Influence of kinetics of gastric emptying on hepatic IGF1 production in young calves.

Five preruminant calves, fitted with chronically indwelling catheters in the hepatic and portal veins and hepatic artery, were fed two kinds of diet (a conventional clotting milk diet and an incurdled milk diet) or fasted for 24 h. The statistical analysis of the plasma growth hormone (GH; nmol/l) or IGF1 (nmol/l) daily secretory profiles indicated that hormonal levels were very high in fed calves (GH mean values: 0.40 +/- 0.11 and 0.36 +/- 0.10, respectively; IGF1 mean values: 6.65 +/- 0.57 and 7.54 +/- 0.33, respectively). The GH secretory profile observed over a 24-hour period showed 7 secretory spikes without periodicity in both diets. In fasting animals, plasma GH and IGF1 concentrations were very low (0.17 +/- 0.08 and 3.08 +/- 0.36, respectively) and stable, they increased with refeeding (1.51 +/- 0.05 and 7.36 +/- 0.55, respectively). Hepatic IGF1 production (micrograms/kg body weight/day) was 195 +/- 7 in standard milk fed calves and -255 +/- 30 in others. Our results demonstrated that nutritional factors, such as different kinetics of gastric emptying or fasting, may influence hepatic IGF1 production in the conscious newborn calf.

Steroid hormone may modulate hepatic somatomedin C production in newborn calves.

We have studied the possible influence of steroid hormones and a beta-agonist (clembuterol) on hepatic insulin-like growth factor 1 (IGF1) production in young calves. For this purpose nine 20- to 40-day-old Holstein X Friesian male calves were fitted with chronically indwelling catheters in hepatic and portal veins and hepatic artery. Estradiol induced a simultaneous increase in plasma growth hormone (GH nmol/l) and IGF1 (nmol/l) levels (0.35 +/- 0.05 vs. 0.10 +/- 0.01 in control calves; 9.5 +/- 1.0 vs. 5.9 +/- 0.5 in controls, respectively). In the same way, 90 min after starting testosterone treatment, plasma GH levels increased from 0.21 +/- 0.08 to 1.30 +/- 0.40 while plasma IGF1 concentrations began to rise only 240 min after starting infusion (8.4 +/- 1.0) to reach maximal values at 300 min (10.7 +/- 1.1). Cortisol and clembuterol did not significantly modify either plasma GH levels or plasma IGF1 concentrations. Our results indicate that in young calves gonadal steroids exert their anabolic action through GH and IGF1.

Nutrient effects on the hepatic production of somatomedin C (IGF1) in the milk-fed calf.

The present study was aimed at determining the influence of nutrients supplied by a milk diet (glucose, amino acids, triglycerides) on hepatic somatomedin C (IGFi) production in vivo in four 30-d-old milk-fed calves fitted with chronically indwelling catheters in hepatic (HV), portal (PV) and mesenteric veins and in the hepatic artery (HA), and with electromagnetic flow-meters on HA and PV. Fasting for 16 h induced a decrease (P less than 0.01) in hepatic IGF1 production (nmol/kg body-weight (BW) for 6 h) (1.1 (SE 0.2) v. 6.6 (SE 0.7) in control animals). Infusion of glucose (1.8 g/kg BW for 4 h) or a mixture of amino acids (Azonutril; R. Bellon, Neuilly sur Seine; 62.5 mg nitrogen/kg BW for 3 h) in a mesenteric vein led to no significant effect on hepatic IGF1 production for 6 h (1.2 (SE 0.3) and 0.7 (SE 0.3) nmol/kg BW respectively) compared with fasted calves. Infusion of chylomicrons purified from milk-fed calves (10.5 mg/h per kg BW, i.e. 0.16 mg triglycerides/kg BW per min) enhanced significantly (P less than 0.01) the hepatic production of IGF1 (mean value for 6 h: 5.3 (SE 0.8) nmol/kg BW). Infusion of Intralipid (7 mg triglycerides/kg BW per min) induced a slight but significant hepatic IGF1 production which amounted to 3.5 (SE 0.4) nmol/kg BW (P less than 0.1 compared with chylomicron treatment) and it began only 5 h after starting the infusion. Neither triglyceride nor chylomicron infusion significantly modified hepatic blood flow. Thus, these results demonstrate for the first time the role of lipids in the regulation of hepatic IGF1 production in vivo.

Systemic bone growth factors concentrations in calves during the perinatal period.

Plasma somatostatin (SRIF), growth hormone (GH), somatomedin C (IGF1), osteocalcin (BGP), 1,25-dihydroxyvitamin D (1,25-(OH)2D), calcium and inorganic phosphorus were measured in 10 chronically-catheterized fetal calves and in their dams during the two last months of gestation. Thus fetal life is associated with high levels of GH (1.53 +/- 0.14 nmol.l-1), BGP (64 +/- 4 nmol), Ca (2.90 +/- 0.06 nmol.l-1) compared to the results obtained in the pregnant cows. The first week of postnatal life was associated with a tremendous increase in plasma SRIF concentration (from 36 +/- 5 to 106 +/- 15 pmol.l-1; P less than 0.01). These results agree with an intense bone growth during the end of fetal life in the bovine species. However, IG 1 might not play a major role in the regulation of fetal skeletal growth during this period.

Excessive growth in a child with craniopharyngioma and growth hormone deficiency.

In a 5-year-old boy presenting with clumsiness and excessive growth, a large craniopharyngioma was diagnosed. Biochemically, there was a deficiency of growth hormone, a hypothalamic hypothyroidism and hypocorticalism, a thyroxine binding globulin elevation, an abnormal gonadotropin secretion and a mild hyperprolactinaemia. After removal of the tumour growth stopped almost completely. Plasma insulin-like growth factor (IGF)-I was in the lower normal range. Plasma IGF-II decreased after tumour removal. It is speculated that the tumour produced a growth factor causing excessive growth.

Regulation of growth hormone release in fetal calves.

Plasma GH and IGF1 concentrations were measured during the last 2 months of gestation in 9 chronically catheterized fetal calves under basal conditions or following growth-hormone-releasing factor (GRF), thyrotropin-releasing hormone (TRH) or SRIF intravenous cotyledonnary injections. Plasma GH concentrations were higher in fetuses (1.40 +/- 0.10 nmol/l) than in dams (0.14 +/- 0.01 nmol/l). Plasma GH secretory profile was pulsatile. The number of secretory pulses, as well as their magnitude and mean baseline values decreased from 220 to 270 days of gestation. Synthetic 1-29 GRF or TRH increased fetal plasma GH concentration at 250 and 270 days of gestation but was devoid of any significant effect at 220 days. SRIF injection decreased plasma GH concentration in 270-day-old fetuses. Plasma IGF1 concentrations were lower in fetuses than in dams. No treatment had a significant effect on fetal and maternal IGF1 levels.

Plasma somatotropin and somatomedin C concentrations following GRF or TRH injections in newborn calves.

Plasma somatotropin (GH) and somatomedin C (IGF1) concentrations were measured by radioimmunoassay in 3-dy old and 10-day old calves intravenously injected with growth hormone releasing factor (GRF) 1-44, GRF 1-29 or thyrotropin-releasing hormone (TRH). In 3 day old animals the increase in plasma GH concentration was GRF 1-44 dose-related (50, 100, 200 pmoles . kg(-1) of body wt). In four 10-day old calves injected with the lowest dose, the increase in plasma GH concentration was not different from that observed in four 3-day old animals treated with 200 pmoles . kg-1 of body weight. However, the response observed in four 3-day old calves injected with GRF 1-29 (50 pmoles . kg-1 of body wt) was not different from that observed following the same treatment in four 10-day old calves. In four 3-day old calves TRH (10 nmoles . kg-1 of body wt) induced a significant rise in plasma GH, prolactin (Prl), thyroxine (T4) and triiodothyronine (T3) concentrations. The same dose of TRH injected into four 10-day old calves elicited a similar rise in plasma T3 and T4 concentrations, but plasma GH and Prl increased less than in 3-day old animals. In three 3-day old or 10-day old calves born spontaneously before term (258-260 days of gestation), the increase in plasma Prl and GH concentrations following TRH was not different from that observed in mature calves of the same postnatal age. Neither GRF 1-44, GR 1-29 nor TRH elicited any significant change in plasma IGF1, insulin or glucose concentration in any group of calves.

Characterization of specific receptors for leukotriene D4 on human alveolar macrophages.

Using 3H-leukotriene D4, a specific receptor assay has been developed for human alveolar macrophages, obtained by broncho-alveolar lavage of patients undergoing fiberoptic bronchoscopy because of suspected bronchial carcinoma. Lavage was performed in a carcinoma-free lobe of the lung and alveolar macrophages were subsequently isolated and incubated for binding studies. 3H-Leukotriene D4 was found to bind specifically with high affinity (Kd = 3.8 nM), in a saturable manner (Bmax = 90 fmol/10(6) cells), reversible and selective. Specific binding was linear with protein concentration and equilibrium binding at 4 degrees C was reached at 50 min. Scatchard and Hill analysis revealed a single class of binding sites with no cooperativity among the sites. Displacement studies with LTD4, the selective SRS-A antagonist FPL 55712 and with leukotriene C4 revealed respective Ki values of 3.4; 16; and 110 nM. The data suggest that human alveolar macrophages may contain a specific receptor type for LTD4, which has a relatively low affinity for LTC4, and are discussed in relation to modulatory processes in the lung, apart from direct actions of LTD4 on smooth muscle receptors. From the data here acquired, it may be apparent that the study of characteristics of receptors specific for a broncho-active substance like LTD4 on human alveolar macrophages, which play an important role in immuno-inflammatory processes seen in many chronic lung diseases, may yield major insights into the pathogenesis and therapy decisions involved in these diseases.

Regulation of prostaglandin E2 receptors in vivo by dietary fatty acids in peritoneal macrophages from rats.

Groups of rats were pretreated with 4-week diets containing 12.5% corn oil or linseed oil. At the end of this period peritoneal macrophages were elicited and isolated. These cells were used for binding experiments with 3H-PGE2 and for estimation of prostaglandin-stimulated cAMP production. Specific binding of 3H-PGE2 was saturable, reversible, protein-dependent, and correlated with stimulation of cAMP production, indicating that specific binding referred to receptor binding. PGE1 and PGI2 were far less effective than PGE2 in competition of binding with 3H-PGE2, indicating receptor selectivity for PGE2. Scatchard analysis of the specific binding data revealed a high affinity component (Kd 17 nM) and low affinity component. The total number of high- and low-affinity binding sites, respective Kd values, and PG stimulation of cAMP production of cells from rats fed the linseed oil diet were comparable to controls. The corn oil diet, however, resulted in a twofold increase in total number of high- and low-affinity binding sites, while respective Kd values were unchanged. This enhancement of binding capacity could be explained by an increased density of binding sites on the cells, and may itself be responsible for the increased sensitivity of the macrophages in this diet group for PG-stimulated cAMP production. The data suggest a regulatory mechanism at the receptor level and are discussed in terms of possible altered bioavailability of arachidonic acid-derived PGE2.

Competition for adenyl cyclase coupled (3H)-prostacyclin binding sites with prostaglandin E2 in rat peritoneal macrophages.

Prostaglandins regulate macrophage function by their action on membrane-associated adenyl cyclase. In order to define more directly macrophage-prostaglandin interactions, a binding assay has been developed for macrophage receptors using (3H)-PGI2 as ligand. (3H)-PGI2 binding was specific, saturable and reversible. Moreover, specific binding showed to be enriched in a membrane-enriched fraction of the cells. The assay conditions ensured stability of (3H)-PGI2 during incubations and should exclude intracellular accumulation of the ligand in macrophages. Unlabelled PGE2 and PGI2 competed for (3H)-PGI2 specific binding in both macrophages and membrane preparations. PGE2 showed to be more potent in this respect than PGI2, a phenomena which was also observed for prostaglandin activation of cAMP production in macrophages. The data suggest an interaction at receptor level of endogenously released PGE2 and PGI2 by peritoneal macrophages in vivo and provide support for a previously proposed mechanism of action of low concentrations of PGE2, counteracting stimulation of cAMP production by PGI2 in macrophages.

Direct evidence for the presence of selective binding sites for (3H) prostaglandin E2 on rat peritoneal macrophages.

A method is presented which provides for a simple and rapid determination of PGE2 receptors on viable peritoneal macrophages. Incubation of the harvested cells with (3H)PGE2 revealed specific binding of (3H)PGE2 by use of the Millipore filter assay system. Maximum binding was attained in the presence of 1 mM EDTA. Specific binding was saturable at 65 fmol/mg protein with an equilibrium dissociation constant (Kd) of 3.2 X 10(-8)M. Inhibition of (3H)PGE2 binding with unlabelled prostaglandins revealed a potency series of PGE2 greater than PGE2 greater than PGI2. The PGE2 concentration which displaced 50% of the labelled ligand was 10(-7)M. Comparable kinetic data were obtained for adenylate cyclase stimulation, since the concentration which showed a halfmaximal stimulation of cAMP production was 2 X 10(-7)M of PGE2. Since PGE1 and PGI2 compete with (3H)PGE2 binding in a non-parallel manner compared to PGE2 itself, it is proposed that macrophages possess different types of PG receptors.

Interaction of calcium with 3H-morphine binding to synaptosomes of the guinea-pig ileum.

Specific binding sites for 3H-morphine were assayed in subcellular fractions of a crude mitochondrial P2 pellet from the guinea-pig ileum longitudinal muscle-myenteric plexus, prepared by discontinuous sucrose-gradient fractionation. The highest specific binding (ca. 100 fmol/mg protein) was obtained in the fraction containing synaptosomes, as examined by electron microscopy. A synaptosomal fraction prepared from guinea-pig brain had comparable specific binding (ca 130 fmol/mg) to that of the ileal synaptosomal fraction. Addition of calcium (10 mM) to the binding assay medium resulted in a marked decrease in particular of the specific 3H-morphine binding. Detailed analysis of the specific 3H-morphine binding. Detailed analysis of the specific 3H-morphine binding in the synaptosomal fraction of the ileum revealed that 1) a saturable component of specific opiate binding was present between 0.34 and 21.74 nM of 3H-morphine; 2) in the presence of 3 and 10 nM of calcium similar decreases of specific 3H-morphine binding were obtained, indicating that this binding was maximally inhibited already by 3 mM of calcium; 3) both in the absence and presence of calcium the KD of specific 3H-morphine binding was about 38 nM, indicating a non-competitive nature of the calcium inhibition; 4) addition of magnesium exhibited a similar effect as that of calcium, although magnesium appeared to be less potent than calcium in this respect. The data are discussed in the context of previously observed calcium effects on opioid actions in the electrically stimulated guinea-pig ileum bioassay and may contribute to the evidence that the interaction of calcium and opioids on the stimulus-release coupling mechanism at the neuromuscular junction of the guinea-pig ileum is occurring beyond opioid receptor activation as well.

beta-Endorphin proteolysis by guinea-pig ileum myenteric plexus membranes: increased gamma-endorphin turnover after chronic exposure to morphine.

The influence of chronic morphine exposure in vitro on the biotransformation of beta-endorphin (beta E) was investigated using the myenteric plexus-longitudinal muscle of guinea-pig ileum. A membrane preparation was incubated with beta E and the degradation of beta E as well as the accumulation of several beta E fragments in the incubation medium were followed with time. The levels of peptides were determined by specific radioimmunoassays after separation by high-pressure liquid chromatography. It was found that exposure to morphine did not affect the disappearance of beta E, but altered the time course of accumulation of beta E fragments. In fact, the accumulation of gamma-endorphin, alpha-endorphin and des-tyrosine1-alpha-endorphin was enhanced, while that of des-tyrosine1-gamma-endorphin was not change. Additionally, the disappearance of gamma-endorphin appeared to be stimulated by morphine exposure. These data provide evidence that the fragmentation of beta E is changed by chronic morphine exposure in such a way that the turnover of gamma-endorphin is increased.

Gamma-melanotropin and brain function.

In view of the close structural similarity between the pro-opiocortin fragment, gamma-MSH, and ACTH/MSH-type peptides, the behavioural profile of gamma-MSH was explored. Attention was first focused on behavioural procedures in which ACTH/MSH-related neuropeptides have been found effective. It was found that gamma-MSH and ACTH-like neuropeptides had opposite effects on avoidance behaviour. In this respect the activity of gamma-MSH resembles that of opiate antagonists rather than that of beta-endorphin. Accordingly, ACTH(1-24) induced excessive grooming which is blocked by opiate antagonists and is attenuated by gamma-MSH. In addition, gamma-MSH injected into the periaqueductal grey matter of the brainstem of opiate-naive rats elicited symptoms reminiscent of those seen after opiate withdrawal. Gamma-MSH attenuated several effects of intracerebroventricularly administered beta-endorphin (e.g. antinociception, hypothermia, alpha-MSH release) and decreased the acquisition of heroin self-administration. Although gamma-MSH at rather high doses displaced naloxone from its specific binding sites in brain homogenates, it did not interfere with beta-endorphin-induced effects on in vitro muscle preparations (guinea-pig ileum; rat rectum). Interestingly, gamma-MSH induced relaxation of the rat rectum in vitro. It is postulated that gamma-MSH may attenuate beta-endorphin-induced effects by acting via gamma-MSH receptor sites (functional antagonism), although a pharmacological antagonism cannot be excluded as yet.

Differential involvement of calcium in acute and chronic opioid action in the guinea-pig ileum in vitro.

The role of calcium in acute and chronic opioid action was studied by measuring the inhibitory response of electrically stimulated (0.1 Hz) myenteric plexus-longitudinal muscle strips of guinea-pig ileum in vitro subsequent to a test dose of morphine or to high frequency stimulation (10 Hz for 1 min) in the presence of graded concentrations of calcium in the buffer surrounding the strips. The inhibitory response induced by both procedures was enhanced in low calcium and diminished in high calcium medium. After the inhibitory response was established, an increase of the exogenous calcium concentration completely antagonized the inhibition. Additional experiments, in which the magnesium content of the medium was varied or the calcium-chelator [ethylenebis(oxyethylenenitrilo)tetraacetate] was used, pointed to an important role of endogenous calcium in the action of morphine and of opioid material released by high frequency stimulation. The degree of tolerance induced in vitro by incubating strips with morphine for 20 hr was not affected by omitting calcium in the incubation medium. It is suggested that endogenous calcium plays a modulatory role in processes which are intermediary between opioid receptor activation and the subsequent effect on neurotransmission (i.e., acetylcholine in the guinea-pig ileum). In contrast to the acute action of opioids, calcium seems to be less important for the development of tolerance in vitro.

ACTH-induced lipolysis in rat adipocytes: structure-activity relationships.

The lipolytic action of natural porcine ACTH1(-39) and of a number of highly purified synthetic ACTH peptide fragments was studied using rat adipocytes. Of the analogues tested, only ACTH1(-24) exhibited full lipolytic activity with respect to intrinsic activity and affinity. Several shorter fragments appeared to be full agonists but had lower affinity. Fragments ACTH5(-10) and ACTH7(-10) were inactive. No antagonistic effects against the lipolytic action of ACTH could be demonstrated with substimulatory doses of ACTH1(-16), ACTH1(-10), ACTH7(-24) and ACTH11(-24). Based on the relative potency derived from dose-response curves, a more refined model with respect to the active centers being encoded in various sequences of the hormone, is proposed.