Simultaneous effects of the platelet 5-HT2 and alpha 2-adrenergic receptor populations on phosphoinositide hydrolysis.

The interaction of multiple receptor populations on a common second messenger system is a critical aspect of cell function and may be involved in pathology. We studied the interactions of the 5-HT2, alpha 2-adrenergic and prostaglandin (PGI2) receptors on phosphoinositide (PI) turnover in human platelets. Serotonin and epinephrine (EPI) stimulated PI hydrolysis in a dose-dependent manner. The PI turnover response to serotonin was mediated by the 5-HT2 receptor. The PI response to EPI was mediated by alpha 2-adrenergic receptors. An additive PI turnover response was generated by the combination of 5-HT and EPI. The sum of the maximal responses to 5-HT (72.5 +/- 4.9%) and EPI (56.0 +/- 4.2%) approximated the maximal response (129.3 +/- 9.5) to the combination. Prostacyclin (PGI2) at 1 microgram/mL reduced PI turnover by 21.8 +/- 1.1%. The PI response to 5-HT and EPI was not significantly altered once the reduction in the baseline PI turnover by PGI2 is taken into account. Similarly, PGI2 did not reduce PI hydrolysis stimulated by a combination of 5-HT (0.2 mM) and EPI (0.1 mM) once the decrease in baseline was taken into account (p greater than 0.20). The summation of serotonin stimulation of PI turnover by a combination of both epinephrine and serotonin was blocked by either yohimbine or ketanserin. These studies indicate: (1) the pool of phospholipases appears to exceed the maximal capacity of the individual alpha 2-adrenergic and 5-HT2 receptor populations to activate this second messenger system. (2) inhibition of serotonin or epinephrine-stimulated PI turnover by prostacyclin is due to a lowering of basal PI turnover. Future studies should examine other cell systems to assess the generalizability of these findings regarding the differences in effects on a second messenger system when activated by one receptor population as opposed to two different receptor types.

Ontogeny of PCP and sigma receptors in rat brain.

Phencyclidine (PCP) binds with high affinity to two receptors in rat brain--the PCP receptor and the Sigma receptor. Although both receptors are present prenatally, and their number increases postnatally, their rate of increase, compared to the increase in brain protein, is quite different, yielding distinct ontogenic profiles. Thus, PCP receptors are present on prenatal day 2 and show a further 15-fold increase by postnatal day 28. In contrast, Sigma receptors are present at their highest density during the perinatal period, and decline thereafter. The Kd of the PCP receptor for TCP remains constant throughout development, whereas the Kd of the Sigma receptor for (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine decreases 40% postnatally. On postnatal day 6, both PCP and Sigma receptors display a pharmacological profile similar to that observed in adult animals.

Expression of (+)-3-PPP binding sites in the PC12 pheochromocytoma cell line.

Phencyclidine binds with high affinity to both PCP and sigma receptors. We investigated whether the clonal cell line PC12 expressed either of these receptors, and found that these cells contain a haloperidol-sensitive (+)-[3H]3-PPP binding site with a KD of 56 nM, but no PCP binding sites. The (+)3-PPP binding sites in PC12 cells displayed a reversed stereoselectivity for the benzomorphan opiates compared to CNS sigma receptors. Neither nerve growth factor nor sodium butyrate treatment affected the expression of either (+)-3-PPP or TCP binding sites in PC12 cells.

Influence of molecular structure on half-life and hydrolysis of dipeptides in plasma: importance of glycine as N-terminal amino acid residue.

To investigate the effect of molecular structure on plasma disappearance and metabolism of dipeptides, rats were injected intravenously with individual dipeptides, and at various intervals after injection, dipeptide and amino acid concentrations were measured in plasma, tissues, and urine. In addition, plasma hydrolase activity against individual dipeptides was investigated. The half-lives of Ala-Leu, Ala-Tyr, and Ala-Gln were shorter than those of dipeptides with glycine substituting for alanine. Furthermore, the increases in plasma concentrations of leucine, tyrosine, and glutamine and rates of dipeptide hydrolysis by plasma enzymes were far greater with alanyl than glycyl dipeptides. In fact, Ala-Leu behaved like a mixture of corresponding free amino acids in raising the plasma concentration of leucine while Gly-Leu did not. There was no significant difference in either plasma half-life or hydrolysis when Leu-Gly and Leu-Ala were used as substrates, but both had rapid rates of hydrolysis in plasma. In comparison to Gly-Leu, Phe-Leu and Arg-Leu had shorter half-lives and greater rates of hydrolysis in plasma. On the other hand, Asp-Leu had a slower rate of plasma hydrolysis than Gly-Leu, but its excretion in the urine was much greater than that of Gly-Leu. In contrast to Gly-Leu and Ala-Leu, Gly-Pro was detected intracellularly in liver, muscle, and particularly, kidney. In fact, the intracellular concentration of Gly-Pro in kidney was either equal to or greater than Gly-Pro concentration in plasma. Increases in intracellular amino acid concentration after injection of individual dipeptides were considerably greater in the kidney than in either liver or muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

A concentrated mixture of amino acids and dipeptides for total parenteral nutrition.

Using a subhuman primate (baboon) we have investigated the utility of a 20% mixture of amino acids and dipeptides as the nitrogen source for total parental nutrition. The mixture, besides containing all 8 essential amino acids and a number of non-essential amino acids (glutamate, aspartate, arginine, histidine, serine, ornithine and alanine), contained 6 dipeptides (Gly-Ile, Gly-Leu, Gly-Val, Gly-Tyr, Gly-Gln, and Ala-Gln) and acetyl-cysteine. A week of total parenteral nutrition was preceded by one week of oral feeding. The caloric intake and composition during the two periods was identical except for the nitrogen source, which was intact protein during the oral period, and the mixture of amino acids and dipeptides during the parenteral period. There was no significant difference between gain in body weight or nitrogen balance during the two periods. There were selective increases in plasma and muscle concentrations of amino acids during the parenteral period, which appeared to reflect the amino acid enrichment of the nitrogen source. The efficient utilization of dipeptides was evidenced by their small concentrations in plasma and urine. The urinary excretion of dipeptides was about 1% of the amount infused. This efficiency of dipeptide utilization persisted even when the infusion rate of the amino acid and dipeptide mixture was increased by 7-fold. There was no alteration in liver, kidney, and immune function during the parenteral period. The data indicate the efficacy and safety of the mixture of amino acids and dipeptides as the nitrogen source for parenteral nutrition.

Protein nutrition and amino acid metabolism after 4 weeks of total parenteral nutrition with a mixture of 14 dipeptides: serendipitous observations on effects of sepsis in baboons.

We investigated parameters of nutrition, metabolism, and organ function after 4 wk of total parenteral nutrition (TPN) in baboons receiving either dipeptides or amino acids as the nitrogen source. The two groups showed no significant difference with respect to gain in body weight, nitrogen balance, plasma and muscle concentrations of amino acids, plasma concentrations of proteins, and leucine incorporation into muscle protein. All dipeptides were efficiently utilized as evidenced by trace concentrations of dipeptides in plasma and urine; they produced no deleterious effect on the function of liver, kidney, or immune system. Development of infection in several baboons increased urinary excretion of urea nitrogen but had no effect on urinary excretion of dipeptides and amino acids with the single exception of taurine, which was greatly increased. In conclusion, the data show long-term efficacy and safety of the dipeptide mixture as the sole nitrogen source for TPN.

Efficacy of a synthetic dipeptide mixture as the source of amino acids for total parenteral nutrition in a subhuman primate (baboon). Plasma concentration, metabolic clearance, and urinary excretion of a series of dipeptides.

To assess the efficacy and safety of oligopeptides as substrates for total parenteral nutrition, we investigated the effects of intravenous infusion of a synthetic dipeptide mixture, as compared with a corresponding amino acid mixture, on a range of parameters of nutrition, metabolism, and organ function in baboons. In all respects the two periods of total parenteral nutrition, each lasting for 1 wk, were identical except for the difference between the forms of amino acids in the parenteral solutions, being in free form in one period and in dipeptide form in the other. The dipeptide mixture was composed of a series of 12 dipeptides each containing glycine in the N-terminal position and either an essential or nonessential amino acid in the carboxyl position. The infusion of the dipeptide mixture and the amino acid mixture had similar effects on parameters of protein nutrition (e.g., nitrogen balance, plasma aminogram, urinary excretion of 3-methylhistidine) and metabolism (such as plasma concentrations of insulin, glucose, lipids). During infusion of the dipeptide mixture, rapid metabolic clearance resulted in a barely detectable concentration of most dipeptides in plasma. Total loss of dipeptides was 1.3% +/- 0.1% of the infused amount. The functions of liver, kidney, bone marrow, and the immune system remained the same before and during the two periods of treatment. In conclusion, the data showed that (a) there was efficient utilization of dipeptides when infused as a mixture and (b) parameters of nutrition, metabolism, and organ function were well maintained after 1 wk of total parenteral nutrition with the dipeptide mixture acting as the sole nitrogen source.

Combined effects of calcium and dibutyryl cyclic AMP on germinal vesicle breakdown in the mouse oocyte.

Mouse oocytes were cultured in the presence of dibutyryl cyclic AMP (dbcAMP) and various agents that affect cytoplasmic calcium concentrations. Treatment that inhibited calcium uptake potentiated the inhibitory effect of dbcAMP and treatments which stimulated cellular calcium uptake overcame the effect of dbcAMP. Elevated extracellular calcium (greater than 10 mM) significantly decreased the inhibitory effect of concentrations of dbcAMP up to 150 microM when compared to control levels of calcium (1.7 mM). In addition, the calcium ionophore A23187 (greater than 1 microM) significantly overcame the effect of dbcAMP in media that contained 1.7 or 20 mM calcium. In the presence of 41 microM-dbcAMP the calcium antagonist verapamil increased (in a dose-dependent fashion) the percentage of oocytes blocked at the germinal vesicle stage, from 21% with 10 microM-verapamil to 99% with 200 microM. A similar dose-dependent, reversible potentiation of the effect of dbcAMP was found with tetracaine, which also lowers cytoplasmic calcium concentrations. These results suggest that a minimum level of cytoplasm calcium is required for the initiation of germinal vesicle breakdown and that the action of dbcAMP is mediated by its effect upon this calcium.

The effect of calcium on the first meiotic division of the mammalian oocyte.

In the mammalian ovary, fully grown oocytes are arrested at the diplotene stage of the first meiotic division. While oocytes in vivo resume meiosis only in response to a preovulatory surge of gonadotropic hormones, oocytes isolated from the ovaries and cultured in vitro will spontaneously resume meiosis. Both in vivo and in vitro, meiotic maturation proceeds through the extrusion of the first polar body, where it is again arrested until fertilization. We have used the spontaneous, in vitro maturation of the mouse oocyte to examine the role of calcium in germinal vesicle breakdown (GVBD) and polar body extrusion. In calcium-free medium or in the presence of concentrations of lanthanum greater than 0.5 mM, the oocytes degenerate rapidly. However, there does not appear to be any effect upon maturation that can be distinguished from a general toxicity of these media. In contrast, two treatments that are known to inhibit transmembrane calcium movements, verapamil and tetracaine each, individually, inhibit polar body formation. They have no effect on GVBD. In addition, oocytes cultured in media containing a higher calcium concentration than control media (greater than 10 mM versus 1.71 mM) show a significantly higher percentage of polar body formation. We conclude that, in these culture conditions, extracellular calcium is not required for GVBD but is required for the completion of the first meiotic division of a mammalian oocyte.