The effect of angiotensin-converting enzyme inhibitors of left atrial pressure in dogs with mitral valve regurgitation.

BACKGROUND: Despite many epidemiological reports concerning the efficacy of angiotensin-converting enzyme (ACE) inhibitors in dogs with mitral regurgitation (MR), the hemodynamic effects of ACE inhibitor administration have not been fully evaluated. OBJECTIVES: To document left atrial pressure (LAP) in dogs with MR administered ACE inhibitors, in order to obtain interesting information about daily LAP changes with administration of ACE inhibitors. ANIMALS: Five healthy Beagle dogs weighing 9.8 to 14.2 kg (2 males and 3 females; aged 2 years). METHODS: Experimental, crossover, and interventional study. Chordae tendineae rupture was induced, and a radiotelemetry transmitter catheter was inserted into the left atrium. LAP was recorded for 72 consecutive hours during which each of 3 ACE inhibitors--nalapril (0.5 mg/kg/d), temocapril (0.1 mg/kg/d), and alacepril (3.0 mg/kg/d)--were administered in a crossover study. RESULTS: Averaged diurnal LAP was significantly, but slightly reduced by alacepril (P = .03, 19.03 +/- 3.01-18.24 +/- 3.07 mmHg). The nightly drops in LAP caused by alacepril and enalapril were significantly higher than the daily drops (P = .03, -0.98 +/- 0.19 to -0.07 +/- 0.25 mmHg, and P = .03, -0.54 +/- 0.21-0.02 +/- 0.17 mmHg, respectively), despite the fact that the oral administrations were given in the morning. Systolic blood pressure (122.7 +/- 14.4-117.4 +/- 13.1 mmHg, P = .04) and systemic vascular resistance (5800 +/- 2685-5144 +/- 2077 dyne x s/cm5, P = .03) were decreased by ACE inhibitors. CONCLUSIONS AND CLINICAL IMPORTANCE: ACE inhibitors decrease LAP minimally, despite reductions in left ventricular afterload. ACE inhibitors should not be used to decrease LAP.

Heterogeneity of glycine receptors and their messenger RNAs in rat brain and spinal cord.

Messenger RNAs isolated from adult or newborn rat spinal cord were fractionated in a sucrose gradient. The fractions were injected into Xenopus oocytes to determine their potencies for expression of glycine receptors (GlyRs), which were then examined electrophysiologically. The sedimentation profiles disclosed two classes of GlyR mRNAs, one heavy and the other light. The adult spinal cord was rich in heavy GlyR mRNA, whereas the light GlyR mRNA was more abundant in neonatal spinal cord and in adult cerebral cortex. Glycine receptors encoded by heavy and light mRNAs of adult spinal cord showed some electrophysiological differences. Thus there are two types of GlyRs encoded by mRNAs of different sizes, and the expression of these mRNAs is developmentally regulated. A tissue- and age-dependent distribution of heterogeneous GlyR mRNAs may imply diverse roles of the GlyRs in neuronal function in the central nervous system.

Functional correlation of fetal and adult forms of glycine receptors with developmental changes in inhibitory synaptic receptor channels.

Functional maturation of the nicotinic acetylcholine receptor is executed by its gamma-to-epsilon subunit switching. The glycine receptor also has fetal (alpha 2) and adult (alpha 1) isoforms. However, whether subunit switching is responsible for developmental changes in glycine receptor function is not known. We recorded single-channel currents from homomeric glycine receptors expressed in Xenopus oocytes with cRNAs encoding the alpha 2 or alpha 1 subunits and compared them with those recorded from native glycine receptors in rat spinal neurons at various ontogenic periods. The mean channel life times of the alpha 1 and mature glycine receptors were equally short, whereas both the alpha 2 and fetal receptors showed a significantly longer open time. Consistent with these results, the decay time of the glycinergic inhibitory postsynaptic currents (IPSCs) in spinal neurons became shorter during postnatal development. We conclude that developmental switching of alpha subunits may accelerate the kinetics of IPSCs.

Expression of inflammatory mediators in the otitis media induced by Helicobacter pylori antigen in mice.

Helicobacter pylori is a Gram-negative bacterium that is recognized as one of the key factors in gastric diseases such as gastritis, peptic ulcer and gastric cancer. Recent studies have shown relationships between H. pylori and extra-digestive diseases, and the presence of H. pylori in the middle ear and upper respiratory tract has been reported. However, the role of H. pylori in middle ear disease remains unclear. The present study demonstrated that H. pylori whole-cell protein directly induces macrophage migration inhibitory factor, macrophage inflammatory protein 2, interleukin 1 beta and tumor necrosis factor alpha in middle ear epithelium in mice, and severe proliferation of inflammatory cells was observed in middle ear cavity inoculated with H. pylori whole-cell protein. In addition, trans-tympanic injection of macrophage migration inhibitory factor up-regulated expression of macrophage inflammatory protein 2 in the middle ear. These findings indicate that H. pylori infection causes immunological inflammation in middle ear epithelium, and H. pylori may play a significant role in otitis media.

Marked differences in the carcinogenic activity of optically pure (+)- and (-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene in newborn mice.

Optically pure (+)- and (-)-trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrenes [(+)- and (-)-BP 7,8-dihydrodiol] were tested for carcinogenicity by giving newborn mice i.p. injections of 20, 40, and 80 nmol of compound on Days 1, 8, and 15 of life. The animals were killed at 17 weeks of age. Control mice had 0.10 pulmonary adenoma per mouse, whereas animals treated with (+)-BP 7,8-dihydrodiol and (-)-BP 7,8-dihydrodiol had 0.16 and 9.28 pulmonary adenomas per mouse, respectively. When a 5-fold higher dose was administered according to the above dosage schedule, (+)-dihydrodiol caused 2.34 pulmonary adenomas per mouse and (-)-BP 7,8-dihydrodiol caused 32.2 pulmonary adenomas per mouse. When 200, 400, and 800 nmol of benzo(a)pyrene or (+)-BP 7,8-dihydrodiol were administered sequentially on Days 1, 8, and 15 of life, 4.13 and 18.5 pulmonary adenomas per mouse, respectively, were observed when the mice were 17 weeks of age. This high dose of (-)-BP 7,8-dihydrodiol killed most of the mice. Administration of (-)-BP 7,8-dihydrodiol caused a high incidence of malignant lymphomas, whereas (+)-BP 7,8-dihydrodiol and benzo(a)pyrene had little or no ability to cause malignant lymphomas.

Mutagenicity and tumor-initiating activity of cyclopenta(c,d)pyrene and structurally related compounds.

The biological activities of benzo(a)pyrene, cyclopenta(c,d)pyrene, and 12 other structurally related compounds were assessed by mutagenicity studies with bacterial and mammalian cells and/or skin tumorigenicity studies with mice. The ability of the parent hydrocarbons to be metabolically activated to mutagenic products was examined in strains TA98 and TA100 of Salmonella typhimurium, using 3 experimental protocols. In each case, cyclopenta(c,d)pyrene was metabolically activated to products mutagenic to the bacteria to a greater extent than was benzo(a)pyrene. However, 7,8-dihydrobenzo(a)pyrene and 0,10-dihydrobenzo(e)pyrene were the best substrates for metabolic activation to bacterial mutagens. Highly purified epoxide hydrase added to a purified and reconstituted monooxygenase system readily abolished the mutagenic activity observed in strain TA100 of S. typhimurium when cyclopenta(c,d)pyrene was the substrate, but not when benzo(a)pyrene was the substrate. Inherent mutagenicity of several epoxides of the hydrocarbons generally paralleled the ability of their potential metabolic precursors to be activated to mutagens. 1-Pyrenyloxirane and 10,11-dihydrocycloheptapyrene 8,9-oxide were highly mutagenic in strains TA98 and TA100 of S. typhimurium, and in the former strain these activities were comparable to that observed with 9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, 4-Pyrenyloxirane was significantly less mutagenic than was 1-pyrenyloxirane in both strains of bacteria and in mammalian cells. Benzo(a)pyrene was over 20 times more tumorigenic than was cyclopenta-(c,d)pyrene, and it was the most potent of the 11 compounds tested for tumor-initiating activity in 2-stage initiation-promotion experiments on the skin of mice. Cyclopenta(c,d)pyrene had tumor-initiating activity comparable to that of benzo-(a)anthracene, but it was significantly less active than chrysene. Thus, contrary to inferences made from its high mutagenic activity, cyclopenta(c,d)pyrene is a weak tumor initiator on mouse skin.

Isolation of cDNA for an NADP-malic enzyme from Aloe arborescens.

NADP-malic enzyme catalyzes the reaction of decarboxylation from malate. In CAM plants, functions of this enzyme diverged to include both photosynthetic and non-photosynthetic roles. A full length cDNA for an NADP-malic enzyme was isolated from an 'obligate' CAM plant aloe (Aloe arborescens). The cDNA contains an ORF encoding 592 amino acid residues, whose sequence is highly homologous to the known plant NADP-malic enzymes. This gene is constitutively expressed in all organs in a low level. The amount of the transcript exhibited no diurnal variation, suggesting that this gene is not involved in photosynthetic functions.

An isozyme of the NADP-malic enzyme of a CAM plant, Aloe arborescens, with variation on conservative amino acid residues.

In Aloe arborescens, an obligate CAM plant, Western analysis detected three major isoforms of NADP-malic enzyme (NADP-ME), 72kDa with a pI of 6.0, 65kDa with a pI of 5.6 and 65kDa with a pI of 5.5. Among them, the 65kDa protein with a pI of 5.5 was leaf-specific, and the 65kDa protein with a pI of 5.6 was found only in roots, whereas the 72kDa protein was uniformly detected in both organs. Activity staining indicated enzyme activity of both 65kDa NADP-MEs but little activity of the 72kDa protein. A cDNA clone encoding a leaf-abundant NADP-ME, AME1, was isolated. Deduced amino acid sequence of AME1 showed a high degree of homology to known NADP-MEs, but it was also found that AME1 contained substitutions on five conservative amino acid residues, some of which have been predicted to be important for their enzyme activity. Transgenic rice carrying the aloe AME1 gene efficiently produced an additional 65kDa protein with a pI of 5.5 as an active NADP-ME. These results indicate that AME1 corresponds to the leaf-specific 65kDa NADP-ME, which may be involved in CAM photosynthesis. It was also shown that substitutions of these conservative amino acid residues identified in AME1 still allowed it to give enzyme activity.

Cloning of a glycine receptor subtype expressed in rat brain and spinal cord during a specific period of neuronal development.

Complementary (c) DNAs encoding a glycine receptor (GlyR) isomer were cloned from libraries constructed in lambda ZAPII with poly (A)+ RNA of neonatal rat spinal cord. Northern blot analysis revealed that RNA hybridized to the cloned cDNA is detectable only for a period of late embryonic/early postnatal stage of the spinal cord. Moreover, other central nervous tissues, such as hippocampus and cerebral cortex, in the infant rats are also rich in this message. The 'neonatal (N) GlyR' has 71% homology to that of another GlyR isoform in which adult rad cord is rich (AGlyR). Injection of a single RNA transcribed from the NGlyr-cDNA into Xenopus oocyte induced functional formation of glycine-gated Cl- channels, however, its pharmacological property differed from that of AGlyR.

GABAergic modulation of a substance P-mediated reflex of slow time course in the isolated rat spinal cord.

The effects of gamma-aminobutyric acid (GABA) and other drugs which interact with GABA receptors were studied on a reflex of slow time course in the spinal cord preparation isolated from the neonatal rat. A single shock to a dorsal root (L3-L5) elicited a stereotyped series of reflexes, consisting of fast and slow components, recorded from the contralateral ventral root of the corresponding segment. The slow component, i.e. the contralateral slow ventral root potential (v.r.p.) had a time-to-peak of 2-5 s and lasted 20-30 s. Bath-application of GABA (5-20 microM) or muscimol (0.05-0.5 microM) caused a decrease in the amplitude of the contralateral slow v.r.p. without producing any change in the d.c. potential recorded from the ventral root. The monosynaptic reflex recorded from the ipsilateral ventral root was not changed by the drugs at these concentrations. Diazepam (0.1-1 microM) potentiated the depolarizing response of the dorsal root to GABA and markedly depressed the contralateral slow v.r.p. Neither the d.c. potential of the ventral root nor the dorsal root was changed by diazepam. The monosynaptic reflex was also unaffected by the drug. Bicuculline (1 microM) suppressed the GABA-induced depolarization recorded from the dorsal root whilst it markedly potentiated the contralateral slow v.r.p. Baclofen at concentrations from 0.01 to 0.1 microM reduced the contralateral slow v.r.p. The inhibitory action of baclofen on the contralateral slow v.r.p. was more marked than on the monosynaptic reflex. 7 The depolarization of the ventral root induced by a brief application of substance P (SP) was depressed by muscimol, diazepam and baclofen, whereas the depolarization was potentiated by bicuculline. 8 The present results suggest that an intraspinal GABAergic inhibitory mechanism plays a role in the modulation of certain slow spinal reflexes. They also support the hypothesis that SP released from certain primary afferent fibres is a neurotransmitter involved in the contralateral slow v.r.p.

Identification of two taurine receptor subtypes on the primary afferent terminal of frog spinal cord.

1. Effects of taurine on primary afferent terminals in the frog spinal cord were examined by a sucrose-gap method applied to a dorsal root (9th or 10th segment). 2. In a normal Ringer solution, taurine (1 mM, applied for 5s at a rate of 0.04 ml s-1, 0.2 mumol) caused a hyperpolarization, but a higher concentration (10 mM, applied at the same rate, 2.0 mumol) caused a biphasic response consisting of a hyperpolarization followed by a slow onset depolarization. A similar biphasic response could also be observed in tetrodotoxin-treated preparations. 3. When the concentration of extracellular Mg2+ was increased up to 9.0 mM, the depolarizing response to taurine was augmented. The rate of the augmentation was dependent upon the extracellular Mg2+ concentration. 4. The depolarizing effect was selectively antagonized by bicuculline in concentrations (10-30 microM) that had no significant antagonizing action on gamma-aminobutyric acid (GABA)-induced depolarization. On the other hand the hyperpolarizing effect of taurine was selectively reduced by strychnine (0.1 microM) which had no antagonizing effect on responses to glycine. 5. These results suggest that in the frog spinal cord there are at least two subtypes of taurine receptor whose pharmacological profiles resemble GABA and glycine receptors in the mammalian central nervous system, and whose sensitivity may be modulated by extracellular Mg2+.

The role of substance P as a neurotransmitter in the reflexes of slow time courses in the neonatal rat spinal cord.

In order to reveal the spinal reflexes involving the transmitter action of substance P (SP), the effects of capsaicin and an SP antagonist on the isolated spinal cord of the neonatal rat studied. When a single shock stimulus was given to a dorsal root (L3-L5) or a sciatic nerve, depolarizing responses of various time courses were recorded extracellularly from both ipsi- and contra-lateral ventral roots of the corresponding segments. The reflex response recorded from the contralateral ventral root consisted of fast and slow components, which will be referred to as contralateral fast and slow ventral root potentials (v.r.ps). The latter contralateral slow v.r.p. had a time-to-peak of 2-5 s and lasted 10-30 s. The threshold for the contralateral slow v.r.p. was about two times higher than that for the monosynaptic reflex, and it coincided with the threshold for activating the slow-conducting afferent fibres. The contralateral slow v.r.p. was abolished after the spinal cord was treated with capsaicin (1 microM for 30 min) in vitro. The contralateral slow v.r.p. was absent in the spinal cord derived from 4-day-old rats that had received capsaicin (50 mg kg-1, s.c.) on the 2nd day of life. The contralateral fast v.r.p. and other reflexes of fast time courses remained unaltered after treatment with capsaicin in vitro or in vivo. Administration of an SP antagonist, [D-Arg1, D-Pro2, D-Trp7,9 Leu11]-SP in concentrations of 5-16 microM depressed the contralateral slow v.r.p., but did not affect the monosynaptic reflex, the dorsal root potential and the contralateral fast v.r.p. [D-Arg1, D-Pro2, D-Trp7,9, Leu11]-SP (5 microM) markedly depressed the SP-induced depolarizing response recorded from the ventral root whereas the responses to noradrenaline, 5-hydroxytryptamine, neurotensin and thyrotrophin releasing hormone (TRH) were unaffected by the SP antagonist. The response of the ventral root to acetylcholine was slightly depressed by the antagonist. The SP antagonist at 5-10 microM did not exert any agonist action on the motoneurones. The present results in conjunction with those of previous studies support the hypothesis that SP released from certain primary afferent fibres acts as a neurotransmitter, producing in dorsal horn neurones slow excitatory postsynaptic potentials which lead to the generation of the contralateral slow v.r.p.

Actions of 3-[2-phosphonomethyl[1,1-biphenyl]-3-yl]alanine (PMBA) on cloned glycine receptors.

1. PMBA is a novel antagonist of strychnine-sensitive glycine receptors in the rat spinal cord, however, its mode of action is unknown. The actions of PMBA on rat glycine receptor alpha1 and alpha2 homomers in Xenopus oocytes were studied under two-electrode voltage-clamp. 2. Co-application of PMBA and glycine to both alpha1 and alpha2 homomers yielded inward currents which decayed to a steady-state. Responses rose slowly to the same steady-state amplitude following a 2 min pre-incubation in PMBA. Strychnine, but not picrotoxinin, showed similar antagonism to PMBA. The potency of PMBA was independent of membrane potential between -100 and 0 mV. 3. When tested against EC50 concentrations of glycine, PMBA was almost equally potent on alpha1 (IC50, 406+/-41 nM: Hill coefficient, 1.5+/-0.2) and alpha2 (IC50, 539+/-56 nM; Hill coefficient, 1.4+/-0.2) homomers. 4. PMBA (1-I0 microM) and strychnine (200 nM) reduced the potency of glycine and the amplitude of the maximal agonist response of alpha1 and alpha2 homomers. In 10 microM PMBA, two distinct classes of glycine response were observed on alpha2, only a single class of responses were observed on alpha1. 5. There are similarities in PMBA and strychnine antagonism, although these compounds are structurally distinct. The possibility that PMBA interacts at two binding sites which differ in alpha1 and alpha2 subunits is discussed. PMBA may provide a lead structure for novel antagonists with which to investigate structural differences in glycine receptor at alpha1 and alpha2 subunits.

Down-regulation of glycine receptor channels by protein kinase C in Xenopus oocytes injected with synthetic RNA.

Interaction of protein kinase C (PKC) with glycine receptor channels was examined using Xenopus oocytes expressing homomeric alpha 1 glycine channels. 4 beta-Phorbol 12-myristate 13-acetate (4 beta-PMA), an activator of PKC, reduced the response to glycine; this effect was inhibited in the presence of staurosporine, a PKC inhibitor. By contrast, 4 alpha-PMA, a poor PKC stimulant, did not affect the glycine currents. Thus, the PKC system is involved in negative-regulation of the glycine receptor channels. The results obtained from experiments with mutant receptors suggest that phosphorylation of the intracellular serine residue at 419 may relate to modification of the channel function.

Mortality and survival for Minamata disease.

Analysis of mortality of 439 deaths that occurred among 1483 patients with Minamata disease (MD) in Kumamoto Prefecture, Japan was performed from the end of 1981. Causes of death and survival rates were studied by means of the standardized mortality ratio (SMR) and life-table technique. Of the 439 deaths (29.6%) in MD cases, the first death occurred in 1954. There was a first peak incidence in 1956 when MD was initially reported, however, the majority of deaths occurred after 1972 when a second and much larger peak was evident. In 1970 an important milestone occurred when the Public Nuisance Relief Law (an anti-pollution law) was enacted. Among its provisions, this law required and enabled verification of MD among people suspected of having been exposed. In contrast to the early cases, later cases of MD were older and their mean age-at-death was not different from that of the general population. The mortality rate for all causes of death was significantly higher in both sexes compared to the general population. Significantly lower survival rates were noted for older patients. The cause-specific mortality rates also showed significantly increased SMRs for liver diseases and nephritis-nephrosis-nephrotic syndrome in male patients, and for nephritis-nephrosis-nephrotic syndrome and other diseases in females. On the other hand, the SMR for senility without mention of psychosis was significantly lower than expected in both sexes.

Distribution patterns of mRNAs encoding glycine receptor channels in the developing rat spinal cord.

The developmental changes in the expression of mRNAs encoding the alpha 1 and alpha 2 subunits of inhibitory glycine receptors in the spinal cord of fetal and postnatal rats were examined by in situ hybridization. During embryonic periods (E11-18), the mantle zone was scarce in the alpha 1 mRNA, but the germinal zone (matrix layer) at E11-14 expressed higher levels of the message. At postnatal day 0 (P0), the alpha 1 signals became manifested throughout the gray matter of the spinal cord. The intensities of the signals were increased to reach a maximal level at P21. By contrast, the spinal tissues at P0 exhibited the highest levels of alpha 2 mRNA, which decreased with the postnatal development. In P50 rats, the alpha 2 mRNA was barely expressed in the ventral horn, but a significant number of grains could still be detectable in a population of cells in the dorsal horn. During postnatal development from P0 to P10, the spinal tissues were rich in the alpha 1 and alpha 2 mRNAs, both of which were detected in the presumed motoneurons. The coexistence of the two subunits in single neurons might correlate with the modification of the glycine receptor function during the development of the spinal cord.

Functional properties of strychnine-sensitive glycine receptors expressed in Xenopus oocytes injected with a single mRNA.

Mature rat spinal cord cDNA libraries constructed in lambda gt10 and lambda ZAPII were screened with an oligonucleotide probe (39 mer), and 4 clones that possess DNA-inserts encoding a glycine receptor subunit were obtained. The cloned cDNAs were used to reconstruct the nucleotide sequence of the full-length open reading frame consisting of 1350 base pairs (bp) as well as the 5'-(184 bp) and 3'-(591 bp) non-coding regions. Synthetic RNA transcribed in vitro from the glycine receptor cDNA induced Xenopus oocytes to synthesize functional glycine receptor that generated large Cl- currents. The electrophysiological properties of the wild-type receptor and some mutant receptors produced by site-directed mutagenesis were analyzed.

Pericardial hood repair of cardiac rupture secondary to extended myocardial infarction.

A surgical technique for simple and safe repair of oozing-type postinfarction cardiac rupture secondary to extended myocardial infarction is described. A hood-shaped pericardium was glued with gelatin-resorcinol and formaldehyde glue to cover the extended oozing infarcted myocardium. This technique was used on 3 elderly patients with good results.

Opposite actions of forskolin at pre- and postsynaptic sites in rat sympathetic ganglia.

In isolated rat superior cervical ganglia, forskolin, a powerful activator of adenylate cyclase, augmented the amplitude of fast excitatory postsynaptic potentials. Quantal analysis showed that forskolin acts presynaptically to facilitate the release of the transmitter. The time course of the presynaptic action of forskolin paralleled that of the increase in cyclic AMP level in the ganglia. In addition, forskolin exerted a postsynaptic action on the nicotinic acetylcholine receptor so that the acetylcholine-induced depolarization was depressed. The action of forskolin on the nicotinic acetylcholine receptor seems to be unrelated to the cyclic AMP system.

A potent metabotropic glutamate receptor agonist: electrophysiological actions of a conformationally restricted glutamate analogue in the rat spinal cord and Xenopus oocytes.

The (2S,3S,4S) isomer of alpha-(carboxycyclopropyl)glycine (L-CCG-I), a conformationally restricted glutamate analogue, caused a marked depolarization of motoneurons in the isolated rat spinal cord, which was almost insensitive to CPP and CNQX. Depolarizing responses to L-CCG-I were markedly decreased by reducing the temperature of the bathing fluid. Similar results were obtained in the case of trans-ACPD, which is a metabotropic glutamate receptor agonist, but the depolarizing action of L-CCG-I was more potent than that of trans-ACPD. In Xenopus oocytes injected with poly(A)+ mRNA extracted from the rat brain, L-CCG-I induced significant oscillatory chloride currents, suggesting that L-CCG-I is a potent agonist for metabotropic-type glutamate receptors.