Gábor Oroszi

Release of somatostatin and its role in the mediation of the anti‐inflammatory effect induced by antidromic stimulation of sensory fibres of rat sciatic nerve

1 The effect of antidromic stimulation of the sensory fibres of the sciatic nerve on inflammatory plasma extravasation in various tissues and on cutaneous vasodilatation elicited in distant parts of the body was investigated in rats pretreated with guanethidine (8u2003mgu2003kg−1, i.p.) and pipecuronium (200u2003μgu2003kg−1, i.v.). 2 Antidromic sciatic nerve stimulation with C‐fibre strength (20u2003V, 0.5u2003ms) at 5u2003Hz for 5u2003min elicited neurogenic inflammation in the innervated area and inhibited by 50.3±4.67% the development of a subsequent plasma extravasation in response to similar stimulation of the contralateral sciatic nerve. Stimulation at 0.5u2003Hz for 1u2003h also evoked local plasma extravasation and inhibited the carrageenin‐induced (1%, 100u2003μlu2003s.c.) cutaneous inflammation by 38.5±10.0% in the contralateral paw. Excitation at 0.1u2003Hz for 4u2003h elicited no local plasma extravasation in the stimulated hindleg but still reduced the carrageenin‐induced oedema by 52.1±9.7% in the paw on the contralateral side. 3 Plasma extravasation in the knee joint in response to carrageenin (2%, 200u2003μl intra‐articular injection) was diminished by 46.1±12.69% and 40.9±4.93% when the sciatic nerve was stimulated in the contralateral leg at 0.5u2003Hz for 1u2003h or 0.1u2003Hz for 4u2003h, respectively. 4 Stimulation of the peripheral stump of the left vagal nerve (20u2003V, 1u2003ms, 8u2003Hz, 10u2003min) elicited plasma extravasation in the trachea, oesophagus and mediastinal connective tissue in rats pretreated with atropine (2u2003mgu2003kg−1, i.v.), guanethidine (8u2003mgu2003kg−1, i.p.) and pipecuronium (200u2003μgu2003kg−1, i.v.). These responses were inhibited by 37.8±5.1%, 49.7±9.9% and 37.6±4.2%, respectively by antidromic sciatic nerve excitation (5u2003Hz, 5u2003min) applied 5u2003min earlier. 5 Pretreatment with polyclonal somatostatin antiserum (0.5u2003ml/rat, i.v.) or the selective somatostatin depleting agent cysteamine (280u2003mgu2003kg−1, s.c.) prevented the anti‐inflammatory effect of sciatic nerve stimulation (5u2003Hz, 5u2003min) on a subsequent neurogenic plasma extravasation of the contralateral paw skin. The inhibitory effect of antidromic sciatic nerve excitation on plasma extravasation in response to vagal nerve stimulation was also prevented by somatostatin antiserum pretreatment. 6 Cutaneous blood flow assessment by laser Doppler flowmetry indicated that antidromic vasodilatation induced by sciatic nerve stimulation was not inhibited by excitation of the sciatic nerve of the contralateral leg (1u2003Hz, 30u2003min) or by somatostatin (10u2003μg/rat, i.v.) injection. 7 Plasma levels of somatostatin increased more than 4 fold after stimulation of both sciatic nerves (5u2003Hz, 5u2003min) but the stimulus‐evoked increase was not observed in cysteamine (280u2003mgu2003kg−1, s.c.) pretreated rats. 8 These results suggest that somatostatin released from the activated sensory nerve terminals mediates the systemic anti‐inflammatory effect evoked by stimulating the peripheral stump of the sciatic nerve.

Systemic anti-inflammatory effect induced by counter-irritation through a local release of somatostatin from nociceptors

1 Neurogenic plasma extravasation evoked by topical application of 1%u2003vv−1 mustard oil on the skin of the acutely denervated rat hindleg (primary reaction) inhibited the development of a subsequent oil‐induced plasma extravasation induced in the skin of the contralateral hindleg by 49.3±7.06% (n=9) and in the conjunctival mucosa due to 0.1%u2003wv−1 capsaicin instillation by 33.5±10.05% (n=6). The primary reaction also inhibited the non‐neurogenic hindpaw oedema evoked by s.c. injection of 5%u2003wv−1 dextran into the chronically denervated hindpaw by 48.0±4.6% (n=5). 2 Capsaicin injection (100u2003μgu2003ml−1 in 50u2003μl, s.c.) into the acutely denervated hindleg caused 56.5±4.0% (n=5) inhibition in the intensity of plasma extravasation elicited by 1%u2003vv−1 mustard oil smearing on the contralateral side. After chronic denervation, subplantar injection of 5%u2003wv−1 dextran elicited a non‐neurogenic inflammatory response with intensive tissue oedema without causing any systemic anti‐inflammatory effect. Bilateral adrenalectomy did not inhibit the mustard oil‐induced anti‐inflammatory effect in the contralateral hindleg. 3 Pretreating the rats with polyclonal somatostatin antiserum (0.5u2003ml rat−1, i.v.) or with the somatostatin depleting agent cysteamine (280u2003mgu2003kg−1, s.c.) prevented the inhibitory action of mustard oil‐induced inflammation on subsequent neurogenic plasma extravasation and strongly diminished the inhibition of non‐neurogenic oedema formation evoked by dextran. 4 Exogenous somatostatin (10u2003μgu2003kg−1, i.p.) caused a 30.3±8.3% (n=6) inhibition of plasma extravasation caused by mustard oil smearing on the acutely denervated hindleg and this inhibitory effect was abolished by somatostatin antiserum (0.5u2003ml rat−1, i.v.). The plasma level of somatostatin‐like immunoreactivity (SST‐LI) increased by 40.03±6.8% (n=6) 10u2003min after topical application of 1%u2003vv−1 mustard oil on the acutely denervated hindpaws compared to the paraffin oil treated control group. Chronic denervation of the hindlegs or cysteamine (280u2003mgu2003kg−1, s.c.) pretreatment prevented the mustard oil‐induced elevation of SST‐LI in plasma. 5 It is concluded that chemical excitation of the capsaicin‐sensitive sensory receptors not only induces local neurogenic plasma extravasation but also inhibits the development of a subsequent inflammatory reaction at remote sites of the body in the rat. A role for somatostatin in this systemic anti‐inflammatory effect is suggested.

Anti‐inflammatory effect of synthetic somatostatin analogues in the rat

Somatostatin (6.11u2003nmolu2003kg−1 i.p.) inhibited neurogenic plasma extravasation evoked by 1% mustard oil and non‐neurogenic oedema induced by 5% dextran in the rat skin. Cyclic synthetic octapeptide (TT‐248 and TT‐250) and heptapeptide (TT‐232) somatostatin analogues proved to be more effective in reducing neurogenic and non‐neurogenic inflammatory reactions but octreotide had no influence on either neurogenic or non‐neurogenic inflammation. TT‐232 administered i.p. or i.v. (1.06u2003–u200342.40u2003nmolu2003kg−1) inhibited in a dose‐dependent manner the plasma extravasation evoked by mustard oil in the rats paw. Neither diclofenac (15.78u2003–u2003315.60u2003μmolu2003kg−1) nor the selective COX‐2 inhibitor meloxicam (2.95u2003–u2003569.38u2003μmolu2003kg−1) attenuated the mustard oil‐induced neurogenic plasma extravasation. TT‐232, diclofenac and meloxicam dose‐dependently diminished non‐neurogenic dextran‐oedema of the paw the ED35 values were 1.73u2003nmolu2003kg−1 for TT‐232 and 34.37u2003μmolu2003kg−1 for diclofenac. TT‐232 inhibited in the dose range of 1.06u2003–u200321.21u2003nmolu2003kg−1 the bradykinin‐induced plasma extravasation in the skin of the chronically denervated paw. Mustard oil‐induced cutaneous plasma extravasation was dose‐dependently diminished by s.c. TT‐232 1, 2, 4, 6 or 16u2003h after the treatment. TT‐232 (2×106, 2×212 and 2×530u2003nmolu2003kg−1 per day s.c. for 18 days) caused dose‐dependent inhibition of chronic Freund adjuvant‐induced arthritis during the experimental period. TT‐232 (200 and 500u2003nM) inhibited the release of SP, CGRP and somatostatin from the rat isolated trachea induced by electrical field stimulation (40u2003V, 0.1u2003ms, 10u2003Hz, 120u2003s) or by capsaicin (10−7u2003M), but did not influence the basal, non‐stimulated peptide release. It is concluded that somatostatin analogues without endocrine functions as TT‐232 are promising compounds with a novel site of action for inhibition of non‐neurogenic and neurogenic inflammatory processes.

Role of voltage-gated cation channels and axon reflexes in the release of sensory neuropeptides by capsaicin from isolated rat trachea.

In order to reveal the role of axon reflexes and sensory receptors in sensory neuropeptide release in response to capsaicin, liberation of substance P, calcitonin gene-related peptide and somatostatin from isolated rat tracheae was investigated in the presence of voltage-sensitive Na(+) and Ca(2+) channel blocking agents. Neuropeptide release induced by capsaicin (10 nM) remained unchanged in the presence of 25 mM lidocaine, 1 microM tetrodotoxin or the N-type Ca(2+) channel inhibitor, omega-conotoxin GVIA (100-300 nM). Peptide release by 100 pulses of 2 Hz field stimulation was prevented by lidocaine or tetrodotoxin. Omega-agatoxin TK (250 nM) significantly inhibited and Cd(2+) (200 microM) prevented capsaicin-induced neuropeptide release. These results suggest that chemical stimulation-induced neuropeptide release does not involve activation of fast Na(+) channels or N- and P-type voltage-dependent Ca(2+) channels, but contribution of Q-type Ca(2+) channels is possible. Sensory neuropeptides are released by capsaicin from sensory receptors without axon reflexes.

Functional and biochemical evidence for capsaicin-induced neural endothelin release in isolated working rat heart.

In isolated working rat heart, capsaicin elicited a concentration-dependent constriction of coronary arteries accompanied by decline of all cardiac parameters recorded (heart rate, coronary and aortic flow, left ventricular developed pressure, and first derivative of left ventricular developed pressure). The following evidence suggests that capsaicin-induced changes are mediated by endothelin of neural origin: (1) the capsaicin (10 nM)-evoked decrease in coronary flow resulting in deterioration of cardiac functions was mimicked by endothelin (0.1 nM); (2) the selective endothelin ET(A) receptor antagonist, cyclo (D-alpha-aspartyl-L-propyl-D-valyl-L-leucyl-D-tryptophyl) (1 microM), abolished the cardiac effects provoked by capsaicin (10 nM); (3) reduction of extracellular Ca2+ concentration from 2.4 to 1.2 or 0.6 mM inhibited the cardiac effects of capsaicin (10 nM) but not those induced by endothelin (0.1 nM); (4) perfusion of the heart with 0.1% (v/v) Triton X-100 damaged the endothelium and reversed the enhancement of coronary flow evoked by bethanechol (1 microM), decreased the basal flow, but was without effect on capsaicin-induced coronary constriction; (5) in response to capsaicin challenge (10-100 nM), the endothelin concentration measured in coronary effluent by means of radioimmunoassay increased up to sevenfold but remained unchanged in the presence of 0.6 mM Ca2+; (6) no reduction of coronary flow was induced by capsaicin (100 nM) applied to the heart of rats which were desensitised by capsaicin (150 mg/kg). It is concluded that, in the rat heart, capsaicin acting on VR1 capsaicin receptors elicits a release of endothelin from the sensory nerve terminals.

125I-labeling and purification of peptide hormones and bovine serum albumin

The iodination and separation of various diagnostically and/or experimentally important peptides including (Tyr1)-somatostatin-14, rat Tyr-a-calcitonin gene-related peptide (23-37), motilin and vasoactive intestinal peptide, furthermore bovine serum albumin are described. All species were iodinated by the iodogen method. The 125I-labeled peptide products were separated by reversed-phase HPLC, the specific activities of mono-iodinated forms are near identical with the theoretical value. The labeled bovine serum albumin was separated by Sephadex G-100 gel filtration.

Decreased sensory neuropeptide release from trachea of rats with streptozotocin-induced diabetes

We studied the release of somatostatin, calcitonin gene-related peptide (CGRP) and substance P in response to electrical field stimulation from isolated tracheas of rats following 4 weeks of streptozotocin (50 mg/kg i.v.)-induced diabetes. Field stimulation (40 V, 0.1 ms, 10 Hz for 120 s) increased the release of somatostatin, CGRP and substance P from the baseline 0.18+/-0.029, 0.17+/-0.027, and 1.77+/-0.086 to 0.51+/-0.022, 0.69+/-0.115, and 5.96+/-0.377 in control preparations and 0.31+/-0.081, 0.41+/-0.142, and 3.14+/-0.443 fmol/mg wet tissue weight in preparations from diabetic rats as measured by radioimmunoassay (control vs. diabetic P<0.01 for each). The results show a simultaneous decrease in release of the three sensory neuropeptides and an enhanced plasma somatostatin level in rats with streptozotocin-induced diabetes.

Interaction between capsaicin and nitrate tolerance in isolated guinea-pig heart

Capsaicin-induced increases in heart rate and coronary flow were blocked by N(G)-nitro-L-Arg-methyl ester (30 mM) in Langendorff-perfused guinea-pig hearts. Neither heart rate nor coronary flow changed by capsaicin in hearts from animals made tolerant to the hypotensive effect of 30 microg/kg nitroglycerin by the administration of 50 mg/kg nitroglycerin subcutaneously 4 times a day over 3 days. We conclude that the effector function of sensory nerves may deteriorate in nitrate tolerance.

Endothelin release by capsaicin in isolated working rat heart

Capsaicin (1 nM-1 microM) induced a concentration-dependent decrease in heart rate, coronary flow, aortic flow, left ventricular developed pressure and its first derivative, dP/dt(max) in isolated working rat heart. The effect of 10 nM capsaicin was mimicked by 0.1 nM endothelin. PD142893 (200 nM), a non-selective endothelin receptor blocking agent antagonized the effect of either endothelin (0.1 nM) or capsaicin (10 nM). We conclude that the majority of the effects of capsaicin in the rat heart are mediated by neural endothelin release.

Preparation of mono‐125I‐labelled gastrin‐17 for radioimmunoassay measurements

In the present work the efficiency of two oxidising agents (chloramine-T and iodogen) for human gastrin-17 labelling is investigated, whilst the purification of ion-exchange chromatography is compared with that of reversed-phase HPLC. In the Iodogen method the extent of radioactive iodine incorporation can be as high as 95-98 % in contrast to 60-65 % attained using the chloramine-T method. Reversed-phase HPLC purification proved to be superior to ion-exchange chromatography on the basis of a more distinct separation of mono-iodinated gastrin underlain by specific activity value (73 to 75 TBq/mmol) near identical with the theoretical one.