Randall B. Murphy

Stimulation of astroglial 5-HT1A receptors releases the serotonergic growth factor, protein S-100, and alters astroglial morphology

Stimulation of astroglial 5-HT1A receptors causes astroglial cells to acquire a more mature morphology and to release a factor (or factors) which promotes growth of serotonergic neurons. By using an antibody-blocking approach, we have shown that at least one of the growth-promoting factors thus released is the astroglial-specific protein S-100. This may be a particularly important observation, in view of studies implicating S-100 in both Downs syndrome and Alzheimers disease.

Modulation of [3H]-dopamine binding by cholecystokinin octapeptide (CCK-8)

Cholecystokinin-octapeptide (CCK-8) is a putative neurotransmitter which has been demonstrated previously to occur in midbrain dopamine neurones. We observe that CCK-8 causes changes in both the affinity and density of binding sites for [3H]-dopamine in rat striatal homogenates, in vitro, upon incubation with the peptide at a concentration of 1 micromolar. A dose-response study of the competetion of CCK-8 with [3H]-dopamine binding indicates an IC50 for the peptide of 450 nM; desulfated CCK-8 and the related peptide caerulin are at least 4-fold less active than CCK-8. CCK-8 was also administered to rats in a separate study; the binding of [3H]-dopamine was evaluated to homogenates of striata and olfactory tubercles obtained from these animals, which had been treated with systemic injection at a dose of 20 micrograms/kg, daily, for four days. A decrease in the number of striatal binding sites for the radioligand was observed, with a concomitant increase in the number of binding sites in the olfactory tubercle. These data collectively suggest a possible regulatory role for CCK-8 in the ascending dopamine systems.

MDMA (Ecstasy) effects on cultured serotonergic neurons: evidence for Ca2+-dependent toxicity linked to release

Animal studies have established a correlation between release of 5-hydroxytryptamine (5-HT) and the long-term reduction of 5-HT (toxicity) by 3,4-methylenedioxymethamphetamine (MDMA) with the S(+) enantiomer being more active than the R(-). Using a microculture system of fetal raphe neurons, the enantiomers of MDMA were tested to determine if a similar difference in potency existed. The results showed that the development of the uptake capacity of [3H]5-HT in 4-day cultures was half-maximally inhibited by a single application at time of plating of 5 X 10(-6) M S(+)-MDMA and 5 X 10(-5) M R(-)-MDMA. In order to determine if the Ca2(+)-independent release (chemically induced through the transporter protein and inhibited by reuptake blockers) or the Ca2(+)-dependent release (K(+)-induced and inhibited by presynaptic receptors) contributed to the toxicity, fluoxetine and D1 and alpha 2 agonists were studied. The results showed that both forms of release were involved in the loss of [3H]5-HT uptake capacity, with the direct MDMA-induced Ca2(+)-independent (fluoxetine-sensitive) release being the first step. Evidence from binding studies indicates that MDMA has a micromolar affinity for the 5-HT2 receptor, and our studies in culture showed that ketanserin, a specific 5-HT2 antagonist, was effective at attenuating the effects of S(+)-MDMA on the development of the [3H]5-HT uptake capacity by the cultured raphe neurons. The 5-HT2 receptor is linked to increased intracellular Ca2+ through a second messenger phosphatidylinositol (PI)-hydrolysis mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Lateralization of striatal dopamine (D2) receptors in normal rats.

A highly significant endogenous lateral asymmetry in striatal dopamine (D2) receptors was found utilizing the binding of [3H]spiroperidol (0.6 nM) to homogenates prepared from the left and the right striata of male rats individually assayed. The mean level of binding was 23% greater in left than in right striatum, while in mesolimbic terminal zones 10% more binding was observed on the right than on the left. These data provide evidence for an intrinsic lateralization of neuroleptic (D2) receptors in the rat brain, and are consistent with prior determinations of endogenous asymmetries in neurochemical and behavioral indices of dopaminergic function in the nigrostriatal system.

Solvent-free lipid bimolecular membranes of large surface area

The formation of a solvent-free lipid bimolecular membrane of large surface area (approx. 2 mm2) by the successive transfer of two monolayers upon an aperature of a closed chamber has been demonstrated. The electrical parameters of the membrane appear to be similar to the conventional Montal-Mueller solvent-free membrane. The use of a closed chamber greatly increases the stability of the membrane to mechanical disturbances and produces hydrostatic equilibrium necessary for electrical measurements.

Hydrostatic stabilization of solvent-free lipid bimolecular membranes

Abstract The technique of obtaining hydrostatically stabilized solvent-free lipid bimolecular membranes is described. The technique involves the preparation of a solvent-containing or an essentially solvent-free membrane upon an aperture of a hydrostatically closed chamber combined with a system for automatic digital control of the transmembrane pressure difference. The electrochemical behavior of the resultant systems is investigated and the advantages of the hydrostatically stabilized membrane are analyzed.

Evidence that oocyte maturation induced by an oncogenic ras-p21 protein and insulin is mediated by overlapping yet distinct mechanisms

We have recently shown that a peptide (residues 35-47) from a functional region of the ras p21 protein, thought to be involved in the binding of p21 to GTPase activating protein, the antibiotic azatyrosine, known to induce the ras-recision gene, and the selective protein kinase C inhibitor, CGP 41,251, all inhibit oncogenic p21 protein-induced maturation of oocytes in a dose-dependent manner. We now show that these three agents only partially inhibit insulin-induced oocyte maturation, known to be dependent on activation of cellular p21 protein. On the other hand, the anti-p21 protein antibody Y13-259 completely inhibits both insulin- and oncogenic p21 protein-induced maturation as does a tetrapeptide, CVIM, known to block the enzyme farnesyl transferase which covalently attaches the farnesyl moiety to the p21 protein allowing it to attach to the cell membrane. Our results suggest that while the oncogenic and insulin-activated normal p21 proteins share certain elements of their signal transduction pathways in common, these pathways diverge and allow for selective inhibition of the oncogenic pathway.

A peptide from the gap-binding domain of the ras-p21 protein as well as azatyrosine block ras-induced maturation of Xenopus oocytes

The ras-oncogene-encoded p21 protein is known to produce malignant transformation of NIH 3T3 cells as well as maturation of Xenopus oocytes when microinjected into these cells. p21 protein is known to bind a GTPase activating protein (GAP) intracellularly; residues 32-45 have been implicated in interacting with GAP. We demonstrate here that a peptide corresponding to residues 35-47 of p21 as well as the antibiotic azatyrosine inhibit the ras-induced maturation of Xenopus oocytes in a dose-related manner upon microinjection. We have previously shown that this p21 peptide and azatyrosine could inhibit the effects of p21 protein on cell transformation and pinocytosis in NIH 3T3 cells. In the present study, in which we have extended these results to the oocyte system, we also demonstrate that both partially inhibit insulin-induced oocyte maturation, a process which is thought to involve activation of endogenous p21 protein; on the other hand, both agents fail to inhibit oocyte maturation induced by progesterone, which is known not to act through p21 protein activation. Control studies with other peptides and tyrosine analogues support the selective nature of these events. These results suggest that both the p21-related peptide and azatyrosine have potent anti-ras effects intracellularly.

Irreversible blockade of striatal dopamine receptors in vivo by a derivative of α-flupenthixol

Flupenthixyl chloride (FPT-Cl), a derivative of the dopamine (DA) receptor antagonist and neuroleptic α-flupenthixol possessing a cloroethyl side chain, has been prepared and evaluated for use in vivo in affinity labeling of DA receptors. Binding of [3H]spiperone to rat striatal DA receptors was markedly altered up to 12 h after intraventricular injection of FPT-Cl as compared with controls, while Scatchard plots of [3H]spiperone binding obtained on rat striatal homogenates 24 and 48 h after injection of FPT-Cl had values of Bmax significantly lower than in controls. The results suggest that the administration of FPT-Cl leads to irreversible and possibly covalent blockade of a portion of the spiperone binding sites in rat striatum. A second population of receptors appears to be blocked reversibly and presumably noncovalently by FPT-Cl, and these spiperone binding sites are partially reactivated in vivo after 48 h.

Comparative X-ray crystallographic evidence for a β-bend conformation as the active structure for peptide T in T4 receptor recognition

A sequence similarity has been found between two segments of endothiapepsin (acid proteinase, 2APE), bovine pancreatic ribonuclease A, and peptide T, a segment of the gp120 protein of human inmmune deficiency virus (HIV), which has been implicated in blocking viral attachment to the T4 receptor. The two similar sequences of the acid proteinase enzyme are Leu-Ile-Asp-Ser-Ser-Ala-Tyr-Thr (residues 169–176) and Tyr-Thr-Gly-Ser-Leu-Asn-Tyr-Thr (residues 175–182). Since the X-ray crystallographic structures of the acid proteinase and ribonuclease are known, it has been possible to determine whether the three-dimensional structures of the segments are similar. Portions of both of the segments of acid proteinase are directly superimposable on the structure of the RNase A 19–26 segment. The fact that the three similar sequences from two completely unrelated proteins give rise to almost identical structures raises the possibility that these segments may be involved in nucleating the folding of these proteins. In addition, this provides further support for the concept that the octapeptide sequence of peptide T of HIV, which is also similar in sequence to the 19–26 sequence of RNase A, is also structurally similar to these residues, which adopt a β-bend conformation. Furthermore, comparison of similarities and differences in the structure of these similar sequences provides an explanation for alterations in the biological activity of various truncated or substituted derivatives of peptide T and additional confirmation of the structural requirements for peptide T in T4-receptor recognition.