Invertebrate connectin spans as much as 3.5 microm in the giant sarcomeres of crayfish claw muscle.

In crayfish claw closer muscle, the giant sarcomeres are 8.3 microm long at rest, four times longer than vertebrate striated muscle sarcomeres, and they are extensible up to 13 microm upon stretch. Invertebrate connectin (I-connectin) is an elastic protein which holds the A band at the center of the sarcomere. The entire sequence of crayfish I-connectin was predicted from cDNA sequences of 53 424 bp (17 352 residues; 1960 kDa). Crayfish I-connectin contains two novel 68- and 71-residue repeats, and also two PEVK domains and one kettin region. Kettin is a small isoform of I-connectin. Immunoblot tests using antibody to the 68-residue repeats revealed the presence of I-connectin also in long sarcomeres of insect leg muscle and barnacle ventral muscle. Immunofluorescence microscopy demonstrated that the two repeats, the long spacer and the two PEVK domains contribute to sarcomere extension. These regions rich in charged amino acids, occupying 63% of the crayfish I-connectin molecule, may allow a span of a 3.5 microm distance as a new class of composite spring.

Evaluation of high through-to-peak ratio of perindopril in SHR.

The present study was designed to evaluate trough-to-peak ratio (T/P) of ACE inhibitors in spontaneously hypertensive rats (SHR) by a continuous monitoring of ambulatory blood pressure for 24 hours with a biotelemetric system. Blood pressure was recorded uninterruptedly with a battery-operated transmitter connected to a sensor catheter. Perindopril (3 mg/kg), trandolapril (1 mg/kg), quinapril (10 mg/kg) and enalapril (6 mg/kg) were given once a day for 7 days. On the first day of the treatment these ACE inhibitors equally decreased blood pressure by 20 mmHg at each peak. The peak and trough blood pressure decreased steadily until day 4, and then they were constant until the end of experiment (day 7). T/P for each inhibitor also increased until day 4, and the ratios in systolic blood pressure at the end of experiments (day 7) were as follows, perindopril: 0.63, trandolapril: 0.62, quinapril: 0.41, enalapril: 0.27. The T/P of perindopril was significantly higher than that of enalapril. The results of the present studies testing four ACE inhibitors are well consistent with those in clinical trials. Thus, the measurement of T/P in SHR would provide a meaningful information for the evaluation of antihypertensive agents like ACE inhibitors.

Effect of blending tricalcium phosphate on hydrolytic degradation of a block polyester containing poly(L-lactic acid) segment.

The effect of blending tricalcium phosphate (TCP) on hydrolytic degradation of a new type of poly(L-lactic acid)/poly(ethylene:hexamethylene/sebacate) block polyester (60: 40 wt%) was studied. 100- and 250-microm film specimens blended with 0, 10, and 30 wt% TCP were immersed in phosphate buffered saline (pH 7.4) at 37 degrees C for up to 80-104 weeks. At appropriate intervals, water absorption, dry and wet tensile strength, molecular weight, and thermal properties of the specimens were measured by weighing, tensile strength testing, size exclusion chromatography, and differential scanning calorimetry, respectively. Some samples were characterized by 1H NMR spectroscopy. Blending of TCP with the block polyester was effective in retarding degradation. The blended TCP was thought to retard degradation for the most part by neutralizing the lactic acid oligomers produced by hydrolysis of the poly(lactic acid) part during the initial stage of degradation.

Asp278 of human beta-adrenergic receptor kinase 1 is essential for phosphorylation activity.

Asp278 of beta-adrenergic receptor kinase 1 (betaARK1) was suggested to play a key role in substrate recognition of beta2-adrenergic receptors in our previous study, in which a three-dimensional model of betaARK1 was studied in comparison with a crystal structure of PKA-PKI5-24 complex. In the present study, to confirm the molecular recognition mechanism at Asp278 of betaARK1, two mutants of betaARK1, D278R and D278A, were designed based on molecular modeling studies and produced by Sf-9 cells. As predicted by the molecular modeling study, the mutants showed no kinase activities while wild type betaARK1 phosphorylated beta2-adrenergic receptors in a concentration-dependent manner. These results strongly suggest the involvement of Asp278 in substrate recognition by betaARK1. The results also suggest a high reliability of the three-dimensional model of betaARK1.

A new natriuretic peptide isolated from cardiac atria of trout, Oncorhynchus mykiss.

Atrial and brain natriuretic peptides (ANP and BNP, respectively) are two cardiac natriuretic peptides (NPs) found in tetrapods from amphibians to mammals, whereas ANP and ventricular NP (VNP) have been identified in eel hearts. Because VNP has also been found in the rainbow trout ventricle, we attempted to isolate NP from trout cardiac atria in order to determine whether ANP and VNP are common cardiac NPs in teleosts. In the present experiments, we isolated VNP and a novel atrial NP consisting of 29 amino acid residues from the atria. This new trout NP exhibited similar sequence identity to mammalian ANP and BNP (50-60%). Its homology to eel ANP was low (52%) compared with high homology of trout and eel VNP (78%). Based on yield, the content of this new NP in trout atria may be even smaller than that of VNP. The new trout atrial NP exhibited low relaxant activity in the chick rectum (only 1/10 of that of trout VNP), and extremely low vasorelaxant activity in the rat aortic strip (only 1/400 of that of human ANP). However, the new trout NP was equipotent with trout VNP and human ANP in relaxing trout epibranchial artery. Based on the sequence similarity with other NPs and on atrial content, the new NP isolated from trout atria cannot yet be assigned to a known member of the NP family.

Role of bradykinin-NO pathway in prevention of cardiac hypertrophy by ACE inhibitor in rat cardiomyocytes.

To examine whether the bradykinin-nitric oxide (NO) pathway directly participates in the antihypertrophic property of angiotensin-converting enzyme (ACE) inhibitors in congestive heart failure, the effects of bradykinin were studied in rat cultured heart cells. Bradykinin (0.1, 1 nM) prevented the phenylephrine-induced increase in protein/DNA content, an index of hypertrophy of heart cells, and amplified the nitrite/nitrate content in the medium. Perindoprilat (1 microM), an ACE inhibitor, also restrained the progression of cardiac hypertrophy and augmented NO release. These effects of perindoprilat were abolished by HOE-140 (kinin B2 antagonist), N omega-nitro-L-arginine (NO synthase inhibitor), and methylene blue (guanylate cyclase inhibitor). Furthermore, there was a significant correlation between protein/DNA content and nitrite/nitrate content. These results indicate that bradykinin inhibits the progression of cardiac hypertrophy due to the increase in NO release and that perindoprilat produces beneficial effects on cardiac hypertrophy by stimulating the bradykinin-NO pathway.

B-type natriuretic peptide isolated from frog cardiac ventricles.

A new natriuretic peptide with 27 amino acid residues has been isolated from cardiac ventricles of the bullfrog, Rana catesbeiana. Since this ventricular peptide had high sequence identity to B-type (brain) natriuretic peptide (BNP), especially to chicken BNP (74%), we named it bullfrog BNP. Thus, semi-aquatic amphibians have tetrapod-type BNP, but do not seem to have fish-type ventricular natriuretic peptide (VNP) in their ventricles. Compared with other known BNPs, the C-terminus of bullfrog BNP was elongated by two amino acid residues and was not amidated. Bullfrog BNP dose-dependently decreased arterial blood pressure in the bullfrog with a potency twofold greater than that of human ANP. Bullfrog BNP also exhibited vasodepressor, natriuretic and diuretic activities in the rat, but it was 1/3, 1/7, and 1/17 as potent as human ANP in this mammalian species.

Pharmacological profile of semotiadil fumarate, a novel calcium antagonist, in rat experimental angina model.

1. The aim of the present study was to determine whether antianginal efficacy of semotiadil fumarate (SD-3211), a structurally novel calcium antagonist, is distinct from those of diltiazem, nifedipine and nisoldipine. 2. First, the duration of the inhibitory effects of semotiadil was compared with that of other Ca2+ antagonists in rat experimental angina evoked by vasopressin. Semotiadil (10 mg kg-1, p.o.) was effective for at least 9 h in the anginal model and those effects of semotiadil were longer-lasting than those of diltiazem (30 mg kg-1, p.o.), nifedipine (10 mg kg-1, p.o.), and nisoldipine (3 mg.kg-1, p.o.). 3. Second, the selectivity of actions of these Ca2+ antagonists for the coronary arteries and myocardium was evaluated in rat isolated perfused hearts. Diltiazem (10(-6) M) reduced cardiac contractility without inhibiting the elevation of perfusion pressure evoked by acetylcholine. Semotiadil (10(-7) M) significantly suppressed cardiac contractility and inhibited the coronary response to acetylcholine. In contrast, nifedipine (3 x 10(-9)-3 x 10(-8) M) and nisoldipine (3 x 10(-10)-10(-8) M) did not reduce cardiac contractility at concentrations which significantly inhibited the increase in perfusion pressure to acetylcholine. 4. The selectivity of semotiadil for coronary artery and myocardium is intermediate between diltiazem and dihydropyridines tested in the present study. 5. These findings suggest that semotiadil has an advantage of diltiazem, nifedipine, and nisoldipine in the treatment of angina with regard to long-lasting action and selectivity for coronary artery and myocardium.

Chemical structure of the carbohydrate moiety of fucose-rich glycopeptides from human pancreatic juice.

Human pancreatic juice, obtained from nine patients after partial excision of the pancreas for bile duct cancer, was fractionated in order to isolate its glycopeptides. Three glycopeptides were purified employing ion-exchange chromatography and gel filtration. All the glycopeptides were found to be free of sialic acid and galactosamine but to have an unusually high content of L-fucose. The chemical structures of the three glycopeptides were determined using 500-MHz [1H]-NMR spectroscopy. One of them, glycopeptide, GP-4, possessed a biantennary structure with three L-fucose residues. The second glycopeptide, GP-3, had a triantennary structure with four L-fucose residues, and the third one, G-2, had a tetra-antennary structure with five L-fucose residues. The chemical compositions of these glycopeptides, including the absence of sialic acid and the high L-fucose content, indicate that they represent a new class of glycopeptide present in the normal human pancreas.

Long-lasting ex vivo inhibition by perindopril of rat vascular response to angiotensin I.

The ex vivo effects of perindopril and enalapril, inhibitors of angiotensin-converting enzyme (ACE), were studied on rat aortae and perfused hearts to clarify the inhibition of vascular response to angiotensin I. The duration of the effects of these inhibitors was also studied. One hour after oral administration of perindopril (0.1-30 mg/kg) or enalapril (0.3-100 mg/kg), the aortae and hearts were isolated for the measurement of isometric force and coronary flow, respectively. In aortae, perindopril and enalapril dose-dependently inhibited the maximal contractions to angiotensin I (1-1000 nM). In isolated perfused hearts, the compounds inhibited the decrease in coronary flow induced by angiotensin I (100 ng). In other experiments, the inhibitory effects of perindopril lasted for 24 h in both aortae (3 mg/kg, p.o.) and hearts (10 mg/kg, p.o.). In contrast, the effects of enalapril disappeared within 6 h in aortae (3 mg/kg, p.o.) and 12 h in hearts (100 mg/kg, p.o.). These results demonstrate that oral administration of ACE inhibitors reduce the ex vivo vascular response to angiotensin I and suggest that perindopril is a longer-lasting inhibitor than enalapril on vascular contraction to locally generated angiotensin II.

Lack of correlation of hypotensive effects with prevention of cardiac hypertrophy by perindopril after ligation of rat coronary artery.

1. The present study was designed to test the hypothesis that beneficial effects of angiotensin converting enzyme (ACE)inhibitors are independent of a fall in blood pressure in rat experimental heart failure following coronary ligation. 2. The animals were assigned randomly to six groups; sham operation, controls subjected to coronary ligation (control), coronary ligation plus chronic treatment with ACE inhibitors at non- and hypotensive doses; perindopril (0.2 or 2 mg kg-1 day-1) or enalapril (2 or 20 mg kg-1 day-1) for three weeks starting one week after the ligation. 3. Systemic blood pressure was measured every week during the experiments. At the end of the treatments, cardiac function and heart weight (an index of myocardial hypertrophy) were determined. In the other animals, ACE activities in plasma and tissues including heart, kidney, lung and blood vessels were measured. 4. In the controls, cardiac ACE activity, weight of right ventricle and left ventricular end-diastolic pressure (LVEDP) were higher compared to those in the sham-operated animals four weeks after the coronary ligation. However, ACE activities were not changed in plasma, kidney, lung and aorta by ligation of the coronary artery. 5. The chronic treatment with perindopril at a dose of 0.2 mg kg-1 day-1 inhibited the increase in ACE activity in cardiac tissue and suppressed the right ventricular hypertrophy without affecting systemic haemodynamics. In contrast, enalapril at a dose of 20 mg kg-1 day-1, but not 2 mg kg-1 day-1, prevented the development of the right ventricular hypertrophy. Enalapril at 20 mg kg-1 day-1 also lowered systemic blood pressure. 6. There is no significant correlation between systemic blood pressure and right ventricular hypertrophy at the end of the treatment with perindopril (r = 0.06) or enalapril (r = 0.1).7. These findings demonstrate that perindopril, an ACE inhibitor, prevents cardiac hypertrophy without affecting systemic blood pressure in the rat with heart failure after coronary ligation, and suggest that selective augmentation of ACE activity in cardiac tissue is involved in the progression of hypertrophy in this model.