Formula alpha-linolenic (18:3(n - 3)) and linoleic (18:2(n - 6)) acid influence neonatal piglet liver and brain saturated fatty acids, as well as docosahexaenoic acid (22:6(n - 3)).

Saturated fatty acids can be synthesized de novo and play a role in determining properties of structural membranes. The effect of dietary essential fatty acids, linoleic acid (18:2(n - 6)) and alpha-linolenic acid (18:3(n - 3)), on the saturated fatty acid content of membrane phospholipid has not previously been considered in newborn nutrition. The studies report the effect of low (1% fatty acids) or high (4%) formula 18:3(n - 3) with low (16%) or high (30-35%) formula 18:2(n - 6) on the saturated and unsaturated fatty acid composition of liver and brain structural lipid of piglets fed formula from birth for 15 days. A significant inverse relationship between the formula % 18:3(n - 3), but not 18:2(n - 6), and the liver phospholipid palmitic acid (16:0) was found. This may indicate a possible effect of dietary 18:3(n - 3) on de novo synthesis of 16:0 and requires further investigation. Monounsaturated fatty acids in both liver and brain were significantly lower in response to high 18:3(n - 3) and to high 18:2(n - 6) plus low 18:1(n - 9) in the formula. Liver phospholipid and brain total lipid % docosahexaenoic acid (22:6(n - 3)) were significantly higher when formula containing 4% rather than 1% 18:3(n - 3) was fed, suggesting that 1% 18:3(n - 3) may limit tissue (n - 3) fatty acid accretion. These results suggest that future studies of essential fatty acid requirements, specifically 18:3(n - 3), should consider possible influences on the saturated fatty acids which also play a functional role in tissue structural lipids.

Short-term feeding of a diet enriched in phospholipids increases bile formation and the bile acid transport maximum in rats.

Earlier studies suggested that the secretory rate maximum (SRm) of bile acid and the cholestasis which occurs after the SRm is reached may be determined by the hepatic or extrahepatic biliary phospholipid pool. We therefore investigated whether bile formation and the bile acid SRm could be influenced by feeding a diet enriched in phospholipids. Male rats were fed phospholipid (PLD) or triacylglycerol (TgD)-enriched diet for 3 days, and bile formation as well as biliary lipid output were measured on the 4th day. In other similarly fed groups, cholic acid was infused in stepwise increasing doses to determine the effect of PLD on the SRm of cholic acid. The plasma lipid levels were significantly lower in PLD and TgD diets compared to basal diet. But, while the levels of total cholesterol (CH), HDL-CH, and phospholipid (PH) were not significantly altered by PLD compared to TgD, the triacylglycerol levels were markedly increased by PLD. In the liver of PLD fed rats, triacylglycerol and CH ester contents decreased by 39 and 62%, respectively, while free CH and PH contents were not significantly changed. The PLD significantly augmented spontaneous bile flow, bile acid, PH and CH secretion rates compared to TgD diet (65, 124, 164 and 654%, respectively). The enhanced biliary secretory function was associated with an increase in pericanalicular vacuoles and diverticuli in centrilobular hepatocytes. Compared to TgD fed rats, PLD rats showed a 2-fold decrease in the ratio of cholic acid/chenodeoxycholic acid in bile and a significant decrease in the % contribution of taurine conjugated BA. The PH fatty acids in bile were similar in both groups except that in PLD group the % contribution of C18:2 was higher than in TgD group. No differences were found in plasma membrane CH/PH content or total fatty acid composition. During bile acid infusion, the SRm and the total cholic acid secreted were significantly higher in the PLD than in the TgD rats. Moreover, the cholestatic response observed after high bile acid dose was markedly reduced by PLD. The results show that short-term feeding of PLD induces changes in CH and bile acid metabolism which result in enhanced biliary output of CH and PH. The enhanced pool of biliary lipid may protect plasma membranes from the deleterious effects of high bile acid concentrations.

Arachidonic acid concentrations in plasma and liver phospholipid and cholesterol esters of piglets raised on formulas with different linoleic and linolenic acid contents.

The influence of sow milk or infant formulas containing 18:2n-6 and 18:3n-3 (% fatty acids) at 30/1, 16/1, 35/4, or 16/4 on plasma and liver phospholipid (PL) and cholesterol ester (CE) arachidonic acid (20:4n-6) was studied in piglets fed from birth for 15 d. Piglets fed the 35/4, 16/1, and 16/4 formulas had a significantly lower percentage of plasma 20:4n-6 in PL than did piglets fed sow milk or the 30/1 formula. The lowest plasma PL 20:4n-6 was in the 16/4 group, the only group in which liver PL 20:4n-6 was significantly reduced. This suggests competitive inhibition of synthesis or acylation of 20:4n-6 when formula with a high content of 18:3n-3 is fed in conjunction with a low content of 18:2n-6. The percentage of plasma CE 20:4n-6 was not altered by formula feeding. In contrast, the liver CE 20:4n-6 was significantly lower in all formula-fed animals than it was in sow-milk-fed animals. These studies confirm that 20:4n-6 metabolism is altered in artificially fed neonates. Liver and plasma cholesterol concentrations were also significantly lower in all formula-fed than in milk-fed piglets. The potential relationship of the decrease in cholesterol to n-6 fatty acids in CE is unknown.

Feeding formula without arachidonic acid and docosahexaenoic acid has no effect on preferential looking acuity or recognition memory in healthy full-term infants at 9 mo of age.

Preferential looking acuity and novelty preference (a test of recognition memory) were determined by using Teller Acuity Cards and the Fagan Test of Infant Intelligence, respectively, for 399-433 healthy full-term infants at 39 +/- 1 wk of age. Duration of breast-feeding and age of infant at introduction and amount and type of formula were determined by questionnaire. Seventy-four infants (17%) were never breast-fed; another 92 infants (21%) were still receiving breast milk as the milk source at 39 wk of age. There were no differences in visual acuity or novelty preference among the infants when they were stratified by incidence or duration of breast-feeding. The formulas met current Canadian guidelines with > or = 0.7% of energy as linolenic acid, but had no docosahexaenoic or arachidonic acid. The studies indicate that formulas containing adequate linoleic and linolenic acids, without arachidonic or docosahexaenoic acid, impose no measurable deficits in performance in these visual and cognitive developmental tests at 9 mo of age in healthy full-term infants.

Synthesis of peptides by the solid-phase method. 6. Neurotensin, fragments, and analogues.

Neurotensin (NT) and 24 related compounds, including fragments or analogues modified at the C-terminal end of the parent molecule, have been prepared by the solid-phase method. After purification by cation-exchange chromatography, the compounds were characterized by thin-layer chromatography, amino acid analysis, elemental analysis, and high-pressure liquid chromatography. The stimulating effects of the peptides were evaluated in rat stomach strips, in isolated spontaneously beating atria of guinea pigs, and in the coronaries of perfused rat hearts. The differences between the biological activities of these compounds are discussed.

Effects of capsaicin desensitization on the stimulatory effect of kinins, prostaglandins, biogenic amines and various drugs in guinea-pig isolated atria.

1. A simple desensitization protocol was set up using capsaicin and isolated, spontaneously beating atria of guinea-pigs to assess the possible participation of cardiac, capsaicin-sensitive, substance P (SP)- and calcitonin gene-related peptide (CGRP)-containing sensory nerve fibres, in the cardiac stimulatory effects of bradykinin (Bk), kallidin (Kd), 5-hydroxytryptamine (5-HT), histamine, prostaglandin E2 (PGE2), prostaglandin E1 (PGE1), prostaglandin F2 alpha, (PGF2 alpha), adrenaline (Ad), glucagon, nicotine and angiotensin II (AII). 2. The positive chronotropic and inotropic effects of Bk, Kd and 5-HT were markedly reduced in capsaicin-desensitized atria compared to control. The percentage inhibition of the chronotropic and inotropic responses to the three agonists seemed to be inversely related to the concentration of agonist used and to vary also with the type of cardiac effect produced by the drug (for Bk the percentage inhibition was: 36-81% (chronotropic effect) and 62-86% (inotropic effect); for Kd: 61-78% (chronotropic effect) and 53-77% (inotropic effect); for 5-HT: 25-66% (chronotropic effect) and 40-64% (inotropic effect]. 3. The positive chronotropic and inotropic effects of histamine, PGE1, PGE2, PGF2 alpha, glucagon and AII had similar amplitudes in capsaicin-desensitized and control atria. 4. The positive chronotropic and inotropic effects of Ad and nicotine were differentially affected by capsaicin desensitization. The inotropic effects of 7.5 x 10(-7) and 7.5 x 10(-6) M Ad were reduced by 41 and 27% respectively, in capsaicin-desensitized atria compared to control. The chronotropic effects of 1.54 x 10(-5) and 6.17 x 10(-5) M nicotine were inhibited by 57 and 26% respectively, by capsaicin desensitization. On the other hand, the chronotropic effect of Ad and the inotropic action of nicotine were of similar amplitude in capsaicin-desensitized and control atria. 5. These results were taken as an indication that a substantial part of the chronotropic and inotropic effects of Bk, Kd or 5-HT in guinea-pig atria, unlike those of histamine, PGE1, PGE2 PGF2 alpha, glucagon and AII, might be the result of stimulation of capsaicin-sensitive, SP- and CGRP- containing sensory nerve fibres. The slight, differential inhibition of the chronotropic and inotropic effects of Ad and nicotine by capsaicin desensitization suggests a minor contribution by cardiac, capsaicin-sensitive sensory nerve fibres to the effects of nicotine and Ad in guinea-pig atria.

Haemodynamic and abdominal motor reflexes elicited by neurotensin in anaesthetized guinea-pigs.

1. Single intraperitoneal (i.p.) injections of neurotensin (NT) (0.14- 140 nmol kg-1) in anaesthetized guinea-pigs were found to trigger transient abdominal wall contractions (TAWC) accompanied by relatively sustained increases of systemic blood pressure (BP) and heart rate (HR). The modification of the latter NT effects by various drugs and surgical manipulations was examined to obtain some insight into the nature of, and possible relationship between, these responses. 2. The abdominal motor response (i.e. TAWC) to i.p. NT (14 nmol kg-1) was inhibited by prior i.v. injection of the guinea-pigs with pancuronium (0.27 mumol kg-1), morphine (1.5 and 15 mumol kg-1), clonidine (0.34 mumol kg-1), by concomitant i.p. injection of procaine 2% w/v, or by acute spinalization. It was potentiated by naloxone (2.8 and 28 mumol kg-1), but not affected by i.v. injection of autonomic drugs (i.e. pentolinium, prazosin, yohimbine and atropine), by capsaicin desensitization, or by acute bilateral cervical vagotomy. In spinalized animals a sustained abdominal wall contraction (SAWC) was unmasked, which was resistant to i.v. morphine, clonidine or baclofen but suppressed by i.v. pancuronium or i.p. lignocaine 2% w/v. 3. Haemodynamic responses to i.p. NT were not affected by i.v. pancuronium, morphine, naloxone, atropine, or by vagotomy. They were inhibited by i.v. pentolinium or clonidine (BP, HR), i.v. prazosin (BP), i.p. procaine 2% w/v (BP, HR), capsaicin desensitization or acute spinalization (BP, HR). Yohimbine (i.v.) potentiated BP and HR increases caused by i.p. NT.4. These results suggest that: (1) the haemodynamic and TAWC responses to i.p. NT in this animal model, are two independent, neurally-mediated reflexes which are likely to originate from the abdominal cavity and require a functionally intact spinal cord for their full expression; (2) the neural pathways subserving both types of responses appear to be different from each other. The nature and time-response characteristics of the reflexes caused by i.p. NT, raise the possibility that i.p. NT is a noxious stimulus, at least in guinea-pigs.

A new bioassay for glucagon.

1 The relaxant action of glucagon has been studied in strips of rabbit renal arteries partially contracted by a low concentration (1 ng/ml) of noradrenaline.2 The preparation was relaxed in a dose-dependent manner by concentrations of glucagon varying between 25 ng/ml and 420 ng/ml.3 The relaxant effect of glucagon (0.1 mug/ml approximately ED(60)) on this preparation was not affected by propranolol (5.0 mug/ml), cimetidine (10 mug/ml), diphenhydramine (10 mug/ml), indomethacin (5.0 mug/ml), phentolamine (1.2 mug/ml), atropine (10 mug/ml) and 8-Leu-AT(II) (1.0 mug/ml) but was slightly potentiated by Des-Arg(9) Leu-OMe(8)-Bk (25 mug/ml) and indomethacin (50 mug/ml).4 The dose-response curve to glucagon remained parallel in the presence of papaverine (2.5 mug/ml) but was shifted to the left by a factor of 2.5 to 2.8. Theophylline (250 mug/ml) also potentiated the vascular relaxation induced by glucagon.5 Insulin (10 mug/ml) did not influence the relaxant effect of glucagon.6 The removal of the N-terminal amino acid (His) of glucagon reduced by 89% the biological activity of this fragment on the vascular preparation. The removal of the C-terminal amino acids Met-27, Asn-28 and Thr-29 of glucagon resulted in a fragment which was inactive either as an agonist or as an antagonist when tested at concentrations as high as 925 ng/ml.7 It is concluded that the relaxation of partially contracted strips of rabbit renal arteries by glucagon constitutes a simple, sensitive, relatively specific and reliable bioassay which may be useful for the determination of glucagon in biological materials and for structure-activity relationship studies with this hormone.

Studies on the mechanism of action of glucagon in strips of rabbit renal artery.

1 The vasodilator effects of glucagon and adenosine cyclic 3',5'-monophosphate (cyclic AMP) were evaluated in strips of rabbit renal artery contracted with noradrenaline (NA) in the absence and presence of phosphodiesterase inhibitors or calcium (Ca(2+)) antagonists.2 The vascular relaxant effect of glucagon was markedly potentiated by various concentrations of four different phosphodiesterase inhibitors (papaverine, theophylline, 3-isobutyl-l-methylxanthine (IBMX) and indomethacin), while that of cyclic AMP was potentiated by only two of them (papaverine and indomethacin) and inhibited by the others (theophylline and IBMX).3 Amongst the four phosphodiesterase inhibitors, IBMX (10 mug/ml) was found to produce the largest potentiation (e.g. the sensitivity increased by a factor of 10) of glucagon-induced vascular relaxations (ED(50) of glucagon in the presence of IBMX = 9.2 +/- 1.0 ng/ml).4 Ca(2+) antagonists such as verapamil and SKF 525A produced a dose-dependent inhibition of the vasodilator action of glucagon. Verapamil (2.5 mug/ml) also antagonized cyclic AMP-induced vascular relaxations.5 The vasodilator effect of verapamil was inhibited dose-dependently by raising the concentration of extracellular Ca(2+) from 0.05 to 0.2 g/l (or 1.25 to 5.0 mM) while those elicited by glucagon or cyclic AMP were not influenced, thus suggesting that the latter two drugs do not interfere with Ca(2+) influx.6 Disodium edetate (Na(2)EDTA, 210 to 840 mug/l) produced a dose-dependent vasodilator effect which was attributed to the facilitation of Ca(2+) extrusion from the smooth muscle cells and/or Ca(2+) binding to the cell membrane. The relaxation produced by Na(2)EDTA was significantly blocked by verapamil (10 mug/ml) or SKF 525A (10 mug/ml).7 The results were taken as an indication that glucagon produces at least a fraction of its vasodilator effect by promoting Ca(2+) extrusion from the vascular smooth muscle cells and/or Ca(2+) binding to or sequestration into intracellular sites, presumably via a cyclic AMP-dependent mechanism.

Studies on the mechanism of action of various vasodilators.

1 The vascular relaxant effects of histamine, adenosine, isoprenaline nitroglycerine, papaverine and 3-isobutyl-l-methylxanthine (IBMX) were assessed individually, in strips of rabbit renal artery moderately contracted with noradrenaline (NA) in the absence or presence of phosphodiesterase inhibitors (papaverine and IBMX) or verapamil, a Ca(2+) antagonist.2 The vasodilator effect of histamine was potentiated by papaverine (6.1 x 10(-7) M) and IBMX (4.4 x 10(-5) M) but inhibited dose-dependently by verapamil (5.1 and 51.0 x 10(-7) M).3 Adenosine-induced vascular relaxations were greatly increased in the presence of papaverine (6.1 x 10(-7) M) but significantly reduced in the presence of IBMX (4.4 x 10(-5) M) or verapamil (5.1 and 51.0 x 10(-7) M).4 The vasodilatation produced by isoprenaline was increased in the presence of IBMX (4.4 x 10(-5) M) or papaverine (6.1 x 10(-7) M), but inhibited by verapamil (5.1 and 51.0 x 10(-7) M).5 The vascular relaxant effects of nitroglycerine and papaverine were inhibited in the presence of IBMX (4.4 x 10(-5) M) or verapamil (5.1 and 51.0 x 10(-7) M). Papaverine (6.1 x 10(-7) M) also antagonized nitroglycerine-induced vascular relaxation.6 The vasodilator effect of IBMX was greatly reduced in the presence of papaverine (6.1 x 10(-7) M) or verapamil (5.1 and 51.0 x 10(-7) M).7 The vascular relaxant effect of verapamil was reduced proportionally by raising the extracellular Ca(2+) concentration from 1.25 to 5.0 mM while those elicited by histamine, adenosine, isoprenaline, nitroglycerine, papaverine and IBMX were not modified by this procedure.8 These results were taken as an indication that several vasodilators (e.g. histamine, adenosine, isoprenaline, nitroglycerine, papaverine and IBMX), but not a Ca(2+) antagonist such as verapamil, produce a fraction of their vasodilator effects by promoting Ca(2+) extrusion from and/or Ca(2+) sequestration into the vascular smooth muscle cells, via a cyclic adenosine 3',5'-monophosphate-dependent mechanism.

Mode of action of thrombin in the rabbit aorta.

1. Thrombin is a vasoactive protease that elicits the contraction of the rabbit aorta by activating a G-protein coupled receptor through cleavage of its N-terminal extracellular domain. Synthetic peptides corresponding to the newly exposed N-terminus, following thrombin cleavage, have been shown to reproduce some of the activities of thrombin in the rabbit aorta. 2. Intracellular pathways involved in the contractile response of the rabbit aorta to thrombin and synthetic peptides were examined by use of a series of inhibitors. A similar method was applied to characterize the mitogenic effect of thrombin on cultured smooth muscle cells (SMCs) derived from the same tissue. 3. Results from this study indicate that the contractile response of the rabbit aorta to thrombin is dependent on the activation of protein kinase C (PKC) and independent of extracellular calcium. The contractile response to thrombin can be fully reproduced by peptide agonists related to the N-terminal receptor sequence. However, subtle differences seem to exist between the mechanism of the contractile effect of thrombin and of the synthetic peptides, as both PKC activation and extracellular calcium were found to participate in the contractile effect of the synthetic peptides. 4. In cultured SMCs, both thrombin and the synthetic peptides increased inositol phosphate turnover; however, only thrombin elicited a mitogenic effect, which occurs at thrombin concentrations well below those needed to increase inositol phosphate turnover significantly. Activation of a tyrosine kinase pathway is involved in the mitogenic effect of thrombin on aortic SMCs. 5. Altogether these results suggest the existence of subtle differences between the mode of action of thrombin and of synthetic peptides related to the N-terminal thrombin receptor sequence, in the rabbit aorta.

Characterization of angiotensin receptors in vascular and intestinal smooth muscles.

1. A series of analogues of angiotensin II (AT(II)) has been used in the present experiments to characterize receptors for AT(II) in intestinal (rat stomach strip, rat colon) and vascular (rabbit aorta) smooth muscles. Two types of compounds have been chosen: (a) agonists with reduced potency, in which 4-Tyr, 6-His or 7-Pro had been substituted with L-Ala or with Gly and 1-amino-cyclopentane carboxylic acid (Acpc) and (b) competitive antagonists (8-Gly-AT(II), 8-Leu-AT(II)).2. Replacement of 4-Tyr, 6-His and 7-Pro with L-Ala decreases the potency, but does not influence the maximum effect of the analogue, while substitution of the same residue with Gly and Acpc reduces both potency and maximum effect.3. Compounds showing full size maximum responses were chosen to establish the following order of potency on the three preparations: AT(II)>4-Phe-AT(II)>7-Ala-AT(II)>6-Ala-AT(II)>4-Ala-AT(II).4. The four derivatives of AT(II) were completely inactive on tissues desensitized with AT(II). The responses to 5-hydroxytryptamine, acetylcholine and noradrenaline were not significantly modified, except for the rat colon.5. pA(2) values for the two competitive antagonists against AT(II) and 4-Phe-AT(II) were estimated in the three preparations by the use of the cascade superfusion technique. For comparison, pA(2) values were also estimated in rat stomach strips and rabbit aortas suspended in a normal organ bath, according to the method of Schild (1947). The similarities of the pA(2) values obtained in two series of experiments indicate that (a) the cascade superfusion technique is suitable for this type of study and (b) the receptor for AT(II) in the three tissues may be the same.6. It is suggested that receptors for AT(II) in intestinal and vascular smooth muscles may be the same, because (a) the order of potency of various agonists follows the same pattern, (b) the agonists are inactive on tissues desensitized with AT(II), (c) pA(2) values for competitive antagonists are similar in the three preparations.

Vascular mode of action of kinin B1 receptors and development of a cellular model for the investigation of these receptors.

1. Kinins exert a contractile effect on rabbit aortic rings via the stimulation of B1 receptors. Des-Arg9-bradykinin (BK) is more potent than BK on this receptor type. The mode of action of des-Arg9-BK on rabbit aortic tissue has been studied by both the aortic ring contractility assay and a cellular model using cultured aortic smooth muscle cells (SMCs). 2. The des-Arg9-BK-induced contractions in rabbit aortic rings were unaffected by pretreatments with nifedipine, indomethacin, REV-5901 (a 5-lipoxygenase blocker) and LY-83583 (a guanylyl cyclase inhibitor); however, the protein kinase inhibitors H-7 and H-9 significantly reduced the maximal effect of des-Arg9-BK. 3. The contractile responses to des-Arg9-BK in calcium-free Krebs solution were slightly but not significantly attenuated in amplitude, as compared to paired control tissues bathed in Krebs solution, and sustained plateaus of contraction were observed in the absence of Ca2+. However, Ca2+ replenishment further increased the kinin-induced contraction measured in Ca(2+)-free bathing fluid. 4. Despite the lack of evidence of a mediating role for prostaglandin in the mechanical response to des-Arg9-BK, the kinin stimulated the release of prostacyclin from rabbit aorta rings measured as immunoreactive 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha). 5. Smooth muscle cells (SMCs) derived from the rabbit aorta exhibit functional responses to des-Arg9-BK in acute release of 6-keto-PGF1alpha and of inositol phosphate turnover which were inhibited by pretreatment with the B1 receptor antagonist, Lys[Leu8]des-Arg9-BK, but not by the B2 receptor antagonist, Hoe-140. Preincubation of the cells with interleukin- 1 (IL-1) 20 h before stimulation with the kinin had no effect on basal inositol phosphate turnover, but potentiated the acute effect of des-Arg9-BK.6. These results suggest that second mesengers derived from the action of phospholipase C are produced by SMCs when B1 receptors are activated in rabbit aortic tissue. Intracellular calcium stores are primarily mobilized by des-Arg9-BK, although receptor-controlled calcium influx has not been ruled out, and may contribute to initiate the contractile responses. The maintenance of the contractile state involves protein kinase C activity and is consistent with a current model of SMC function. The cell model retains some of the cardinal properties of B1 receptor-mediated vascular responses: endothelium independent PGI2 release and up-regulation by the cytokine IL-1. PGI2 is not involved in the mechanical response, possible because the rabbit aorta is refractory to this prostaglandin.

Structure-activity studies with neurotensin: analysis of positions 9, 10 and 11.

1 The stimulant effects of neurotensin (NT) and NT analogues modified in positions 9, 10 or 11 were evaluated and compared in two pharmacological preparations: the rat stomach strip and the isolated spontaneously beating atria of guinea-pig. 2 The data derived from our structure-activity study suggest that Arg9 and Pro10 mainly contribute to the affinity of neurotensin for its cardiac and smooth muscle receptors. Tyr11 seems to be more closely involved in the process of receptor activation by NT. 3 The order of potency of some NT analogues modified in position 11 (e.g. [D-Phe11]-NT, [D-Tyr11]-NT) was strikingly different from that described in other systems (e.g. hypothermia test and specific mast cell binding). The importance of this observation is discussed.

The stimulatory effects of neurotensin and related peptides in rat stomach strips and guinea-pig atria.

1 The stimulatory effects of neurotensin (NT) and several NT fragments were evaluated in two pharmacological preparations: rat stomach strips and isolated spontaneously beating atria of guinea-pigs.2 In rat stomach strips, NT elicited a dose-dependent contractile effect in concentrations varying between 1.3 x 10(-9) and 5.4 x 10(-7) M.3 The contractile effect of NT (1.3 and 5.4 x 10(-8) M) in this tissue was not modified by atropine (3.4 x 10(-7) M), methysergide (2.0 x 10(-6) M), a mixture of cimetidine (8.0 x 10(-6) M) and diphenhydramine (7.8 x 10(-6) M), indomethacin (1.4 x 10(-5) M), 8-Leu-angiotensin II (1.0 x 10(-6) M), glucagon (2.0 x 10(-6) M) or somatostatin (3.0 x 10(-7) M).4 Rat stomach strips desensitized by bradykinin (6.1 x 10(-6) M) or substance P (7.4 x 10(-6) M) maintained their sensitivities to NT (1.3 and 5.4 x 10(-8) M).5 In guinea-pig atria, NT produced a dose-dependent positive inotropic action in concentrations varying between 5.4 x 10(-10) and 2.7 x 10(-7) M.6 The inotropic effect of NT (2.7 x 10(-9) M) was not influenced by methysergide (2.8 x 10(-6) M), atropine (3.4 x 10(-7) M), practolol (1.5 x 10(-5) M), 8-Leu-angiotensin II (1.0 x 10(-6) M), or indomethacin (1.4 x 10(-5) M), but it was reduced by 37% by cimetidine (4.0 x 10(-5) and 2.0 x 10(-4) M). A combination of cimetidine (4.0 x 10(-5) M) and diphenhydramine (3.9 x 10(-6) M) did not produce a greater inhibition of NT than cimetidine alone.7 Atria desensitized by bradykinin (6.1 x 10(-6) M) or glucagon (2.0 x 10(-6) M) maintained their sensitivities to NT (2.7 x 10(-9) M). Substance P was inactive both as an agonist or antagonist of NT.8 These results suggest the existence of specific NT receptors in rat stomach strips and guinea-pig atria.9 The data derived from our structure-activity study suggest that the minimum structure required for the full stimulation of NT receptors in these two preparations is H-Arg(9)-Pro(10)-Tyr(11)-Ile(12)-Leu(13)-OH. The sequence PyroGlu(1)-Leu(2)-Tyr(3)-Glu(4)-Asn(5)-Lys(6)-Pro(7)-Arg(8)- and the amino acids Ile(12) and Leu(13) appear to contribute mainly to the affinity or binding of NT to its receptor. The chemical groups responsible for the full activation (intrinsic activity) of NT receptors seem to be located in the sequence -Arg(9)-Pro(10)-Tyr(11).

An analysis of the negative inotropic action of somatostatin.

1 Somatostatin (SS) was evaluated as a chronotropic and inotropic agent in isolated spontaneously beating auricles of rats, rabbits and guinea-pigs.2 SS was completely inactive in rat and rabbit auricles but exerted a dose-dependent, negative inotropic effect in guinea-pig auricles in concentrations between 1.5 x 10(-8) to 1.2 x 10(-6) M.3 The negative inotropic effect of SS (6.0 x 10(-8) and 3.0 x 10(-7) M) was not inhibited by a mixture of antagonists containing practolol (7.9 x 10(-6) M), phentolamine (3.5 x 10(-7) M), methysergide (2.8 x 10(-7) M), diphenhydramine (3.9 x 10(-5) M), cimetidine (4.0 x 10(-5) M) atropine (3.4 x 10(-7) M) and indomethacin (1.4 x 10(-5) M).4 The negative inotropic effect of SS was greatly potentiated by reduction in the Ca(2+) concentration of the medium from 5.0 to 1.25 mM.5 On a molar basis, SS was equipotent with acetylcholine (ACh) as a negative inotropic agent in the guinea-pig auricles.6 SS (6.0 x 10(-8) and 6.0 x 10(-7) M) was found to inhibit selectively the positive inotropic action of neurotensin (NT) in guinea-pig but not in rat auricles.7 The inhibitory action of SS against NT was independent of its negative inotropic action.8 These results suggest that SS produces its negative inotropic action by interacting with specific receptors presumably located in the cell membrane of guinea-pig atria. The interaction between SS and its receptor may cause a decreased Ca(2+) diffusion and/or transport into the atrial cells. The physiological and pharmacological significance of these results is discussed.

Regulation of kinin-induced contraction and DNA synthesis by inflammatory cytokines in the smooth muscle of the rabbit aorta.

1. In rabbit aortic rings, the contractile response to kinins is mediated by the B1 receptors for kinins; the response is upregulated from an initial null level in a time- and protein synthesis-dependent manner. Incubation (3 h) with human recombinant interleukin-1 beta (IL-1 beta) selectively amplified the contractile response to the B1 receptor agonist Sar-[D-Phe8]des-Arg9-BK, while it did not affect the contractile effect of other agents (angiotensin II, endothelin-1, phenylephrine). 2. Oncostatin M (OSM), but not macrophage migration inhibitory factor (MIF), increased the contractile response to the B1 receptor agonist, des-Arg9-bradykinin (des-Arg9-BK). 3. Cultured smooth muscle cells derived from the rabbit aorta exhibit a significant des-Arg9-BK-induced increase in [3H]-thymidine incorporation if pretreated with a cyclo-oxygenase inhibitor (diclofenac) and concomitantly treated with the cytokines IL-1 or OSM. Angiotensin II, endothelin-1 or phenylephrine, alone or in the presence of IL-1 beta, exerted little effect on DNA synthesis in these cells. 4. The pharmacological characterization of the mitogenic response to kinins using a set of agonist and antagonist analogues is consistent with mediation by B1 receptors. Des-Arg9-BK-induced DNA synthesis is suppressed by prostaglandin E2 by a prostacyclin mimetic (iloprost), by the Ser/Thr protein kinase inhibitor, H-7, and by a tyrosine kinase inhibitor (i.e. an erbstatin analogue). 5. B1 receptor-mediated responses and their capacity to be regulated by cytokines, are retained in rabbit aortic smooth muscle cells. Such responses could be relevant to tissue repair mechanisms and hypertrophic medial responses to injury in arteries.

Synthetic C5a receptor agonists. Pharmacology, metabolism and in vivo cardiovascular and hematologic effects.

Recent investigations have produced novel compounds that act on the receptor for anaphylatoxin C5a. These products are C-terminal analogues of C5a, some of which are modified extensively. We have measured the receptor affinities of such analogues in a binding assay on human neutrophils (PMNs). We have also characterized their pharmacological profiles in vitro on the isolated rabbit portal vein and pulmonary artery, on superoxide release by PMNs as well as in vivo in the anesthetized rabbit (acute hypotensive and neutropenic effects). The metabolic resistance of these analogues was also evaluated in the presence of different peptidases. One of these compounds, MePhe-Lys-Pro-D-Cha-Phe-D-Arg, behaved as an antagonist on the release of superoxide by neutrophils while exerting agonist activity in all other assays. Its partial agonist status was documented in a receptor down-regulation experiment on PMNs where its activity was compared with those of recombinant C5a and of protamine which behaves as a competitive antagonist on these cells. Degradation studies indicated that the discrepancy between the affinity of certain analogues in vitro and their potency in vivo was probably linked to their metabolic stability.

Cholesterol and fatty acid metabolism in piglets fed sow milk or infant formula with or without addition of cholesterol.

Several studies have reported that plasma cholesterol and phospholipid (PL) levels of arachidonic acid (20:4n-6) are lower and PL levels of linoleic acid (18:2n-6) are higher in infants fed formula than in infants fed human milk. Plasma cholesterol level and possibly the dietary intake of cholesterol could be related to plasma PLn-6 fatty acid metabolism because plasma PL 18:2n-6 is used for esterification of plasma free cholesterol. Whether the low cholesterol content of infant formula as compared with human milk is related to the difference in plasma n-6 fatty acid levels between infants fed human milk and infants fed formula is not known. This study determined the effect of feeding formula with 0.05 mmol cholesterol/L, formula with 1.09 mmol cholesterol/L, or sow milk with 0.34 mmol cholesterol/L on plasma, liver, and bile lipid fatty acid levels and liver low-density lipoprotein (LDL) receptor mass in piglets. Liver microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase activity and plasma lathosterol were assayed as indices of liver and body cholesterol synthesis, respectively. Formula with or without cholesterol added, or sow milk, was fed from birth to 18 days of age. Providing cholesterol in the formula did not correct the significantly lower plasma cholesterol or plasma and liver PL 20:4n-6 levels associated with formula feeding. The liver total cholesterol and cholesteryl esters (CE), biliary bile acid, and PL concentrations were significantly higher and the liver HMG CoA reductase activity and plasma lathosterol:cholesterol ratio were significantly lower in piglets fed the formula with cholesterol than in piglets fed the formula without cholesterol added.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiovascular and abdominal motor responses evoked by intravenous neurotensin in guinea pigs.

The intravenous (IV) infusion of neurotensin (NT) in anesthetized guinea pigs was found to elicit dose-dependent increases of systemic blood pressure (BP) and of heart rate (HR), accompanied by abdominal motor responses consisting in transient, twitch-like contractions of the abdominal wall (TAWC), and a slowly developing, relatively sustained increase of the basal abdominal wall tension (AWT). The TAWC responses were inhibited in animals pretreated with pancuronium, morphine, clonidine, and CP-96,345 [a neurokinin (NK) antagonist], were potentiated by naloxone, but were not modified by atropine or prior (30 s) intraperitoneal (IP) injection of lidocaine. The BP increases caused by IV NT were reduced by clonidine and by IP lidocaine only. The HR increases were attenuated by morphine and clonidine only. Increases of the basal AWT were resistant to all drug treatments and were attributed to a passive stretch of the abdominal wall caused by cecal distension. No defecation was observed in any of the animals given IV NT. These results were interpreted as an indication that the pressor and TAWC responses to IV NT represent an integrated nociceptive response likely to be triggered in part by NT-induced activation of abdominal visceral afferents. A NK acting through NK-1 receptors may participate in TAWC responses to IV NT.