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To investigate structure-function relationships in gamma-glutamyl carboxylases, the enzyme from Drosophila melanogaster was characterized. Four cysteine residues were shown to be important determinants for enzymatic activity. Native Drosophila substrates have not yet been identified, but propeptides of human prothrombin and factor IX are recognized by the Drosophila enzyme. The presence of the propeptide region increased apparent affinity by approximately 200-fold, and mutation of a hydrophobic residue of factor IX propeptide (F-16A) decreased carboxylation by 90%, as in the human enzyme. Substrate recognition appears to be highly conserved between the human and Drosophila gamma-glutamyl carboxylases. Inactivation of Drosophila gamma-glutamyl carboxylase by non-sense mutations or insertional mutagenesis by P-element insertion have no apparent effects on growth and fertility under laboratory conditions.

We have studied the hemodynamic effects of neuropeptide Y (NPY) and its COOH-terminal fragment NPY-(18–36) in conscious rats. Intra-arterial injection of NPY rapidly elevated systemic vascular resistance (SVR), which remained high for greater than 30 min. Cardiac output (CO) decreased, and it remained low for greater than 30 min. Accordingly, blood pressure rose only transiently and returned to base-line values within 5 min. The reduction of CO could be attributed to a decreased stroke volume with an only marginal reduction of heart rate. Thus a direct cardiodepressive effect of NPY rather than baroreflex activation appears to be the major cause of the reduced CO. In vitro experiments excluded the possibility that NPY has direct negative inotropic effects and suggest that its cardiodepressive action is caused by coronary vasoconstriction or by presynaptic inhibition of norepinephrine release. Intra-arterial injections of NPY-(18-36) caused different hemodynamic effects. NPY-(18–36) decreased CO in a manner similar to that seen with NPY but initially did not elevate SVR, resulting overall in a reduced blood pressure. Only later, when blood pressure was reduced, was an elevation of SVR observed, which could be associated with increased plasma levels of catecholamines, angiotensin II, vasopressin, and NPY. Thus NPY-(18–36) mimics the cardiac effects of NPY but does not elicit its vascular effects. As NPY-(18–36) discriminates between NPY receptor subtypes in vitro, we conclude that the cardiac and vascular effects of NPY are mediated by distinct receptor subtypes.

Somatostatin was purified from anglerfish pancreatic islets using acetic acid extraction, gel filtration (Bio-Gel P-10), ion exchange chromatography (CM Bio-Gel A), and reversed phase high pressure liquid chromatography. The resulting peptide was characterized by RIA, bioassay, and determination of amino acid composition. Anglerfish islet somatostatin was found to possess an amino acid composition and immunological and biological activities equivalent to synthetic somatostatin. Sequence analyses revealed that the primary structure was H-Ala-Gly-cyclo-[Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys]-OH. These results demonstrate that anglerfish islet somatostatin has the same primary structure as somatostatin from all other sources characterized to date.

The central nervous system effects of thyrotropin-releasing hormone (TRH) on proximal duodenal bicarbonate secretion were studied in freely moving rats. Cerebroventricular administration of TRH (0.5-5.0 nmol) significantly stimulated basal duodenal bicarbonate secretion, whereas intravenous administration of TRH did not. Ganglionic blockade with chlorisondamine and truncal vagotomy abolished TRH-induced bicarbonate secretion, whereas atropine significantly attenuated the response. The vasoactive intestinal peptide (VIP) receptor antagonist, (4Cl-D-Phe6, Leu17) VIP given intravenously completely prevented the stimulatory effect of central TRH on duodenal bicarbonate secretion. In contrast, hypophysectomy, adrenalectomy, opiate and noradrenergic blockade, or indomethacin did not affect the TRH-induced bicarbonate response. Intravenous administration of VIP and carbachol significantly stimulated bicarbonate outputs, and these responses were blocked by the VIP antagonist and atropine, respectively. These results indicate that TRH may serve as a central nervous system mediator that stimulates duodenal bicarbonate secretion in rats by increasing vagal outflow. Vagal stimulation induced by TRH increases duodenal bicarbonate secretion by the release of VIP and, in part, by activation of a muscarinic pathway but not by pituitary, adrenal, and noradrenergic pathways or endogenous opiates and prostaglandins. The actions of peripheral VIP and carbachol appear to be mediated by specific VIP and muscarinic receptors, respectively.

While chronic treatment with LHRH agonists interferes with reproductive functions (Rivier et al., 1978, 1979; Rivier and Vale, 1979, 1981b; Vale et al., 1979), it is at first associated with an increase in gonadotropin and steroid secretion. This initial stimulation may be considered undesirable under some clinical circumstances. Additionally, LHRH agonists have a multiplicity of sites of action in rats which, if verified in humans, may also prove them unacceptable for human use.

Bullfrog sympathetic ganglion neurons have a “late, slow” excitatory postsynaptic potential (EPSP) that is mediated by a peptide similar to mammalian luteinizing hormone-releasing hormone (M-LHRH) (Jan, Y. N., L. Y. Jan, and S. W. Kuffler (1979) Proc. Natl. Acad. Sci. U. S. A. 76: 1501–1505). On biochemical evidence (Eiden, L. E., E. Loumaye, N. Sherwood, and R. L. Eskay (1982) Peptides 3:323–327) the endogenous LHRH-like peptide in the ganglion may be identical to teleost LHRH (T- LHRH; [Trp7,Leu8]M-LHRH) (Sherwood, N., L. Eiden, M. Brownstein, J. Speiss, J. Rivier, and W. Vale (1983) Proc. Natl. Acad. Sci. U. S. A. 80: 2794–2798). We have found that T-LHRH acts qualitatively like M- LHRH on bullfrog ganglion cells, but it is quantitatively more potent. As for M-LHRH, the primary action of T-LHRH under single-electrode voltage clamp is to inhibit the M-current, a voltage-dependent potassium current that is active in the region between rest and threshold in these cells. The M-current is also inhibited during the late, slow EPSP. Both T-LHRH and the late, slow EPSP can also induce an additional inward current, associated with an increased conductance, in some cells. An LHRH antagonist is effective against both T-LHRH and the late, slow EPSP. These results are consistent with a role for T-LHRH as the natural transmitter for the late, slow EPSP in bullfrog sympathetic ganglia.

A symposium report on the synthesis and structural characterization both by NMR in CD3CN solution. and by x-ray diffraction of the cyclic tetrapeptides cyclo(b-Ala-L-Pro-b-Ala-Aaa) (Aaa = L-Pro, L-Val) in order to verify the usefulness of the sequence b-Ala-Pro-b-Ala as molecular tool to force the peptide in a gamma-turn conformation.