Analysis of new imidazoline derivative-induced increase in the maximum response to norepinephrine in the rat vas deferens

The effects of newly synthesized 5-imidazoline derivatives on the dose-response relationship to norepinephrine were investigated in the normal and denervated vasa deferentia of the rat. Three derivatives (K-3827, K-4011 and K-4300) exerted alpha-antagonistic action, the potency of which was similar to that of tolazoline. The pA2 values of these derivatives and currently known alpha-antagonists (tolazoline, phentolamine and prazosin, but not yohimbine) in the denervated tissue were slightly but significantly larger than those in the normal tissue. All imidazoline derivatives and alpha-antagonists produced an increase in the maximum response to norepinephrine in the normal vas deferens. In the denervated tissue, however, K-3827, K-4011 and alpha-antagonists caused only a rightward shift of the dose-response curve to norepinephrine, but not an increase in the maximum response, i.e., relatively pure alpha-antagonism. In contrast, the other 3 imidazoline derivatives, K-4299 and K-6342 which exhibited neither alpha-agonistic nor antagonistic action and K-4300, increased the maximum response to norepinephrine even after denervation. Their effects were nonspecific in that they also potentiated acetylcholine-induced contractions in both normal and denervated tissues. These 3 imidazoline derivatives antagonized the action of diltiazem. The effects of imidazoline derivatives and alpha-antagonists were discussed in relation to those of denervation, and the drug enhancement by 3 imidazoline derivatives was analyzed from the viewpoint of calcium movement.     

Influence of ouabain on the tracheal musculature of the dog

The effects of ouabain on the smooth muscles of the airway were investigated in anesthetized, paralyzed and artificially ventilated mongrel dogs. Ouabain (30 micrograms/kg, i.v.) caused a constriction of the tracheal smooth muscle which was followed by bradycardia. When ouabain was infused at a rate of 2 micrograms/kg/min (i.v.), the tracheal constriction was induced by a total dose of 45.0 +/- 5.5 micrograms/kg, while the bradycardia appeared with a total dose of 54.4 +/- 6.1 micrograms/kg. The ouabain-induced tracheal constriction was inhibited by bilateral vagotomy. The tracheal constriction induced by i.a. infusion of 10 microM ouabain into the bilateral cranial thyroid arteries was inhibited by bilateral vagotomy, but it was not completely blocked. With bilateral vagotomy, the tracheal constriction induced by i.a. infusion of ouabain was unaffected by 3 microM hexamethonium, but it was significantly inhibited by 1 microM atropine. These results suggest that ouabain may induce tracheal constriction by a neurogenic action in addition to its action via the augmentation of the vagal reflex, and the neurogenic action of ouabain may be related, in large part, to the release of acetylcholine from the presynapses of vagus nerves in dogs.     

Similarities between the relaxations induced by vasoactive intestinal peptide and by stimulation of the non-adrenergic non-cholinergic neurons in the rat stomach

Summary The characteristics of the non-adrenergic, noncholinergic inhibitory response of the rat stomach fundus to transmural nerve stimulation were compared with the relaxation induced by vasoactive intestinal polypeptide (VIP). Treatment with -chymotrypsin (5 U/ml) or VIP antiserum (1:200) significantly reduced the relaxation induced by transmural nerve stimulation at 30 Hz, indicating that the possible transmitter in the non-adrenergic, non-cholinergic nerves is a peptide and may be VIP or a closely related peptide. VIP was able to relax, fully and dose-dependently, the stomach fundus that had previously been constricted by treatment with 10^–6 M serotonin, and the IC₅₀ value for VIP was 2.4 × 10^–9 M. VIP elevated levels of cyclic AMP in a dose-dependent manner and the EC₅₀ value was 2.8 × 10^–9 M in the presence of 10^–6 M atropine and 10^–6 M guanethidine. The stomach fundus was relaxed by transmural nerve stimulation (30 Hz, 50 mA) and transmural nerve stimulation also caused production of cyclic AMP in the rat stomach in the presence of atropine and guanethidine. The basal level of cyclic AMP in the stomach was 8.7 ± 0.26 pmole/mg protein. When transmural nerve stimulation was applied for 5 min, the contraction of the stomach, induced by 10^–6 M serotonin, was inhibited by 54% in the presence of atropine and guanethidine and the level of cyclic AMP was increased to 13.0 ± 0.73 pmol/mg protein. Apamin inhibited the transmural nerve stimulation-induced relaxation and shifted the dose-response curve for VIP to the right. These results suggest that one of the putative neurotransmitter from non-adrenergic, non-cholinergic nerves in the rat stomach is VIP and that VIP-induced relaxation may be mediated by the production of cyclic AMP and by the opening of apamin-sensitive K⁺-channels.Send offprint requests to K. Kamata at the above address     

Radiotelemetry recording of electroencephalogram in piglets during rest

A wireless recording system was developed to study the electroencephalogram (EEG) in unrestrained, male Landrace piglets. Under general anesthesia, ball-tipped silver/silver chloride electrodes for EEG recording were implanted onto the dura matter of the parietal and frontal cortex of the piglets. A pair of miniature preamplifiers and transmitters was then mounted on the surface of the skull. To examine whether other bioelectrical activities interfere with the EEG measurements, an electrocardiogram (ECG) or electromyogram (EMG) of the neck was simultaneously recorded with the EEG. Next, wire electrodes for recording movement of the eyelid were implanted with EEG electrodes, and EEG and eyelid movements were simultaneously measured. Power spectral analysis using a Fast Fourier Transformation (FFT) algorithm indicates that EEG was successfully recorded in unrestrained piglets, at rest, during the daytime in the absence of interference from ECG, EMG or eyelid movements. These data indicate the feasibility of using our radiotelemetry system for measurement of EEG under these conditions.     

Expression and characterization of mouse angiotensin II type 1a receptor tagging hemagglutinin epitope in cultured cells

The octapeptide angiotensin II mediates the physiological actions of the renin-angiotensin system through activation of several angiotensin II receptor (AT) subtypes, in particular AT1 (AT1a and AT1b in the case of rodents). Although we and others have generated mutant mice in which the AT1a gene was disrupted, the function of mouse AT1 remains to be fully elucidated, due to the lack of effective tools involving antibodies against AT1 for detecting biological responses in cellular conditions. To avoid these problems, we constructed the hemagglutinin (HA)-tagged mouse AT1a, and stably introduced this recombinant receptor into human embryonic kidney 293-T cells. Radioligand binding of [(125)I] angiotensin II to AT1a was specific, saturable, and reversible. Scatchard analysis demonstrated that the transfected receptor had a dissociation constant of 1.7 nM with a density of 1.2 x 10(5) sites/cells. Angiotensin II stimulated a rapid increase in cytosolic free calcium, and angiotensin II-induced phosphorylation of extracellular signal-regulated kinases (Erk) was found in a dose-dependent manner. After solubilization, Western blot analysis showed specific interactions between an anti-HA antibody and HA-tagged mouse AT1a. Furthermore, a significant proportion of HA-tagged mouse AT1a was specifically immunoprecipitated with this antibody. In the immunocytochemical and electronmicroscopic studies, treatment of this cell line with angiotensin II resulted in decrease in signals of the surface receptors. Based on these results, the cell line established here provides an excellent tool for studying angiotensin II actions mediated through mouse AT1a, at sub-nanomolar concentrations.     

Boy with a chromosome del (3)(q12q23) and Blepharophimosis syndrome

We report on a 6-year-old boy with de novo 46, XY, del(3)(q12q23) and bilateral blepharo-phimosis, ptosis, epicanthus inversus, in addition to multiple other anomalies. Since 4 previously reported cases of interstitial deletion of 3q involving 3q23 band are clinically similar, we propose this blepharophimosis sequence due to 3q23 deletion as a further “contiguous gene syndrome”.     

The actions of ivermectin on cultured chick spinal cord neurons

The effects of ivermectin (IVM) on cultured chick spinal neurons sensitive to GABA agonists were investigated. IVM caused a change of membrane potential which was associated with an increase in membrane conductance, apparently reversed at about -40 mV and weakened by repetitive application. Furthermore, this agent did not have any effects on either the actions of GABA agonists on neurons or [3H]GABA efflux from those cells. These results suggest that IVM increases membrane conductance through sites which are different from GABA recognition sites.     

p53 protein overexpression and K-rascodon 12 mutation in pancreatic ductal carcinoma: Correlation with histologic factors

The correlation of p53 protein overexpression and the K-ras codon 12 mutatlon wlth histologlc type, grade of cytologic atypla, depth of lnvasion and other histologlc prognostic factors was studied In paraffin sectlons from 43 ductectatic-and 70 solid-type pancreatic ductal carcinomas. Overexpression of p53 was found in 23.3% (10143) of ductectatic carcinomas (17.2% of intraductal and 35.7% of lnvaslve carclnomas) and in 61.4% (43/70) of solid carcinomas. In ductectatic cancers, p53 overexpression was detected In 14.8% (4/27) of carcinomas wlth lowgrade atypla (CAL), 50.0% (5110) of carcinomas wlth high-grade atypla (CAH) and in 16.7% (In) of mixed low- and hlgh-grade cancers. In the last group, expression was restricted to an area of CAH. In solld cancers, p53 overexpression did not dlffer by histologic type or grade. Overexpresslon of p53 and K-ras mutatlons did not correlate with histologlc prognostic factors (lymphatic, venous and perineural Invasion, and lymph node metastasls) in ductectatlc and solld cancers or depth of invasion of solld carclnomas. Our data suggest that p53 alteratlon occurs at an early intraductal stage of solld carcinoma, irrespectlve of cellular atypla, but Is low in ductectatic CAL and becomes hlgher In ductectatlc CAH. K-ras mutatlon, present In a high percentage of tumors of all groups and not correlating with the factors above, showed no changes In frequency with tumor progression.     

New method for quantitative measurement of N-nitrosodimethylamine formation in the whole mouse

A simple method for the quantitative estimation of the formation of N-nitrosodimethylamine (NDMA) in mice has been developed. Mice were frozen in liquid nitrogen and homogenized. NDMA was then extracted and analyzed by a gas chromatograph equipped with a thermal energy analyzer. In normal mice NDMA (100 nmole) administered orally was rapidly metabolized and recovery of NDMA was about 10% after 60 min. However, when pyrazole (300 mg/kg) was injected i.p. to mice 60 min before the administration of NDMA, more than 80% of the administered NDMA could be recovered within 60 min. This result suggested that in pyrazole pretreated mice the accurate amount of NDMA formed could be estimated. Therefore the NDMA formation was measured in the pyrazole pretreated mice. When 0.25 mole of aminopyrine and from 0.25 to 2.0 umole of sodium nitrite were simultaneously administered orally, the amount of the NDMA formation in 20 min was found to be from 8.2 to 60.3 nmole. These values are equal to about from 30 to 200 g/kg of body weight which are nearly daily doses expected to cause the carcinogenic effect on mice or rats. This method of measuring NDMA in pyrazole pretreated mice appears to be useful for investigating the in vivo formation of NDMA quantitatively.     

Two cases of methicillin-resistant Staphylococcus aureus (MRSA) sepsis following craniotomy

We report here two cases of MRSA sepsis following craniotomy. In case 1, a petroclival meningioma was subtotally removed and lumbar drainage was inserted postoperatively to prevent cerebrospinal fluid leakage. Ventriculo-peritoneal shunt was performed after meningitis was treated with vancomycin and panipenem/betamipron. Two weeks after the procedure, the patient revealed continuous spiking fevers related to MRSA sepsis, which did not improve with vancomycin and arbekacin administration. The focus of infection was found by scintigraphy and CT by 67Ga to be spondylo-diskitis at the level of L2-L3. The lesion was removed and bone from the iliac crest grafted. In case 2, seven days after surgery for multiple meningioma, the patient exhibited spiking fevers and swelling in the left leg. The central venous catheter was removed from the left femoral vein and MRSA was found from blood culture. The patient was treated with arbekacin (200 mg/day). Venous thrombosis diagnosed by CT was treated with heparin. Symptoms related to the infection and laboratory data did not improve because the concentration of arbekacin in the blood did not reach an effective level. The symptoms markedly improved when the dose of arbekacin was doubled (400 mg/day).