross-linking agent disuccinimidyl suberate to attach covalently 125I-labelled ovine prolactin or human growth hormone to binding sites from (1) liver from pregnant rats and (2) the rat-derived Nb2 lymphoma cell line. Analysis by SDS/polyacrylamide-gel electrophoresis of the proteins cross-linked to labelled hormone in rat liver indicated a major specifically-labelled complex with an Mr of 68,000-72,000, when run under reducing or non-reducing conditions. With Nb2 cells a major specifically-labelled complex with an Mr of 97,000-110,000 was identified, but only when electrophoresis was run using reducing conditions. Assuming one hormone molecule (Mr 22,000-24,000) per hormone-receptor complex, then the receptor proteins have an Mr of 44,000-50,000 for rat liver and 73,000-88,000 for the Nb2 cells. For both cell types the receptors were of lactogenic specificity; lactogenic hormones competed for binding whereas somatogenic hormones did not. These studies suggest that the lactogenic receptors in rat liver membranes and Nb2 cells differ in two respects. Firstly, the Mr of the labelled receptor protein in Nb2 cells is greater than that of the corresponding receptor protein in rat liver membranes; secondly, the Nb2 cell receptor appears to exist as a disulphide-linked oligomer whereas the receptor in rat liver membranes does not.

Animals↗
PubMed1988

The molecular basis of growth hormone deficiency.

A well-known law states that 'if a thing can go wrong it will go wrong'. This clearly applies to the hypothalamic-pituitary-somatic axis as to many other physiological and biochemical systems. Defects of this axis, giving rise to stunted growth, can occur at several different points, as has been discussed in detail in this review. Defects at the level of the brain can lead to inadequate production or secretion of the factors that control growth hormone secretion. Defects at the level of the pituitary can lead to failure to produce or secrete adequate quantities of growth hormone, or to production of inactive hormone. Defects at the level of target organs can lead to inability to respond to growth hormone or somatomedins. The axis involved in the production and effects of growth hormone is a complex one, and defects have been identified at most of the points that 'could go wrong', although in many cases the molecular details are far from fully understood. Increased understanding of the biochemistry and physiology of the hormonal control of growth, and of the impairments to which it is subject, should provide an improved basis for treatment of growth defects. Nevertheless, there remain many points at which our knowledge is very incomplete. The field is a rapidly moving one and further developments in both basic understanding and clinical treatment are to be expected during the next few years.

Animals↗
PubMed1988

Mechanisms involved in the effects of TRH on GHRH-stimulated growth hormone release from ovine and bovine pituitary cells.

Incubation of cultured ovine pituitary cells with growth hormone-releasing hormone (GHRH) (10(-12)-10(-7) M) stimulated growth hormone secretion up to 3-fold. At a maximal stimulatory concentration of GHRH (10(-10) M), thyrotropin-releasing hormone (TRH) (10(-7) M) caused an inhibition of growth hormone release to approx. 50% of the response obtained with GHRH alone (during a 15 min incubation period). TRH also caused a small inhibition of the GHRH-stimulated cellular cyclic AMP level but this effect was only significant at a relatively high concentration of GHRH (10(-9) M). Incubation of cultured bovine pituitary cells with GHRH (10(-11)-10(-8) M) plus TRH (10(-7) M) caused a significant stimulation of growth hormone release by up to 40%, compared with the response obtained with GHRH alone (at all concentrations of GHRH). TRH (10(-7) M) had no effect on GHRH (10(-8) M)-stimulated cellular cyclic AMP levels in a partially purified bovine pituitary cell preparation. The effects of varying extracellular [Ca2+] (0.1-10 mM) on intracellular [Ca2+] and on the responsiveness to releasing hormones were also determined using ovine pituitary cells. GHRH (10(-10) M)-stimulated growth hormone release was inhibited when cells were incubated at both high (10 mM) and low (0.1 mM) [Ca2+] (compared with 1 mM or 3 mM Ca2+) with or without TRH (10(-7) M). At 1 mM Ca2+, TRH produced a synergistic effect with GHRH to stimulate growth hormone release. However, at 3 mM Ca2+ TRH inhibited GHRH-stimulated growth hormone release.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Regulation of growth hormone secretion and cyclic AMP metabolism in ovine pituitary cells: interactions involved in activation induced by growth hormone-releasing hormone and phorbol esters.

Growth hormone-releasing hormone (GHRH) and the phorbol ester tetradecanoylphorbol acetate (TPA) each stimulated a rapid and extensive (up to 15-fold) increase in the secretion of growth hormone from cultured ovine anterior pituitary cells. Effects of the releasing hormone on growth hormone secretion were associated with a concurrent, large increase in cellular cyclic AMP accumulation. TPA induced a much smaller (26-78%), though still significant, increase in cellular cyclic AMP levels. Forskolin and isobutylmethylxanthine (IBMX) also stimulated growth hormone secretion and cyclic AMP accumulation. When combined with a maximally effective concentration of GHRH these compounds did not further elevate growth hormone secretion even though they induced further increases in cyclic AMP concentration; this is consistent with activation occurring via a common cyclic AMP-dependent pathway. In contrast TPA when combined with maximally effective concentrations of either GHRH, forskolin or IBMX caused additional release of growth hormone, suggesting that the TPA-induced secretion involved a cyclic AMP-independent process. However, TPA also markedly potentiated the cellular cyclic AMP accumulation due to each of these agents. That TPA induced stimulation of basal and GHRH-stimulated cyclic AMP levels measured in the presence of IBMX suggests an action affecting cyclic AMP synthesis. Carbachol had no effect on basal or GHRH-stimulated growth hormone secretion or cyclic AMP levels. The two actions of TPA, one on secretion and one on cyclic AMP metabolism, may result from activation of some common event possibly involving protein kinase C. Our results suggest that GHRH and TPA activate independent pathways regulating growth hormone secretion.

1-Methyl-3-isobutylxanthine↗
PubMed1988

Actions of pertussis toxin on the inhibitory effects of dopamine and somatostatin on prolactin and growth hormone release from ovine anterior pituitary cells.

Forskolin and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate stimulate prolactin and GH release from ovine anterior pituitary cells cultured in vitro. Dopamine and somatostatin inhibit release of prolactin and GH respectively, after stimulation by these agents, but without effects on intracellular cyclic AMP concentrations. In each case the inhibitory effects were reversed by pretreatment of cells with pertussis toxin, in a dose-related fashion (1-100 ng/ml), again without affecting cyclic AMP levels. The results suggest that the inhibitory effects of dopamine and somatostatin in this system are mediated by one or more pertussis toxin-sensitive G proteins, and that these act by a mechanism which does not involve inhibition of adenylate cyclase.

Adenylate Cyclase Toxin↗
PubMed1987

Monoclonal antibodies to bovine growth hormone potentiate effects of the hormone on somatomedin C levels and growth of hypophysectomized rats.

Bovine growth hormone (50 micrograms/day) elevated somatomedin C levels in plasma of hypophysectomized rats: the effect was seen 12 h after the first injection and was sustained throughout a 7-day treatment period. When monoclonal antibodies to bovine growth hormone were administered with the hormone the stimulation of somatomedin C levels was markedly enhanced, as were effects on body weight. Each one of a panel of 7 monoclonal antibodies potentiated the actions of the hormone on growth and somatomedin C levels, though the extent and pattern of potentiation varied considerably from one antibody to another. Effects on growth and somatomedin C levels correlated fairly well, though there were some discrepancies.

Animals↗
PubMed1987

Heterogeneity of growth-hormone receptors detected with monoclonal antibodies to human growth hormone.

The specificity of hormone-receptor interactions has been examined with the aid of monoclonal antibodies (MABs) (EB1, EB2, QA68 and NA71) defining four non-overlapping antigenic determinants on human growth hormone (hGH). The results indicate that growth-hormone receptors in liver obtained from different sources differ with regard to their affinities and relative numbers; they may also differ with respect to the region of the growth-hormone molecule to which they bind. Antibody NA71 effectively inhibited hormone binding to all receptor preparations tested, although with various degrees of potency. Monoclonal antibody EB1 demonstrated a graded inhibition with respect to its ability to block 125I-hGH binding to receptors from various sources, the maximum inhibition being seen in receptor preparations from mouse and ovine liver and the minimum in rat liver. MABs EB2 and QA68 also showed various abilities to inhibit hormone-receptor interaction, depending on the origin of the receptor preparation. Furthermore, the receptor-binding characteristics of hormone-antibody complexes were dependent on whether the binding-site preparation was derived from pregnant, lactating or 'normal' animals. A particularly striking difference between the ability of hormone-MAB complexes to bind to receptors from different sources was seen for microsomes (microsomal fractions) derived from livers of animals of the 'Little' mouse strain. These animals become progressively obese, and it was shown that MABs were considerably more effective in inhibiting 125I-labelled hGH binding to microsomes from phenotypically obese mice than to those derived from their non-obese littermates. The results can be explained by the presence of multiple receptor types for GH, the relative proportions of which vary according to the physiological state of the animal, and possibly between species.

Animals↗
PubMed1987

In-vitro evidence for the autoregulation of prolactin secretion at the level of the pituitary gland in the rat.

The autoregulation of rat prolactin secretion at the level of the pituitary gland was investigated, using a static incubation system. The rate of prolactin secretion from the female anterior pituitary gland in vitro was found to be constant when the medium was changed at 20-min intervals. However, when the medium was left unchanged and secretory products were allowed to accumulate, prolactin secretion began to decline within 60 min. This effect was not observed with the male tissue, where the level of accumulated prolactin did not reach that at which the inhibition occurred using female tissue. The nature of the putative secretory product causing the inhibition of prolactin secretion was investigated. Exogenous bovine prolactin (1-4 mg/l) caused an inhibition of endogenous rat prolactin secretion. Inclusion of monoamine oxidase in unchanged medium, to prevent dopamine accumulation in the medium (a possible consequence of co-storage and co-secretion with prolactin granules), did not prevent the inhibition observed in the control incubation. We therefore conclude that in-vitro autoregulation of prolactin secretion can occur at the level of the pituitary gland, probably due to the accumulated prolactin having a feedback action on the lactotroph. This might be of physiological significance if localized concentrations of the hormone within the gland are high.

Animals↗
PubMed1987

Purification of ovine somatotrophs using a combination of density gradient centrifugation and short-term culture.

Freshly dispersed or cultured (18 h) ovine anterior pituitary cells were fractionated on 55% sigmoidal Percoll gradients. This resulted in the separation of two sub-populations of cells at densities of 1.075 g/ml (peak I) and 1.055 g/ml (peak II). Radioimmunoassay of fractions from a gradient on which cells were separated before culture showed the profiles of GH and prolactin to be virtually superimposable. After culture, however, the lactotroph population exhibited an apparent shift in its density profile, the lighter population increasing at the expense of the denser one. Immunocytochemistry of the cells remaining in the denser peak (peak I) showed that it consisted of about 65% somatotrophs which was approximately a three-fold enrichment over the proportion of somatotrophs present in the original cell preparation (24%). Refractionation studies indicated that the change in the density profile of the lactotrophs was due to an actual reduction in their density rather than a loss of viability of the denser sub-population. Secretion data showed the purified somatotrophs to be more responsive than the crude cell preparation to the regulatory factors, GH-releasing hormone and somatostatin. This enriched cell population should prove useful in the detailed study of GH secretion.

Animals↗
PubMed1986

Use of quin 2 to measure calcium concentrations in ovine anterior pituitary cells and the effects of quin 2 on secretion of growth hormone and prolactin.

Intracellular free Ca2+ concentrations [Ca2+]i were measured in ovine anterior pituitary cells using the quin 2 technique. Thyrotropin-releasing hormone (TRH) increased, dopamine decreased and growth hormone-releasing hormone (GHRH) had no detectable effect on [Ca2+]i. Loading the cells with quin 2, at an intracellular concentration less than that used during calcium determination, reduced both basal growth hormone (GH) and (to a small extent) prolactin secretion. Loading cells with quin 2 also markedly reduced GHRH-stimulated GH secretion. However, TRH-stimulated prolactin secretion was 3-times basal irrespective of quin 2 loading. The results indicate that the use of quin 2 to measure [Ca2+]i in some cell types may be complicated by actions of quin 2 on cellular function.

Aminoquinolines↗
PubMed1986

The specificity of binding of growth hormone and prolactin to purified plasma membranes from pregnant-rabbit liver.

The binding of 125I-labelled human growth hormone (hGH) to a purified plasma membrane preparation from the liver of pregnant rabbit, and to receptors solubilized from this fraction with Triton X-100, was dependent on time, temperature, the cations used and the receptor concentration. Solubilization did not affect the binding properties of the receptors at low concentrations of Triton X-100. Some somatogenic hormones, such as bovine GH, and some lactogenic hormones, such as ovine prolactin, displaced 125I-labelled hGH from purified plasma membranes and solubilized receptor preparations, but GHs and prolactins from various other species were rather ineffective. The results indicate that although there are binding sites for hGH in these pregnant rabbit liver membranes, few of these are specifically somatogenic or lactogenic. The binding properties of the purified plasma membranes are similar to those of a microsomal preparation studied previously, suggesting that the complex nature of the binding of hGH is not due to the heterogeneity of cellular membranes used to study binding, but is a property of the receptors associated with plasma membranes.

Animals↗
PubMed1986

Effects of dopamine and somatostatin on phorbol ester-stimulated prolactin and growth hormone secretion.

Incubation of cultured ovine pituitary cells with the tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) (0.1-100 nM), caused a dose-related stimulation of both growth hormone (ED50 approximately 4 nM) and prolactin (ED50 approximately 14 nM) secretion. Stimulation by TPA (100 nM) produced a substantial 10-fold increase in growth hormone with a smaller, 2-fold rise in prolactin secretion over 30 min; significant effects on the release of both hormones occurred within 2 min. Treatment with TPA also produced a small, time- and concentration-dependent rise in cellular cyclic AMP content which reached, at maximum, a level 20-30% over basal values. Non-tumor-promoting phorbol esters did not stimulate the secretion of either growth hormone or prolactin. In the presence of TPA (10 nM), dopamine (1-1000 nM) suppressed prolactin secretion to a level close to that observed for maximal inhibition of unstimulated cells. At high concentrations (0.1-1.0 microM) dopamine also partially attenuated (by 43%) the TPA-induced stimulation of growth hormone secretion. Somatostatin (0.01-1.0 microM) completely inhibited the substantial (approximately 9-fold) TPA-induced stimulation of growth hormone secretion (inhibitory ED50 approximately 47 nM), and also suppressed TPA-stimulated prolactin secretion to the control level. Our results suggest that activation of protein kinase-C may be involved in the stimulatory regulation of both growth hormone and prolactin secretion in sheep pituitary cells. Failure of TPA to attenuate the inhibitory activity of dopamine and somatostatin suggests that inhibitory regulation occurs at, or beyond, the point in the secretory process regulated by protein kinase-C.

Animals↗
PubMed1986

Dopamine and somatostatin inhibit forskolin-stimulated prolactin and growth hormone secretion but not stimulated cyclic AMP levels in sheep anterior pituitary cell cultures.

Forskolin, an activator of adenylate cyclase, has been used to investigate the effects of raising pituitary cell cyclic AMP concentrations on prolactin and growth hormone secretion and to examine the role of cyclic AMP in the inhibitory actions of dopamine and somatostatin. Incubation of cultured ovine pituitary cells with forskolin (0.1-10 microM; 30 min) produced a modest dose-related increase in prolactin release (120-140% of basal) but a much greater stimulation of growth hormone secretion (170-420% of basal). Cellular cyclic AMP concentrations were only increased in the presence of 1 and 10 microM forskolin (2-5.5 times basal). A study of the time course for forskolin (10 microM) action showed that stimulation of prolactin (1.5-fold) and growth hormone (4.7-fold) secretion occurred over 15 min; subsequently (15-60 min) the rate of prolactin secretion from forskolin-treated cells was equivalent to that measured in controls, while growth hormone release remained elevated. Cellular cyclic AMP concentrations were also rapidly stimulated by forskolin (10 microM); they reached a maximum (12 times control) within 15 min, and then declined (15-60 min) but remained elevated relative to those in untreated cells (4.9 times control at 60 min). Dopamine (0.1 microM) inhibited basal secretion of both prolactin and growth hormone. In the presence of forskolin (0.1-10 microM), dopamine (0.1 microM) inhibited prolactin secretion to below the basal level and considerably attenuated the stimulation of growth hormone secretion. Similarly, somatostatin suppressed both basal and forskolin-induced prolactin and growth hormone secretion. However, neither dopamine nor somatostatin significantly decreased the stimulatory effect of forskolin on cellular cyclic AMP accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗