D. Mitolo-Chieppa

Upregulation of Proinflammatory Proteins Through NF-κB Pathway by Shed Membrane Microparticles Results in Vascular Hyporeactivity

Objective—Microparticles are membrane vesicles with procoagulant and proinflammatory properties released during cell activation, including apoptosis. The present study was designed in dissecting the effects evoked by microparticles on vascular reactivity. Methods and Results—Microparticles from either apoptotic T lymphocytic cells or from plasma of diabetic patients with vascular complications induced vascular hyporeactivity in response to vasoconstrictor agents in mouse aorta. Hyporeactivity was reversed by nitric oxide (NO) synthase plus cyclooxygenase-2 inhibitors, and associated with an increased production of vasodilatory products such as NO and prostacyclin. Microparticles induced an upregulation of proinflammatory protein expressions, inducible NO-synthase and cyclooxygenase-2, mainly in the medial layer of the vessels as evidenced by immunochemical staining. In addition, microparticles evoke NF-&kgr;B activation probably through the interaction with the Fas/Fas Ligand pathway. Finally, in vivo treatment of mice with lymphocyte-derived MPs induces vascular hyporeactivity, which was reversed by the combination of NO and cyclooxygenase-2 inhibitors. Conclusion—These data provide a rationale to explain the paracrine role of microparticles as vectors of transcellular exchange of message in promoting vascular dysfunction during inflammatory diseases.

Interleukins 1 beta and 6 induce functional alteration of rat colonic motility: an in vitro study

Background In rodents, interleukins administration induces intestinal changes similar to those found in inflammatory bowel disease. We investigated the effects of in vivo subchronic treatment with IL‐1 beta and IL‐6 on rat colonic mucosa and circular smooth muscle.

Functional characterization of endothelin receptors in hypertensive resistance vessels.

OBJECTIVE The physiological and pathophysiological functions of endothelin-1 in modulating the regional blood flow of normal and spontaneously hypertensive rats (SHR) were studied in the perfused mesenteric vascular bed, a useful model for investigating resistance vessels. DESIGN AND METHODS We used 12-week-old SHR and Wistar-Kyoto (WKY) rats. Endothelin A (ETA) receptor responsiveness was evaluated by endothelin-1 (0.2-2 mumol/l) concentration-response curves, and repeated in the presence of indomethacin and the ETA and endothelin B (ETB) receptor antagonists BQ-485 and BQ-788, respectively. ETB receptor responsiveness was tested by sarafotoxin S6c concentration-response curves, obtained in the noradrenaline-precontracted mesenteric vascular bed, and repeated after treatment with BQ-788 and after endothelial denudation. RESULTS In both groups, endothelin-1 induced concentration-dependent contraction; SHR exhibited a markedly increased maximal effect compared with WKY rats (P < 0.01). BQ-485 produced a shift to the right for endothelin-1 concentration-response curves in both groups, with a higher pA2 (negative common logarithm of the antagonist that produces an agonist dose ratio of 2) value in SHR than in WKY rats (P < 0.01). The increase in the maximal effect produced by endothelin-1 in SHR was prevented by indomethacin, which also induced a significant increase in the endothelin-1 concentration producing the half-maximal response (EC50) in SHR (P < 0.05). Sarafotoxin S6c produced an ETB-dependent endothelium-mediated relaxant effect in WKY rats, which was not observed in SHR. CONCLUSIONS The higher vasoconstriction induced by endothelin-1 in SHR may be related to a greater number of available ETA receptors, due to the presence of an ETA receptor subtype. This mechanism may be linked to the production of prostanoids that add to the direct endothelin-1-evoked vasoconstriction. These results, together with the lack of relaxation in response to sarafotoxin S6c in SHR, suggest that an imbalance in the endothelin-1 ability to induce both contraction and relaxation is present in SHR with sustained hypertension, manifesting as a greater contractile effect evoked in this strain.

Hyporeactivity of mesenteric vascular bed in endotoxin-treated rats

Vascular reactivity and activation of the nitric oxide (NO) pathway were investigated in perfused mesenteric vascular bed removed from rats 5 h after i.p. injection of bacterial lipopolysaccharide (E. coli lipopolysaccharide, 30 mg kg -1). Lipopolysaccharide treatment induced hyporesponsiveness to noradrenaline. Maximal noradrenaline-induced vasoconstriction was significantly reduced in lipopolysaccharide-treated vs. untreated preparations. Continuous infusion of L-arginine (L-Arg) (0.2 mM) enhanced noradrenaline hyporeactivity of lipopolysaccharide-treated rats. N omega-Nitro-L-arginine methyl ester (L-NAME) (0.2 mM), a non-selective inhibitor of NO synthase, failed to completely restore the noradrenaline hyporeactivity of lipopolysaccharide-treated + L-Arg-infused mesenteric vascular bed. After L-NAME treatment. Methylene blue (10 microM), a guanylate cyclase inhibitor, produced no additional increase of noradrenaline vasoconstriction in lipopolysaccharide-treated + L-Arg-infused mesenteric vascular bed, suggesting that an NO-independent activation of guanylate cyclase may be excluded. In lipopolysaccharide-treated preparations, L-Arg (0.2 mM) elicited a significant increase in nitrite production, which was antagonized by L-NAME. In conclusion, lipopolysaccharide-induced noradrenaline hyporesponsiveness of rat resistance vessels can only be partially explained by NO overproduction. Other mechanisms, probably related to vasoconstriction, may be involved.

Mechanism of action of ketamine in the current and voltage clamped myelinated nerve fibre of the frog

1 The effects of the general anaesthetic ketamine, on the frog isolated node of Ranvier, were studied under current and voltage clamp conditions. 2 Ketamine (0.5 and 1 mM) reversibly decreased the amplitude of the action potential and increased both the duration of the action potential and the threshold potential. When the K current was blocked, spontaneous action potentials appeared after washout of the drug. 3 Ketamine rapidly blocked the Na current and more slowly modified a fraction of Na channels (about 10%) to give rise to a non‐inactivatable (late) Na current. After washout of the drug, the block reversed more rapidly than the ketamine‐induced late Na current disappeared. 4 Steady‐state outward, peak Na and ketamine‐induced late Na currents were rapidly and reversibly blocked by ketamine with an apparent dissociation constant of 0.7 mM. 5 Both peak Na and ketamine‐induced late Na currents were reversibly blocked by procaine.

Idiopathic chronic constipation: tachykinins as cotransmitters in colonic contraction

Background Tachykinins (TKs) have been shown to be involved in the excitatory enteric motor pathway. This study aimed to examine the direct and nerve‐mediated effect of specific NK1, NK2 and NK3 receptor agonists and antagonists in colonic preparations from control subjects and patients with idiopathic chronic constipation (ICC).

INHIBITION OF IONIC CURRENTS IN FROG NODE OF RANVIER TREATED WITH NALOXONE

1 Myelinated nerve fibres of frog sciatic nerve were investigated under current and voltage clamp conditions. In the presence of 68 μM external naloxone, the action potential was completely, though progressively, blocked within 15 min of drug superfusion. The resting potential remained constant. 2 Under voltage clamp conditions both peak Na+ and steady‐state K+ currents were decreased reversibly by external naloxone. Both currents were reduced in a dose‐dependent manner but, whereas sodium current was affected by the smallest concentrations of naloxone (1.3 up to 12.5 μm), potassium current was decreased only by higher concentrations (25 up to 112 μm). 3 The time‐course of development of the effect on both Na+ and K+ currents after exposure to 112 μm naloxone (a concentration giving more than 50% of decrease) showed that the effect develops quickly within the first 2 min of exposure to the drug, but afterwards both currents continue to fall more slowly, though progressively. 4 Experiments with constant test pulses to Em = −10 mV and conditioning prepulses of various amplitudes, showed that the Na inactivation curve, h∞ (Em), was shifted in a negative direction along the potential axis; the shape of the curve was also slightly changed in the presence of naloxone since the shift was larger near the top of the curve. All the observed effects were reversible after returning to the standard Ringer solution. 5 Internal naloxone (< 0.2 mm) reduced the amplitude of the action potential as well as peak Na+ and steady‐state K+ currents; the sodium inactivation curve, h∞ (Em), was shifted to more negative potentials. 6 A possible anaesthetic‐like activity of naloxone on the nodal membrane is discussed.

Electrophysiological studies of the effects of the general anaesthetic etomidate on frog myelinated nerve fibre

1 The effects of the general anaesthetic etomidate (0.1 to 1 mmM) upon the node of Ranvier of frog isolated nerve fibres were investigated under current and voltage clamp conditions. 2 When added to the external solution, etomidate reversibly decreased the amplitude of the action potential. The action potential block, induced by the drug, was reversed by increasing the membrane potential. 3 Etomidate rapidly and reversibly blocked the Na current with an apparent dissociation constant of 0.6 mM. In the presence of the drug, the steady‐state inactivation‐voltage curve of the Na current was shifted towards negative voltages. 4 The block of Na current by etomidate was partially removed by repetitive depolarization preceded by a 50 ms period of hyperpolarization. In contrast, the block was enhanced when the repetitive depolarization was not preceded by hyperpolarization. This suggests that Na channels were preferentially blocked by the drug in the inactivated state. 5 The K current was reversibly blocked by etomidate with an apparent dissociation constant of 0.2 mM. In the presence of the drug, the K current showed an apparent fast inactivation suggesting that K channels were blocked in the open state. 6 We conclude that at higher concentrations than those attainable in the mammalian brain following single anaesthetic doses the general anaesthetic etomidate has a ‘local anaesthetic‐like’ action on the peripheral nervous system.

Endothelin-1-receptor-mediated responses in resistance vessels of young and adult spontaneously hypertensive rats.

Objective To assess whether primary changes in endothelin-1 (ET-1) receptor responsiveness or secondary vessel functional modifications could characterize the effects evoked by ET-1 in the mesenteric vascular bed (MVB) of prehypertensive 5-week-old and 12-week-old spontaneously hypertensive rats (SHRs). Design and methods We used male 5-week-old and 12-week-old SHRs and sex- and age-matched Wistar-Kyoto (WKY) rats as controls. ET-1 receptor responsiveness was evaluated by ET-1 (0.04–2 μmol/l) concentration-response curves and repeated with indomethacin and BQ-123 (0.1–0.5 μmol/l), the latter a selective ETA receptor antagonist. ETB receptor responsiveness was tested by sarafotoxin S6c (1–100 nmol/l) and IRL-1620 (0.1–10 nmol/l) concentration-response curves, obtained in the noradrenaline-precontracted MVB. Results At 5 weeks of age, ET-1 induced a similar concentration-dependent contraction in SHRs and WKY rats, with an overlapping BQ-123 pA2 value (negative common logarithm of the antagonist that produces an agonist dose ratio of 2) in the two strains. Indomethacin was ineffective in both groups. Sarafotoxin S6c and IRL-1620 both evoked an ETB-mediated, significant relaxation, only in WKY rats. In 12-week-old SHRs, ET-1 evoked a markedly increased maximal effect compared with the response in WKY rats (P < 0.01); this was prevented by treatment with indomethacin. The BQ-123 pA2 value was higher in SHRs than in WKY rats (P < 0.01). Both sarafotoxin S6c and IRL-1620 evoked a significant concentration-dependent relaxation in WKY rats, which was not detected in SHR preparations. Conclusions Our results could suggest that the different responses evoked by ET-1 in the MVB of SHRs during the onset of hypertension may be related partially to primary alterations in the ET-1 receptorial pattern and partially to the onset of high blood pressure, leading to an impairment in the haemodynamic balance.

Block of sodium current in myelinated nerve fibre with enkephalins

The effects of leu- and met-enkephalin were investigated on the node of Ranvier of isolated nerve fibers of frog under current and voltage clamp conditions. When added to the external solutions, enkephalins (1--5 mM) caused a slight decrease in peak Na+ and steady state K+ currents. The action potential was not significantly affected. When added to the internal medium (by diffusion from the two cut ends of the fibre), enkephalins (less than 5 mM) drastically reduced the peak Na+ current without significantly affecting the steady state K+ current. The block of Na+ current was greatly accelerated and enhanced by repetitive depolarizations. The sodium current slowly recovered after the end of pulsing. The extent of the block and rate of accumulation increased with increasing magnitude and frequency of the depolarizing pulses. Internal applications of enkephalins induced a blockade of the action potential when the fibre was stimulated at frequencies above 0.1 Hz. The results suggest that during depolarizations, enkephalin molecules plug the inner end of Na+ channels or immobilize the channel gates leaving the channels in a closed configuration, and remain in or near the pores for a long time after the end of depolarizations. Possible physiological significance and molecular mode of action of enkephalins on myelinated nerve fibres are discussed.