Agonist and antagonist sensitivity of non-selective cation channel currents evoked by muscarinic receptor stimulation in bovine ciliary muscle cells.

1 In the bovine ciliary muscle, stimulation of muscarinic receptors with carbachol (CCh) opens two types of non-selective cation channels (NSCCS and NSCCL) with widely different unitary conductances (100 fS and 35 pS). Here we examined the dependence of the activity of NSCCS on the agonist (CCh) concentration by whole-cell voltage clamp in freshly isolated bovine ciliary muscle cells. We also examined the sensitivity of CCh-evoked NSCCS currents to several muscarinic receptor antagonists. 2 The voltage clamp experiments were carried out using Ba2+ as the charge carrier, as this divalent cation is the most permeant for NSCCS of the alkali and alkaline earth metal ions hitherto examined, whereas it is relatively impermeant to NSCCL. For the dose-activation relationship obtained, the apparent dissociation constant K was estimated to be 0.5 +/- 0.2 microm (n = 31), a value of an order of magnitude smaller than the one reported for CCh-evoked NSCCL currents in our previous experiments. 3 In the dose-inhibition experiments we observed that the CCh-evoked NSCCS currents were inhibited by the muscarinic antagonists with the following potency sequence: atropine approximately 4-DAMP >> pirenzepine > AF-DX116, indicating that the activation of NSCCS by CCh is mediated by an M3 muscarinic receptor. 4 We have previously shown by reverse transcriptase-polymerase chain reaction that the bovine ciliary muscle contains mRNAs for several transient receptor potential channel homologues (TRPC1, TRPC3, TRPC4 and TRPC6) which are attracting attention as molecular candidates for receptor-operated NSCCs. In the present experiments, we succeeded in visually identifying these TRPCs in the plasma membrane of cultured bovine ciliary muscle cells by immunofluorescence microscopy.

Choroidal blood flow in the foveal region in eyes with rhegmatogenous retinal detachment and scleral buckling procedures.

AIMS: To investigate changes in choroidal blood flow (ChBF) in the foveal region of the human eye with rhegmatogenous retinal detachment induced by scleral buckling. METHODS: ChBF was measured in the foveal region using laser Doppler flowmetry in patients with a rhegmatogenous retinal detachment and no macular involvement before and after scleral buckling. The ChBF ratio was evaluated (ChBF of the affected eye to ChBF of the fellow control eye) to minimise individual variations. RESULTS: Retinal reattachment was confirmed by 2 weeks after scleral buckling in all patients. The ChBF in the foveal region of the affected eyes did not differ from the fellow eyes before scleral buckling. The ChBF ratio significantly (p<0.05) decreased 2 and 4 weeks after scleral buckling compared with that before scleral buckling and returned to baseline 12 weeks after scleral buckling. CONCLUSIONS: The results suggest that ChBF in the foveal region transiently decreases after scleral buckling and recovers to the baseline level within 12 weeks in patients with a retinal detachment and no macular involvement.

Peroxynitrite decomposition catalyst, FP15, and poly(ADP-ribose) polymerase inhibitor, PJ34, inhibit leukocyte entrapment in the retinal microcirculation of diabetic rats.

PURPOSE: Oxidative and nitrosative stress and activation of poly(ADP ribose) polymerase (PARP) play a role in the pathogenesis of diabetic complications. We evaluated the effectiveness of the peroxynitrite decomposition catalyst, FP15, and the PARP inhibitor, PJ34, in the treatment of leukocyte entrapment in the retinal microcirculation of diabetic rats. METHODS: Diabetes was induced in rats by intraperitoneal injection of 60 mg/kg of streptozotocin. Rats were divided into four groups: controls; untreated diabetes; diabetes treated with FP15 (10 mg/kg oral gavage twice daily) and diabetes treated with PJ34 (10 mg/kg oral gavage twice daily). All experiments were performed 4 weeks after initiation of treatment. Leukocyte entrapment in the retinal microcirculation was quantitatively evaluated in vivo with acridine orange digital fluorography. RESULTS: The density of leukocytes trapped in the retinal microcirculation 30 minutes after dye injection was significantly greater in untreated diabetes (32.1 +/- 4.7 cells/mm2) than in controls (11.3 +/- 4.5 cells/mm2) (p < 0.05). Compared with untreated diabetes, the density of trapped leukocytes significantly decreased in diabetes treated with FP15 (14.5 +/- 5.1 cells/mm2) (p < 0.0001) and diabetes treated with PJ34 (24.1 +/- 4.2 cells/mm2) (p < 0.05). CONCLUSIONS: Treatment with FP15 and PJ34 decreased enhanced leukocyte entrapment in the retinal microcirculation during the early diabetic period. The current study suggests a role for peroxynitrite production and for PARP activation in the pathogenesis of retinal microvascular leukostasis in early diabetes.

Alteration of choroidal circulation in the foveal region in patients with type 2 diabetes.

AIM: To investigate changes in choroidal blood flow (CBF) in the foveal region in patients with type 2 diabetes. METHODS: Laser Doppler flowmetry was used to determine the CBF in the foveal region in 70 patients with type 2 diabetes and 36 age and sex matched healthy subjects (control group). The patients were classified into three groups: 33 patients (33 eyes) with no diabetic retinopathy (NDR), 20 patients (20 eyes) with non-proliferative diabetic retinopathy and no macular oedema (NPDR/MO-), and 17 patients (17 eyes) with NPDR and MO (NPDR/MO+). Optical coherence tomography was also used to measure the foveal thickness. RESULTS: The group averaged CBF values were 13.5 (4.9), 9.4 (2.5), 10.8 (4.8), and 5.6 (2.0) (arbitrary units) in the control, NDR, NPDR/MO-, and NPDR/MO+ groups, respectively. The group averaged CBF values in the NDR group decreased (30.2%; p<0.01) compared with the control group. The average CBF value in the NPDR/MO+ group was also significantly lower (48.2%; p<0.01) compared with that in the NPDR/MO- group. CONCLUSION: The CBF in the foveal region significantly decreases in patients with diabetes, especially those with macular oedema.

Features of abnormal choroidal circulation in central serous chorioretinopathy.

AIMS: To evaluate abnormalities in the choroidal circulation in cases of central serous chorioretinopathy (CSC). METHODS: A complete clinical ophthalmological examination was performed using simultaneous fluorescein and indocyanine green (ICG) angiography with a confocal scanning laser ophthalmoscopy and the digital images analysed in 36 consecutive patients with acute CSC. To quantify the choroidal circulation, the foveal choroidal blood flow was measured in 11 patients using laser Doppler flowmetry. RESULTS: Fluorescein angiography showed focal leakage from the retinal pigment epithelium in all patients. ICG angiography revealed delays in arterial filling in 27 eyes (75%), and fluorescein angiography showed small hypofluorescent points around the leakage in 27 eyes (75%). Abnormal choroidal hyperfluorescence was observed in 30 eyes (83%). The choroidal blood flow in eyes with CSC was 45% lower than in fellow eyes (p<0.01). CONCLUSION: Decreased choroidal blood flow in CSC was demonstrated for the first time. The decreased choroidal blood flow might be correlated with the small, localised hypofluorescent areas, which may indicate non-perfused areas of the choriocapillaris that are frequently seen during ICG angiography.

Taste responses to the optical isomers of potassium warfarin and dipotassium glutamyl glutamate and the synergistic response to a mixture of both substances in mice.

The taste responses to the optical isomers of potassium warfarin, dipotassium glutamyl glutamate, and a mixture of both the substances in laboratory mice were studied using licking activity as an indicator of taste effectiveness. The offensive effectiveness of the optical isomers of potassium warfarin was the same. Every optical isomer of dipotassium glutamyl glutamate was avoided, the taste avoidance of dipotassium D-glutamyl D-glutamate being the strongest. The taste avoidance of potassium warfarin was greatly increased by the addition of every isomers of dipotassium glutamyl glutamate, and its cause was related to synergistic effects.

Structure-activity relationship between the hydrophobicity of alkali metal salts of warfarin [3-(alpha-acetonyl-benzyl)-4-hydroxycoumarin] and the effectiveness of the taste response to these salts in mice.

The taste responses to five alkali metal salts of warfarin, aqueous rodenticides, in laboratory mice were observed at a short measurement time of 20 min by the two-bottle preference test using a licking time monitoring system newly developed. It was made clear that the order of taste avoidance to the warfarin salts was as follows: cesium salt > rubidium salt > lithium salt > sodium salt > potassium salt. The relationship between the hydrophobicity of the warfarin salts and the effectiveness of the taste avoidance was analyzed. The effectiveness of the taste avoidance largely depended on the hydrophobicity.

Phase-only computer-generated hologram produced by an ion-exchange technique.

We have produced a highly efficient phase-only hologram, in which the refractive index of the substrate is modified by using an ion-exchange method. This binary phase-only hologram is produced using a conventional binary hologram as a mask for the ion exchange. A simplified theory of the phase-only hologram is presented along with a description of the fabrication method and the experimental test results.

Novel responses of peripheral lymphocytes of cancer patients to chemical induction of sister chromatid exchanges.

Sensitivities to sister chromatid exchange (SCE) induction by chemicals of peripheral lymphocytes from 26 cancer patients were estimated under conditions identical to those for healthy humans which had been reported (Cancer Res., 43: 439-442, 1983). The sensitive individual was defined as one whose cells give a mean induced SCE frequency more than 2 standard deviation units above the population mean of induced SCEs in cells from the healthy humans. When cells were treated with 3-amino-1-methyl-5H-pyrido[4, 3-b]indole in the presence of rat liver S9 mix, 8 in 10 stomach cancer patients, 4 in 4 colon cancer patients, 3 in 9 lung cancer patients, 0 in 3 patients bearing other cancers, and 0 in 9 non-cancerous individuals were sensitive. The corresponding frequency of individuals in the healthy population, reported previously, was 1 in 33 persons. Thus, the frequency of sensitive individuals in the combined group of stomach and colon cancer patients was very significantly higher than were frequencies in control groups. Three in 10 patients with stomach cancer and 4 in 16 patients with other cancers were sensitive to induction of SCE by methyl methanesulfonate. Six in these 7 methyl methanesulfonate-sensitive patients were also 3-amino-1-methyl-5H-pyrido[4,3-b]indole sensitive. The frequency of methyl methanesulfonate-sensitive individuals in the healthy populations was 2 in 50. There was no patient who was sensitive to SCE induction by 4-nitroquinoline 1-oxide. The frequency was not significantly different from the healthy population, in which 3 in 50 persons were sensitive. These results suggest that a particular cancer correlates with the sensitivity of peripheral lymphocytes to SCE induction by particular chemicals.

Studies on beta-turn of peptides. IX. Effect of 1st and 14th amino acids of tetrapeptide sequences on their beta-turn preferences studied by CD spectra of their chromophoric derivatives.

The effect of changing 1st and 4th amino acid residues on beta-turn preference of tetrapeptide sequences was studied by use of CD spectra of th chromophoric derivatives, which have Dnp- and pNA-groups as the amino and carboxyl substituents, respectively. The effect was examined with the tetrapeptides having such sequences at the 2nd and 3rd positions as -L-Pro-L-Asn-, -L-Pro-Gly-, -L-Pro-D-Ala-, -L-Ala-D-Leu-, -L-Ala-L-Pro-, and -D-Ala-L-Pro-. The beta-turn preferences estimated from the CD intensities of the bands due to exciton interaction were found to depend largely on the configurations of the 1st and 4th amino acid residues. When 1st and 2nd (or 3rd and 4th) residues had the same configuration, decreased intensity of the CD band was observed even if the internal sequence had high beta-turn preference. Terminal Gly residues were favorable for the beta-turn conformation in many of the tetrapeptide sequences examined.

Antagonizing effect of 3-aminoharman on induction of sister-chromatid exchanges by mutagens.

3-Aminoharman (3AH, 3-amino-1-methyl-9H-pyrido[3,4-b]indole), which has been reported as a novel substance with an antagonistic effect on induction of sister-chromatid exchange (SCE) by polycyclic mutagens in the presence of the metabolic activation system, was examined with a cultured human lymphoblastoid cell line, NL3, for its effect on SCE induction by direct-acting mutagens such as mitomycin C (MMC), nitrogen mustard N-oxide (NMO), methyl methanesulfonate (MMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitroquinoline 1-oxide (4NQO) and 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (OH-Trp-P-2), and also by ultraviolet light (UV) irradiation. The results obtained on simultaneous treatment with 3AH and mutagens were as follows: (1) 3AH suppressed more than 50% of SCEs induced by MMC, NMO and OH-Trp-P-2; (2) 4NQO- and MNNG-induced SCEs were also suppressed by 3AH but to a lesser degree; (3) MMS-induced SCEs were not, however, altered by 3AH; and (4) the suppression of SCE by 3AH was dose-dependent. Treatment of cells with 3AH for 2 h immediately before MMC exposure suppressed SCE induction to a significant degree similar to the simultaneous treatment, but post-treatment with 3AH was much less effective. 3AH inhibited SCE induction by NMO when 3AH treatment was carried out either before or after NMO treatment, to an extent similar to the simultaneous treatment. Treatments with 3AH either before or after UV exposure did not change the UV-induced SCEs. Results with these direct-acting mutagens ruled out the relevance of metabolic activation as a necessary step for the antagonizing effect of 3AH.

Actions of amino-beta-carbolines on induction of sister-chromatid exchanges.

Synthetic 3-aminoharman and 3-aminonorharman (amino-beta-carbolines) caused slight but definite induction of sister-chromatid exchanges (SCEs) in human lymphoblastoid cells NL3 and Chinese hamster cells CHO-K1. These amino-beta-carbolines are ranked between 2-amino-alpha-carboline and 2-amino-6-methyl-9a-aza-delta-carboline (Glu-P-2) and much lower than 3-amino-gamma-carbolines (Trp-P-1 and 2) in inductive activity. 1-Amino-beta-carboline, harman and norharman had very weak, if any, SCE-inducer activity. Norharman had a synergistic effect with aromatic amines such as Trp-P-2 and aniline on SCE induction, while 3-aminoharman suppressed SCE induction by more potent inducers such as Trp-P-2 and benzo[a]pyrene.