Suppression of PU.1-linked TLR4 expression by cilostazol with decrease of cytokine production in macrophages from patients with rheumatoid arthritis.

BACKGROUND AND PURPOSE: The present study assessed the effects of cilostazol on LPS-stimulated TLR4 signal pathways in synovial macrophages from patients with rheumatoid arthritis (RA). These effects were confirmed in collagen-induced arthritis (CIA) in mice. EXPERIMENTAL APPROACH: Expression of TLR4, PU.1, NF-κB p65 and IκBα on synovial fluid macrophages from RA patients was determined by Western blotting, and cytokines were measured by ELISA. Anti-arthritic effects were evaluated in CIA mice. KEY RESULTS: Intracellular cAMP was concentration-dependently raised by cilostazol (1-100 µM). Cilostazol significantly suppressed LPS-stimulated increase of TLR4 expression by blocking PU.1 transcriptional activity in RA macrophages. In addition, cilostazol decreased LPS-induced myeloid differentiation factor 88 (MyD88) expression, but not that of TNF receptor-associated factor 6 (TRAF6). Cilostazol also suppressed IkBα degradation and NF-κB p65 nuclear translocation. Moreover, LPS-induced increase of cytokine production (TNF-α, IL-1ß) was inhibited by cilostazol, an effect which was accompanied by suppression of IκBα degradation, and NF-κB p65 nuclear translocation. However, expression of anti-inflammatory IL-10 was elevated by cilostazol and forskolin/IBMX. In mice with CIA, post-treatment with cilostazol (30 mg kg⁻¹ day⁻¹) decreased expression of TLR4 in knee joints in association with decreased recruitment of macrophages. Consequently, synovial inflammation, proteoglycan depletion and bone erosion were significantly inhibited by cilostazol treatment. CONCLUSIONS AND IMPLICATIONS: Cilostazol down-regulated LPS-stimulated PU.1-linked TLR4 expression and TLR4/MyD88/NF-κB signal pathways, and then suppressed inflammatory cytokine production in synovial macrophages from RA patients. Also cilostazol markedly inhibited the severity of CIA in mice.

Epitope mapping of Porphyromonas gingivalis heat-shock protein and human heat-shock protein in human atherosclerosis.

To identify T- and/or cross-reactive B-cell epitopes of P. gingivalis and human heat-shock protein (HSP)60 in atherosclerosis patients, we synthesized 104 overlapping synthetic peptides spanning whole molecules of P. gingivalis HSP60 and human HSP60, respectively. T-cell epitopes of P. gingivalis HSP were identified with the use of previously established P. gingivalis HSP-reactive T-cell lines. B-cell epitopes of P. gingivalis HSP60 and human HSP60 were identified by the use of patients' sera. Anti-P. gingivalis, anti-P. gingivalis HSP60, or anti-human HSP60 IgG antibody titers were higher in the atherosclerosis patients compared with the healthy subjects. Five immunodominant peptides of P. gingivalis HSP60, identified as T-cell epitopes, were also found to be B-cell epitopes. Moreover, 6 cross-reactive B-cell epitopes of human HSP60 were identified. It was concluded that P. gingivalis HSP60 might be involved in the immunoregulatory process of atherosclerosis, with common T- and/or B-cell epitope specificities and with cross-reactivity with human HSP60.

Establishment of Porphyromonas gingivalis heat-shock-protein-specific T-cell lines from atherosclerosis patients.

Human atherosclerotic plaques contain heat-shock proteins which may serve as potential targets of the immune response in atherosclerosis. Since periodontal infections are suggested as risk factors for the development of cardiovascular diseases, we undertook the present study to evaluate the T-cell immune responses specific to Porphyromonas gingivalis (P. gingivalis) heat-shock protein (hsp)60 in patients suffering from atherosclerosis. Anti-P. gingivalis hsp60 IgG antibody titers were elevated in all patients. We could establish P. gingivalis hsp-specific T-cell lines from the atheroma lesions and the peripheral blood. The T-cell lines were a mixture of CD4+ and CD8+ cells producing the cytokines characteristic of both Th1 and Th2 subsets. The present findings suggest that the T-cell immune response specific to P. gingivalis hsp60 may be involved in the immunopathologic process of atherosclerotic diseases.

Restoration of vasodilation and CBF autoregulation by genistein in rat pial artery after brain injury.

This study determined whether, after fluid percussion injury (FPI), tyrosine kinase activation is coupled to inhibition of K(+) channels and alteration in cerebral blood flow (CBF) autoregulation in the rat pial artery. Injury of moderate severity (2--2.5 atm) was produced by FPI in anesthetized rats equipped with a closed cranial window. The suppressed vasodilation of the pial arterioles to calcitonin gene-related peptide (CGRP) and levcromakalim (LMK) and altered lower limit of CBF autoregulation after FPI were restored by genistein but not by daidzein, an inactive analog. Vasodilation to S-nitroso-N-acetyl penicillamine (0.1--10 micromol/l) was, however, little influenced after FPI. The restored vasodilation was decreased by sodium orthovanadate, suggesting the reciprocal action of tyrosine phosphorylation and dephosphorylation. After FPI, CGRP-induced vasodilation restored by genistein (10 micromol/l) was strongly antagonized by iberiotoxin but not by glibenclamide, whereas LMK-induced vasodilation was, in contrast, inhibited by glibenclamide but not by iberiotoxin. Taken together, we suggest that, after FPI, activation of tyrosine kinase links the inhibition of K(+) channels to impaired autoregulatory vasodilation in response to acute hypotension and alteration in CBF autoregulation in the rat pial artery.

SKP-450 inhibits migration and DNA synthesis stimulated by oxidized low density lipoprotein in smooth muscle cells.

This study was carried out to examine the inhibitory effects of SKP-450 (2-[2"(1", 3"-dioxolone)-2-methyl]-4-(2'-oxo-1'-pyrrolidinyl)-6-nitro-2H-1-be nzo pyran), a potassium channel opener, on the proliferation and migration stimulated by oxidized low density lipoprotein (LDL) of cultured smooth muscle cells of Wistar Kyoto rat aorta. SKP-450 (10(-7) and 10(-6) M) as well as probucol (10(-7)-10(-5) M) reduced the production of thiobarbituric acid reactive substances from LDL submitted to CuSO(4) (10 microM). The increased [3H]thymidine incorporation and migration (chemotactic and wound-edge) of the cultured smooth muscle cells in association with increased production of platelet-derived growth factor (PDGF)-BB-like immunoreactivity stimulated by oxidized LDL were significantly reduced by SKP-450 (10(-7)-10(-6) M). Inhibition by SKP-450 of the oxidized LDL-stimulated [3H]thymidine incorporation was antagonized by iberiotoxin (10(-7) M), but not by glibenclamide (10(-6) M), suggestive of mediation of Ca(2+)-activated K(+) channel opening in the action of SKP-450. Taken together, SKP-450 inhibited the proliferation and migration of the smooth muscle cells as well as PDGF production stimulated by oxidized LDL, accompanying with its antiperoxidative action.

Metabolism of cAMP to adenosine: role in vasodilation of rat pial artery in response to hypotension.

The purpose of this experiment was to examine whether the cAMP-adenosine pathway is implicated in the autoregulatory vasodilation in response to hypotension. Suffusion with cAMP (1-100 micromol/l) or adenosine (0.01-10 micromol/l) caused a sustained vasodilation of the resting pial arteries in a concentration-dependent manner. In contrast, N6,2'-O-dibutyryl-cAMP and 8-bromo-cAMP exerted a weak dilation at high concentration (100 micromol/l). The vasodilation to cAMP (1-100 micromol/l), adenosine (0.01-10 micromol/l), and hypotension was significantly reduced by pretreatment with 3,7-dimethyl-1-propargylxanthine (1 micromol/l), an A2 receptor antagonist, as well as 3-isobutyl-1-methylxanthine (3 micromol/l), an inhibitor of endo- and ectophosphodiesterase, 1, 3-dipropyl-8-p-sulfophenylxanthine (100 micromol/l), an inhibitor of ecto-5'-phosphodiesterase, or alpha,beta-methylene-adenosine 5'-diphosphate (100 micromol/l), an inhibitor of ecto-5'-nucleotidase. However, 8-cyclopentyltheophylline (1 micromol/l), an A1 antagonist, did not elicit a similar response. The increased release of adenosine when the cortical surface was suffused with cAMP (100 micromol/l) was significantly reduced by 3-isobutyl-1-methylxanthine, 1,3-dipropyl-8-p-sulfophenylxanthine, and alpha,beta-methylene-adenosine 5'-diphosphate (each 100 micromol/l). These results indicate that the cAMP-adenosine pathway as a viable metabolic mechanism is implicated in the production of adenosine in the rat pial artery and contributes to the regulation of vasodilation in response to hypotension.

Effect of omega-conotoxin GVIA and omega-agatoxin IVA on the capsaicin-sensitive calcitonin gene-related peptide release and autoregulatory vasodilation in rat pial arteries.

This study assesses the effect of neuronal voltage-sensitive Ca2+ channel blockers, omega-conotoxin GVIA (CTX), and omega-agatoxin IVA (AgTX) on the vasodilation and release of calcitonin gene-related peptide (CGRP), both of which were induced by either application of capsaicin or acute stepwise hypotension. Changes in pial arterial diameter were determined directly through a closed cranial window. The vasodilation of pial artery induced by either CGRP (0.1 micromol/L) or capsaicin (0.3 micromol/L) was significantly inhibited by CGRP(8-37) (0.1 micromol/L) (P < 0.05 and P < 0.05, respectively). The autoregulatory vasodilation to acute stepwise hypotension was severely attenuated by pretreatment with either CTX or AgTX. When the hypotension was kept for 2, 4, and 10 minutes, the releasable CGRP-like immunoreactivity (CGRP-LI) level (vehicle, 13.4+/-1.5 fmol/mm2/30 min) by 10 micromol/L capsaicin from the isolated pial arteries was significantly reduced in the 4- and 10-minute hypotension groups (11.3+/-1.2 fmol/mm2/30 min, P < 0.05, and 11.1+/-1.5 fmol/mm2/30 min, P < 0.05, respectively), but not in 2-min group. Moreover, the CGRP-LI level released by 10 micromol/L capsaicin (13.7+/-0.9 fmol/mm2/30 min) also was significantly depressed by pretreatment with 1 micromol/L CTX to 10.4+/-1.0 fmol/mm2/30 min (P < 0.01) and with 0.1 micromol/L AgTX to 8.7(1.7 fmol/mm2/30 min (P < 0.001), as well as by pretreatment with 10 micro-mol/L capsaicin (6.0+/-1.6 fmol/ mm2/30 min, P < 0.001). These results suggest that the neuronal N- and P-type voltage-sensitive Ca2+ channels are implicated in the release of CGRP from capsaicin-sensitive perivascular sensory nerves in response to acute hypotension, and that the released CGRP may contribute to the autoregulatory vasodilation in the cerebral microcirculation.

Effect of rebamipide on liver damage and increased tumor necrosis factor in a rat model of endotoxin shock.

We investigated the effect of rebamipide, a novel antiinflammatory agent, on liver damage in a rat model of circulatory shock induced by bacterial endotoxin (E. coli lipopolysaccharide, LPS). Endotoxemia for 6 hr resulted in a 5.9-fold rise in the serum levels of nitrite (P < 0.05) with a significant rise in the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactic dehydrogenase (LDH), suggestive of liver dysfunction. The increased activities of serum ALT, AST, and LDH, but not serum nitrite were significantly inhibited by rebamipide (100 mg/kg, orally for five days). Myeloperoxidase activity in the liver was significantly elevated in the rats with endotoxemia by 2.4-fold (P < 0.05), which was also significantly inhibited by rebamipide. Upon LPS injection, serum TNF-alpha levels peaked at 1 hr after LPS (from 167.4 +/- 20.0 to 1570.0 +/- 100.0 pg/ml) and thereafter rapidly declined. The increased TNF-alpha level measured at 1 hr was significantly inhibited by pretreatment with rebamipide (100 mg/kg for five days). It is suggested that rebamipide exerts a strong protective effect on the LPS-induced liver damage through inhibition of activation of neutrophils and TNF-alpha production.

Decreased CGRP level with increased sensitivity to CGRP in the pial arteries of spontaneously hypertensive rats.

It was aimed to investigate the importance of calcitonin gene-related peptide (CGRP) in maintenance of normal cerebral microcirculation. We examined both the functional (in vivo) and biochemical effects (in vitro) of CGRP on the pial arteries of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). When mock cerebrospinal fluid containing capsaicin (3 x 10(-7) M) was suffused over the cortical surface, the diameter of pial arteries of SHR was transiently increased and rapidly returned to the baseline level, while the capsaicin-induced increase in pial arterial diameters of WKY was large and sustained for a longer duration (> 10 min). Capsaicin-induced vasodilation was significantly attenuated by pretreatment with CGRP8-37, a CGRP1, receptor antagonist, in both WKY and SHR. On the other hand, cortical suffusion with CGRP (10(-9) approximately 10(-6) M) exerted a larger enhancement in the vasodilation of pial artery of SHR than WKY. The CGRP-induced vasodilation was significantly antagonized by CGRP8-37 in both WKY and SHR. The released level of CGRP-like immunoreactivity (CGRP-LI) from the pial artery was significantly lower in SHR (12.3 +/- 1.2 fmol/mm2/hr) than that in WKY (24.5 +/- 3.9 fmol/mm2/hr). CGRP (10(-6) M)-induced stimulation of cyclic AMP formation was rather larger in the pial arteries from SHR (50.2 +/- 5.8 fmol/mm2/30 min, p < 0.05) than those from WKY (34.5 +/- 3.8 fmol/mm2/30 min). These data suggest that, in the pial arteries of SHR, the transient vasodilation to capsaicin and enhanced vasodilation to CGRP are related to the decreased CGRP level in the cerebral microvascular beds, consequently leading to increased sensitivity of the CGRP receptors to CGRP.

Pharmacological coupling and functional role for CGRP receptors in the vasodilation of rat pial arterioles.

In this study, we investigated the signal transduction underlying the vasodilator action of calcitonin gene-related peptide (CGRP) in the rat pial arterioles. In an in vivo experiment, changes in pial arterial diameters (20.2 +/- 1.9 microns) were observed under suffusion with mock cerebrospinal fluid containing CGRP (10(-9)-10(-7) M) directly through a closed cranial window. Changes in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in response to CGRP and levcromakalim were measured in the pial arterioles in an in vitro experiment. CGRP-induced vasodilation and cAMP production were significantly inhibited by specific CGRP antibody serum and CGRP-(8-37) fragment, suggesting involvement of the CGRP1 receptor subtype. Vasodilation and increase in cAMP production evoked by CGRP were inhibited not only by glibenclamide (ATP-sensitive K+ channel blocker) but also by charybdotoxin (large-conductance Ca(2+)-activated K+ channel blocker), but this was not the case for the isoproterenol-induced vasodilation and cAMP production. These findings implicate the ATP-sensitive K+ channels and the large-conductance Ca(2+)-activated K+ channels in the CGRP receptor-coupled cAMP production for vasodilation. Further study is required to identify whether the cAMP-dependent K+ channel activation is related to CGRP-induced vasorelaxation of the rat pial arterioles.

Characterization of PD 121981- and CGP 42112-induced unmasking of low concentration effects of angiotensin II in rabbit abdominal aorta.

The unmasking of the low concentration effect of angiotensin II (AII) was identified within the concentration ranges of 10(-13) to 10(-11) M of AII by PD 121981 (5-diphenylacetyl-1-(4-methoxy-3-methylbenzyl)- 4,5,6,7-tetrahydro-1H-imidazo[4,5-c]-pyridine-6-carboxylic acid) and 10(-12) to 3 x 10(-10) M of AII by CGP 42112 (nicotinic acid-Tyr-(N alpha-benzyl-oxycarbonyl-Arg)Lys-His-Pro-IIe-OH), AT2 antagonists, in association with the ordinary contraction curve, i.e., high concentration effect (at 3 x 10(-10)-10(-6) M of AII), in the rabbit abdominal aorta. Thus, they showed clear biphasic features of AII-induced contraction curves. However, this was not the case for angiotensin I and angiotensin III. This PD 121981-evoked low concentration effect of AII was selectively inhibited by DuP 753 (0.01-1 nM), dithiothreitol (10 and 100 microM), pertussis toxin (50 and 300 ng/ml, for 2 hr), nifedipine (1 and 10 microM) and 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (1 and 3 microM), which suggests the receptors were the AT1 subtype. However, the high concentration effect of AII was not affected by these drugs within the concentration ranges used in the present studies. These myographic results were almost consistent with the features of the intracellular Ca++ changes. Thus, it was concluded that the receptors that mediate the low concentration effect of AII belong to the AT1 subtype. However, the current study did not determine the mechanism by which PD 121981 and CGP 42112 evoked the up-regulation of the AT1 receptors.

Relaxant effects of cromakalim and ATP depletion in dog and rat mesenteric arteries--species differences.

In the present study, the effects of cromakalim on tension and 86Rb+ efflux rate were evaluated in strips of dog and rat mesenteric arteries and compared with the variables obtained from ATP-depleted strips of both species. The cromakalim-induced relaxation was competitively antagonized by glibenclamide, with similar pA2 values, in both dog and rat mesenteric arteries. Glibenclamide caused an enhancement of the precontraction or a reversal of the cromakalim-induced inhibition in the mesenteric arteries of both species when cromakalim was applied prior to or during phenylephrine-contraction. The 86Rb+ efflux rate from the mesenteric arteries was significantly increased in both species after application of cromakalin (10 microM). However, in the ATP-depleted mesenteric artery (verified by high performance liquid chromatography), an increase in 86Rb+ efflux and a glibenclamide-induced enhancement of contraction were observed in the rat, but not in the dog. Taken together, between dog and rat mesenteric arterial strips, a differential effect of ATP depletion with 2-deoxyglucose plus oligomycin was identified in the activation of ATP-sensitive K+ channels, but not of cromakalim.

Relaxation by cromakalim and pinacidil of isolated smooth muscle cells from canine coronary artery-multiple sites of action.

Dispersed cells were isolated from the canine coronary artery by enzymatic digestion. Their contraction and relaxation were determined by measuring their length using a video microscaler system. The cells remained structurally intact when examined by Trypan blue exclusion and electron microscopy. The cells showed a concentration-dependent contraction (EC50: 2.3 +/- 0.36 x 10(-12) M) to phenylephrine. The phenylephrine-induced contraction of the intact cells was inhibited by cromakalim (IC50: 1.24 +/- 0.27 x 10(-10) M) and pinacidil (IC50: 6.8 +/- 1.89 x 10(-10) M). The sensitivity of the dispersed cells to cromakalim was approximately 3 orders of magnitude larger than that of the muscle strips (EC50: 1.94 +/- 0.22 x 10(-7) M). Glibenclamide (a selective inhibitor of the ATP-sensitive K+ channel in pancreatic beta-cells) competitively antagonized the cromakalim-induced inhibition of the phenylephrine-contraction in intact cells (pA2:9.12; slope: 1.13) as well as in muscle strips (pA2: 7.84; slope: 0.95). Permeabilized cells were made by a brief exposure of the cells to saponin and were suspended in a buffer medium containing 100 mM KCl and 0.18 microM Ca++. The cells showed a concentration-dependent contraction to phenylephrine (EC50:2.2 +/- 0.40 x 10(-12) M) and inositol 1,4,5-triphosphate (EC50: 5.3 +/- 1.05 x 10(-11) M). These contractions were concentration-dependently inhibited by cromakalim and pinacidil. The inhibition by cromakalim of the inositol-induced contraction was markedly antagonized by apamin and, to a lesser extent, by glibenclamide. Thus, it is suggested that cromakalim and pinacidil exert a potent relaxation by acting on multiple sites: the glibenclamide-sensitive K+ channels of the plasma membrane and the intracellular site sensitive to inositol and apamin.

Differential signal transduction pathways in cat lower esophageal sphincter tone and response to ACh.

Lower esophageal sphincter (LES) basal tone and contraction in response to maximally effective doses (Emax) of acetylcholine (ACh) may be mediated by different intracellular transduction pathways. In the basal state resting tone, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] formation and levels of diacylglycerol (DAG) (C. Hillemeier, K. N. Bitar, and P. Biancani, unpublished data) are higher in LES circular muscle than in esophageal muscle, which does not maintain tone. In vitro resting tone and spontaneously elevated formation of Ins(1,4,5)P3 in LES circular muscle strips decrease in a dose-dependent manner in response to the phospholipase C antagonist 1-[6-([(17-beta)-3-methoxyestra-1,3, 5(10)-trien-17-yl]amino)hexyl]-1H-pyrrole-2,5-dione (U-73122). Basal Ins(1,4,5)P3 formation, however, is submaximal, since it can be increased by cholinergic stimulation. These data suggest that LES tone is associated with partial activation of phospholipase C. We therefore tested submaximal doses of Ins(1,4,5)P3 and DAG in permeabilized LES muscle cells and found that they act synergistically; their interaction depends on calcium release and is mediated through a protein kinase C (PKC)-dependent pathway. In contrast, we have previously shown that contraction induced by Emax of ACh is mediated through calmodulin-dependent mechanisms (14). To investigate these differences, we tested high and low doses of ACh. Contraction induced by high doses of ACh was inhibited by calmodulin but not by PKC antagonists, as previously reported, but low ACh doses were preferentially inhibited by PKC antagonists. Similarly, low Ins(1,4,5)P3 concentrations activated a PKC-dependent pathway, whereas contraction induced by Emax of Ins(1,4,5)P3 was calmodulin dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological evidence that calcitonin gene-related peptide is implicated in cerebral autoregulation.

In anesthetized rats, we examined the possibility that calcitonin gene-related peptide (CGRP, a neuropeptide) released in response to transient hypotension may contribute to the reflex autoregulation of cerebral blood flow. Changes in pial arterial diameter (mean 33.0 +/- 1.1 microns) with changes in systemic arterial blood pressure (mean 101.9 +/- 2.7 mmHg) were observed directly through a closed cranial window. In capsaicin-treated rats (depletor of CGRP and substance P, 50 nmol capsaicin injected intracisternally 24 h before experiment), vasodilatation, which was evoked on transient hypotension, and vasoconstriction on reverse of hypotension were markedly attenuated or almost abolished. When changes in pial arterial diameter were plotted as a function of changes in blood pressure, the slopes of regression lines for vasodilatation and vasoconstriction were markedly reduced after capsaicin treatment. Similar reductions were evidenced under suffusion of CGRP antibody serum (1:1,000) and after CGRP receptor desensitization but not after substance P receptor desensitization. Pretreatment with glibenclamide, a K(+)-channel antagonist, also caused severe alterations in the autoregulatory vasomotor responses to hypotension and its reverse. Suffusion with mock cerebrospinal fluid, containing either CGRP or cromakalim, a K(+)-channel opener, dilated the pial artery in a concentration-dependent manner, and their effects were antagonized by glibenclamide. Substance P produced a vasodilatation, which was unaffected by glibenclamide.(ABSTRACT TRUNCATED AT 250 WORDS)

Inositol trisphosphate restores impaired human gallbladder motility associated with cholesterol stones.

BACKGROUND: Gallbladder motility is impaired in specimens with cholesterol stones but normal with pigment stones. METHODS: Muscle cells obtained from 19 human gallbladders with cholesterol stones and 11 with pigment stones were enzymatically digested and contracted with cholecystokinin octapeptide (CCK-8), acetylcholine, and KCl. RESULTS: Muscle cells from pigment stones had a greater contraction than cells from cholesterol stones. CCK-8-induced contraction was unaffected by calcium-free media but was blocked by strontium. Potassium-evoked contraction was blocked by a calcium-free media and unaffected by strontium. Inositol triphosphate (IP-3)-induced contraction was similar to the contraction caused by CCK-8 in permeable cells from pigment stones but was greater than the response to CCK-8 in cells from cholesterol stones. CONCLUSIONS: Muscle cells from gallbladders with cholesterol stones contract less than cells from gallbladders with pigment stones; CCK-8-induced contraction only uses stored calcium; and IP-3 causes contractions of equal magnitude in cells from gallbladders with cholesterol and pigment stones. These abnormalities could result from an impaired receptor activation of the mechanism for IP-3 generation and release of stored calcium.

Disturbances in autoregulatory responses of rat pial arteries by sulfonylureas.

It was aimed to test the role of ATP-sensitive K+ channels in the autoregulatory response of cerebral arterioles in vivo. Changes in pial arterial caliber (mean, 43.2 +/- 2.3 microns in diameter) in response to changes in systemic arterial blood pressure (mean, 104.3 +/- 1.4 mmHg) were observed directly through closed cranial windows in anesthetized normotensive rats. During superfusion with vehicle, pial arterial caliber automatically increased in response to hypotension induced by arterial bleeding into a reservoir and decreased on reverse of arterial blood pressure by infusion of blood. After pretreatment with sulfonylureas, glibenclamide (1 and 3 microM) and glipizide (30 and 100 microM), arteriolar dilatation and constriction observed during hypotension and its reverse were disturbed. A similarity was evidenced when hypotension was induced by sodium nitroprusside (750 nmol kg-1min-1, i.v.). Cromakalim, a K+ channel opener, exerted a concentration-dependent vasodilatation of the pial artery and its effect was antagonized by glibenclamide. These data suggest that the endogenous glibenclamide-sensitive K+ channel opener is involved in the modulation of cerebral microvascular autoregulation.

Effect of ouabain on relaxation induced by cromakalim in human and canine mesenteric arteries.

We investigated the effect of cromakalim, a K+ channel opener, that activates indirectly the Na(+)-K+ pump, in association with increased K+ conductance in the mesenteric arteries. In 65% of human mesenteric arteries tested, the concentration-dependent relaxation curves for cromakalim were biphasic: the low concentration (< 10(-7) M) effect was preferentially inhibited by ouabain, whereas the higher concentration effect was significantly inhibited by glibenclamide. In branches of canine mesenteric artery, the cromakalim-induced relaxation was inhibited by pretreatment with ouabain (1 microM) as well as by glibenclamide (1 microM). The reduction in contraction of human and canine mesenteric arterial strips caused by cromakalim was totally reversed by pretreatment with ouabain (1 microM) or glibenclamide (1 microM). On the other hand, in canine mesenteric artery, cromakalim caused a significant stimulation of 22Na+ influx and ouabain-sensitive 86Rb+ uptake in association with increased 86Rb+ efflux, all of which were inhibited by glibenclamide (1 microM). Thus, it is suggested that cromakalim possesses the additional property to stimulate the Na(+)-K+ pump through an elevation in intracellular Na+, resulting in strong relaxation of blood vessels.

Acute experimental esophagitis impairs signal transduction in cat lower esophageal sphincter circular muscle.

It has been previously shown that induction of experimental esophagitis in the cat by esophageal perfusion for 30 minutes with 0.1N HCl for 4 consecutive days results in a significant reduction of in vivo lower esophageal sphincter (LES) resting pressure and in vitro spontaneous tone without affecting esophageal response to KCl. It has also been shown that basal LES tone and LES contraction in response to acetylcholine depend on the release of calcium from intercellular stores, whereas esophageal contraction is mediated by extracellular calcium. The present report shows that esophageal acid perfusion impairs the transduction pathway mediating lower esophageal sphincter contraction in response to acetylcholine through release of intracellular calcium because LES strips and single cells no longer contract in response to acetylcholine if calcium is removed from the physiologic salt solution. This suggests that either the intracellular calcium stores or the release mechanisms that mediate maintenance of tone and contraction in response to acetylcholine may be damaged. However, the acid perfusion has no effect on the acetylcholine response in the esophagus, which is mediated by the influx of extracellular calcium. In the LES circular muscle, the injury results in reduced levels of inositol phosphates without affecting resting levels of 5'-cyclic adenosine monophosphate or 5'-cyclic guanosine monophosphate. The reduced levels of 1,4,5-inositol trisphosphate are consistent with impairment in the mechanisms responsible for release of intracellular calcium, although concurrent damage to calcium stores may also occur.

Similarities between effects of superoxide-mediated endothelium-derived relaxing factor and cromakalim.

A stable endothelium-derived relaxing factor has been reported to be generated on exposure of endothelial cells to the superoxide anion. In this study, we first evaluated the effects of the relaxing factor and cromakalim on mechanical tone and, second, assessed their consequences on the 86Rb efflux rate. On application of hypoxanthine-xanthine oxidase to a bath for generating superoxide anion, the precontracted rabbit mesenteric artery exhibited another transient increase in contraction, followed by sustained relaxation. This relaxation was lost in the K(+)-physiological salt solution (PSS) (greater than 35 mM) and was inhibited by glibenclamide (10 microM) but not by N-methyl-L-arginine or methylene blue. Hypoxanthine-xanthine oxidase application did not increase either basal or stimulated synthesis of guanosine 3',5'-cyclic monophosphate. In the presence of 2 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and 10 mM MgCl2, the relaxing factor caused a significant increase in 86Rb efflux from the aortic and mesenteric arterial segments, as did the cromakalim. The increased 86Rb efflux, either by the relaxing factor or by cromakalim, was wholly inhibited by glibenclamide. These results suggest that superoxide-mediated endogenous relaxing factor may have a similar mechanism of action to cromakalim in vasodilatation.