Comparison of Gd-DTPA-induced signal enhancements in rat brain C6 glioma among different pulse sequences in 3-Tesla magnetic resonance imaging.

BACKGROUND: T1-shortening contrast media are routinely used in magnetic resonance (MR) examinations for the diagnosis of brain tumors. Although some studies show a benefit of 3 Tesla (T) compared to 1.5T in delineation of brain tumors using contrast media, it is unclear which pulse sequences are optimal. PURPOSE: To compare gadopentetate dimeglumine (Gd-DTPA)-induced signal enhancements in rat brain C6 glioma in the thalamus region among different pulse sequences in 3T MR imaging. MATERIAL AND METHODS: Five rats with a surgically implanted C6 glioma in their thalamus were examined. T1-weighted brain images of the five rats were acquired before and after Gd-DTPA administration (0.1 mmol/kg) using three clinically available pulse sequences (spin echo [SE], fast SE [FSE], fast spoiled gradient echo [FSPGR]) at 3T. Signal enhancement in the glioma (E(T)) was calculated as the signal intensity after Gd-DTPA administration scaled by that before administration. Pulse sequences were compared using the Tukey-Kramer test. RESULTS: E(T) was 1.12+/-0.05 for FSE, 1.26+/-0.11 for FSPGR, and 1.20+/-0.11 for SE. FSPGR showed significantly higher signal enhancement than FSE and comparable enhancement to SE. CONCLUSION: FSPGR is superior to FSE and comparable to SE in its ability to delineate rat brain C6 glioma in the thalamus region.

Comparison in prognosis after VATS lobectomy and open lobectomy for stage I lung cancer: retrospective analysis focused on a histological subgroup.

BACKGROUND: Video-assisted thoracoscopic surgery (VATS) has become an attractive surgical procedure, but several issues remain to be resolved. Prognosis after VATS lobectomy is important to evaluate the adequacy of VATS lobectomy as a cancer operation. Interestingly, several investigators, including us, have reported that prognosis after VATS lobectomy was superior to that after open lobectomy in early non-small-cell lung cancer (NSCLC). One of the possible reasons is the low invasiveness of VATS lobectomy. But we considered that patient bias might have some influence favoring VATS lobectomy. To evaluate our hypothesis, we reviewed medical records of stage I NSCLC patients undergoing operation between 1993 and 2002. We compared and evaluated the relationship between patient characteristics and prognosis after VATS and open lobectomy. We focused particularly on histological type, classifying it into four subgroups; (1) bronchioloalveolar carcinoma (BAC), (2) mixed BAC + papillary adenocarcinoma (BAC + Pap), (3) other adenocarcinoma (Other adeno), (4) squamous cell carcinoma + others (Sq + others). RESULTS: A total of 165 patients underwent VATS lobectomy, and 123 patients underwent open lobectomy. The 5-year survival rate of the VATS lobectomy group was 94.5% and that of the open lobectomy group was 81.5%. Univariate Cox regression of survival revealed that male, CEA > 5, Other adeno, Sq + others, open lobectomy, and tumor size > 3 cm were significant negative prognostic variables. Multivariate Cox regression of survival revealed that histological subtype and tumor size were independent prognostic factors, but surgical procedure was not an independent prognostic factor. COMMENTS: Prognosis after VATS lobectomy was superior to that after open lobectomy, but patient bias influenced the prognosis in favor of VATS lobectomy, and the surgical procedure itself was not a prognostic factor.

The actions of azelnidipine, a dihydropyridine-derivative Ca antagonist, on voltage-dependent Ba2+ currents in guinea-pig vascular smooth muscle.

BACKGROUND AND PURPOSE: Although azelnidipine is used clinically to treat hypertension its effects on its target cells, Ca2+ channels, in smooth muscle have not been elucidated. Therefore, its effects on spontaneous contractions and voltage-dependent L-type Ca2+ channels were investigated in guinea-pig portal vein. EXPERIMENTAL APPROACH: The inhibitory potency of azelnidipine on spontaneous contractions in guinea-pig portal vein was compared with those of other dihydropyridine (DHP)-derived Ca antagonists (amlodipine and nifedipine) by recording tension. Also its effects on voltage-dependent nifedipine-sensitive inward Ba2+ currents (IBa) in smooth muscle cells dispersed from guinea-pig portal vein were investigated by use of a conventional whole-cell patch-clamp technique. KEY RESULTS: Spontaneous contractions in guinea-pig portal vein were reduced by all of the Ca antagonists (azelnidipine, Ki = 153 nM; amlodipine, Ki = 16 nM; nifedipine, Ki = 7 nM). In the whole-cell experiments, azelnidipine inhibited the peak amplitude of IBa in a concentration- and voltage-dependent manner (-60 mV, Ki = 282 nM; -90 mV, Ki = 2 microM) and shifted the steady-state inactivation curve of IBa to the left at -90 mV by 16 mV. The inhibitory effects of azelnidipine on IBa persisted after 7 min washout at -60 mV. In contrast, IBa gradually recovered after being inhibited by amlodipine, but did not return to control levels. Both azelnidipine and amlodipine caused a resting block of IBa at -90 mV. Only nifedipine appeared to interact competitively with S(-)-Bay K 8644. CONCLUSIONS AND IMPLICATIONS: These results suggest that azelnidipine induces long-lasting vascular relaxation by inhibiting voltage-dependent L-type Ca2+ channels in vascular smooth muscle.

Comparative studies of AJG049, a novel Ca2+ channel antagonist, on voltage-dependent L-type Ca2+ currents in intestinal and vascular smooth muscle.

BACKGROUND AND PURPOSE: Antagonists of Ca2+ channels reduce contraction of intestinal smooth muscle but also affect vascular smooth muscle. We have therefore examined the effects of AJG049, a newly synthesized antagonist for regulation of gut motility, on voltage-dependent L-type Ca2+ channels, in vascular and intestinal smooth muscle, comparing AJG049 with two other Ca2+ channel antagonists, verapamil and diltiazem. EXPERIMENTAL APPROACH: Affinities of AJG049 for various types of voltage-dependent Ca2+ channels were examined by binding studies. Effects of AJG049 on voltage-dependent inward Ca2+ (or Ba2+) currents (ICa or IBa) in dispersed smooth muscle cells from guinea-pig ileum, colon and mesenteric artery were measured using conventional whole-cell configurations. KEY RESULTS: In binding studies, AJG049 showed a high affinity for the diltiazem-binding site of L-type Ca2+ channels. In whole-cell configuration, AJG049 suppressed ICa in ileal myocytes, with concentration-, voltage-and use-dependencies. AJG049 shifted the steady-state inactivation curve of ICa to the left. The order of potency to inhibit ICa in ileal myocytes was AJG049>verapamil>diltiazem. AJG049 also suppressed IBa in guinea-pig mesenteric arterial myocytes, showing concentration- and voltage-dependencies and the potency order for this action was also AJG049>verapamil>diltiazem. For the relative ratio of Ki values between ileal and mesenteric arterial myocytes, the order was AJG049>diltiazem>>verapamil. CONCLUSIONS AND IMPLICATIONS: These results show that AJG049 inhibits L-type Ca2+ channels mainly through the diltiazem-binding site(s). From our results, AJG049 showed a little selectivity for these Ca2+ channels in intestinal smooth muscle.

Actions of ZD0947, a novel ATP-sensitive K+ channel opener, on membrane currents in human detrusor myocytes.

BACKGROUND AND PURPOSE: ATP-sensitive K+ channels (K(ATP) channels) play important roles in regulating the resting membrane potential of detrusor smooth muscle. Actions of ZD0947, a novel KATP channel opener, on both carbachol (CCh)-induced detrusor contractions and membrane currents in human urinary bladder myocytes were investigated. EXPERIMENTAL APPROACH: Tension measurements and patch-clamp techniques were utilized to study the effects of ZD0947 in segments of human urinary bladder. Immunohistochemistry was also performed to detect the expression of the sulphonylurea receptor 1 (SUR1) and the SUR2B antigens in human detrusor muscle. KEY RESULTS: ZD0947 (> or = 0.1 microM) caused a concentration-dependent relaxation of the CCh-induced contraction of human detrusor, which was reversed by glibenclamide. The rank order of the potency to relax the CCh-induced contraction was pinacidil > ZD0947 > diazoxide. In conventional whole-cell configuration, ZD0947 (> or = 1 microM) caused a concentration-dependent inward K+ current which was suppressed by glibenclamide at -60 mV. When 1 mM ATP was included in the pipette solution, application of pinacidil or ZD0947 caused no inward K+ current at -60 mV. Gliclazide (< or =1 microM), a selective SUR1 blocker, inhibited the ZD0947-induced currents (Ki = 4.0 microM) and the diazoxide-induced currents (high-affinity site, Ki1 = 42.4 nM; low-affinity site, Ki2 = 84.5 microM) at -60 mV. Immunohistochemical studies indicated the presence of SUR1 and SUR2B proteins, which are constituents of KATP channels, in the bundles of human detrusor smooth muscle. CONCLUSIONS AND IMPLICATIONS: These results suggest that ZD0947 caused a glibenclamide-sensitive detrusor relaxation through activation of glibenclamide-sensitive KATP channels in human urinary bladder.

Effects of tyrosine kinase inhibitors on voltage-dependent Ba(2+) currents in the guinea-pig gastric antrum.

We have investigated whether tyrosine kinases modify the activity of voltage-dependent Ba(2+) currents (I(Ba)) recorded from guinea-pig gastric myocytes by use of patch-clamp techniques. All experiments were carried on single smooth muscle cells, dispersed from the circular layer of the guinea-pig gastric antrum. Genistein ( > or = 10 microM), a specific tyrosine kinase inhibitor, reduced the peak amplitude of I(Ba) in a voltage- and concentration-dependent manner. Daidzein ( > or = 30 microM), an inactive analog of genistein, also inhibited I(Ba) in a concentration-dependent manner. Similarly, other types of tyrosine kinase inhibitors (lavendustin A and tyrphostin 23) suppressed the peak amplitude of I(Ba) in a concentration-dependent manner. These results indicate that tyrosine kinases may be essential to regulate Ca(2+) mobilization through voltage-dependent Ca(2+) channels in gastric myocytes.

LMP-1, the Epstein-Barr virus-encoded oncogene with a B cell activating mechanism similar to CD40.

Many details in the expression pattern of Epstein-Barr virus (EBV)-encoded proteins, their role in blast and growth transformation in infected B lymphocytes are known, but 'black holes' still exist. The two main types of virus-B lymphocyte interactions are denoted as Type I and Type III. These are characterized by the difference in the EBV protein expression which is related to the phenotype of the cell. The difference was first detected in comparisons between Burkitt lymphoma cells (BL) and lymphoblastoid cell lines, LCLs, which arise from normal B lymphocytes after experimental infection and are growth transformed by EBV. A third type of interaction can be seen in B-CLL cells which carry the EBV receptor CD21 and can be thus infected with the virus in vitro. The infected cells express the EBV-encoded proteins with a pattern which is different from the above mentioned two types, in that they express the nuclear proteins but not the membrane localized LMP-1. Importantly, the infected B-CLL cells do not enter DNA synthesis and they do not growth transform. Normal B lymphocytes with similar expression pattern have been seen in analysis of the lymphoreticular tissues of patients which responded to the primary EBV infection with development of the infectious mononucleosis symptoms.

Activation by levcromakalim and metabolic inhibition of glibenclamide-sensitive K channels in smooth muscle cells of pig proximal urethra.

1. The effects of levcromakalim (BRL 38227) on ionic currents recorded from pig proximal urethra were investigated by use of tension measurement and patch clamp techniques (conventional whole-cell configuration, nystatin perforated patch, and cell-attached configuration). 2. Levcromakalim (1 microM) caused a relaxation in the resting tone. This levcromakalim-induced relaxation was inhibited by the pretreatment with 1 microM glibenclamide. 3. The resting membrane potential recorded from single cells in current-clamp mode was-36.1 +/- 4.4 mV (n = 5). 4. Levcromakalim induced a concentration-dependent hyperpolarization with a maximum (at > or = 10 microM) close to the theoretical equilibrium potential of potassium (EK). The membrane hyperpolarization caused by 1 microM levcromakalim (24.7 +/- 5.8 mV, n = 4) was abolished by 1 microM glibenclamide. 5. Levcromakalim (100 microM) caused an outward K current in whole-cell recordings which was unaffected by iberiotoxin (300 nM) but abolished by glibenclamide (10 microM). 6. In cell-attached patches, levcromakalim activated a 43 pS K channel which was inhibited by the application of glibenclamide. 7. The metabolic poison, cyanide (CN), also activated a 43 pS K channel which was suppressed by the application of 10 microM glibenclamide. 8. These results indicate that levcromakalim and metabolic inhibition activate the same 43 pS K channel in pig proximal urethra. The resultant urethral hyperpolarization might reduce the usefulness of K channel openers in the treatment of detrusor instability, but be of value in treating outflow obstruction.

Properties and pharmacological modification of ATP-sensitive K(+) channels in cat tracheal myocytes.

The effects of levcromakalim and nucleoside diphosphates (NDPs) on both membrane currents and unitary currents in cat trachea myocytes were investigated by use of patch-clamp techniques. In conventional whole-cell configuration, levcromakalim produced a concentration-dependent K(+) current which was suppressed by additional application of 5 microM glibenclamide at -70 mV. When 3 mM ATP was added in the pipette solution, the peak amplitude of the levcromakalim-induced current was much smaller than that in the absence of ATP. When 3 mM uridine 5'-diphosphate (UDP) was included in the pipette solution, much higher concentrations of glibenclamide (>/=50 microM) were required to suppress the 100 microM levcromakalim-induced membrane current in comparison with those in the absence of UDP. In the cell-attached patches, levcromakalim activated a 40 pS K(+) channel which was inhibited by additional application of glibenclamide in symmetrical 140 mM K(+) conditions. UDP (>/=0.1 mM) was capable of reactivating the channel in inside-out patches in which the glibenclamide-sensitive K(+) channel had run down, in the presence of levcromakalim. The K(+) channel reactivated by UDP was suppressed by additional application of either intracellular 3 mM ATP or 100 microM glibenclamide. These results demonstrate that smooth muscle cells in the cat trachea possess ATP-sensitive 40 pS K(+) channels which are blocked by glibenclamide (i.e. K(ATP)) and can be activated by levcromakalim and that intracellular UDP causes a significant shift of the glibenclamide-sensitivity of these channels.

The effects of nifedipine and other calcium antagonists on the glibenclamide-sensitive K+ currents in smooth muscle cells from pig urethra.

1. The effects of nifedipine on both levcromakalim-induced membrane currents and unitary currents in pig proximal urethra were investigated by use of patch-clamp techniques (conventional whole-cell configuration and cell-attached patches). 2. Nifedipine had a voltage-dependent inhibitory effect on voltage-dependent Ba2+ currents at - 50 mV (Ki=30.6 nM). 3. In current-clamp mode, subsequent application of higher concentrations of nifedipine (> or =30 microM) caused a significant depolarization even after the membrane potential had been hyperpolarized to approximately -82 mV by application of 100 microM levcromakalim. 4. The 100 microM levcromakalim-induced inward current (symmetrical 140 mM K+ conditions, -50 mV) was inhibited by additional application of three different types of Ca antagonists (nifedipine, verapamil and diltiazem, all at 100 microM). In contrast, Bay K 8644 (1 microM) possessed no activating effect on the amplitude of this glibenclamide-sensitive current. 5. When 100 microM nifedipine was included in the pipette solution during conventional whole-cell recording at -50 mV, application of levcromakalim (100 microM) caused a significant inward membrane current which was suppressed by 5 microM glibenclamide. On the other hand, inclusion of 5 microM glibenclamide in the pipette solution prevented levcromakalim from inducing an inward membrane current. 6. The levcromakalim-induced K+ channel openings in cell-attached configuration were suppressed by subsequent application of 5 microM glibenclamide but not of 100 microM nifedipine. 7. These results suggest that in pig proximal urethra, nifedipine inhibits the glibenclamide-sensitive 43 pS K+ channel activity mainly through extracellular blocking actions on the K+ channel itself.

Multiple effects of mefenamic acid on K(+) currents in smooth muscle cells from pig proximal urethra.

The effects of mefenamic acid on both membrane potential and K+ currents in pig urethral myocytes were investigated using patch-clamp techniques (conventional whole-cell, cell-attached, outside-out and inside-out configuration). In the current-clamp mode, mefenamic acid caused a concentration-dependent hyperpolarization, which was inhibited by preapplication of 1 microm glibenclamide. In the voltage-clamp mode, mefenamic acid induced an outward current that was blocked by glibenclamide even in the presence of iberiotoxin (IbTX, 300 nm) at -50 mV. ATP-sensitive K+ channels (KATP channels) could be activated in the same patch by mefenamic acid and levcromakalim, with the same unitary amplitude and the similar opening gating at -50 mV in cell-attached configuration. In outside-out recording, external application of mefenamic acid activated intracellular Ca2+-activated IbTX-sensitive large-conductance K+ channels (BKCa channels). Mefenamic acid (or=100 microm) increased sustained outward currents, diminishing the activity of STOCs. Over the whole voltage range, mefenamic acid caused opposite effects on the membrane currents in the absence and presence of 5 microm glibenclamide. In the presence of 10 mm 4-aminopyridine (4-AP), mefenamic acid only increased the outward currents. These results indicate that mefenamic acid increases the channel activities of two distinct types of K+ channels (i.e. BKCa channels and KATP channels) and decreased 4-AP-sensitive K+ channels in pig urethral myocytes.

Effects of levcromakalim and nucleoside diphosphates on glibenclamide-sensitive K+ channels in pig urethral myocytes.

1. Effects of levcromakalim and nucleoside diphosphates (NDPs) on both membrane currents and unitary currents in pig proximal urethra were investigated by use of patch clamp techniques (conventional whole-cell configuration, nystatin perforated patch, cell-attached configuration and inside-out patches). 2. Levcromakalim produced a concentration-dependent outward current at a holding potential of -50 mV. The peak current amplitude showed little variation when measured by either conventional whole-cell or nystatin perforated patch configurations. 3. In conventional whole-cell configuration, the levcromakalim (100 microM)-induced outward current decayed by about 90% in 18 min. In contrast, with the nystatin perforated patch, approximately 86% of the levcromakalim-induced outward current still remained after 18 min. 4. The peak amplitude of the levcromakalim (100 microM)-induced outward membrane current recorded by the conventional whole-cell configuration was greatly reduced by inclusion of 5 mM EDTA in the pipette. The much smaller but significant outward membrane current remaining was abolished by glibenclamide. 5. In conventional whole-cell recordings, inclusion of an NDP in the pipette solution induced a small outward current which slowly reached a maximal amplitude (in 2 to 10 min) and was suppressed by glibenclamide. Addition of 100 microM levcromakalim after the NDP-induced current had peaked activated a further outward current which was larger than that recorded in the absence of NDPs. Approximately 50% of this current still remained at 18 min, even when conventional whole-cell configuration was used. 6. In the cell-attached mode in symmetrical 140 mM K+ conditions, glibenclamide inhibited the 100 microM levcromakalim-activated 43 pS K+ channel in a concentration-dependent manner, showing an inhibitory dissociation constant (Ki) of approximately 520 nM. 7. In inside-out patches in which the glibenclamide-sensitive K+ channel had run down after exposure to levcromakalim, both uridine 5'-diphosphate (UDP) and MgATP were capable of reactivating the channel. Further application of Mg2+ to the UDP-reactivated K+ channels enhanced the channel activity reversibly. 8. In inside-out patches UDP was capable of activating the glibenclamide-sensitive K+ channel without levcromakalim, providing that there was free Mg2+ present (either UDP in 5 mM EGTA or UDP in 5 mM EDTA with Mg2+). Additional application of levcromakalim caused a further reversible activation of channel opening. 9. In the presence of levcromakalim, application of adenosine 5'-triphosphate (ATP) to the inner surface of the membrane patch inhibited UDP-reactivated channel opening in a concentration-dependent manner. 10. Addition of an untreated cytosolic extract of pig proximal urethra reactivated the glibenclamide-sensitive K+ channel in the presence of 100 microM levcromakalim in inside-out patches. 11. These results demonstrate the presence in the pig proximal urethra of a glibenclamide-sensitive K+ channel that is blocked by intracellular ATP and can be activated by levcromakalim. Intracellular UDP can reactivate the channel after rundown. Additionally, intracellular Mg2+ may play an important role in regulating the channel activity.

The effects of flecainide on ATP-sensitive K(+) channels in pig urethral myocytes.

The effects of the antiarrhythmic drug flecainide on levcromakalim-induced hyperpolarization, macroscopic and unitary K(+) currents in pig urethra were investigated using patch-clamp techniques. The effects of flecainide were also examined on currents in inside-out patches of COS7 cells expressing carboxy terminus truncated inwardly rectifying K(+) channel (Kir6.2) subunits (i.e. Kir6.2DeltaC36) which form ATP-sensitive K(+) channels (K(ATP) channels). In current-clamp mode, application of flecainide (> or =100 microM) caused a significant depolarization after the membrane potential had been hyperpolarized by levcromakalim. In voltage-clamp experiments, the levcromakalim-induced outward current was suppressed by 300 microM flecainide in quasi-physiological K(+) conditions (K(i)=51 microM). In contrast, approximately 20% of the levcromakalim-induced inward current still remained even after application of 300 microM flecainide in symmetrical 140 mM K(+) conditions (K(i)=51 microM). In contrast, approximately 20% of the levcromakalim-induced inwar=126 microM). In cell-attached configuration, the channel activity of the levcromakalim-induced K(ATP) channels was reversibly inhibited by flecainide (> or =30 microM) at -50 mV. Their activity was also suppressed by either disopyramide or cibenzoline. Flecainide reversibly inhibited the channel activity of Kir6.2DeltaC36 expressed in COS7 cells using inside-out configuration. Inhibitory effects of flecainide on the levcromakalim-induced currents became more potent when the value of external pH increased, although this slightly reduced the proportion of drug molecules carrying a positive charge. These results suggest that flecainide inhibits channel activity through blocking the pore site of the K(ATP) channel in pig urethra.

The effects of caffeine on ATP-sensitive K(+) channels in smooth muscle cells from pig urethra.

The effects of caffeine on both levcromakalim-induced macroscopic and unitary currents in pig proximal urethra were investigated by the use of patch-clamp techniques (conventional whole-cell configuration and cell-attached configuration). The effects of caffeine were also examined on currents in inside-out patches of COS7 cells expressing carboxy terminus truncated inwardly rectifying K(+) channel (Kir6.2) subunits (i.e. Kir6.2DeltaC36) which form ATP-sensitive K(+) channels (K(ATP) channels). In conventional whole-cell configuration, the levcromakalim (100 microM)-induced inward current (symmetrical 140 mM K(+) conditions) was inhibited by caffeine (> or =1 mM) at a holding potential of -50 mV. In contrast, ryanodine (10 microM) caused no significant inhibitory effect on the gradual decay of the levcromakalim-induced current at -50 mV. The amplitude of the 30 microM levcromakalim-induced current was enhanced by 3-isobutyl-1-methylxanthine (IBMX, 100 microM). In cell-attached configuration, the levcromakalim-induced K(+) channel openings were inhibited by subsequent application of 10 mM caffeine, decreasing the channel open probability at -50 mV. Reverse transcriptase-polymerase chain reaction (RT - PCR) analysis revealed the presence of Kir6.2 transcript in pig urethra. Caffeine (> or =3 mM) inhibited the channel activity of Kir6.2DeltaC36 expressed in COS7 cells (3 mM caffeine, 65+/-6%, n=4; 10 mM caffeine, 29+/-2%, n=4). These results suggest that caffeine can inhibit the activity of K(ATP) channels through a direct blocking effect on the pore-forming Kir subunit.

The involvement of L-type Ca(2+) channels in the relaxant effects of the ATP-sensitive K(+) channel opener ZD6169 on pig urethral smooth muscle.

1. The effects of ZD6169, a novel ATP-sensitive K(+) channel (K(ATP) channel) opener, were investigated on membrane currents in isolated myocytes using patch-clamp techniques. Tension measurement was also performed to study the effects of ZD6169 on the resting tone of pig urethral smooth muscle. 2. Levcromakalim was more potent than ZD6169 in lowering the resting urethral tone. Relaxation induced by low concentrations of ZD6169 (< or =3 microM) was completely suppressed by additional application of glibenclamide (1 microM). In contrast, glibenclamide (1-10 microM) only partially inhibited the relaxation induced by higher concentrations of ZD6169 (> or = microM). 3. Bay K8644 (1 microM) reduced the maximum relaxation produced by ZD6169 (> or =10 microM). 4. In whole-cell configuration, ZD6169 suppressed the peak amplitude of voltage-dependent Ba(2+) currents in a concentration- and voltage-dependent manner, and at 100 microM, shifted the steady-state inactivation curve of the voltage-dependent Ba(2+) currents to the left at a holding potential of -90 mV. 5. In cell-attached configuration, open probability of unitary voltage-dependent Ba(2+) channels (27 pS, 90 mM Ba(2+)) was inhibited by 100 microM ZD6169 and by 10 microM nifedipine. 6. Reverse transcriptase-polymerase chain reaction (RT - PCR) analysis revealed the presence of the transcript of the alpha(1C) subunit of L-type Ca(2+) channels in pig urethra. 7. These results demonstrate that ZD6169 causes urethral relaxation through two distinct mechanisms, activation of K(ATP) channels at lower concentrations and inhibition of voltage-dependent Ca(2+) channels at higher concentrations (about 10 microM).

Dual action of ZD6169, a novel K(+) channel opener, on ATP-sensitive K(+) channels in pig urethral myocytes.

1. The effects of ZD6169, a novel K(+) channel opener, on both membrane and unitary currents in pig urethra were investigated using patch-clamp techniques. Its effect was also examined on currents in inside-out patches of COS7 cells expressing carboxy terminus truncated inwardly rectifying K(+) channel (Kir6.2) subunits (Kir6.2C36) which form ATP-sensitive K(+) channels (K(ATP) channels). 2. In current-clamp mode, ZD6169 (< or = 10 microM) induced a concentration-dependent membrane hyperpolarization. Higher concentrations (> or = 30 microM) caused a transient membrane hyperpolarization, followed by a gradual membrane depolarization. On removal of ZD6169, an after hyperpolarization was observed. 3. In conventional voltage-clamp configuration, at -50 mV in symmetrical 140 mM K(+) conditions, ZD6169 (100 microM) caused a transient inward current which gradually decayed. Removal of ZD6169 evoked a much larger amplitude K(+) current with a similar time course. 4. ZD6169 produced an inward glibenclamide-sensitive K(+) current, demonstrating a bell-shaped concentration-response relationship. 5. In cell-attached configuration in symmetrical 140 mM K(+) conditions, ZD6169 (< or = 30 microM) activated an K(ATP) channel which was reversibly suppressed by application of glibenclamide. In contrast, ZD6169 (100 microM) inhibited the activity of the levcromakalim-induced K(ATP) channels. 6. ZD6169 (100 microM) had no significant effect on the channel activity of Kir6.2C36 in inside-out configuration, although cibenzoline greatly suppressed the channel activity. 7. These results demonstrate that ZD6169 possesses a dual effect on the activity of the K(ATP) channel; activating at low concentration and inhibiting at higher concentration.

Promotional effects of azathioprine on peripheral B cell lymphomas in BALB/c mice induced by administration of 7,12-dimethylbenz[a]anthracene.

BALB/c mice either were injected subcutaneously with 25 micrograms/g or 50 micrograms/g body weight 7,12-dimethyl-benz[a]anthracene (DMBA) at the age of 13 weeks, or received repeated s.c. injections of azathioprine (0.1 mg/g) starting from the age of 6 or 18 weeks, or they received treatments with both agents. Of the 317 mice treated, 69 developed lymphomas by the age of 50 weeks. None of the 31 non-treated mice developed lymphomas during this period. The lymphomas, classified according to Pattengale-Taylor's classification for murine lymphomas/leukemias, were examined immunohistochemically and immunogenetically. The lymphomas induced with DMBA showed a male preponderance and a higher incidence of the B cell type, especially in the lymph node and spleen. Azathioprine induced predominantly T-lymphoblastic lymphomas in the thymus and these were found more frequently in females. Treatment with both DMBA and azathioprine remarkably increased the incidence of B lymphomas, especially follicular center cell lymphomas in the spleen, which are the predominant type of spontaneous lymphomas occurring in older BALB/c mice. Some of the treated mice also developed prelymphomatous lesions.

Animal in vivo models of EBV-associated lymphoproliferative diseases: special references to rabbit models.

Animal models of human EBV-associated diseases are essential to elucidate the pathogenesis of EBV-associated diseases. Here we review those previous models using EBV or EBV-like herpesviruses and describe the details on our two newly-developed rabbit models of lymphoproliferative diseases (LPD) induced by simian EBV-like viruses. The first is Cynomolgus-EBV-induced T-cell lymphomas in rabbits inoculated intravenously (77-90%) and orally (82-89%) during 2-5 months. EBV-DNA was detected in peripheral blood by PCR from 2 days after oral inoculation, while anti-EBV-VCA IgG was raised 3 weeks later. Rabbit lymphomas and their cell lines contained EBV-DNA and expressed EBV-encoded RNA-1 (EBER-1). Rabbit lymphoma cell lines, most of which have specific chromosomal abnormality, showed tumorigenicity in nude mice. The second is the first animal model for EBV-infected T-cell LPD with virus-associated hemophagocytic syndrome (VAHS), using rabbits infected with an EBV-like herpesvirus, Herpesvirus papio (HVP). Rabbits inoculated intravenously with HVP-producing cells showed increased anti-EBV-VCA-IgG titers, and most (85%) subsequently died of fatal LPD and VAHS, with bleeding and hepatosplenomegaly, during 22-105 days. Peroral spray of cell-free HVP induced viral infection with seroconversion in 3 out of 5 rabbits, with 2 of the 3 infected rabbits dying of LPD with VAHS. Atypical T lymphocytes containing HVP-DNA and expressing EBER-1 were observed in many organs. Hemophagocytic histiocytosis was observed in the lymph nodes, spleen, bone marrow, and thymus. These rabbit models are also useful and inexpensive alternative experimental model systems for studying the biology and pathogenesis of EBV, and prophylactic and therapeutic regimens.

Simultaneous detection of two independent antigens by double staining with two mouse monoclonal antibodies.

Simultaneous detection of two antigens by immunostaining usually requires primary antibodies from two different species or a hapten modification of one of the antibodies if they are from the same species. A novel double staining method is described for immunodetection of two independent antigens using two mouse monoclonal antibodies. The principle of the method is the following: The first antigen is detected by a monoclonal antibody that is diluted so extensively that it cannot be recognized with conventional detection systems. A highly sensitive biotin-tyramide amplification system is used to identify this antibody. The second antigen is stained with a monoclonal antibody by dilution and detected by conventional immunostaining. The method was tested for both alkaline-phosphatase staining on paraffin sections and immunofluorescence staining on cultured cells in cytospin preparation. The absence of cross-reaction in the former system was demonstrated by the mutually exclusive detection of T- and B-cells in human lymph nodes or T-cells and carcinoma cells in nasopharyngeal carcinoma biopsies. Similarly, the EBV encoded EBNA2 and ZEBRA proteins showed a mutually exclusive staining by immunofluorescence on B95-8 cells. The method could be used to demonstrate co-expression of two independent antigens in the same cells, such as PCNA and keratin in carcinoma cells in paraffin sections and for EBNA2 and LMP1 EBV proteins in immunofluorescence preparations of B95-8 cells.

Glibenclamide-sensitive K+ channels underlying levcromakalim-induced relaxation in pig urethra.

To investigate the possible mechanisms involved in the stable and long-lasting levcromakalim-induced relaxation of the resting urethral tone, we have performed mechanical and voltage-clamp experiments using intact tissue and isolated cells from pig urethra, respectively. At negative membrane potentials, levcromakalim induced time- and voltage-independent membrane currents in whole-cell configurations. In cell-attached patches, levcromakalim not only increased the open-state probability (the NP(0) value) of the glibenclamide-sensitive 43 pS K+ channel (K(GS)) in a concentration-dependent manner, but also activated K(GS) with a time- and voltage-independence. During long burst-like channel activity, neither the mean open lifetime nor the mean closed time of K(GS) exhibited voltage-dependency between -100 and - 40 mV. It is concluded that levcromakalim causes a stable and potent relaxation of pig urethra through opening of K(GS) which possesses time- and voltage-independent activating mechanisms.