Wiktor Koziołkiewicz

The 3-7 fragment of angiotensin II is probably responsible for its psychoactive properties

The abilities of angiotensin II-(3-7)-pentapeptide (A-II-(3-7), 1 nmol) and angiotensin II (A-II, 1 nmol) to influence rats psychomotor and cognitive behaviours were compared. Both peptides, given intracerebroventricularly (i.c.v.), 15 min before the experiment, increased number of crossings, rearings and bar approaches in the open field. A-II-(3-7) as well as A-II, at the same doses and routes, significantly intensified stereotypy produced by apomorphine (1 mg/kg) and amphetamine (6.5 mg/kg), both given intraperitoneally. The 3-7 fragment of A-II and A-II in equimolar doses (1 nmol, i.c.v.) were similarly effective in improving learning of conditioned avoidance responses and recall of a passive avoidance behaviour. Taken together, these data and our previous findings indicate that, in rats, the 3-7 fragment of A-II is responsible for the psychoactive properties of angiotensins.

Comparison of platelet aggregability and P-selectin surface expression on platelets isolated by different methods.

Three methods commonly used for isolation of blood platelets from plasma were compared. Platelets were isolated by: 1) a washing method; 2) a method of metrizamide-gradient centrifugation; 3) a modified method of gel-filtration. The last method employed BSA-Sepharose gel instead of routinely used Sepharose gel saturated with BSA. BSA-Sepharose gel was prepared by covalent binding of thermally deactivated BSA to CNBr-activated Sepharose 2B. In contrast to platelets isolated by the other methods, an aggregability of the gel-filtered platelets and control platelets in plasma, both activated with ADP, were comparable. When expression of P-selectin on the surface of freshly isolated platelets was examined, the gel-filtered platelets exhibited the same extent of fluorescence signal as platelets in the citrated blood, whereas platelets isolated by the other methods exhibited twice the extent of the signal. The methods involving the centrifugation process cause a low but a significant platelet activation.

Platelet membrane lipid fluidity and intraplatelet calcium mobilization in type 2 diabetes mellitus.

Abstract: The aim of the present study was to relate the impairments in calcium mobilization and/or release to the altered membrane dynamics in platelets from patients with type 2 diabetes mellitus. Higher expression of P‐selectin (1.4‐fold, NS) and the reduction in GPIbα expression (by 27.8 ± 16.7%, p < 0.0002), as well as the increased fractions of platelet microparticles (p < 0.03), reflected more intensified platelet release reaction in diabetic platelets. Overall, diabetic platelets appeared more vulnerable to stimuli facilitating calcium mobilization (by 41%, p < 0.01) and less susceptible to preventive effects of the agents hampering calcium release from intraplatelet storage pools (by 38%, p < 0.01). Both the increased calcium mobilization from intraplatelet storage pools and higher levels of intracellular free calcium in the presence of procaine in diabetic platelets correlated with the reduced platelet membrane lipid fluidity (resp. pR < 0.03 and pR < 0.015). We conclude that the biophysical state of platelet membrane components in diabetes mellitus is the crucial determinant of platelet hyperfunction and probably contributes to the intensified calcium mobilization in diabetic platelets. The depressed preventive effects of procaine on platelet release reaction and calcium mobilization in diabetic platelets may result from the primary dislocations and/or distortions of membrane components caused by the diabetic state.

Release of calcium and P-selectin from intraplatelet granules is hampered by procaine

The inhibition of platelets by some local anaesthetics has been related to the modulation of platelet membrane lipid fluidity, and one of these compounds, procaine, has been proven to be particularly effective inhibitor. In the present study, we examined the effect of procaine on the mobilization of intracellular granule contents in isolated washed platelets. We revealed that the presence of 10 mg/ml procaine significantly hampered platelet release reaction, as demonstrated by the significant reduction in the expression of platelet P-selectin (CD62) on one hand, and significantly enhanced expression of GPIb alpha (CD42b) antigen on the other, following either 1 hour incubation of washed platelets at room temperature (%CD62: 37.1+/-6.8% of control incubated without procaine, p<<0.0001; %CD42b: 116.2+/-6.3% of control, p<0.0001) or activation of whole blood platelets with ADP, TRAP, or thrombin. Procaine, which acted as a rigidizer, significantly decreased platelet membrane fluidity (ESR h(+1)/h0 ratio of 5-DOXYL-Ste reduced down to 93.1+/-3.7% of control, p<0.001). In washed Fura-2-loaded platelets procaine not only brought about the significantly reduced Ca2+ release from intraplatelet storage pools after platelet stimulation with 15 micromol/l ADP (25.3+/-12.5% of control, p<0.001), but also it significantly increased the reduction in Ca2+ concentration upon the addition of Ca2+ chelator, EDTAK2 (48.9+/-13.5% vs. 40.9+/-12.1% of initial [Ca2+]i concentration, p(1,alpha)<0.025). Overall, procaine considerably reduced calcium mobilization from intraplatelet storage pools and Ca2+ efflux across platelet membrane. Based on these data, we suggest that the preventive effects of procaine on platelet release reaction and calcium mobilization might relate to the changes in the organization of membrane components embedded into a lipid bilayer, which are crucial in triggering of platelet release reaction. Procaine-mediated dislocations of some membrane components and/or distortion of lipid-protein interactions could generate a steric hindrance, which might interfere with platelet signal transduction, thus leading to impaired mobilization of Ca2+ and other components from intraplatelet storage pools.

Inhibition of tongue reflex in rats by tooth pulp stimulation during cerebral ventricle perfusion with (6–11) substance P analogs

The effect of 12 C-terminal hexapeptide analogs of substance P perfused into the cerebral ventricles on the trigemino-hypoglossal reflex caused by incisor pulp stimulation in rats was investigated. A substitution of the L-isomer by D at position 6, 7, or 8 of the substance P analogs used in this study caused the most pronounced inhibition of the trigemino-hypoglossal reflex. A correlation between the degree of inhibition of the reflex and concentration of the peptide used was observed.

Concentration of RGDS-containing degradation products in uremic plasma is correlated with progression in renal failure

A concentration of protein degradation products containing the RGDS sequence, which could contribute to a lower reactivity of uremic platelets, has been estimated in both uremic (n = 16) and control (n = 7) plasmas. Degradation products and other small molecules were separated from plasma by filtration through AMICON YM-10 filter. RGDS antigen was determined in filtered material using the radioimmunoassay method based on monospecific anti-RGDS rabbit polyclonal antibodies. The concentration of RGDS-containing degradation products in uremic plasma ranged from 0.8 to 353 nM with mean value 58.6 +/- 24.9 nM and was higher than in control (0.7 to 5.9 nM, mean value 2.1 +/- 0.9 nM). Moreover, the level of RGDS-antigen positively correlated with plasma creatinine concentration (R = 0.87, p < 0.001). The filtered material showed an inhibitory effect on fibrinogen binding to control platelets in respect to RGDS-antigen concentration. We conclude that the elevated concentration of RGDS-containing degradation products in uremic plasma is partially responsible for bleeding tendency in renal failure.

Platelet membrane fluidity and intraplatelet Ca2+ mobilization are affected in uraemia

Abstract:  In present investigations, platelet membrane fluidity and intraplatelet Ca2+ mobilization were analysed in uraemic platelets by fluorescence techniques. Thirteen non‐dialyzed uraemic patients and 16 control subjects were examined. Anisotropy of DPH‐probe, measured at 37°C, was significantly higher in control (0.2236 ± 0.0050) than in uraemic platelets (0.1969 ± 0.0082; p < 0.01). There was no difference between control (109.8 ± 6.0 nm) and uraemic platelets (100.0 ± 7.3 nm) when the basal [Ca2+]i in resting platelets was determined. Activation of platelets by ADP (12.5 μm) or by thrombin (0.1 U/ml) resulted in an increase in [Ca2+]i. It was significantly higher (p* < 0.003 for ADP and p* < 0.009 for thrombin, respectively) in control platelets (383.6 ± 56.3 nm and 2031.0 ± 298.8 nm, respectively) than in uraemic ones (191.0 ± 21.3 nm and 838.7 ± 144.1 nm, respectively). The amount of released Ca2+ was higher in control platelets activated by both ADP and thrombin (157.6 ± 21.4 nm and 409.3 ± 71.0 nm, respectively) than in uraemic platelets (76.7 ± 15.7 nm and 203.0 ± 29.3 nm, respectively) and the differences were significant (p < 0.01 and p* < 0.01, respectively). These results indicate an abnormal intracellular Ca2+ mobilization in uraemic platelets. Both increased membrane fluidity and decreased Ca2+ mobilization should be considered as a possible reason of reduced fibrinogen receptor exposure on uraemic platelets.

Synthesis and biological activity of Substance P C-terminal hexapeptide analogues: Structure-activity studies

The synthesis of six hexapeptide analogues of C-terminal Substance P fragment containing alpha, beta-dehydrophenylalanine (delta Phe) in the position 7 or 8 is described. The effect of the structural changes on the hypotensive activity and antigenic properties of analogues was compared. It was found that substitution of delta Phe in various analogues of C-terminal hexapeptide of Substance P resulted in different effects on the hypotensive activity. The analogues [Glp6, delta Phe7]SP6-11 and [Glp6, delta Phe8]SP6-11 retained 70% and 45% of hypotensive activity of the C-terminal hexapeptide of Substance P, respectively but they showed a completely destroyed antigenic determinant. The analogues containing additionally Sar or His in the position 9 showed a complete lack of both: hypotensive activity and expression of the antigenic determinant of Substance P.

Vascular endothelial growth factor-D modulates oxidant–antioxidant balance of human vascular endothelial cells

Vascular endothelial growth factor‐D (VEGF‐D) is an angiogenic and lymphangiogenic glycoprotein that facilitates tumour growth and distant organ metastasis. Our previous studies showed that VEGF‐D stimulates the expression of proteins involved in cell–matrix interactions and promoting the migration of endothelial cells. In this study, we focused on the redox homoeostasis of endothelial cells, which is significantly altered in the process of tumour angiogenesis. Our analysis revealed up‐regulated expression of proteins that form the antioxidant barrier of the cell in VEGF‐D‐treated human umbilical endothelial cells and increased production of reactive oxygen and nitrogen species in addition to a transient elevation in the total thiol group content. Despite a lack of changes in the total antioxidant capacity, modification of the antioxidant barrier induced by VEGF‐D was sufficient to protect cells against the oxidative stress caused by hypochlorite and paraquat. These results suggest that exogenous stimulation of endothelial cells with VEGF‐D induces an antioxidant response of cells that maintains the redox balance. Additionally, VEGF‐D‐induced changes in serine/threonine kinase mTOR shuttling between the cytosol and nucleus and its increased phosphorylation at Ser‐2448, lead us to the conclusion that the observed shift in redox balance is regulated via mTOR kinase signalling.

2-Hydroxyimino-2-phenylacetonitrile active esters in peptide synthesis

Abstract A number of 2-hydroxyimino-2-phenylacetonitrile esters of acylamino acids have been synthesized. These compounds react readily with amino acid or peptide esters to elongate the peptide chain.