Teratogenicity and maternal effects of Lactobacillus brevis KB290 in rats and rabbits.

Lactobacillus brevis KB290 (KB290), a plant-derived probiotic lactic acid bacterium, improves gut health and stimulates immune function. Here we extensively investigated the teratogenicity of KB290 in rats and rabbits. We observed no adverse maternal or fetal effects and concluded that the no observable adverse effect level for maternal general toxicity, maintenance of pregnancy, and teratogenicity should be ≥ 10(10) cfu/kg/day. Our results suggest that KB290 would be safe for pregnant females and their offspring.

Safety assessment of Lactobacillus brevis KB290 as a probiotic strain.

Lactobacillus brevis KB290 (KB290), a plant-derived probiotic lactic acid bacterium, reportedly improves gut health and stimulates immune function. Here we extensively investigated the geno-, acute, subacute, and subchronic toxicity of KB290 and its bacterial translocation potential. KB290 was non-mutagenic in the bacterial reverse mutation assay by the preincubation method. In the single oral dose toxicity test, KB290 at 10(9) cfu/ml was nontoxic at maximum capacity (20 ml/kg). When 10(8), 10(9), or 10(10) cfu/kg was administered daily to rats by gavage for 2 weeks (subacute assay), we observed no clear treatment-related effect and no evidence of bacterial translocation from the gastrointestinal tract. When it was administered for 13 weeks (subchronic assay), we again observed no clear treatment-related effect and no significant toxicological effect. Based on those results, we consider 10(10) cfu/kg per day, the highest dose tested, to be the no observed adverse effect level (NOAEL). These results suggest that KB290 is safe for human consumption.

Biosynthetic characterization and biochemical features of the third natural nisin variant, nisin Q, produced by Lactococcus lactis 61-14.

AIMS: To characterize the genetic and biochemical features of nisin Q. METHODS AND RESULTS: The nisin Q gene cluster was sequenced, and 11 putative orfs having 82% homology with the nisin A biosynthesis gene cluster were identified. Nisin Q production was confirmed from the nisQ-introduced nisin Z producer. In the reporter assay, nisin Q exhibited an induction level that was threefold lower than that of nisin A. Nisin Q demonstrated an antimicrobial spectrum similar to those of the other nisins. Under oxidizing conditions, nisin Q retained a higher level of activity than nisin A. This higher oxidative tolerance could be attributed to the presence of only one methionine residue in nisin Q, in contrast to other nisins that contain two. CONCLUSIONS: The 11 orfs of the nisin producers were identical with regard to their functions. The antimicrobial spectra of the three natural nisins were similar. Nisin Q demonstrated higher oxidative tolerance than nisin A. SIGNIFICANCE AND IMPACT OF THE STUDY: Genetic and biochemical features of nisin Q are similar to those of other variants. Moreover, owing to its higher oxidative tolerance, nisin Q is a potential alternative for nisin A.

Fine positioning of a poloidal probe array.

Multipoint detection is an essential requirement for investigating plasma turbulence which is a highly nonlinear phenomenon in space and time. We have fabricated an array of 64-channel poloidal probes surrounding the linear cylindrical plasma named LMD-U in order to study turbulence properties, particularly the nonlinear mode couplings, in the domain of poloidal wave number and frequency. However, misalignments of probe tips produce spurious modes, which do not exist in the real plasma, to distort the precise wave number measurements. The paper presents the description of the 64-channel poloidal probe array with means to adjust the probe positions, with discussion on the effects of the misalignments on the wave number measurements.

Confinement of pure-electron plasmas in a toroidal magnetic-surface configuration.

A pure-electron plasma has been confined in a toroidal magnetic-surface configuration for as long as classical diffusion time due to neutral collisions. By controlling the potential of the internal conductor, long-term stable confinement of electrons has been achieved in a toroidal geometry.

Injection of electron beam into a toroidal trap using chaotic orbits near magnetic null.

Injection of charged particle beam into a toroidal magnetic trap enables a variety of interesting experiments on non-neutral plasmas. Stationary radial electric field has been produced in a toroidal geometry by injecting electrons continuously. When an electron gun is placed near an X point of magnetic separatrix, the electron beam spreads efficiently through chaotic orbits, and electrons distribute densely in the torus. The current returning back to the gun can be minimized less than 1% of the total emission.

Effects of SK&F 96365 and mefenamic acid on Ca2+ influx in stimulated endothelial cells and on endothelium-derived hyperpolarizing factor-mediated arterial hyperpolarization and relaxation.

This study was undertaken to assess how Ca2+ influx into endothelial cells via Ca2+-permeable nonselective cation channels (NSCCs) is important in vascular responses mediated by endothelium-derived hyperpolarizing factor (EDHF). In cultured porcine aortic endothelial cells, the sustained increases in the intracellular Ca2+ concentration ([Ca2+]i) elicited by bradykinin and cyclopiazonic acid, which were strongly dependent on the presence of extracellular Ca2+, were suppressed by the NSCC blockers, SK&F 96365 and mefenamic acid. In porcine coronary artery with intact endothelium, bradykinin elicited a rapid fall in the membrane potential, followed by sustained hyperpolarization with a slow decay. In the presence of SK&F 96365 or mefenamic acid, the peak amplitude was severely reduced and the decay phase of hyperpolarization to bradykinin was greatly accelerated, which was apparently similar to the response obtained in Ca2+-free medium. Cyclopiazonic acid caused sustained hyperpolarization in an extracellular Ca2+-dependent manner, an effect which was markedly diminished by SK&F 96365 and mefenamic acid. In rings of coronary artery precontracted with U46619, bradykinin and cyclopiazonic acid produced endothelium-dependent relaxations even in the presence of N(G)-nitro-L-arginine and indomethacin. SK&F 96365 and mefenamic acid significantly attenuated the relaxant responses. These results indicate that the increase in [Ca2+]i of endothelial cells due to Ca2+ entry via NSCCs plays a crucial role in the maintenance of the EDHF-mediated vascular responses.

Role of endothelial Ni(2+)-sensitive Ca(2+) entry pathway in regulation of EDHF in porcine coronary artery.

Elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in endothelial cells is proposed to be required for generation of vascular actions of endothelium-derived hyperpolarizing factor (EDHF). This study was designed to determine the endothelial Ca(2+) source that is important in development of EDHF-mediated vascular actions. In porcine coronary artery precontracted with U-46619, bradykinin (BK) and cyclopiazonic acid (CPA) caused endothelium-dependent relaxations in the presence of N(G)-nitro-L-arginine (L-NNA). The L-NNA-resistant relaxant responses were inhibited by high K(+), indicating an involvement of EDHF. In the presence of Ni(2+), which inhibits Ca(2+) influx through nonselective cation channels, the BK-induced EDHF relaxant response was greatly diminished and the CPA-induced response was abolished. BK and CPA elicited membrane hyperpolarization of smooth muscle cells of porcine coronary artery. Ni(2+) suppressed the hyperpolarizing responses in a manner analogous to removal of extracellular Ca(2+). EDHF-mediated relaxations and hyperpolarizations evoked by BK and CPA in porcine coronary artery showed a temporal correlation with the increases in [Ca(2+)](i) in porcine aortic endothelial cells. The extracellular Ca(2+)-dependent rises in [Ca(2+)](i) in endothelial cells stimulated with BK and CPA were completely blocked by Ni(2+). These results suggest that Ca(2+) influx into endothelial cells through nonselective cation channels plays a crucial role in the regulation of EDHF.

Alterations in EDHF-mediated hyperpolarization and relaxation in mesenteric arteries of female rats in long-term deficiency of oestrogen and during oestrus cycle.

This study was undertaken to determine whether endothelium-dependent relaxations are altered in mesenteric arteries from young female rats during oestrus cycle and after castration. The contractile response to phenylephrine (Phe) was significantly enhanced in arteries from rats subjected to ovariectomy than in those from sham-operated (control) rats. Treatment of ovariectomized rats with 17beta-oestradiol returned the Phe response to the control level. Arteries from rats at the diestrus stage also exhibited greater contraction in response to Phe. In the presence of 100 microM N(G)-nitro-L-arginine (L-NOARG), the enhancement of the Phe contractile response associated with oestrogen deficiency was not observed. Endothelium-dependent relaxations elicited by acetylcholine (ACh) in arteries precontracted with Phe were significantly reduced in ovariectomized and diestrus rats regardless of whether endothelium-derived nitric oxide (NO) was blocked with L-NOARG. Treatment with 17beta-oestradiol prevented the reduced vascular relaxant response to ACh in ovariectomized rats. The reduction in the ACh responses observed in ovariectomized and diestrus rats was eliminated when 500 nM apamin and 100 nM charybdotoxin were present. ACh-induced endothelium-dependent hyperpolarizations were depressed in arteries from ovariectomized and diestrus rats. The hyperpolarizing response to ACh was significantly improved when ovariectomized rats were treated with 17beta-oestradiol. The resting membrane potentials and pinacidil-induced hyperpolarizations were unaffected by ovariectomy or the diestrus stage. These results suggest that oestrogen-deficient states of both short and long duration reduce the basal release of NO from the endothelium and specifically attenuate endothelium-dependent hyperpolarization and relaxation transduced by endothelium-derived hyperpolarizing factor.

Myosin light-chain kinase regulates endothelial calcium entry and endothelium-dependent vasodilation.

Activation of smooth muscle myosin light-chain kinase (MLCK) causes contraction. Here we have proven that MLCK controls Ca2+ entry (CE) in endothelial cells (ECs): MLCK antisense oligonucleotides strongly prevented bradykinin (BK)- and thapsigargin (TG)-induced endothelial Ca2+ response, while MLCK sense did not. We also show that the relevant mechanism is not phosphorylation of myosin light-chain (MLC): MLC phosphorylation by BK required CE, but MLC phosphorylation caused by the phosphatase inhibitor calyculin A did not trigger Ca2+ response. Most important, we provide for the first time strong evidence that, in contrast to its role in smooth muscle cells, activation of MLCK in ECs stimulates the production of important endothelium-derived vascular relaxing factors: MLCK antisense and MLCK inhibitors abolished BK- and TG-induced nitric oxide production, and MLCK inhibitors substantially inhibited acetylcholine-stimulated hyperpolarization of smooth muscle cell membrane in rat mesenteric artery. These results indicate that MLCK controls endothelial CE, but not through MLC phosphorylation, and unveils a hitherto unknown physiological function of the enzyme: vasodilation through its action in endothelial cells. The study discovers a counter-balancing role of MLCK in the regulation of vascular tone.

Regulation of BK(Ca) channels expressed in human embryonic kidney 293 cells by epoxyeicosatrienoic acid.

Epoxyeicosatrienoic acids (EETs) are arachidonic acid metabolites of cytochrome P450 monooxygenase, which are released from endothelial cells and dilate arteries. Dilation seems to be caused by activation of large-conductance Ca2+ activated K+ channels (BK(Ca)) leading to membrane hyperpolarization. Previous studies suggest that EETs activate BK(Ca) channels via ADP-ribosylation of the G protein Galphas with a subsequent membrane-delimited action on the channel [Circ Res 78:415-423, 1996; 80:877-884, 1997; 85:349-356, 1999]. The present study examined whether this pathway is present in human embryonic kidney (HEK) 293 cells when the BK(Ca) alpha-subunit (cslo-alpha) is expressed without the beta-subunit. 11,12-EET increased outward K+ current in whole-cell recordings of HEK293 cells. In cell-attached patches, 11,12-EET also increased the activity of cslo-alpha channels without affecting unitary conductance. This action was mimicked by cholera toxin. The ADP-ribosyltransferase inhibitors 3-aminobenzamide and m-iodobenxylguanidine blocked the stimulatory effect of 11,12-EET. In inside-out patches 11,12-EET was without effect on channel activity unless GTP was included in the bathing solution. GTP and GTPgammaS alone also activated cslo-alpha channels. Dialysis of cells with anti-Galphas antibody completely blocked the activation of cslo-alpha channels by 11,12-EET, whereas anti-Galphai/o and anti-Gbetagamma antibodies were without effect. The protein kinase A inhibitor KT5720 and the adenylate cyclase inhibitor SQ22536 did not reduce the stimulatory effect of 11,12-EET on cslo-alpha channels in cell-attached patches. These data suggest that EET leads to Galphas-dependent activation of the cslo-alpha subunits expressed in HEK293 cells and that the cslo-beta subunit is not required.

Relaxation in different-sized rat blood vessels mediated by endothelium-derived hyperpolarizing factor: importance of processes mediating precontractions.

To clarify the mechanisms involved in relaxations mediated by endothelium-derived hyperpolarizing factor (EDHF), acetylcholine (ACh)-induced endothelium-dependent relaxations and hyperpolarizations were examined in the rat aorta, the main branch of the mesenteric artery (MBMA) and the first branch of the mesenteric aftery (FBMA). In the presence of 100 microM N(G)-nitro-L-arginine (L-NNA) and 10 microM indomethacin, ACh (1 nM to 100 microM) produced no relaxation in the phenylephrine-precontracted aorta. The L-NNA-resistant relaxations by ACh in MBMA precontracted with phenylephrine were eliminated in the presence of 1 microM nifedipine where contractions were independent of L-type Ca(2+) channel activation. In FBMA precontracted with phenylephrine, the L-NNA-resistant relaxations were only partially inhibited by nifedipine. When vessels had been contracted with 300 nM phorbol-12,13-dibutyrate in the presence of nifedipine, ACh-induced L-NNA-resistant relaxations were observed in FBMA only. Pinacidil produced relaxations in all different-sized blood vessels, although sensitivity was inversely related to vessel size. The extent of the ACh hyperpolarizing responses was much smaller than that by pinacidil in the aorta. The membrane potential changes by ACh and pinacidil were almost the same in FBMA. These results indicate that the contribution of EDHF to endothelium-dependent relaxations increases as the vessel size decreases. This may be partly explained by precontractile processes dependent on Ca(2+) entry through L-type Ca(2+) channels, because Ca(2+) channel deactivation seems to be involved as a major mechanism of EDHF-mediated vasorelaxations. However, EDHF may also generate vasorelaxations by an additional mechanism, probably a reduced Ca(2+) sensitivity of contractile elements, as proposed for ATP-sensitive K(+) channel openers.

Effect of prolonged treatment with amlodipine on enhanced vascular contractility in cardiomyopathic hamsters.

This study examined the effects of prolonged treatment with amlodipine on the enhanced vascular contractions in dilated cardiomyopathic (CM) hamsters. From the ages of 5 to 20 weeks, CM hamsters (BIO 53.58) orally received amlodipine. Then we compared the contractile responses to vasoconstrictors in aortas and mesenteric arteries from CM hamsters with or without treatment with those in the arteries from controls (F1b). We also investigated the effect of amlodipine treatment on the Ca2+ sensitivity of tension in beta-escin-skinned smooth muscle of mesenteric artery. The contractile responses to phenylephrine, angiotensin II, and high K+ in both aorta and mesenteric artery were greatly enhanced in CM hamsters compared with controls. Amlodipine treatment slightly but significantly inhibited the enhanced responses in aorta but did not alter the responses in mesenteric arteries. The Ca2+ sensitivity of tension was significantly increased in CM hamster preparations, which was unaffected by amlodipine treatment. These data indicate that amlodipine treatment differentially affects the enhanced responses to vasoconstrictors between large and small blood vessels from CM hamsters. The lack of effect of amlodipine treatment on the responsiveness of CM mesenteric artery leads to the suggestion that the preventive effect of amlodipine on focal myocytolytic necrosis of cardiomyocytes, which was previously reported to be the main cause of cardiomyopathy, results from an action on cardiomyocytes.

Relationship between NaF- and thapsigargin-induced endothelium-dependent hyperpolarization in rat mesenteric artery.

1. In isolated rat mesenteric artery with endothelium, NaF caused slowly developing hyperpolarization. The hyperpolarizing effect was unchanged in the presence of N(G)-nitro-L-arginine (L-NOARG) and indomethacin, but was markedly reduced by high K+. In Ca2+ -free medium or in the presence of Ni2+, NaF failed to produce hyperpolarization. 2. NaF-induced hyperpolarization was substantially unaffected by deferoxamine, an Al3+ chelator, okadaic acid and calyculin A, phosphatase inhibitors, and preincubation with pertussis toxin, suggesting that neither the action of fluoroaluminates as a G protein activator nor inhibition of phosphatase activity contributes to the hyperpolarizing effect. 3. The selective inhibitors of the Ca2+ -pump ATPase of endoplasmic reticulum, thapsigargin and cyclopiazonic acid, elicited hyperpolarization, whose properties were very similar to those of NaF. When intracellular Ca2+ stores had been depleted with these inhibitors, NaF no longer generated hyperpolarization. 4. In Ca2+ -free medium, NaF (or thapsigargin) caused a transient increase in the cytosolic Ca2+ concentration ([Ca2+]i) in cultured porcine aortic endothelial cells, and subsequent application of thapsigargin (or NaF) failed to increase [Ca2+]i. 5. In arterial rings precontracted with phenylephrine, NaF produced endothelium-dependent relaxation followed by sustained contraction even in the presence of L-NOARG and indomethacin. The relaxant response was abolished by high K+ or cyclopiazonic acid. 6. These results indicate that NaF causes endothelium-dependent hyperpolarization, thereby leading to smooth muscle relaxation of rat mesenteric artery. This action appears to be mediated by the promotion of Ca2+ influx into endothelial cells that can be triggered by the emptying of intracellular Ca2+ stores, as proposed for those of thapsigargin and cyclopiazonic acid.

Cyclic GMP-dependent protein kinase activates cloned BKCa channels expressed in mammalian cells by direct phosphorylation at serine 1072.

NO-induced activation of cGMP-dependent protein kinase (PKG) increases the open probability of large conductance Ca2+-activated K+ channels and results in smooth muscle relaxation. However, the molecular mechanism of channel regulation by the NO-PKG pathway has not been determined on cloned channels. The present study was designed to clarify PKG-mediated modulation of channels at the molecular level. The cDNA encoding the alpha-subunit of the large conductance Ca2+-activated K+ channel, cslo-alpha, was expressed in HEK293 cells. Whole cell and single channel characteristics of cslo-alpha exhibited functional features of native large conductance Ca2+-activated K+ channels in smooth muscle cells. The NO-donor sodium nitroprusside increased outward current 2.3-fold in whole cell recordings. In cell-attached patches, sodium nitroprusside increased the channel open probability (NPo) of cslo-alpha channels 3.3-fold without affecting unitary conductance. The stimulatory effect of sodium nitroprusside was inhibited by the PKG-inhibitor KT5823. Direct application of PKG-Ialpha to the cytosolic surface of inside-out patches increased NPo 3.2-fold only in the presence of ATP and cGMP without affecting unitary conductance. A point mutation of cslo-alpha in which Ser-1072 (the only optimal consensus sequence for PKG phosphorylation) was replaced by Ala abolished the PKG effect on NPo in inside-out patches and the effect of SNP in cell attached patches. These results indicate that PKG activates cslo-alpha by direct phosphorylation at serine 1072.

A role of myofilament Ca2+ sensitivity in enhanced vascular reactivity in cardiomyopathic hamsters.

We compared the contractile responses to vasoconstrictors in aortas from 20- to 22-week old cardiomyopathic hamsters, BIO 53.58 strain, and age-matched F1b strain controls. Aortas from cardiomyopathic hamsters exhibited greater contractions in response to phenylephrine, angiotensin II, and high K+ than did the controls. Neither endothelium removal nor the presence of indomethacin and N(omega)-nitro-L-arginine (L-NNA) affected the enhanced contractile responses to these vasoconstrictors, indicating no involvement of endogenous prostanoids and nitric oxide from the endothelium. The contractile response to phorbol-12,13-dibutyrate (PDB) was also more markedly increased in cardiomyopathic aortas regardless of whether extracellular Ca2+ was present. The contractile response of cardiomyopathic aorta to phenylephrine was more sensitive to the inhibitory actions of the protein kinase C inhibitors staurosporine and calphostin C than was that of control aorta. These results suggest that activation of protein kinase C is partly involved in the enhanced phenylephrine response of cardiomyopathic aorta. None of nifedipine, ryanodine, and cyclopiazonic acid modified the maximum contractions induced by phenylephrine in either cardiomyopathic aortas or controls. The Ca2+ sensitivity of tension was significantly increased in beta-escin-skinned smooth muscle of mesenteric artery from cardiomyopathic hamsters compared to that of controls. PDB induced Ca2+ sensitization, but significantly only in cardiomyopathic hamsters. We propose that the enhanced vascular reactivity in cardiomyopathic hamsters may primarily result from increased Ca2+ sensitivity of contractile proteins. In addition, protein kinase C-mediated Ca2+ sensitization may further contribute to the enhanced vascular response to agonists.

Alterations in endothelium-dependent hyperpolarization and relaxation in mesenteric arteries from streptozotocin-induced diabetic rats.

1. The aim of this study was to determine whether endothelium-dependent hyperpolarization and relaxation are altered during experimental diabetes mellitus. Membrane potentials were recorded in mesenteric arteries from rats with streptozotocin-induced diabetes and age-matched controls. The resting membrane potentials were not significantly different between control and diabetic mesenteric arteries (-55.3 +/- 0.5 vs -55.6 +/- 0.4 mV). However, endothelium-dependent hyperpolarization produced by acetylcholine (ACh; 10(-8)-10(-5) M) was significantly diminished in amplitude in diabetic arteries compared with that in controls (maximum -10.4 +/- 1.1 vs -17.2 +/- 0.8mV). Furthermore, the hyperpolarizing responses of diabetic arteries were more transient. 2. ACh-induced hyperpolarization observed in control and diabetic arteries remained unaltered even after treatment with 3 x 10(-4) M N(G)-nitro-L-arginine (L-NOARG), 10(-5) M indomethacin or 60 u ml (-1) superoxide dismutase. 3. Endothelium-dependent hyperpolarization with 10(-6) M A23187, a calcium ionophore, was also decreased in diabetic arteries compared to controls (-8.3 +/- 1.4 vs -18.0 +/- 1.9 mV). However, endothelium-independent hyperpolarizing responses to 10(-6) M pinacidil, a potassium channel opener, were similar in control and diabetic arteries (-20.0 +/- 1.4 vs - 19.2 +/- 1.1 mV). 4. The altered endothelium-dependent hyperpolarizations in diabetic arteries were almost completely prevented by insulin therapy. Endothelium-dependent relaxations by ACh in the presence of l0(-4) M L-NOARG and 10(-5) M indomethacin in diabetic arteries were also reduced and more transient compared to controls. 5. These data indicate that endothelium-dependent hyperpolarization is reduced by diabetes, and this would, in part, account for the impaired endothelium-dependent relaxations in mesenteric arteries from diabetic rats.

Sources of Ca2+ in relation to generation of acetylcholine-induced endothelium-dependent hyperpolarization in rat mesenteric artery.

1. The aim of the present study was to identify the sources of Ca2+ contributing to acetylcholine (ACh)-induced release of endothelium-derived hyperpolarizing factor (EDHF) from endothelial cells of rat mesenteric artery and to assess the pathway involved. The changes in membrane potentials of smooth muscles by ACh measured with the microelectrode technique were evaluated as a marker for EDHF release. 2. ACh elicited membrane hyperpolarization of smooth muscle cells in an endothelium-dependent manner. The hyperpolarizing response was not affected by treatment with 10 microM indomethacin, 300 microM NG-nitro-L-arginine or 10 microM oxyhaemoglobin, thereby indicating that the hyperpolarization is not mediated by prostanoids or nitric oxide but is presumably by EDHF. 3. In the presence of extracellular Ca2+, 1 microM ACh generated a hyperpolarization composed of the transient and sustained components. By contrast, in Ca(2+)-free medium, ACh produced only transient hyperpolarization. 4. Pretreatment with 100 nM thapsigargin and 3 microM cyclopiazonic acid, endoplasmic reticulum Ca(2+)-ATPase inhibitors, completely abolished ACh-induced hyperpolarization. Pretreatment with 20 mM caffeine also markedly attenuated ACh-induced hyperpolarization. However, the overall pattern and peak amplitude of hyperpolarization were unaffected by pretreatment with 1 microM ryanodine. 5. In the presence of 5 mM Ni2+ or 3 mM Mn2+, the hyperpolarizing response to ACh was transient, and the sustained component of hyperpolarization was not observed. On the other hand, 1 microM nifedipine had no effect on ACh-induced hyperpolarization. 6. ACh-induced hyperpolarization was nearly completely eliminated by 500 nM U-73122 or 200 microM 2-nitro-4-carboxyphenyl-N, N-diphenylcarbamate, inhibitors of phospholipase C, but was unchanged by 500 nM U-73343, an inactive form of U-73122. Pretreatment with 20 nM staurosporine, an inhibitor of protein kinase C, did not modify ACh-induced hyperpolarization. 7. These results indicate that the ACh-induced release of EDHF from endothelial cells of rat mesenteric artery is possibly initiated by Ca2+ release from inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pool as a consequence of stimulation of phospholipid hydrolysis due to phospholipase C activation, and maintained by Ca2+ influx via a Ni(2+)- and Mn(2+)-sensitive pathway distinct from L-type Ca2+ channels. The Ca(2+)-influx mechanism seems to be activated following IP3-induced depletion of the pool.

Evidence against a role of cytochrome P450-derived arachidonic acid metabolites in endothelium-dependent hyperpolarization by acetylcholine in rat isolated mesenteric artery.

1. In rat mesenteric artery, acetylcholine (ACh) causes endothelium-dependent hyperpolarization by releasing endothelium-derived hyperpolarizing factor (EDHF). Recent evidence suggests that EDHF may be a cytochrome P450-derived arachidonic acid metabolite. The aim of the present study was to investigate whether such a metabolite is indeed contributing to ACh-induced hyperpolarization observed in rat mesenteric artery. 2. The phospholipase A2 inhibitor quinacrine (30 microM) nearly completely eliminated ACh-induced hyperpolarization. However, the hyperpolarizing effect of pinacidil was also abolished in the presence of quinacrine. 3. The imidazole antimycotic agents ketoconazole (50 microM), clotrimazole (30 microM) and miconazole (10 microM), which bind to the heme moiety of cytochrome P450, eliminated not only ACh-induced hyperpolarizations but also those induced by pinacidil. SKF525A (30 microM), a prototype inhibitor of the enzyme, also abolished the hyperpolarizing responses to both agents. In contrast, neither 17-octadecynoic acid (10 microM), a mechanism-based inhibitor of cytochrome P450 metabolism of fatty acids, nor eicosatetraynoic acid (20 microM), an inhibitor of all arachidonic acid metabolic pathways, altered ACh-induced hyperpolarization. Furthermore, the hyperpolarization was unaffected by the preferential inhibitors of specific cytochrome P450 isozymes, alpha-naphtoflavone (1 microM), diedthyldithiocarbamate (50 microM), metyrapone (20 microM) and troleandomycin (10 microM). 4. Pretreatment of rats with lipopolysaccharide (2 mg kg-1) and exposure to nitroprusside (10 microM), both of which are expected to inhibit cytochrome P450 activity due to nitric oxide overproduction, were without effect on ACh-induced hyperpolarization. Pretreatment of rats for 3 days with pentobarbitone (80 mg kg-1 day-1), a cytochrome P450 inducer, also did not affect the hyperpolarizing response to ACh. 5. Arachidonic acid in concentrations up to 100 microM had no detectable effect on smooth muscle membrane potential. 11, 12-Epoxyeicosatrienoic acid (EET, 10 microM), one of cytochrome P450-derived epoxygenase metabolites of arachidonic acid, elicited a small endothelium-independent membrane hyperpolarization. The hyperpolarizing response to EET was blocked by glibenclamide (30 microM), in contrast to the response to ACh. 6. These results suggest that the contribution of a cytochrome P450-derived metabolite of arachidonic acid to ACh-induced hyperpolarization via EDHF release is minimal or absent in rat mesenteric artery.

Diminishment of contractions associated with depolarization-evoked activation of Ca2+ channels in diabetic rat aorta.

Alterations in contractile responses of aortic rings mediated by activation of Ca2+ channels were investigated in rats with streptozotocin-induced diabetes. Eight to 12 weeks of diabetes resulted in a marked decrease in the contractile response of aortic rings to high K+. In contrast, diabetic aortas exhibited significantly greater contractions in response to noradrenaline compared to age-matched controls. In the presence of 15 mM K+, the Ca2+ channel agonist Bay K 8644 consistently produced concentration-dependent contractions in control aortas. On the other hand, the half of diabetic aortas did not respond to Bay K 8644. As a result, the contractile response to Bay K 8644 was significantly less in diabetic aortas compared to controls. There was no significant difference in basal 45Ca2+ uptake between control and diabetic aortas. However, the uptake of 45Ca2+ induced by high K+ was significantly less in diabetic aortas than in controls, and pretreatment with the Ca2+ channel antagonist nifedipine abolished both responses. The resting membrane potentials were not significantly different between control and diabetic aortas. Furthermore, no difference was found in the magnitude of depolarization evoked by increasing K+ concentrations between the two groups of tissues. There were no significant differences in the density and the dissociation constant for [3H]-(+)-PN200-110, a radiolabeled Ca2+ channel antagonist, between aortic membranes from control and diabetic rats. These data indicate that the contractile responses to high K+ and Bay K 8644 are specifically diminished in diabetic aortas. These diminished responses are not due to a difference in the number of Ca2+ channels, but may be related to an alteration in activation of the channels by membrane depolarization.