Action of ryanodine on neurogenic responses in rat isolated mesenteric small arteries

1. Rat mesenteric arteries (∼250 μm) were set up in a single-channel isometric myograph designed to allow fluorescence measurements concurrent with field stimulation of intramural nerves. Vessels were loaded with 6 μM fura-2AM for 2 h and simultaneous recordings of neurogenic contraction (force) and intracellular calcium [Ca²⁺]_i were obtained. In other experiments, arteries were loaded with 1 μCi ml⁻¹ [³H]-noradrenaline (NA) for 30 min in order to measure release of [³H]-NA in response to field stimulation to examine whether ryanodine directly inhibited neuronal release of NA.
2. Arteries were activated by single intermittent field stimulation or continuously to excite intrinsic sympathetic nerves, or by cumulative addition of noradrenaline (1 nM–10 μM) to the bathing solution.
3. Pre-incubation with ryanodine markedly inhibited the contraction and [Ca²⁺]_i release in response to single-pulse nerve stimulation. Ryanodine also inhibited an early phasic component of the response to continuous field stimulation and reduced the rate of rise in force in response to continuous field stimulation. However, stable maximal contraction and [Ca²⁺]_i in response to continuous field stimulation as well as maximal responses to exogenous NA were unaffected. Release of [³H]-NA in response to single intermittent field stimulation was not affected by ryanodine when compared to vehicle.
4. Our results suggest that brief intermittent activation of intramural sympathetic nerves increases [Ca²⁺]_i and contracts small arteries primarily by releasing Ca²⁺ from a ryanodine-sensitive intracellular store. In contrast, the stable rise in tone and [Ca²⁺]_i resulting from continuous nerve stimulation may largely depend on sources of Ca²⁺ other than the ryanodine-sensitive intracellular store.

     

Epinephrine facilitates neurogenic responses in isolated segments of dog mesenteric arteries

Epinephrine (EPI) bitartrate (10(-9)-10(-8) M) significantly enhanced tension development in response to electrical field stimulation in isolated segments of dog mesenteric arteries. Responses to exogenous norepinephrine (NE) were generally unaffected, indicating that the EPI-induced increases in response to field stimulation are not explicable in terms of changes in postjunctional sensitivity. The facilitatory effects of EPI (5 X 10(-9) M) were unaffected by atenolol (10(-6) M) but were completely abolished by timolol (2 X 10(-7) M), suggesting an involvement of beta 2-adrenoreceptors in mediating the EPI-induced facilitation of neurogenic responses. Responses to exogenous NE were usually unaffected by either beta-adrenoreceptor antagonist. The results suggest that release of endogenous sympathetic neurotransmitter, measured in terms of postjunctional effects on vascular smooth muscle tone, appears to be modulated by prejunctional facilitatory beta 2-adrenoreceptors and that these receptors may be a physiologic site of action of EPI.     

Constrictor responses of isolated and perfused monkey coronary arteries to pindolol

We studied the mechanisms of pindolol-induced vasoconstrictions in isolated and perfused monkey coronary arteries. Phentolamine marginally reduced the vasoconstrictions, but bunazosin and DG 5128 did not. Diltiazem and methysergide significantly inhibited them, but atropine, diphenhydramine and cimetidine did not. Diltiazem significantly suppressed KCl-induced vasoconstrictions, but methysergide did not. These results suggest that pindolol-induced vasoconstrictions of monkey coronary arteries are mediated, at least partly, through 5-hydroxytryptamine receptors, but not through alpha-adrenoceptors, and that these vasoconstrictions appear to be associated with an increase of Ca2+ entry into the smooth muscle cell.     

Effects of vagal perineural capsaicin treatment on vagal efferent and airway neurogenic responses in anesthetized rats

The acute effect of vagal perineural capsaicin treatment (VPCT) on parasympathetic bradycardia and tracheal neurogenic protein extravasation was examined. In nine anesthetized male Wistar rats the effect of VPCT on the bradycardia induced by electrical stimulation of the vagus was examined. In 24 anesthetized male Wistar rats the effect of VPCT on the tracheal protein extravasation induced by the inhalation of capsaicin aerosols was also studied. VPCT did not alter the bradycardia induced by vagal stimulation or the tracheal protein extravasation induced by the inhalation of capsaicin aerosol. The results of these studies further demonstrate the selectivity of perineural capsaicin treatment on vagal sensory nonmyelinated fibers in the rat and indicate that it is a useful tool for examining the role of sensory vagal C-fibers in pulmonary and cardiovascular reflexes and in isolating C-fiber-mediated reflex responses from those mediated by the release of neuropeptides.     

Effects of the oestrous cycle and gender on acute vasodilatory responses of isolated pressurized rat mesenteric arteries to 17 beta-oestradiol

The influence of the oestrous cycle and gender on responses of isolated pressurized mesenteric arteries to acute 17 beta-oestradiol was investigated. All vessels, pre-contracted with 60 mM KCl or 10 microM U46619 (9,11 dideoxy-11alpha, 9alpha-epoxy methano-prostaglandin), exhibited concentration-dependent vasodilatory responses to 17 beta-oestradiol (3 - 30 microM). The largest responses were seen in vessels from female rats in pro-oestrous (38.9+/-5.4% U46619 max and 63.1+/-4.0% KCl max for 30 microM oestradiol), the smallest from animals in di-oestrous (20.1+/-3.7% U46619 and 50.1+/-4.5% KCL - both P:<0.05 cf pro-oestrous (all n=8)). Responses of vessels from male rats were similar to those from pro-oestrous rats (41.5+/-9.1% U46619 (n=10) and 54.9+/-2.9% KCl (n =8)). All responsees were unaffected by inhibition of nitric oxide synthase (NOS). Female rats in pro-oestrous had the highest plasma concentrations of 17 beta-oestradiol and testosterone (40.76+/-4.73 pg ml(-1) and 0.29+/-0.05 ng ml(-1) respectively (n=8)) while those in di-oestrous had the lowest (15.24+/-3.94 pg ml(-1) for oestradiol and 0.08+/-0.03 ng ml(-1) for testosterone (n=8)). In male rats the concentration of oestrogen was 10.29+/-1.21 pg ml(-1) (n=7) while that of testosterone was 3.15+/-0.36 ng ml(-1) (n=7). Incubation of arteries isolated from male rats and from female rats in pro-oestrous and di-oestrous with testosterone (1 microM, 3 h) significantly enhanced the subsequent vasodilatory responses to acute 17 beta-oestradiol. Following incubation, the responses to 17 beta-oestradiol were similar in all groups. These observations suggest that gender and the oestrous cycle may influence the vascular responses to acute 17 beta-oestradiol administration.     

Omega-conotoxin GVIA is a potent inhibitor of sympathetic neurogenic responses in rat small mesenteric arteries.

1. We have investigated the effects of the N-type calcium channel blocker, omega-conotoxin GVIA, on contractile responses to nerve stimulation, noradrenaline and KCl in rat small mesenteric arteries. In separate experiments, single and summated excitatory junctional potentials (e.j.ps) evoked by nerve stimulation were recorded with an intracellular electrode in the absence and presence of omega-conotoxin. 2. Electrical field stimulation of intramural sympathetic nerves (30 V; 0.25 ms pulse width; 3 s train length; 4-24 Hz) caused frequency-dependent contractions. Cumulative concentration-response curves for the contractions induced by noradrenaline and KCl were constructed in the same preparations. Stimulation at 0.2 Hz and 10 Hz induced respectively single e.j.ps without contractions and summated e.j.ps associated with a contractile response. 3. omega-Conotoxin (0.1 to 3 nM) inhibited markedly and in a concentration-dependent manner both the contractions and e.j.ps to electrical field stimulation. The concentration-response curves to exogenous noradrenaline and KCl remained unaffected. 4. The time-course for the effects of omega-conotoxin (0.3 to 3 nM) indicated a slow onset of action with at least one hour to achieve an equilibrium. 5. The experiments indicate that omega-conotoxin acts prejunctionally to inhibit sympathetic neurotransmission in rat small arteries presumably by inhibition of noradrenaline release. We suggest that omega-conotoxin could be a useful tool to study the control of vascular tone through the autonomic nervous system.     

G-proteins are involved in contractile responses of isolated mesenteric resistance arteries to agonists

Summary We evaluated whether GTP-binding regulatory proteins (G-proteins) are involved in responses of resistance arterial smooth muscle to contractile agonists. We therefore pretreated isolated sympathectomized mesenteric resistance arteries of the rat with pertussis toxin (PTX) and recorded their contractile responses to aluminium fluoride, endothelin, high potassium, phenylephrine, phorbol myristate acetate, serotonin and vasopressin. PTX reduced contractile responses to agonists with the following order of potency: phenylephrine = serotonin > vasopressin = endothelin. The toxin reduced responses to phenylephrine in both the presence and absence of extracellular Ca²⁺. In Ca²⁺-depleted vessels that were exposed to phenylephrine, PTX virtually abolished responses to Ca²⁺ while hardly affecting responses to Ca²⁺ in the presence of endothelin. Also aluminium fluoride and phorbol myristate acetate induced contractions. These were dependent on extracellular Ca²⁺ and inhibited by felodipine. PTX reduced responses to aluminium fluoride but not those to phorbol myristate acetate. These data indicate that PTX sensitive G-proteins are involved in both influx of Ca²⁺ and release of intracellular Ca²⁺ following ₁-adrenergic and serotonergic stimulation of resistance arteries. The role of G-proteins in stimulated Ca²⁺ influx could involve a direct effect on calcium channels although an indirect effect through protein kinase-C can not be entirely excluded. The persistance of contractile responses to vasopressin and endothelin following PTX suggests that these agonists engage different pathways to induce contraction or have a higher efficacy in activating similar G-proteins. Send offprint requests to: Harrie C. M. Boonen at the above address     

Effect of temperature on responses of dog isolated lingual and mesenteric arteries to vasoactive substances

1. The effects of temperature on submaximal vasoconstriction to an intraluminal administration of noradrenaline (NA), phenylephrine, tyramine and KCl were investigated in canine isolated and perfused lingual and mesenteric arteries, using the cannula-inserting method. 2. In lingual arteries, cooling (from 37 to 27 degrees C) caused significant depression of vasoconstriction to the four vasoactive substances used. Rewarming (to 37 degrees C) induced a significant augmentation of constriction by NA, phenylephrine and KCl, but not tyramine. 3. In mesenteric arteries, cooling depressed tyramine- and KCl-induced constrictions, but had no effect on NA- and phenylephrine-induced vasoconstriction. Only in the case of KCl-induced constrictions did rewarming induce a potentiation of the vasoconstrictor response. 4. We conclude that: (i) cooling induces a depression of voltage-dependent Ca2+ channels and rewarming may induce a potentiation of Ca2+ channels in both arteries; (ii) alpha1-adrenoceptor-operated Ca2+ channels are depressed by cooling in lingual arteries but not in mesenteric arteries; and (iii) cooling may induce an attenuation of the re-uptake function in sympathetic nerve terminals in both arteries and this attenuation may be not rapidly restored by acute rewarming.     

Vascular responses of isolated mesenteric resistance and basilar arteries from short- and long-term diabetic rats

Vascular dysfunctions, e.g. alterations in the reactivity of blood vessels to neurotransmitters and hormones, are a well-established complication of diabetes mellitus. Whether these impairments are a consequence of direct postsynaptic deficits and/or indirect presynaptic deficits remains to be determined. To this end, we investigated the influence of the duration of diabetes on relaxation and contraction responses of isolated mesenteric resistance and equally-sized basilar arteries to postsynaptic activation by various vasoactive agents, using streptozotocin-induced diabetic rats and age-matched controls. Relaxation responses to vasodilator agents were studied in KCl-precontracted arteries. The duration of diabetes (4 or 40 weeks) did not affect the vasodilator responses to sodium nitroprusside or salbutamol in either artery. In mesenteric resistance vessels from short-term (4 weeks) and long-term (40 weeks) diabetic rats the vasoconstrictor responses to KCl, serotonin and vasopressin were the same as those in non-diabetic rats; however, the sensitivity (EC₅₀) to noradrenaline was slightly but significantly enhanced after the long-term diabetic state. In contrast to the mesenteric arteries, noradrenaline did not cause contraction in basilar arteries taken from diabetic and control rats. Thus, there appear to be important differences in the reactivity to noradrenaline of the peripheral and cerebral vasculature. The basilar artery from short-term and long-term diabetic rats did not show different responsiveness to vasopressin whereas to serotonin a significant enhanced and decreased sensitivity (EC₁₀ and EC₅₀) was demonstrated in short-term and long-term diabetes, respectively. Our findings indicate that postsynaptic impairments do not play a major role in the alterations of vasoreactivity to vasodilators, noradrenaline or vasopressin seen in experimental diabetes. However, the duration of the diabetic state may have serious consequences for vasoreactivity of basilar arteries to serotonin and, therefore, warrants further investigations. Received: 17 June 1998 / Accepted: 3 August 1998     

Improved method for the synthesis of Nα-9-fluorenylmethyloxycarbonyl-Nδ,ω bis-adamantyloxycarbonyl-L-arginine

A modified method is reported for the prepartion of N^α-9-fluorenylmethyloxycarbonyl-N^δ,ω bis-adamantyloxycarbonyl-L-arginine, giving an overall yield of 60% over three steps based on N^α-benzyloxycarbonyl-L-arginine. Commercially available adamantyl fluoroformate for guanidine function protection of N^α benzyloxycarbonyl-L-arginine, catalytic transfer hydrogenation with formic acid on palladium black for removal of the benzyloxycarbonyl protecting group, and fluorenylmethylsuccinimidyl carbonate for the final synthesis, were introduced to simplify and reduce the cost of preparation of this arginine derivative. The reaction conditions have been accurately studied at each step in order to optimize the yields.     

Effects of dobutamine on isolated canine cerebral, coronary, mesenteric, and renal arteries

The effects of dobutamine on helical strips of isolated canine cerebral, coronary, mesenteric, and renal arteries was investigated. Dobutamine contracted only renal arterial strips under resting condition. When renal and mesenteric arterial strips were partially contracted with prostaglandin F2 alpha (PGF2 alpha), dobutamine caused further concentration-related contraction, while coronary arterial strips were relaxed. Cerebral arterial strips, on the other hand, did not significantly respond to dobutamine. After treatment with 10(-5) M dl-phenoxybenzamine hydrochloride (POB) for 1 h, dobutamine-induced contractions of partially precontracted mesenteric and renal arterial strips were converted to relaxations. Relaxations of coronary arteries were not potentiated by the alpha-antagonist, but were attenuated by treatment with 10(-6) M propranolol and 10(-6) M metoprolol to a similar extent. On the other hand, relaxations of mesenteric and renal arterial strips were not inhibited by metoprolol but by propranolol. Droperidol (3 X 10(-5) M) failed to significantly alter the concentration-response curve for dobutamine. These results suggest that dobutamine causes vasoconstriction mediated by apha-adrenergic receptor and vasodilatation mediated by beta 1- and beta 2-adrenoceptors. Dobutamine does not appear to act on dopamine receptors.     

Mechanisms of agonist-induced constriction in isolated human mesenteric arteries

We determined the calcium signalling pathways involved in the mechanisms of contraction of the vasoconstrictive agonists KCl, U46619 and PDBu in isolated human mesenteric arteries. The influence of gender, vessel diameter and age of the patients was also investigated. Human mesenteric arteries (n = 76) were loaded in a wire myograph and maintained at a tension equivalent to the in vivo pressure of 100 mm Hg, bubbled with 95%O2/5%CO2 to maintain pH 7.4 in physiological saline solution (PSS). Cumulative concentration-response curves were obtained to KCl (100 microM-100 mM), U46619 (1 nM-1 microM) or PDBu (1 nM-1 microM), before or after a 30 min incubation with either the voltage-gated calcium channel (VGCC) blocker nifedipine (10 microM), the store-operated calcium channel (SOCC) blocker SK&F96365 (50 microM) or in calcium-free PSS (-Ca2+ PSS). The KCl response was abolished in -Ca2+ PSS and with nifedipine. The U46619 response was partially blocked in -Ca2+ PSS and with nifedipine and predominantly blocked by SK&F96365. Incubation in -Ca2+ PSS had no effect on the response to PDBu. Arteries from male patients responded significantly higher to KCl than arteries from female patients. This study demonstrates that KCl induces mesenteric vasoconstriction via activation of VGCCs, U46619 induces mesenteric vasoconstriction via activation of SOCCs, but also VGCCs and PDBu induce mesenteric vasoconstriction via a calcium-independent pathway.     

EMD56431对犬冠脉和肠系膜动脉的作用

目的：研究ＥＭＤ对犬冠脉和肠系膜动脉的松弛作用。方法：用２０和６０ｍｍｏｌ／Ｌ ＫＣｌ激动冠脉（＋Ｅ,－Ｅ）,观察ＥＭＤ的松弛效应。同时观察格列本脲对ＥＭＤ松弛冠脉的影响及ＥＭＤ对ＫＣｌ量效曲线的改变。此外,还测定了ＥＭＤ松驰犬肠系膜动脉和冠脉的ＥＣ５０。结果：０．７μｍｏｌ／Ｌ ＥＭＤ对２０ｍｍｏｌ／Ｌ ＫＣｌ去极化冠脉无效;格列本脲能有效拮抗ＥＭＤ对冠脉的松驰作用;ＥＭＤ使５～３０ｍｍｏｌ／Ｌ     

Vasodilator effects of leptin on canine isolated mesenteric arteries and veins

1. Although leptin increases sympathetic nerve activity and blood pressure, its direct action on large arterial rings is to cause relaxation. However, it is the small resistance arteries and veins that are important in blood pressure control. The effects of leptin on these small vessels has not been reported previously in the canine and the effect of leptin on the capacitance vessels is not known. 2. In the present study, third- or fourth-order canine mesenteric arteries and veins were isolated and placed in a perfusion myograph and preconstricted with noradrenaline. The responses to graded concentrations of leptin were determined and the role of nitric oxide was assessed by administration of N(G)-nitro-l-arginine methyl ester (l-NAME), a blocker of nitric oxide synthase. 3. Leptin induced dose-related dilatations in both arterial and venous segments. The mean (+/-SEM) maximum increases in the diameter of the arteries and veins were 25.0 +/- 4.8 and 29.9 +/- 2.0% of the initial preconstriction, respectively. Relaxations of both arteries and veins were abolished by l-NAME or by endothelium denudation, although dilatations were still obtained to sodium nitroprusside, a nitric oxide donor. 4. These results indicate that leptin dilates canine small mesenteric arteries and veins by a mechanism involving endothelial release of nitric oxide. This observation may result in a decrease of peripheral resistance and venous return and, hence, counteract the leptin-induced neurally mediated vasoconstriction that has been reported previously.     

Shear stress-dependent effects of lysophosphatidic acid on agonist-induced vasomotor responses in rat mesenteric artery

We have previously shown that lysophosphatidic acid (LPA), a bioactive plasma lysophospholipid, markedly accelerates shear stress-induced Ca2+ responses in cultured vascular endothelial cells (ECs). This study aimed to demonstrate the impact of LPA and luminal shear stress on vasomotor regulation in the isolated rat mesenteric artery (MA) using a videomicroscopic technique. Although the addition of LPA to the perfusate in a concentration range of 0.03-0.3 μM had no significant effect on the basal MA tone, LPA in a similar concentration range led to increased phenylephrine-induced MA contraction and reduced acetylcholine-induced MA relaxation under physiological shear conditions. These vasomodulatory actions of LPA, which vanished upon removal of ECs, were positively dependent on luminal shear stress levels and were markedly inhibited by the LPA receptor antagonist Ki16425, the cyclooxygenase inhibitor indomethacin, and the thromboxane A2 receptor antagonist SQ29548. These data thus suggest that LPA can modify the agonist-induced vasomotor responses in MAs in a shear stress-dependent manner. This effect of LPA was mediated through ECs, the LPA receptor, and cyclooxygenase/thromboxane A2 signaling.     

Pharmacological features of vascular responses of isolated dog and monkey lingual arteries to vasoactive substances.

Using a perfusion technique of isolated vessels, vasoconstrictor responses to alpha-adrenoceptor agonists (norepinephrine [NE], phenylephrine [PE], clonidine, xylazine and tyramine) and KCl were investigated in isolated, perfused dog and monkey lingual arteries. A stainless steel cannula was inserted into the lingual artery segment and perfused with Krebs-Henseleit solution at a constant flow rate. In dog lingual arteries, the agonists induced vasoconstrictions with the following order of potency: NE greater than PE greater than tyramine much greater than clonidine greater than xylazine greater than KCl. In monkey preparations, the order was NE greater than PE much greater than clonidine greater than or equal to tyramine greater than xylazine greater than KCl. In both preparations, NE- and PE-induced constrictions were blocked by bunazosin (an alpha-1 adrenoceptor antagonist), but not influenced by midaglizole (a potent alpha-2 antagonist). Diltiazem (a Ca entry blocker) significantly attenuated NE-induced vasoconstrictions in dog lingual arteries, but did not significantly influence these in monkey preparations. These results suggest that: [1] these arteries contain mostly alpha-1 but scarcely any alpha-2 adrenoceptors; [2] in dog preparations, tyramine induced a marked vasoconstriction which may contribute to investigation on the mechanisms of catecholamine releases from sympathetic nerve terminals; and [3] different blocking effects of diltiazem may indicate that extracellular Ca++ influx may have varying degrees of importance in alpha-1 adrenoreceptor-mediated constrictions in different species, although participation of an intracellular mechanism might not be ruled out.     

Synthesis and Antiplatelet Effects of ω-Aminoalkoxylxanthones

A series of ω-aminoalkoxylxanthones were synthesized and tested in-vitro for their ability to inhibit aggregation of rabbit washed platelets and human platelet-rich plasma (PRP) induced by various inducers. Nine of these compounds showed more potent antiplatelet effects than natural norathyriol tetraacetate on collagen-induced aggregation. The various ω-aminoalkoxyl side chains of the synthesized compounds modified the antiplatelet effects. All the compounds tested in human PRP showed significant inhibition of secondary aggregation induced by adrenaline, suggesting that the antiplatelet effects of these compounds is mainly due to an inhibitory effect on thromboxane formation. These compounds at high concentration also cause vasorelaxing action in rat thoracic aorta.     

内皮素-3对犬肺动静脉的作用

阐明内皮素 3(ET 3)对肺动静脉的作用机理 .利用犬离体肺动静脉条 ,观察其张力改变 .结果可见 :①ET 3(1～ 30 μmol·L- 1)引起肺动脉舒张 (低浓度 )和收缩 (高浓度 )双向反应 ,ETB 受体激动剂IRL162 0 (1～ 30 μmol·L- 1)只引起舒张反应 ;去内皮 ,ETB 受体阻断剂IRL10 38(1μmol·L- 1)或左旋硝基精氨酸 (L NA ,10 μmol·L- 1)均使ET 3或IRL162 0所致舒张反应减弱或消失 ,ETA 受体阻断剂BQ12 3(10 μmol·L- 1)则使ET 3所致收缩反应翻转为舒张反应 ;②同浓度的ET 3和IRL162 0只引起肺静脉浓度依赖性收缩反应 ;BQ12 3可使ET 3所致收缩反应减弱 ,IRL10 38可使IRL162 0所致收缩反应减弱 ;③在BQ12 3预处理条件下给予第二剂ET 3(30 μmol·L- 1) ,肺静脉表现为舒张反应 ,吲哚美辛 (1μmol·L- 1)可使其舒张反应减弱 .本研究表明 :①存在于肺动脉平滑肌上的ETA 受体参与血管的收缩反应 ,肺动脉内皮上的ETB 受体通过释放NO参与舒张反应 ;②肺静脉平滑肌上的ETA 和ETB 受体均参与收缩反应 ,但ETB 受体所致收缩反应易脱敏 ;③在肺静脉平滑肌上可能还存在非ETA/非ETB 受体 ,通过释放舒张性PG物质参与舒张反应 .     

Effects of topically applied capsaicin cream on neurogenic inflammation and thermal sensitivity in rats

The effects of capsaicin cream on neurogenic inflammation and thermal nociceptive threshold were investigated in rats. Firstly, for topical application of capsaicin cream to hind paw, we shaped boots from dental cement to prevent the animals from licking off the drug. Capsaicin cream (1%) led to significant increases in the amounts of Evans blue and substance P (SP) released into the perfusate, and the former response was significantly suppressed by pretreatment with RP67580, an NK1-receptor antagonist, but not by treatment with an NK2-receptor antagonist. Subsequent electrical stimulation of the sciatic nerve resulted in a significant reduction in Evans blue and SP extravasation 24 h after topical application of capsaicin cream. On the other hand, when capsaicin cream was repeatedly applied to both hind paws once a day, withdrawal latency for noxious heat stimulation decreased after 24 h, and this thermal hyperalgesia was reversed 3 days later. These results suggest that capsaicin cream initially affects neurogenic inflammation mechanisms and then blocks the pain transmission mechanism.