A backbone-cyclic, receptor 5-selective somatostatin analogue: synthesis, bioactivity, and nuclear magnetic resonance conformational analysis.

Cyclo(PheN2-Tyr-D-Trp-Lys-Val-PheC3)-Thr-NH2 (PTR 3046), a backbone-cyclic somatostatin analogue, was synthesized by solid-phase methodology. The binding characteristics of PTR 3046 to the different somatostatin receptors, expressed in CHO cells, indicate high selectivity to the SSTR5 receptor. PTR 3046 is highly stable against enzymatic degradation as determined in vitro by incubation with rat renal homogenate and human serum. The biological activity of PTR 3046 in vivo was determined in rats. PTR 3046 inhibits bombesin- and caerulein-induced amylase and lipase release from the pancreas without inhibiting growth hormone or glucagon release. The major conformation of PTR 3046 in CD3OH, as determined by NMR, is defined by a type II' beta-turn at D-Trp-Lys and a cis amide bond at Val-PheC3.

Neuropeptide Y inhibits sympathetic neurotransmission in ipsilaterally innervated but not contralaterally reinnervated superior tarsal smooth muscle of the rat.

The superior tarsal smooth muscle (STM), which elevates the upper eyelid, normally is innervated by sympathetic neurons from the ipsilateral superior cervical ganglion that are not neuropeptide Y-immunoreactive (NPY-ir). Following neonatal ganglionectomy, this target is reinnervated by sympathetic nerves from the contralateral superior cervical ganglion that are strongly NPY-ir. We examined the effects of exogenously administered NPY on STM tone, response to norepinephrine, and sympathetic neurotransmission in ipsilaterally innervated and contralaterally reinnervated STMs. NPY (2-10 micrograms/kg iv) increased blood pressure but did not alter STM tone. Similarly, contractile responses to co-administered norepinephrine were not affected. These findings imply an absence of direct and indirect postjunctional actions of NPY on STM. Contractions elicited by stimulation of the cervical sympathetic nerve (1.5 Hz) were not affected by NPY on the contralaterally reinnervated side; however, ipsilateral contractions were decreased in a dose-dependent fashion, with an inhibition of about 40% at 10 micrograms/kg. We conclude that while the STM is unresponsive to exogenously administered NPY, this peptide exerts selective inhibitory effects on the ipsilateral NPY-ir-negative but not the contralateral NPY-ir-positive innervation. This suggests that the neonatally denervated STM is reinnervated by contralateral fibers that are functionally different from the normal ipsilateral innervation in being devoid of functional prejunctional NPY receptors.

Binding of phosphate ions to actin.

The decrease of the critical monomer concentration of ADP-actin by millimolar phosphate concentrations has been analysed in terms of equilibrium constants for binding of phosphate ions to ADP-actin. The decrease has been explained by a 10-fold greater affinity of phosphate ions to polymeric ADP-actin (binding constant 100 M-1) than to monomeric ADP-actin (binding constant 10 M-1). Phosphate has an almost identical effect on the critical monomer concentration of the pointed ends of gelsolin-capped actin filaments in the presence of ATP. The phosphate concentration required for half-maximal decrease of the critical monomer concentration of the pointed ends has been determined to be about 15 mM. By using the fluorescent ATP-analogue, 1,N6-ethenoadenosine 5'-triphosphate, phosphate ions have been found to bind also to monomeric ATP-actin, yet with a slightly higher affinity than to monomeric ADP-actin (binding constant 50 M-1).

The actin treadmill.

Actin filaments can assemble at the barbed end and disassemble simultaneously at the pointed end. Prerequisites for this treadmilling reaction are the structural polarity of actin filaments and tight coupling of the actin assembly reaction and the adenosine triphosphate hydrolysis occurring during actin polymerization. In this article, investigations on the actin treadmill are reviewed.

Equilibrium constant for binding of an actin filament capping protein to the barbed end of actin filaments.

Depolymerization of treadmilling actin filaments by a capping protein isolated from bovine brain was used for determination of the equilibrium constant for binding of the capping protein to the barbed ends of actin filaments. When the capping protein blocks monomer consumption at the lengthening barbed ends, monomers continue to be produced at the shortening pointed ends until a new steady state is reached in which monomer production at the pointed ends is balanced by monomer consumption at the uncapped barbed ends. In this way the ratio of capped to uncapped filaments could be determined as a function of the capping protein concentration. Under the experimental conditions (100 mM KCl and 2 mM MgCl2, pH 7.5, 37 degrees C) the binding constant was found to be about 2 X 10(9) M-1. Capping proteins effect the actin monomer concentration only at capping protein concentrations far above the reciprocal of their binding constant. Half-maximal increase of the monomer concentration requires capping of about 99% of the actin filaments. A low proportion of uncapped filaments has a great weight in determining the monomer concentration because association and dissociation reactions occur at the dynamic barbed ends with higher frequencies than at the pointed ends.

Treadmilling of actin.

Actin filaments can assemble at the barbed end and disassemble simultaneously at the pointed end. A higher monomer concentration is required to balance the association of actin monomers and the dissociation of filament subunits at the pointed end than at the barbed end. This treadmilling reaction or disparity of the apparent affinity of the two ends for monomers is caused by a continuous hydrolysis of adenosine triphosphate occurring during the association of a monomer with a filament end. In this article, in vitro investigations on treadmilling are reviewed and emerging physiological implications are discussed.

Similar affinities of ADP and ATP for G-actin at physiological salt concentrations.

The equilibrium constant for the exchange of ATP and ADP at G-actin was determined by fluorimetric titration of G-actin-bound epsilon-ATP by ATP or ADP. The affinity of ATP for G-actin was found to be only about 3-fold higher than the affinity of ADP for G-actin at 37 degrees C, pH 7.5 and physiologically relevant salt concentrations (100 mmol K+/l, 0.8 mmol Mg2+/l, less than 0.01 mmol Ca2+/l).