Martha Knight

Cholecystokinin-octapeptide effects on conditioned-avoidance behavior, stereotypy and catalepsy

The effect of peripherally administered cholecystokinin-octapeptide (CCK8) was tested on signaled-avoidance behavior, apomorphine-induced stereotypy, and catalepsy. Rats were trained to avoid shock in a signaled shuttle-box avoidance task, and then given CCK8, tetragastrin, or haloperidol. CCK8 (20-3840 micrograms/kg i.p.) reduced avoidance in a dose-dependent manner. The impairment at maximal dose levels was approximately 25% from its predrug level compared to 50% with haloperidol (100 micrograms/kg i.p.). Combined injection of CCK8 (320 micrograms/kg i.p.) and haloperidol (75 micrograms/kg i.p.) reduced avoidance significantly more than either drug alone. The effects of CCK8 were relatively brief in that active avoidance was impaired if CCK8 was injected up to 15 min before the avoidance test. CCK8 also facilitated the extinction of avoidance. Tetragastrin (177 micrograms/kg i.p.), which elicits gastrointestinal effects resembling CCK8, did not affect avoidance. In addition, CCK8 diminished apomorphine-induced stereotypy up to 5 min after CCK8 injection. CCK8 (20-1280 micrograms/kg i.p. and s.c.) failed to produce catalepsy in vertical grip tests. These data suggest that peripherally administered CCK8 has sedative and certain neuroleptic-like effects on behavior.

Cardiovascular effects of prorenin blockade in genetically spontaneously hypertensive rats on normal and high-salt diet

Recent reports have demonstrated a potential role of tissue prorenin in the pathogenesis of cardiovascular and renal damage. This study was designed to examine the role of prorenin in the pathogenesis of target organ damage in spontaneously hypertensive rats (SHRs), the best naturally occurring experimental model of essential hypertension. To this end, we studied 20-wk-old male SHRs receiving a normal diet and 8-wk-old male SHRs given food with 8% NaCl. One-half the rats in each group were given prorenin inhibitor (PRAM-1, 0.1 mg.kg(-1).day(-1)) via osmotic minipumps; the other half served as controls. Arterial pressure, left ventricular function, cardiovascular mass indexes, cardiac fibrosis, and renal function were examined at the end of the experiment. Arterial pressure was unaffected by PRAM-1 in rats on either regular or salt-excess diets. In those rats receiving a normal diet, the blockade of prorenin activation consistently reduced left ventricular mass but affected no other variable. Salt-loaded rats given PRAM-1 for 8 wk demonstrated (1) reduced serum creatinine level, (2) decreased left ventricular mass, (3) improved left ventricular function, and (4) reduced left ventricular fibrosis. These data demonstrated that the blockade of nonproteolytic activation of prorenin exerted significant cardiovascular and renal benefit in SHRs with cardiovascular damage produced by salt excess and suggested that the activation of cardiovascular or renal prorenin may be a major mechanism that mediates cardiac and renal damage in this form of accelerated hypertension.

Improved spiral tube assembly for high-speed counter-current chromatography

The original spiral tube support (STS) assembly is improved by changing the shape of the tubing, with 1-cm presses perpendicularly along the length. This modification interrupts the laminar flow of the mobile phase. The tubing in the four return grooves to the center of the rotor is flattened by a specially made pressing tool to increase the number of spiral layers and decrease the dead space volume, thus increasing the column efficiency. The performance of this spiral tube assembly was tested in separations of dipeptides and proteins with suitable polar two-phase solvent systems. The results revealed that the present system yields high partition efficiency with a satisfactory level of stationary phase retention in a short elution time. The present high-speed counter-current chromatographic (HSCCC) system will be efficiently applied to a broad spectrum of two-phase solvent systems including aqueous-aqueous polymer phase systems (TPAS) which are used for separation of biopolymers such as proteins and nucleic acids.

Cholecystokinin-octapeptide fragments: binding to brain cholecystokinin receptors

Structural determinants of cholecystokinin octapeptide (CCK-8) binding to central nervous system receptors have been studied to assess the relative importance of the amino and the carboxyl end of the active peptide sequence, CCK-(26-33). The relative ability to inhibit equilibrium binding of [125I]CCK-33 to guinea pig cortical membranes was determined for a series of amino and carboxyl terminal fragments of CCK-8. While N-acetyl CCK-(26-29), N-acetyl CCK-(26-30) amide and N-acetyl CCK-(26-31) amide were inactive, the N-acetyl CCK-(26-32) amide fragment displayed binding to central receptors. Of the carboxyl terminal peptide fragments, both CCK-(29-33) and CCK-(30-33) bound less potently than CCK-8; CCK-(31-33) interacted more weakly than the tetra- and pentapeptide, but with a higher affinity to brain receptors than to peripheral receptors. The heptapeptide, CCK-(26-32) amide, and the tripeptide, CCK-(31-33), are known to antagonize CCK action at peripheral receptors. The heptapeptide bound to central receptors 25 times more potently than a known peripheral antagonist, dibutyryl cyclic GMP. Thus these peptides may act centrally to oppose CCK-8 mediated functions.

Spiral Tube Assembly for High-Speed Countercurrent Chromatography: Choice of Elution Modes for Four Typical Two-Phase Solvent Systems

Abstract A novel spiral tube support is introduced which allows accommodating a multlilayer spiral column made of a long piece of fluorinated plastic tubing. Two spiral columns tested in this study consist of 1.6 mm ID and 0.85 mm ID FEP (fluorinated ethylenepropylene) and PTFE (polytetrafluoroethylene) tubing with the total capacity of 100 mL and 40 mL, respectively. Performance of these spiral columns was examined with a two phase solvent system composed of 1‐butanol/acetic acid/water at a volume ratio of 4∶1∶5 using tryptophyl‐tyrosine (try‐tyr) and valyl‐tyrosine (val‐tyr) as test samples under various revolution speeds and flow rates. Among 4 different elution modes tested, L‐I‐T (lower phase pumped from the internal head end of the spiral column) and U‐O‐H (upper phase pumped from the external tail of the spiral column) at a low flow rate produced the best results especially for both columns. The highest peak resolution (Rs) of over 3.5 was obtained from U‐O‐H at a flow rate of 1 mL/min with 74% stationary phase retention. At a flow rate of 5 mL/min, the highest revolution speed at 1,200 rpm improved the peak resolution in both elution modes. The present system may be useful for purification of various polar compounds in biomedical researches.Optimal elution modes were determined for four typical two-phase solvent systems each with different physical parameters to achieve the best peak resolution and retention of the stationary phase by spiral tube high-speed countercurrent chromatography using a suitable set of test samples. Both retention of the stationary phase and partition efficiency are governed by an interplay between two forces, i.e., Archimedean Screw force and radial centrifugal force gradient of the spiral channel. In the polar solvent system represented by 1-butanol./acetic acid/water (4:1:5, v/v/v) with settling time of over 30 s, the effect by the radial centrifugal gradient force dominates giving the best separation of dipeptides either by pumping the lower phase from the inner terminal or the upper phase from the outer terminal of the spiral channel. In the moderately hydrophobic two-phase solvent system represented by hexane/ethyl acetate/methanol/0.1 M HCl (1:1:1:1) with settling time of 19 s, and two hydrophobic solvent systems of hexane/ethanol/water (5:4:1, v/v/v) and non-aqueous binary system of hexane/acetonitrile both having settling time of 9, the effect of the Archimedean screw force play a major role in hydrodynamic equilibrium, giving the best separations by pumping the lower phase from the head or the upper phase from the tail of the spiral channel.

CCK26–33 Degrading Activity in Brain and Nonneural Tissue: A Metalloendopeptidase

Abstract: Cholecystokinin octapeptide (CCK26–33) is metabolized by neural membranes with an initial cleavage to CCK29–33 and subsequent breakdown to CCK31–33 and CCK32–33; this pattern of proteolysis occurs on incubation with either P2 or purified lysed synaptosomal membranes. To determine whether the pattern of CCK26–33 proteolysis is unique to the brain and whether regional brain differences in its pathway or rate exist, we analyzed the proteolysis of CCK by synaptic membranes of various brain areas and cellular membranes of peripheral tissue. The pattern of degradation in brain did not differ among the regions studied. The overall proteolysis rate, as measured by the formation of tryptophan, was higher in the striatum than in the cortex, although CCK29–33 was formed at the same rate in both areas. In nonneural tissue, the rate of degradation was highest in liver membranes and lowest in pancreatic acinar cell preparations. Thus, it appears that degradative peptidases are not necessarily colocalized with CCK receptors. The pattern of product formation is the same in peripheral compared with CNS membranes; thus, the degradative pathway does not appear to be unique to brain tissue. The enzyme present in synaptic membranes that is responsible for CCK29–33 formation requires a metal ion and sulfydryl groups for the catalysis and thus is a metalloendopeptidase. Furthermore, its activity is inhibited by Ac‐Gly‐Phe‐Nle‐al, a peptide aldehyde whose sequence bears some homology to the amino acid sequence in the region of CCK26–33 that is cleaved by this enzyme.

Rapid purification of synthetic bombesin by countercurrent chromatography on the multi-layer coil planet centrifuge

Abstract A temperature-controlled multi-layer coil planet centrifuge rapidly yields highly efficient preparative separations of polar compounds. Capability of the method was demonstrated on a one-step purification of crude synthetic bombesin with a two-phase solvent system composed of n-butanol/dichloroacetic acid/water (100:1:100). Under an elevated temperature at 45°C to 50°C, the bombesin peak was eluted within two hours. Reversed phase HPLC analysis of the bombesin fractions showed over 98% purity. The method may be applicable to many other peptides and polar compounds.

Purification of Solid-Phase Synthesized Peptides on the Coil Planet Centrifuge

Abstract The analytical flow-through coil planet centrifuge, an instrument for countercurrent chromatography, performs the preparative purification of synthetic peptides. Various two-phase solvent systems have been tried with either phase mobile to purify many synthesized peptides. A series of N-terminal fragment peptides of cholecystokinin octapeptide (CCK 26–33) were synthesized by solid-phase techniques and purified on the coil planet centrifuge. The peptides were sulfated and chromatographed again. For hydrophobic peptides, purification is effected in solvent systems with a mobile aqueous phase. The n-butanol, acetic acid and water system (4:1:5 by volume) with the lower phase mobile was utilized. For sulfated peptides, the neutral system, 0.2 M ammonium acetate and n-butanol was generally applied.

Tolerance to the anti-avoidance properties of cholecystokinin-octapeptide

Previous investigations have suggested a neuroleptic-like action of cholecystokinin-octapeptide (CCK8) on conditioned-avoidance behavior. This study was initiated to test tolerance to this effect. Rats were trained to avoid electric shock in a shuttle box under a free-operant (Sidman) avoidance paradigm. Each shuttle response postponed a 0.2 sec, 1 mA shock for 20 sec. If the rat failed to respond, shock was delivered every 5 sec until a response occurred. After avoidance training, half of the rats received two daily injections of CCK8 (0.320 mg/kg, IP) and half received saline for 7 days. Rats were then tested on the Sidman avoidance 1 min after receiving CCK8 (0.640 mg/kg, IP) or saline. CCK8 depressed avoidance responding if rats received saline for 7 days prior to the test. Rats pretreated with CCK8 for 7 days were not significantly affected by CCK8 during the avoidance test. Thus, repeated injections of CCK8 result in tolerance to its anti-avoidance properties.

Spiral counter-current chromatography of small molecules, peptides and proteins using the spiral tubing support rotor.

An important advance in countercurrent chromatography (CCC) carried out in open flow-tubing coils, rotated in planetary centrifuges, is the new design to spread out the tubing in spirals. More spacing between the tubing was found to significantly increase the stationary phase retention, such that now all types of two-phase solvent systems can be used for liquid-liquid partition chromatography in the J-type planetary centrifuges. A spiral tubing support (STS) frame with circular channels was constructed by laser sintering technology into which FEP tubing was placed in 4 spiral loops per layer from the bottom to the top and a cover affixed allowing the tubing to connect to flow-tubing of the planetary centrifuge. The rotor was mounted and run in a P.C. Inc. type instrument. Examples of compounds of molecular weights ranging from <300 to approximately 15,000 were chromatographed in appropriate two-phase solvent systems to assess the capability for separation and purification. A mixture of small molecules including aspirin was completely separated in hexane-ethyl acetate-methanol-water. Synthetic peptides including a very hydrophobic peptide were each purified to a very high purity level in a sec-butanol solvent system. In the STS rotor high stationary phase retention was possible with the aqueous sec-butanol solvent system at a normal flow rate. Finally, the two-phase aqueous polyethylene glycol-potassium phosphate solvent system was applied to separate a protein from a lysate of an Escherichia coli expression system. These experiments demonstrate the versatility of spiral CCC using the STS rotor.