[1-deamino-4-cyclohexylalanine] arginine vasopressin: a potent and specific agonist for vasopressin V1b receptors.

To date, there are no vasopressin (VP) agonists that exhibit a high affinity and selectivity for the VP V1b receptor with respect to the V1a, V2, and oxytocin receptors. In this study, we describe the synthesis and pharmacological properties of [1-deamino-4-cyclohexylalanine] arginine vasopressin (d[Cha4]AVP). Binding experiments performed on various membrane preparations revealed that d[Cha(4)]AVP exhibits a nanomolar affinity for V1b receptors from various mammalian species (rat, bovine, human). It exhibits high V1b/V1a and V1b/oxytocin selectivity for rat, human, and bovine receptors. Furthermore, it exhibits high V1b/V2 specificity for both bovine and human vasopressin receptors. Functional studies performed on biological models that naturally express V1b receptors indicate that d[Cha4]AVP is an agonist. Like VP, it stimulated basal and corticotropin-releasing factor-stimulated ACTH secretion and basal catecholamine release from rat anterior pituitary and bovine chromaffin cells, respectively. In vivo experiments performed in rat revealed that d[Cha4]AVP was able to stimulate both ACTH and corticosterone secretion and exhibits negligible vasopressor activity. It retains about 30% of the antidiuretic activity of VP. This long-sought selective VP V1b receptor ligand with nanomolar affinity will allow a better understanding of V1b-mediated VP physiological effects and is a promising new tool for V1b receptor structure-function studies.

Stimulation by leptin of 3H GDP binding to brown adipose tissue of fasted but not fed rats.

OBJECTIVES: To assess the effects of intracerebroventricular (i.c.v.) leptin administration on rats fed ad libitum or fasted on 3H GDP binding to brown adipose tissue (BAT). SUBJECTS: Groups of 5-6 ten-week-old male Wistar rats. EXPERIMENTAL DESIGN: An i.c.v. cannula was inserted and unilateral denervation of interscapular brown adipose tissue (BAT) was performed 5 d before each study. Thereafter, leptin was infused i.c.v. during 72 h while rats were fed ad libitum or fasted. Vehicle-infused, pair-fed or fasted rats were used as controls. MEASUREMENTS: 3H GDP binding to innervated and denervated BAT mitochondria. RESULTS: 3H GDP binding to innervated or denervated BAT of rats fed ab libitum compared to vehicle-infused, pair-fed rats was not increased by i.c.v. leptin. 3H GDP binding was lower in fasted than in fed rats, and the difference was larger in innervated than denervated BAT. I.c.v. leptin increased 3H GDP binding by 30% in innervated, and by 51% in denervated BAT (P < 0.05) in fasted rats. CONCLUSIONS: I.c.v. leptin does not increase 3H GDP binding to BAT of rats fed ad libitum compared to pair-fed (food-restricted) rats. In contrast, i.c.v. leptin produces a mild stimulation of 3H GDP binding to BAT of fasted rats. This effect is not mediated by the sympathetic nervous system, because it is observed in both innervated and denervated BAT. These results are compatible with the concept that, in fasting rats, the decrease in leptin secretion contributes to the reduction in 3H GDP binding to BAT mitochondria.

A phospholipase A2-related snake venom (from Crotalus durissus terrificus) stimulates neuroendocrine and immune functions: determination of different sites of action.

Immune neuroendocrine interactions are vital for the individual's survival in certain physiopathological conditions, such as sepsis and tissular injury. It is known that several animal venoms, such as those from different snakes, are potent neurotoxic compounds and that their main component is a specific phospholipase A type 2 (PLA2). It has been described recently that the venom from Crotalus durissus terrificus [snake venom (SV), in the present study] possesses some cytotoxic effect in different in vitro and in vivo animal models. In the present study, we investigated whether SV and its main component, PLA2 (obtained from the same source), are able to stimulate both immune and neuroendocrine functions in mice, thus characterizing this type of neurotoxic shock. For this purpose, several in vivo and in vitro designs were used to further determine the sites of action of SV-PLA2 on the hypothalamo-pituitary-adrenal (HPA) axis function and on the release of the pathognomonic cytokine, tumor necrosis factor alpha (TNF alpha), of different types of inflammatory stress. Our results indicate that SV (25 microg/animal) and PLA2 (5 microg/animal), from the same origin, stimulate the HPA and immune axes when administered (i.p.) to adult mice; both preparations were able to enhance plasma glucose, ACTH, corticosterone (B), and TNF alpha plasma levels in a time-related fashion. SV was found to activate CRH- and arginine vasopressin-ergic functions in vivo and, in vitro, SV and PLA2 induced a concentration-related (0.05-10 microg/ml) effect on the release of both neuropeptides. SV also was effective in changing anterior pituitary ACTH and adrenal B contents, also in a time-dependent fashion. Direct effects of SV and PLA2 on anterior pituitary ACTH secretion also were found to function in a concentration-related fashion (0.001-1 microg/ml), and the direct corticotropin-releasing activity of PLA2 was additive to those of CRH and arginine vasopressin; the corticotropin-releasing activity of both SV and PLA2 were partially reversed by the specific PLA2 inhibitor, manoalide. On the other hand, neither preparation was able to directly modify spontaneous and ACTH-stimulated adrenal B output. The stimulatory effect of SV and PLA2 on in vivo TNF alpha release was confirmed by in vitro experiments on peripheral mononuclear cells; in fact, both PLA2 (0.001-1 microg/ml) and SV (0.1-10 microg/ml), as well as concavalin A (1-100 microg/ml), were able to stimulate TNF alpha output in the incubation medium. Our results clearly indicate that PLA2-dependent mechanisms are responsible for several symptoms of inflammatory stress induced during neurotoxemia. In fact, we found that this particular PLA2-related SV is able to stimulate both HPA axis and immune functions during the acute phase response of the inflammatory processes.

Effect of chronic intracerebroventricular infusion of insulin on brown adipose tissue activity in fed and fasted rats.

OBJECTIVES: Carbohydrate feeding stimulates, and fasting decreases the sympathetic nervous system activity and brown adipose tissue (BAT) thermogenesis. This study was performed to assess the hypothesis that these effects were secondary to changes in insulin concentrations in the central nervous system. METHODS: BAT sympathetic activity was assessed by comparing 3H-GDP binding to isolated mitochondria of innervated and denervated interscapular BAT of three groups of 10 week old male Wistar rats: food-restricted, 48 h fasted or ad libitum fed. During the three days preceding this measurement, animals received a continuous intracerebroventricular (ivc) infusion of insulin (0.48 U/d) or vehicle. RESULTS: In food-restricted rats, 3H-GDP binding to mitochondria of innervated BAT was 41% higher than that to denervated BAT. Icv insulin did not stimulate 3H-GDP binding in innervated BAT. In 48 h fasted rats, 3H-GDP binding to mitochondria of innervated BAT was reduced by 30-50%, while the activity of denervated BAT was minimally affected. Icv insulin did not prevent this fasting-induced drop in BAT. In rats fed ad libitum, icv insulin decreased food intake by 17% (P < 0.05) and increased 3H-GDP binding to innervated BAT by 27% (P < 0.05). CONCLUSION: Intracerebroventricular insulin stimulates BAT activity in rats fed ad libitum but not in food-restricted or fasted rats. This demonstrates that the decrease in BAT activity observed during fasting is unlikely to be due to a decrease in insulin concentration in the nervous system.

High performance liquid chromatography and pharmacokinetics of the non-steroidal anti-inflammatory drug oxindanac in calves.

A high-performance liquid chromatographic method for the determination of the non-steroidal anti-inflammatory drug, oxindanac, in calf plasma is described. Recoveries over the concentration range 0.375 to 62.5 micrograms/ml were 90.2-107.8% with interassay coefficients of variation of 2.1-22.3%. The limit of detection was estimated as 0.10 micrograms/ml and the limit of quantification calculated to be 0.24 micrograms/ml in a 1 ml plasma sample. This method was used to establish the pharmacokinetics following intravenous (i.v.), intramuscular (i.m.) and oral (p.o.) administration to calves of oxindanac at a dose rate of 2 mg/kg. The elimination t1/2 was long (t1/2 21.2 h after i.v. injection) and absorption was rapid (t1/2a 0.072 h) and complete (F > 100%) following i.m. administration. Bioavailability was incomplete (F = 66.6%) following p.o. administration to calves that had been fed on milk, and Wagner-Nelson analysis revealed two absorption phases (t1/2's 0.20 and 1.9 h). Oxindanac produced long-lasting inhibition of serum TxB2 production, with mean Emax values (% inhibition) of 96.8, 94.1 and 81.3 following i.v., i.m. and p.o. administration, respectively. A single i.v. or i.m. injection of 2 mg/kg oxindanac will probably be active in calves for at least 36-48 h.

A slow release formulation for recombinant bovine interferon alpha I-1.

Recombinant bovine interferon-alpha I1 (rBoIFN-alpha) has known antiviral and immunomodulatory effects which have been exploited to reduce clinical disease in a number of clinical situations including bovine respiratory diseases. A slow release rBoIFN-alpha formulation may be of value to reduce bovine respiratory disease under field conditions by extending the period of protection, and hence improving the prophylactic benefits of rBoIFN-alpha. In this report, we describe a formulation of rBoIFN-alpha in sesame oil containing calcium stearate which can successfully sustain the release of rBoIFN-alpha over an 8-day period. Recombinant bovine IFN-alpha could be measured in serum for 8 days following treatment with an initial burst of release 6 h after injection. After a single subcutaneous depot injection of 50 mg and 100 mg of rBoIFN-alpha, initial serum levels reached 12-15 ng/ml and 25 ng/ml respectively. Correlating with this burst of release, there was a decrease in the number of circulating CD4-CD8- gamma delta+ T lymphocytes, and a slight neutropenia. No alterations in other cell phenotypes tested (CD4, CD8, CD2, CD6, B cells, monocytes or MHC class II) were observed, nor were there changes in lymphokine activated killer (LAK), natural killer (NK) cell activity, or oxygen radical formation (assessed by reduction of nitroblue tetrazolium). However, despite the rapid and short-lived burst of rBoIFN-alpha, levels of 2-5 oligoadenylate (2-5 A) synthetase remained elevated for 8 days. The sustained increase of 2-5 A synthetase was not due to the high initial dose released during the burst 6-12 h after injection, since injection of a bioavailable equivalent dose of interferon induced a significant rise in 2-5 A synthetase activity for 4 days only. As 2-5 A synthetase is known to be a correlate of antiviral activity, we propose that this formulation of rBoIFN-alpha may be one approach to increase the window of protection, leading to more effective prevention of bovine respiratory disease.

Effect of bovine interferon on acute changes in body temperature and serum progesterone concentrations in heifers.

Bovine interferon-alpha I1 has extensive sequence and functional homology with the antiluteolytic protein, bovine trophoblast protein-1. Because of the possible use of interferon-alpha I1 as a drug that supplements embryonic secretion of bovine trophoblast protein-1, interferon-alpha I1 was tested for other biological actions that might affect its usefulness as a fertility-enhancing treatment. Experiments were performed to evaluate whether interferon-alpha I1 causes hyperthermia and an acute depression in circulating concentrations of progesterone. In four experiments, intramuscular administration of interferon-alpha I1 (range 1.25 to 20 mg) caused hyperthermia; average peak body temperatures of 40 to 40.4 degrees C occurred 2.5 to 6 h after injection. Temperatures returned to baseline 12 to 16 h later. The rise in rectal temperature could be reduced, but not totally alleviated, with concomitant administration of an inhibitor of prostaglandin synthesis. The maximal hyperthermic response was similar when interferon-alpha I1 was delivered via osmotic minipumps or through a series of intramuscular injections. The hyperthermic response decreased with repeated daily exposure to interferon-alpha I1. The increase in rectal temperatures was associated temporally with a decrease in serum progesterone. Effects of interferon-alpha I1 on body temperature and circulating progesterone could possibly limit its effectiveness in enhancing fertility.

Induction of circulating tumor necrosis factor cannot be demonstrated during septicemic salmonellosis in calves.

The concentration of tumor necrosis factor in the circulation of calves, which were infected with Salmonella typhimurium and exhibited septicemia as indicated by clinical signs and blood culture, was measured with a radioimmunoassay. These levels were compared with those in calves before infection and in other calves that had received an intravenous dose of gram-negative endotoxin. The tumor necrosis factor levels measured in samples taken during septicemia were not different from those in samples from infected nonsepticemic calves or samples from calves before infection. In contrast, the levels of tumor necrosis factor rose rapidly in calves after treatment with endotoxin by intravenous injection.

Influence of starvation on hormonal control of hypophyseal secretion in rats.

The reduction of hypophyseal hormone secretion during starvation is not completely understood. A previous study showed that the concomitant reduction of plasma TSH and T3 may be related to an increased sensitivity of the thyrotrope cell to T3. This suggests that regulation of hypophyseal secretion by peripheral hormones may be altered in starved rats. As GH and PRL secretion are under the control of thyroid and steroid hormones, the aim of the present study was to investigate the modification of feed-back control by T3 or E2 on hypophyseal secretion during starvation. For this purpose, pituitary GH, PRL and TSH contents and their plasma responses to TRH injection were measured in euthyroid, thyroidectomized (Tx), T3-supplemented Tx and E2-treated male Wistar rats before and after a 3-day starvation. TRH (0.25 micrograms/100 g) was injected iv through a chronically-implanted catheter. Our results show that GH content and GH plasma response to TRH are dramatically increased in T3-treated Tx starved rats, suggesting that starvation also increases the effectiveness of T3 influence on somatotrope cell secretion. By contrast, effects of T3 on PRL secretion remain unchanged during starvation. Furthermore, starvation in E2-treated rats is associated with a marked rise in the PRL and GH responsiveness to TRH without any significant change of hormonal pituitary content. This suggests that, in starved rats, E2 increases the effects of TRH on lactotrope and somatotrope secretion. No significant effect on TSH secretion could be demonstrated. Thus, starvation seems to act differentially on the feed-back mechanisms controlling the hormonal secretion of the three adenohypophyseal target cells to TRH.

Sensitivity of thyrotropin (TSH) secretion to 3,5,3'-triiodothyronine and TSH-releasing hormone in rat during starvation.

The mechanisms by which plasma T3 and TSH decrease after a 3-day starvation period are not completely understood. In this study we tested the hypothesis of a possible modification in the sensitivity of thyrotroph cell to T3 and/or TRH. For that purpose, TRH tests were performed before and after a 3-day starvation in euthyroid, thyroidectomized, and T3-treated (75 or 175 ng/100 g BW) thyroidectomized male Wistar rats. TRH (10 to 500 ng/100 g BW) was injected iv through a chronically-implanted catheter. In another set of experiments, hypophyseal TSH content was also determined. Our results showed that after a 3-day-starvation plasma TSH decreased in all except hypothyroid rats; TSH responsiveness to TRH was unchanged in euthyroid rats but was increased in hypothyroid rats; and the T3-dependent increase in TSH responsiveness to TRH was significantly amplified. Moreover, there was a significant positive correlation between TSH responsiveness to TRH and hypophyseal TSH content. These results suggest that starvation induces an increased sensitivity of thyrotroph cell to T3.

Differential effects of passive immunization with somatostatin antiserum on adenohypophysial hormone secretions in starved rats.

The role of somatostatin (SRIF) on adenohypophysial hormone secretion in starved rats was reassessed by passive immunization. Because of the absence of pulsatile GH secretion in starved rats, the effects of the injection of SRIF antiserum on GH levels can be clearly demonstrated. To determine whether starvation modifies the sensitivity of the adenohypophysis to SRIF, we measured 125I-labelled iodo-N-Tyr-SRIF binding. There was no difference in the dissociation constant (Kd) nor in the maximal binding capacity (Bmax) in fed (n = 15) and starved (n = 15) animals (Kd = 0.38 +/- 0.09 (S.E.M.) and 0.45 +/- 0.09 nmol; Bmax = 204 +/- 39 and 205 +/- 30 fmol/mg protein respectively). Administration of SRIF antiserum resulted in a dose-dependent increase in plasma concentrations of GH, TSH and prolactin. The minimal effective dose of SRIF antiserum was 50 microliters for GH, 100 microliters TSH and 200 microliter for prolactin. Our results show that: starvation does not modify adenohypophysial SRIF-binding sites, in starved male rats endogenous SRIF exerts a negative control on prolactin secretion in vivo and sensitivity to endogenous SRIF seems to be different for each hypophysial cell type.

The Brattleboro rat: normal growth hormone secretion, decreased hepatic growth hormone receptors and low plasma somatomedin activity.

Three-month-old male Brattleboro rats with hereditary diabetes insipidus (DI) present a growth defect; Brattleboro rats were studied together with age-matched Long-Evans (LE) rats. Pituitary growth hormone (GH) content was comparable in both groups of rats. Pulsatile GH release and mean 6 h GH plasma levels did not appear significantly different in chronically catheterized DI and control animals. In parallel with the growth defect, the plasma somatomedin bioactivity was significantly lower in DI than in LE rats. The specific binding of [125I]iodo-hGH to liver microsomal membranes of DI rats was 59.7% that of controls. The number of the GH binding sites rather than the affinity of the binding was decreased. The specific binding of [125I]iodo-insulin was oppositely affected by the DI state: it was 1.5 times higher in liver membranes of DI rats than in membranes of LE rats. These findings make a non-specific effect of the DI state on liver membrane proteins unlikely. The Brattleboro rats present a growth failure without reduction of their GH secretion. The decreased number of the hepatic GH receptors and the subsequent low plasma somatomedin activity could explain the growth retardation of the DI rats.

Involvement of endogenous somatostatin in the regulation of thyrotroph secretion during acute and chronic changes in diet.

The aim of this study was to investigate the involvement of somatostatin (SRIF) in the thyrotroph adaptation to nutritional changes. For this purpose, we studied the effects of passive immunization with SRIF antiserum (A-SRIF) on the reduced basal TSH secretion in rats starved for 72 h and on the plasma TSH surge following carbohydrate (CHO) refeeding. This latter experiment was performed at two different times of the day in order to elucidate whether SRIF may participate in the regulation of the plasma TSH circadian rhythm. In chronically catheterized rats, we observed that A-SRIF injection induced a similar pattern of plasma TSH rise over a sampling period of 5 1/2 h in both fed and rats starved for 72 h (3-way analysis of variance). In morning experiments, CHO refeeding or A-SRIF injection elicited a significant rise in plasma TSH. The amplitude and duration of the response was proportional to the injected dose. In evening experiments, although basal TSH values were significantly lower than those observed in the morning ones, maximal plasma TSH values after A-SRIF injection were not significantly different. At both times of the day, association of refeeding and A-SRIF injection did not stimulate TSH further than either refeeding alone or A-SRIF alone. In conclusion, our data suggest that SRIF cannot account for the differences in serum TSH levels between fed and starved rats; is not responsible for the diurnal difference in basal serum TSH in starved rats, and seems to be involved in the TSH response to refeeding.

Hypothalamo-pituitary regulation of thyrotrophin secretion in chronically catheterized Brattleboro rats.

Plasma TSH rhythms were measured in Brattleboro (DI) and control Long-Evans (LE) rats with an intracardiac catheter allowing repeated sampling in conscious unstressed animals. The TSH response to thyrotrophin-releasing hormone (TRH; 500 ng/100 g body weight) was also determined. Finally, hypothalamic and pancreatic TRH concentrations and TRH-degrading activity (TRH-DA) were measured by specific radioimmunoassay. Long-Evans rats had a 24-h rhythm with a major modulatory 8-h component. In DI rats, only the 24-h rhythm was detected. The mean 24-h rhythm-adjusted mean TSH level was higher in DI than in LE rats (1.38 +/- 0.05 and 1.14 +/- 0.06 micrograms/l respectively, P less than 0.01). The peak TSH response to TRH was significantly increased in DI rats while the pituitary concentration of TSH was also higher (0.93 +/- 0.09 vs 0.39 +/- 0.06 micrograms/mg wet weight in LE, P less than 0.001). Hypothalamic TRH and TRH-DA were similar in both strains. The response to propylthiouracil-induced hypothyroidism was identical in both strains. We conclude that DI rats have a normal pituitary sensitivity to tri-iodothyronine but a central dysfunction in the pituitary environment leading to some alterations of TSH secretion.

Evidence of a thyrotropin-releasing hormone-dependent increase in plasma thyrotropin during refeeding of starved rats.

After 3 days of starvation, refeeding with carbohydrate (CHO) leads to a rapid increase in plasma T4 and T3, suggesting increased TSH secretion. The aim of the present study was to describe the time course of plasma TSH changes during refeeding with CHO and fat. Repetitive blood samples were obtained from freely moving animals by indwelling jugular venous catheters. Refeeding was performed on the fourth day of starvation at either 1100 or 1900 h, and blood was sampled during the preceding hour and during the following 3 h. In control experiments, blood was sampled over 4 h without refeeding. Refeeding with CHO and fat induced a significant increase in plasma TSH in the first hour. This increase could be abolished by a previous injection of an anti-TRH serum, while normal rabbit serum was without effect. Plasma corticosterone, measured hourly, also showed a tendency to increase with refeeding. It is concluded that refeeding of starved rats represents a reproducible stimulus for TSH secretion.

Starvation-induced decreased sensitivity of resting metabolic rate to triiodothyronine.

The decrease in resting oxygen consumption induced by starvation was found to occur not only in euthyroid rats but also in hypothyroid and even in hypothyroid animals treated with triiodothyronine. Furthermore, the effectiveness of triiodothyronine was decreased when given to hypothyroid animals.