Biomedical subjects

T Kitazawa

Publications and source records attributed to T Kitazawa.

At least 19 recordsLinked to original sources

PubMed2000

Agonists trigger G protein-mediated activation of the CPI-17 inhibitor phosphoprotein of myosin light chain phosphatase to enhance vascular smooth muscle contractility.

Myosin light chain phosphatase (MLCP) plays a pivotal role in smooth muscle contraction by regulating Ca(2+) sensitivity of myosin light chain phosphorylation. A smooth muscle phosphoprotein called CPI-17 specifically and potently inhibits MLCP in vitro and in situ and is activated when phosphorylated at Thr-38, which increases its inhibitory potency 1000-fold. We produced a phosphospecific antibody for this site in CPI-17 and used it to study in situ phosphorylation of endogenous CPI-17 in arterial smooth muscle in response to agonist stimulation. In the intact femoral artery, CPI-17 phosphorylation was negligible at the resting state and was not increased during contraction induced by K(+) depolarization. The Ca(2+)-sensitizing agonists histamine and phenylephrine induced nearly equivalent contractions, but histamine generated significantly higher levels of CPI-17 phosphorylation. In alpha-toxin-permeabilized strips at pCa 6.7, contractile force and CPI-17 phosphorylation were proportional in response to histamine, guanosine 5'-O-(gamma-thiotriphosphate), and histamine plus guanyl-5'-yl thiophosphate, implying that histamine increased CPI-17 phosphorylation through activation of G proteins. Inhibitors of Rho-kinase (Y27632) and protein kinase C (PKC; GF109203X) reduced contraction and CPI-17 phosphorylation in parallel, suggesting that CPI-17 functions downstream of Rho kinases and PKC. The results show that agonists such as histamine signal through phosphorylation of CPI-17 to produce Ca(2+) sensitization of smooth muscle contraction.

Amides↗

PubMed2000

The mechanisms of alpha(2)-adrenoceptor agonist-induced contraction in longitudinal muscle of the porcine uterus.

The aim of the present study was to clarify the cellular mechanisms underlying the alpha(2)-adrenoceptor-mediated contraction of porcine myometrium (nonvascular smooth muscle). Acetylcholine (3 nM-1 microM), clonidine (1 nM-10 microM) and 5-bromo-N-[2-imidazolin-2-yl]-6-quinoxalinamine (UK14304) (1 nM-10 microM) in Krebs solution caused a concentration-dependent contraction in the longitudinal muscles of the porcine uterus with similar EC(50) values and maximum responses. A lowered external Ca(2+) concentration and verapamil (10 nM-10 microM) decreased the contractile response to clonidine and UK14304 more markedly than the response to acetylcholine. However, in Kumagai solution, neither clonidine nor UK14304 caused contractile responses, but acetylcholine remained effective. The effects of alpha(2)-adrenoceptor agonists on intracellular Ca(2+) concentration ([Ca(2+)](i)) and smooth muscle force were measured simultaneously using fura-PE3-loaded muscle preparations. Clonidine and UK14304 caused increases in [Ca(2+)](i) and force of the longitudinal muscle. The increases in [Ca(2+)](i) and muscle force were markedly inhibited by verapamil and in Ca(2+)-free solution (EGTA, 1 mM). In the absence of external Ca(2+), clonidine caused only a small increase in [Ca(2+)](i) in Ca(2+)-loaded preparations compared with those increases caused by carbachol, histamine, and oxytocin. Ca(2+) (2.5 mM) caused increases in [Ca(2+)](i) and force of the longitudinal muscles in a Ca(2+)-free high K(+) solution. Clonidine concentration dependently potentiated the Ca(2+)-induced contraction without significantly changing the increase in [Ca(2+)](i), and this potentiation was inhibited by yohimbine. These results suggested that clonidine increases the Ca(2+) sensitivity of the contractile elements through activation of alpha(2)-adrenoceptors. During the development of the contractile response to clonidine (1 microM, 0-5 min), tissue cyclic AMP levels did not change significantly. In vitro treatment with pertussis toxin (1 microg/ml for 2 h) significantly decreased the contraction induced by clonidine without affecting the responses to carbachol and high K(+). The present results indicate that in porcine myometrium, alpha(2)-adrenoceptor stimulation caused contraction of the longitudinal muscles by mechanisms largely dependent on the influx of extracellular Ca(2+), probably through voltage-dependent Ca(2+) channels (VDCCs), and that the potentiation of the Ca(2+) sensitivity of the contractile elements is another mechanism of the contractile responses. These actions involve a pertussis-toxin-sensitive G protein (probably G(i) type) in the signal transduction pathway.

Adrenergic alpha-2 Receptor Agonists↗

PubMed2000

Characterization of functional endothelin receptors in the porcine myometrium.

To characterize the endothelin (ET) receptor that mediates the contraction induced by ET-1 in the porcine myometrium, we carried out a contraction study, radioligand binding study and molecular study (reverse transcription polymerase chain reaction) for detection of ET receptor-coding genes (mRNA). ET-1 (1 nM-1 microM) caused a tetrodotoxin-insensitive contraction in both longitudinal and circular muscles, but the longitudinal muscle was more sensitive to ET-1 than was the circular muscle. On the other hand, ET-3 and sarafotoxin S6c were less effective to cause a contractile response. The contraction induced by ET-1 was markedly inhibited by BQ123 and FR139317, but BQ788 only slightly inhibited the response induced by ET-1. The radioligand binding study indicated the presence of a single class of 125I-ET-1 binding sites with the same Kd value in both muscle layers. However, Bmax in the longitudinal muscle (3252 fmol/mg protein) was significantly higher than that in the circular muscle (1883 fmol/mg protein). ET-1 and FR139317 inhibited the specific 125I-ET-1 binding completely, but ET-3, sarafotoxin S6c and BQ3020 only slightly inhibited the specific binding (inhibition, 10-20%), suggesting that ET(A) is the dominant ET receptor subtype in the porcine myometrium. The results of the molecular study indicated the expression of both ET(A) and ET(B) receptor-coding genes in the porcine myometrium. In conclusion, ET-1 causes contraction of the porcine myometrium through activation of the ET(A) receptor present on smooth muscle cells. There is a marked muscle layer-related difference (longitudinal muscle > circular muscle) in the ET-1-induced contraction and the ET(A) receptor concentration.

Animals↗

PubMed2000

Smooth muscle layer-dependent distribution of 5-hydroxytryptamine(7) receptor in the porcine myometrium.

1. To analyse the mechanisms of muscle layer-dependent inhibition of porcine myometrial contractility by 5-hydroxytryptamine (5-HT), the effects of 5-HT, 5-carboxamidotryptamine(5-CT), 5-methoxytryptamine (5-MeOT), forskolin and cyclic adenosine 3', 5'-monophosphate (cyclic AMP) analogues on spontaneous and stimulant-induced contractions were examined in longitudinal (LM) and circular muscles (CM). In addition, accumulation of cyclic AMP by 5-HT and distribution of 5-HT(7) receptors in LM and CM layers were compared using biochemical and molecular approaches. 2. 5-HT receptor agonists inhibited the spontaneous contractions of LM and CM (5-CT>5-HT>5-MeOT), but CM was more sensitive than was LM. The inhibition by the agonists was antagonized by methiothepin (100 nM). 3. Carbachol-, high-K(+)-, histamine- and Ca(2+)-induced contractions were inhibited by 5-HT with different responses (CM>LM). Even in the presence of 3-isobutyl-1-methylxanthine (IBMX), the inhibition by 5-HT in the CM was still more conspicuous than that in the LM. 4. Compared with the CM, the inhibition of spontaneous contraction by forskolin, dibutyryl-cyclic AMP and 8-bromo-cyclic AMP was marked in the LM. 5. 5-HT (1 nM - 1 microM) increased the cyclic AMP in both muscle layers, but the increment in the CM was higher than that in the LM whether IBMX was present or not. 6. LM and CM layers contained a single class of [(3)H]-5-CT binding sites with a similar K(d) value (0.21 - 0.24 nM). However, B(max) (5-HT(7) receptor concentration) in the CM (120.6 fmol mg(-1) protein) was higher than that in the LM (30.4 fmol mg(-1) protein). 7. The molecular study (reverse transcription polymerase chain reaction) demonstrated the expression of 5-HT(7) receptor mRNA in the CM was higher than that in the LM. 8. These results suggest that the muscle layer-dependent difference in inhibition by 5-HT is not restricted to spontaneous contraction but applies to various contractions in the porcine myometrium. Different inhibition of the contractility by 5-HT is caused by muscle layer-related accumulation of cyclic AMP (CM>LM), due to smooth muscle-layer dependent distribution (CM>LM) of 5-HT(7) receptors.

5-Methoxytryptamine↗

PubMed2000

Studies of delayed systemic effects of ultraviolet B radiation (UVR) on the induction of contact hypersensitivity, 2. Evidence that interleukin-10 from UVR-treated epidermis is the critical mediator.

Acute, low-dose ultraviolet B radiation (UVR) alters cutaneous immunity at the local site as well as systemically. Within 2-3 days of UVR exposure, recipient mice lose their capacity to develop contact hypersensitivity (CH) when hapten is painted on unexposed skin. This loss correlates temporally with a functional deficit among dendritic antigen-presenting cells within non-draining lymph nodes and spleen. In the experiments described, the delayed systemic immune deficiency following acute, low-dose UVR exposure was found to be eliminated with neutralizing anti-interleukin-10 (IL-10) antibody. Intracutaneous injection of IL-10 generated a deficiency of systemic immunity as well as a functional deficit among lymph node dendritic cells that was similar to that induced by UVR. The skin itself was found to be the source of the IL-10 responsible for these defects, and epidermis (presumably keratinocytes) rather than mast cells was found to be the source of IL-10 within UVR-exposed skin. The potential relationships are discussed between the delayed systemic immune deficit created by acute, low-dose UVR, and the systemic immune deficits caused by chronic, high-dose UVR and by a single, high-dose UVR exposure.

Animals↗

PubMed2000

Studies on delayed systemic effects of ultraviolet B radiation on the induction of contact hypersensitivity, 3. Dendritic cells from secondary lymphoid organs are deficient in interleukin-12 production and capacity to promote activation and differentiation of T helper type 1 cells.

Ultraviolet-B radiation (UVR) of mouse skin promotes both local and systemic immune aberrations that are thought to be important in the pathogenesis of cutaneous malignancies. Acute, low-dose UVR regimens inhibit the induction of contact hypersensitivity (CH) in genetically susceptible mice by TNF-alpha-dependent mechanisms. In addition, these regimens also promote the development of tolerance when hapten is applied to the UVR-exposed site at the completion of the radiation treatment protocol. A third immune abnormality is also observed in mice exposed to acute, low-dose UVR. This abnormality, which develops within 48-72 hr of the completion of the UVR regimen, has been described among antigen-presenting cells within secondary lymphoid organs, including lymph nodes that do not drain the site of irradiation. Dendritic cells (DCs) from lymph nodes and spleens of mice exposed to UVR lack the capacity to induce CH if they are derivatized with hapten and injected intracutaneously into naive mice. The DC defect is related to the production of and systemic dissemination of interleukin-10 (IL-10) by keratinocytes within the epidermis of the UVR-exposed skin. We have now examined the nature of the functional aberration that exists among DCs within the secondary lymphoid organs of UVR-exposed mice by examining the capacity of DCs to express co-stimulatory molecules, and their ability to activate ovalbumin (OVA) -specific DO11.10 T-cell receptor transgenic T cells in vitro. Our results indicate that DCs from UVR-exposed mice produced insufficient amounts of IL-12. When pulsed with OVA, these cells were capable of inducing proliferation among DO11.10 T cells in vitro, but the responding cells produced neither IFN-gamma nor IL-10 and IL-4. A similar antigen-presenting cell defect was generated in mice treated with a subcutaneous injection of IL-10. We conclude that acute, low-dose UVR creates an IL-10-dependent functional deficit in DCs in secondary lymphoid organs, and that this defect robs UVR-exposed mice of the capacity to develop CH when hapten is painted epicutaneously.

Animals↗

PubMed2000

In-vivo and in-vitro evidence of a carrier-mediated efflux transport system for oestrone-3-sulphate across the blood-cerebrospinal fluid barrier.

The efflux transport of oestrone-3-sulphate, a steroid hormone sulphate, across the blood-cerebrospinal fluid barrier has been examined following its intracerebroventricular administration. [3H]Oestrone-3-sulphate was eliminated from cerebrospinal fluid (CSF) with an apparent efflux clearance of 205 microL min(-1) per rat. There was 25% of unmetabolized [3H]oestrone-3-sulphate in the plasma 5 min after intracerebroventricular administration, indicating that at least a part of [3H]oestrone-3-sulphate is transported from CSF to the circulating blood across the blood-CSF barrier. This efflux transport was inhibited by co-administration of excess oestrone-3-sulphate (25 mM 10 microL = 0.25 micromol) into rat cerebral ventricle. To characterize the oestrone-3-sulphate transport process, an in-vitro uptake experiment was performed using isolated rat choroid plexus. Oestrone-3-sulphate uptake by isolated rat choroid plexus was found to be a saturable process with a Michaelis-Menten constant (Km) of 18.1 +/- 6.3 microM, and a maximum uptake rate (Vmax) of 48.0 +/- 15.1 pmol min(-1) microL(-1) of tissue. The oestrone-3-sulphate transport process was temperature dependent and was inhibited by metabolic inhibitors such as 2,4-dinitrophenol and rotenone, suggesting an energy dependence. This uptake process was also inhibited by steroid hormone sulphates (1 mM dehydroepiandrosterone sulphate and 1 mM oestrone sulphate), bile acids (1 mM taurocholic acid and 1 mM cholic acid) and organic anions (1 mM sulphobromophthalein and 1 mM phenolsulphonphthalein), whereas 1 mM p-aminohippuric acid, 1 mM p-nitrophenol sulphate, 0.1 mM methotrexate and the cardiac glycoside, 2.5 microM digoxin, had little effect. In conclusion, these results provide evidence that oestrone-3-sulphate is transported from CSF to the circulating blood across the blood-CSF barrier via a carrier-mediated efflux transport system.

2,4-Dinitrophenol↗

PubMed1999

Reconstitution of protein kinase C-induced contractile Ca2+ sensitization in triton X-100-demembranated rabbit arterial smooth muscle.

1. Triton X-100-demembranated smooth muscle loses Ca2+-sensitizing responsiveness to protein kinase C (PKC) activators while intact and alpha-toxin-permeabilized smooth muscles remain responsive. We attempted to reconstitute the contractile Ca2+ sensitization by PKC in the demembranated preparations. 2. Western blot analyses showed that the content of the PKC alpha-isoform (PKCalpha) was markedly reduced and that the smooth muscle-specific protein phosphatase-1 inhibitor protein CPI-17 was not detectable, while the amount of calponin and actin still remained similar to those of intact strips. 3. Unphosphorylated recombinant CPI-17 alone induced a small but significant contraction at constant Ca2+. Isoform-selective PKC inhibitors inhibited unphosphorylated but not pre-thiophosphorylated CPI-17-induced contraction, suggesting that in situ conventional PKC isoform(s) can phosphorylate CPI-17. 4. Exogenously replenishing PKCalpha alone did not induce potentiation of contraction and only slowly increased myosin light chain (MLC) phosphorylation at submaximal Ca2+. 5. PKC in the presence of CPI-17, but not the [T38A]-CPI mutant, markedly induced potentiation of both contraction and MLC phosphorylation. CPI-17 itself was phosphorylated. 6. In in vitro experiments, CPI-17 was a much better substrate for PKCalpha than calponin, caldesmon, MLC and myosin. 7. Our results indicate that PKC requires CPI-17 phosphorylation at Thr-38 but not calponin for reconstitution of the contractile Ca2+ sensitization in the demembranated arterial smooth muscle.

Animals↗

PubMed1999

Characterization of the muscarinic receptor subtype that mediates the contractile response of acetylcholine in the swine myometrium.

The aim of the present study was to characterize the subtype of muscarinic receptor that mediates acetylcholine-induced contractions in the nonpregnant proestrus swine myometrium by means of mechanical, radioligand ([3H]quinuclidinyl benzilate) binding and biochemical (measurement of cyclic AMP) approaches. Acetylcholine (-logEC50, 6.12), oxotremorine-methiodide (6.47), methacholine (6.35), carbachol (6.18) and muscarine (6.33) caused contractile responses of the uterine circular muscle, with a similar maximum amplitude, but pilocarpine and McN-A-343 (4-(m-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium) were ineffective in causing contraction. The contractile response to acetylcholine was antagonized by the following muscarinic receptor antagonists in a competitive manner (with pA2 values in parentheses): atropine (8.95), 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, 8.83), tropicamide (7.07), himbacine (7.01), pirenzepine (6.42) and 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyri do[2,3 b][1,4]benzodiazepin-6-one (AF-DX116, 5.96). Electrical field stimulation (10 Hz) caused tetrodotoxin- and atropine-sensitive contractions in the circular muscle. All muscarinic receptor antagonists decreased the electrical field stimulation-induced contraction in a concentration-dependent manner. The order of inhibition (-logIC50) was 4-DAMP (8.35) > tropicamide (6.72) > himbacine (6.54) > pirenzepine (6.31)> AF-DX116 (6.13). Acetylcholine did not affect the cytoplasmic cyclic AMP level, regardless of the presence or absence of forskolin, suggesting the absence of functional muscarinic M2 and/or M4 receptors in the swine myometrium. The receptor binding study indicated that circular muscle layers of the swine myometrium contained a single class of [3H]quinuclidinyl benzilate binding site (Kd = 0.92 nM; Bmax = 126.6 fmol/mg protein). Specific binding was displaced by muscarinic receptor antagonists in the following order (with pKi value and Hill coefficient in parentheses): atropine (8.22 and 0.93) > 4-DAMP (8.18 and 0.94) > tropicamide (6.78 and 0.93) > pirenzepine (5.46 and 0.92) > AF-DX116 (5.12 and 0.94). The present results suggest that in circular muscle layers of the swine myometrium, exogenous and endogenous acetylcholine cause contraction through activation of muscarinic M3 receptors present on smooth muscle cells.

Acetylcholine↗

PubMed1999

Smooth muscle layer-specific variations in the autonomic innervation of bovine myometrium.

1. To clarify the autonomic innervation regulating longitudinal muscle (LM) and circular muscle (CM) motility in the bovine uterus, functional (nerve stimulation, adrenergic drug responsiveness) and biochemical studies (catecholamine content, radioligand binding) were conducted on parous luteal-phase myometrium. 2. Electrical field stimulation (EFS; 60 V, 0.5-msec duration) caused tetrodotoxin (1 microM)-sensitive contractions in a frequency-dependent manner (0.5-20 Hz) in both LM and CM layers. 3. The EFS-induced LM contractions were potentiated by propranolol and conspicuously decreased by phentolamine, yohimbine, idazoxan or guanethidine, but were unaffected by prazosin or atropine. 4. On the other hand, CM contractions were only slightly decreased by phentolamine, idazoxan, yohimbine and guanethidine, but were insensitive to propranolol, prazosin or atropine. 5. The noradrenaline content in LM was about five times higher than that in CM. 6. Noradrenaline, adrenaline, clonidine, xylazine, UK14,304 and phenylephrine caused concentration-dependent contractions of both smooth muscle layers. 7. Clonidine, UK14,304 and xylazine were more potent contractile agents than noradrenaline and phenylephrine. 8. The contractile response to noradrenaline was competitively antagonized by yohimbine, but not by prazosin. 9. Binding studies using [3H]-prazosin and [3H]-rauwolscine revealed that the bovine myometrium contained both alpha1- and alpha2-adrenoceptors, but the alpha2-type receptor was dominant in both LM (94% of alpha-adrenoceptors) and CM (88%) layers. 10. The distribution of alpha-adrenoceptors was muscle layer-specific; that is, the concentration of alpha1-receptors in LM was the same as in CM, but the concentration of alpha2-receptors in LM was 2.6 times higher than that in CM. 11. The results of the present study indicate that there are layer-specific variations in the functional innervation of the parous bovine myometrium (exclusive adrenergic innervation in LM and adrenergic [minor] plus nonadrenergic, noncholinergic innervation [major] in CM), and that alpha2-adrenoceptors, which were responsive to the excitatory response of endogenous and exogenous noradrenaline, were dominant in both muscle layers of the bovine myometrium.

Adrenergic alpha-Agonists↗

PubMed1999

Inflammatory cell-mediated tumour progression and minisatellite mutation correlate with the decrease of antioxidative enzymes in murine fibrosarcoma cells.

We isolated six clones of weakly tumorigenic fibrosarcoma (QR) from the tumorigenic clone BMT-11 cl-9. The QR clones were unable to grow in normal C57BL/6 mice when injected s.c. (1x10(5) cells). However, they formed aggressive tumours upon co-implantation with a 'foreign body', i.e. a gelatin sponge, and the rate of tumour take ranged from 8% to 58% among QR clones. The enhanced tumorigenicity was due to host cell-mediated reaction to the gelatin sponge (inflammation). Immunoblot analysis and enzyme activity assay revealed a significant inverse correlation between the frequencies of tumour formation by QR clones and the levels of manganese superoxide dismutase (Mn-SOD, P<0.005) and glutathione peroxidase (GPchi, P<0.01) in the respective tumour clones. Electron spin resonance (ESR) revealed that superoxide-scavenging ability of cell lysates of the QR clone with high level of Mn-SOD was significantly higher than that with low level of the antioxidative enzyme in the presence of potassium cyanide, an inhibitor for copper-zinc superoxide dismutase (CuZn-SOD) (P<0.001). Minisatellite mutation (MSM) induced by the inflammatory cells in tumour cells were investigated by DNA fingerprint analysis after QR clones had been co-cultured with gelatin-sponge-reactive cells. The MSM rate was significantly higher in the subclones with low levels of Mn-SOD and GPchi (P<0.05) than in the subclones with high levels of both enzymes. The MSM of the subclones with low levels of both enzymes was inhibited in the presence of mannitol, a hydroxyl radical scavenger. The content of 8-hydroxydeoxyguanosine (8-OHdG) by which the cellular DNA damage caused by active oxygen species can be assessed was significantly low in the tumours arising from the QR clone with high levels of Mn-SOD and GPchi even if the clone had been co-implanted with gelatin sponge, compared with the arising tumour from the QR clone with low levels of those antioxidative enzymes (P<0.001). In contrast, CuZn-SOD and catalase levels in the six QR clones did not have any correlation with tumour progression parameters. These results suggest that tumour progression is accelerated by inflammation-induced active oxygen species particularly accompanied with declined levels of intracellular antioxidative enzymes in tumour cells.

Animals↗

PubMed1999

Mechanisms of 5-hydroxytryptamine-induced inhibition in the porcine myometrium.

The present experiments were designed to clarify the mechanisms of the inhibitory response of 5-hydroxytryptamine (5-HT) in the porcine uterine circular muscle. Inhibitory responses induced by 5-HT (1 nM-1 microM) were not affected by apamin (1 microM), charybdotoxin (100 nM) or glibenclamide (20 microM) but were significantly attenuated by 4-aminopyridine (3 mM) and tetraethylammonium (3 mM). Imidazole (100 microM) decreased but 3-isobutyl-1-methylxanthine (30 microM), milrinone (30 microM) and Ro 20-1724 (10 and 30 microM) potentiated the 5-HT-induced inhibition. On the other hand, zaprinast (3-30 microM) had no significant effect on the inhibitory response of 5-HT. 5-HT caused a time (0-5 min)-and concentration (1 nM-1 microM)-dependent increase in the tissue cyclic AMP level, but had no effect on the tissue cyclic GMP level. A significant correlation (P < 0.05) was observed between the inhibition of contraction and tissue cyclic AMP level. The effect of 5-HT on contractile force and cytosolic Ca2+ level ([Ca2+]i) was investigated using fura-PE3-loaded myometrial strips. A low concentration of 5-HT (< or = 10 nM) inhibited the spontaneous contraction without changing the amplitude of the spontaneous [Ca2+]i increase, but a higher concentration of 5-HT (> or = 100 nM) decreased the resting [Ca2+]i and inhibited both the spontaneous [Ca2+]i increase and spontaneous contraction. High-K+ (50 mM) caused increases in muscle contractile force and [Ca2+]i. 5-HT concentrationdependently inhibited the high-K(+)-induced contraction (EC50, 45 nM) with only a small decrease in [Ca2+]i increase. Carbachol also caused increases in muscle contractile force and [Ca2+]i. 5-HT significantly decreased both the carbachol-induced contraction and [Ca2+]i increase, but was more potent at inhibition of contractile force than [Ca2+]i. In Ca(2+)-loaded myometrial strips, carbachol, but not caffeine, caused a transient increase in [Ca2+]i and contraction in the absence of external Ca2+ (EGTA, 1 mM). 5-HT inhibited both the carbachol-induced increases in [Ca2+]i release and contractile force. In the beta-escin permeabilized myometrium, 5-HT significantly inhibited the Ca(2+)-induced contraction. The present results indicate that 5-HT stimulates tissue cyclic AMP production, and inhibits the porcine uterine muscle contractility by a reduction in [Ca2+]i and in Ca2+ sensitivity of the contractile elements. Activation of K+ channels might be partially involved in 5-HT-induced inhibition of the myometrial contractility.

Animals↗

PubMed1999

Bipedicled submental island flap for upper lip reconstruction.

A case of reconstruction of the upper lip with a bipedicled submental flap is described. Although the mobility of the flap was restricted, the arterial blood supply was abundant by altering the single-pedicled flap into a bipedicled flap. This flap is a good color and texture match to the face, and surpasses other flaps in reconstruction of the mustache or beard in male patients.

Humans↗

PubMed1999

Chondroid syringoma of the orbit.

Chondroid syringoma is a rare subcutaneous tumor that mainly affects the face and the neck. The authors present an occurrence in the orbit, which they believe to be the first reported at that site. Computed tomography and magnetic resonance imaging suggested that the tumor had invaded the globe, but at surgery the mass was well circumscribed and did not invade or adhere to other structures. The tumor was resected easily under general anesthesia. Epithelial cells of characteristic appearance within a chondroid matrix were demonstrated histologically. No recurrence has been evident in more than 1 year of follow-up. Most chondroid syringomas are benign, but because several cases have exhibited malignant behavior, periodic observation following complete resection is required.

Adenoma, Pleomorphic↗

PubMed1999

Ca2+ -induced Ca2+ desensitization of myosin light chain phosphorylation and contraction in phasic smooth muscle.

The temporal relationship between Ca2+ -induced contraction and phosphorylation of 20 kDa myosin light chain (MLC) during a step increase in Ca2+ was investigated using permeabilized phasic smooth muscle from rabbit portal vein and guinea-pig ileum at 25 degrees C. We describe here a Ca2+ -induced Ca2+ desensitization phenomenon in which a transient rise in MLC phosphorylation is followed by a transient rise in contractile force. During and after the peak contraction, the force to phosphorylation ratio remained constant. Further treatment with cytochalasin D, an actin fragmenting agent, did not affect the transient increase in phosphorylation, but blocked force development. Together, these results indicate that the transient phosphorylation causes the transient contraction and that neither inhomogeneous contractility nor reduced thin filament integrity effects the transient phosphorylation. Lastly, we show that known inhibitors to MLC kinase kinases and to a Ca2+ -dependent protein phosphatase did not eliminate the desensitized contractile force. This study suggests that the Ca2+ -induced Ca2+ desensitization phenomenon in phasic smooth muscle does not result from any of the known intrinsic mechanisms involved with other aspects of smooth muscle contractility.

Animals↗

PubMed1999

Transepithelial transport of organic anions across the choroid plexus: possible involvement of organic anion transporter and multidrug resistance-associated protein.

Transport characteristics of 17beta-estradiol 17beta-D-glucuronide (E217betaG), a dual substrate of the transporters for cellular uptake (organic anion-transporting polypeptide 1 or oatp1) and cellular excretion (multidrug resistance-associated protein 1or MRP1), in the rat choroid plexus were studied in vivo and in vitro. The uptake of E217betaG into isolated choroid plexus was mediated by an energy-dependent system with a Km of 3.4 microM. Together with the previous finding that oatp1 is localized on the apical membrane of choroid plexus, these results suggest that oatp1 is responsible for the uptake of this ligand. After intracerebroventricular administration, elimination of E217betaG from cerebrospinal fluid was probenecid sensitive and much more rapid than that of inulin; less than 2% of the administered E217betaG and 40 to 50% of inulin remained in the cerebrospinal fluid 20 min after intracerebroventricular administration. In addition, the amount of E217betaG associated with choroid plexus at 20 min was negligible, suggesting the presence of an efficient excretion system on the basolateral membrane of choroid plexus. Expression of MRP1 was detected in choroid plexus. Semiquantitative reverse transcription-polymerase chain reaction and Western blot analyses indicated that the expression level of MRP1 in choroid plexus is about four or five times higher than that in the lung, one of the tissues exhibiting high expression of MRP1. Together with the in vivo vectorial transport of E217betaG, these results can be accounted for by assuming that there is basolateral localization of MRP1 in choroid plexus. Combined, oatp1 and MRP1 may synergistically mediate the efficient transcellular transport of E217betaG across choroid plexus.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

Possible involvement of the novel CPI-17 protein in protein kinase C signal transduction of rabbit arterial smooth muscle.

1. CPI-17 has recently been identified as a novel protein in vascular smooth muscle. In vitro , its phosphorylation and thiophosphorylation by protein kinase C (PKC) specifically inhibits the type 1 class of protein phosphatases, including myosin light chain (MLC) phosphatase. 2. Both of the phosphorylated CPI-17 states dose-dependently potentiated submaximal contractions at constant [Ca2+] in beta-escin-permeabilized and Triton X-100-demembranated arterial smooth muscle, but produced no effect in intact and less intensely permeabilized (alpha-toxin) tissue. Thiophosphorylated CPI-17 (tp-CPI) induced large contractions even under Ca2+-free conditions and decreased Ca2+ EC50 by more than an order of magnitude. Unphosphorylated CPI-17 produced minimal but significant effects. 3. tp-CPI substantially increased the steady-state MLC phosphorylation to Ca2+ ratios in beta-escin preparations. 4. tp-CPI affected the kinetics of contraction and relaxation and of MLC phosphorylation and dephosphorylation in such a manner that indicates its major physiological effect is to inhibit MLC phosphatase. 5. Results from use of specific inhibitors in concurrence with tp-CPI repudiate the involvement of general G proteins, rho A or PKC itself in the Ca2+ sensitization by tp-CPI. 6. Our results indicate that phosphorylation of CPI-17 by PKC stimulates binding of CPI-17 to and subsequent inhibition of MLC phosphatase. This implies that CPI-17 accounts largely for the heretofore unknown signalling pathway between PKC and inhibited MLC phosphatase.

Animals↗

PubMed1998

Involvement of 5-hydroxytryptamine7 receptors in inhibition of porcine myometrial contractility by 5-hydroxytryptamine.

1 5-Hydroxytryptamine (5-HT; 1 nM - 100 microM) concentration-dependently inhibited the amplitude and frequency of spontaneous contractions in longitudinal and circular muscles of the porcine myometrium. The circular muscle (EC50; 68-84 nM) was more sensitive than the longitudinal muscle (EC50; 1.3-1.44 microM) to 5-HT. To characterize the 5-HT receptor subtype responsible for inhibition of myometrial contractility, the effects of 5-HT receptor agonists on spontaneous contractions and of 5-HT receptor antagonists on inhibition by 5-HT were examined in circular muscle preparations. 2 Pretreatment with tetrodotoxin (1 microM), propranolol (1 microM), atropine (1 microM), guanethidine (10 microM) or L-NAME (100 microM) failed to change the inhibition by 5-HT, indicating that the inhibition was due to a direct action of 5-HT on the smooth muscle cells. 3 5-CT, 5-MeOT and 8-OH-DPAT mimicked the inhibitory response of 5-HT, and the rank order of the potency was 5-CT>5-HT>5-MeOT>8-OH-DPAT. On the other hand, oxymethazoline, alpha-methyl-5-HT, 2-methyl-5-HT, cisapride, BIMU-1, BIMU-8, ergotamine and dihydroergotamine had almost no effect on spontaneous contractions, even at 10-100 microM. 4 Inhibition by 5-HT was not decreased by either pindolol (1 microM), ketanserin (1 microM), tropisetron (10 microM), MDL72222 (1 microM) or GR113808 (10 microM), but was antagonized by the following compounds in a competitive manner (with pA2 values in parentheses): methiothepin (8.05), methysergide (7.92), metergoline (7.4), mianserin (7.08), clozapine (7.06) and spiperone (6.86). 5 Ro 20-1724 (20 microM) and rolipram (10 microM) significantly enhanced the inhibitory response of 5-HT, but neither zaprinast (10 microM) nor dipyridamole (10 microM) altered the response of 5-HT. 6 5-HT (1 nM - 1 microM) caused a concentration-dependent accumulation of intracellular cyclic AMP in the circular muscle. 7 From the present results, the 5-HT receptor, which is functionally correlated with the 5-HT7 receptor, mediates the inhibitory effect of 5-HT on porcine myometrial contractility. This inhibitory response is probably due to an increase in intracellular cyclic AMP through the activation of adenylate cyclase that is positively coupled to 5-HT7 receptors.

Adenylyl Cyclases↗