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Coronary artery occlusion produces myocardial acidosis, which can be attenuated by propranolol or sotalol. The present study was undertaken to determine which beta adrenoceptors, beta-1 or beta-2, contribute to the ischemic myocardial acidosis. Dogs anesthetized with pentobarbital were used. In the first series of experiments, blood flow in the left anterior descending coronary artery was reduced by an occluder to about one-third of the original flow. Myocardial pH was measured by means of a micro pH electrode inserted into the left anterior descending coronary artery area at the depth of about 8 mm. The myocardial pH decreased from 7.44 to 7.55 to 6.73 to 6.89, 30 min after partial occlusion being the time immediately before an i.v. injection of drugs. Atenolol (1 mg/kg) attenuated significantly the decrease in myocardial pH that had been induced by partial occlusion, whereas IPS 339 (360 micrograms/kg) and ICI 118,551 (300 micrograms/kg) did not. The pH effect of atenolol was confirmed even in the paced heart. In the second series of experiments, the antagonistic action of these drugs on the isoproterenol-induced increase in heart rate and myocardial contractile force and the decrease in diastolic blood pressure was investigated. By this experiment, it was confirmed that atenolol has the beta-1 adrenoceptor antagonistic action, and the IPS 339 and ICI 118,551 have the beta-2 antagonistic action. These results suggest that the activation of beta-1 adrenoceptors contribute to the myocardial acidosis during ischemia.

We further characterized the cryptic plasmid pVA318 of Streptococcus mutans. It had a contour length of 5.64 +/- 0.26 kilobases and a guanine-plus-cytosine content of 32 to 34 mol %. Upon cloning the pVA318 plasmid into the vector pBR322 in Escherichia coli, we made the following observations. The expression of tetracycline resistance by HindIII-cloned chimeras, where the insert was in the tetracycline resistance promotor, depended on the orientation of the pVA318 insert. Both HindIII-cloned chimeras segregated from polA(Ts) cells at a nonpermissive temperature. Chimeric molecules cloned with PstI initially showed much instability; the reason for this is unknown, although stable variants were obtained. Both HindIII-cloned variants and a PstI-cloned chimera produced a pVA318-specific protein of approximately 20,000 molecular weight in E. coli minicells. The biological function of this protein is not known; it had no bacteriocin activity against S. mutans or group A Streptococcus indicator strains, and it did not appear in the E. coli periplasm. We constructed a map of pVA318 for restriction endonucleases HindIII, HpaI, PstI, and HaeIII. A previously reported BamHI site in pVA318 did not appear in the pVA318 portion of any of our chimeric clones.

Hemagglutinin is a major glycoprotein of Porphyromonas gingivalis vesicles and likely confers the ability to adsorb and penetrate into host tissue cells. To protect this bacterial invasion, murine monoclonal antibody (MAb) Pg-vc, which inhibited the hemagglutinating activity, was prepared by using P. gingivalis vesicles as an antigen. Western blot analysis revealed that when both MAb Pg-vc and anti-HA-Ag2 antibody raised against the P. gingivalis hemagglutinin adhesin (M. Deslauriers and C. Mouton, Infect. Immun. 60:2791-2799, 1992) were allowed to react with protein blots from P. gingivalis vesicles, a superimposable profile was observed. To obtain a recombinant antibody, cDNAs coding for the variable domains of the L and H chains of MAb Pg-vc were cloned by PCR, and a plasmid specifying a single-chain variable fragment (ScFv) was constructed. Following transformation of Escherichia coli cells, a recombinant ScFv protein was successfully expressed. The immunological properties of this protein were identical to those of the parental murine MAb, specifically recognizing the two proteins (43 and 49 kDa) originating from P. gingivalis vesicles. In addition, the ScFv antibody inhibited the P. gingivalis vesicle-associated hemagglutinating activity. The amino acid sequences deduced from nucleotide sequencing experiments confirmed that variable heavy-chain and variable light-chain regions belonged to VH1 and Vkappa12/13 families, respectively. Since the expression system used in this study can readily provide large quantities of single-chain recombinant antibody, it may be a useful in developing a therapeutic agent for passive immunization in humans.

Effects of four photodegradation products of nifedipine (nitroso-, nitro-, azoxy- and N,N'-dioxide-derivatives) were studied in the rat aortic strip suspended in the bath medium. In the CaCl2-free bath medium containing 40 mM KCl, CaCl2 (2.5 mM) induced contraction, which was relaxed by nifedipine (-log [EC50] = 8.66 M) and all the four photodegradation products (-log[EC50] = 4.15-4.82 M). The EC50 values for all these substances were not affected by denuding the vessel of endothelium. In the bath medium containing KCl (15 mM), Bay K-8644 (2 x 10(-6) M) induced contraction, which was relaxed by nifedipine (10(-9)-10(-6) M) and its four photodegradation products (3 x 10(-6)-10(-4) M). Norepinephrine (10(-7) M) induced contraction, which was relaxed by the nitroso-derivative (10(-4) M) noncompetitively (pIC50 = 4.37) but not by three other photodegradation products. Both methylene blue and hemoglobin had no effect on the relaxation produced by the nitroso-derivative. Phorbol 12-myristate 13-acetate induced contraction, which was inhibited completely by the nitroso-derivative (10(-4) M) but only by 42% by nifedipine (10(-4) M). Prostaglandin F2 alpha (3 x 10(-6) M), endothelin-1 (10(-9) M) and 5-hydroxytryptamine (10(-6) M) also induced contraction, which was relaxed effectively by the nitroso-derivative (10(-4) M) but not by any of the three other photodegradation products.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of dilazep, a potentiator of the adenosine-mediated effects, on the palmitoyl-L-carnitine (PALCAR)-induced mechanical derangement was studied in the isolated rat heart and compared with that of K-7259, a dilazep derivative having less potentiating action on the adenosine-mediated effects. The heart was perfused aerobically by the Langendorff's technique at a constant flow and driven electrically. PALCAR (5 microM) decreased the left ventricular developed pressure and increased the left ventricular end diastolic pressure in the heart (i.e., mechanical dysfunction). These mechanical alterations induced by PALCAR were attenuated by dilazep (1 microM) and K-7259 (1 microM). In contrast, adenosine (10 or 100 microM) did not attenuate the PALCAR-induced mechanical derangement. On the other hand, neither dilazep nor K-7259 modified the mechanical function of the normal (PALCAR-untreated) heart. These results suggest that dilazep and K-7259 attenuate the PALCAR-induced mechanical derangement and that the protective action of both drugs is not due to potentiation of adenosine-mediated effects.

The effect of lidocaine on ischemic myocardial acidosis was investigated in the dog heart, in which the left anterior descending coronary artery was occluded to reduce to about one-third (partial occlusion). Myocardial pH (MpH) was measured by means of a micro glass pH electrode. MpH before partial occlusion was 7.52 to 7.66. Partial occlusion decreased the left anterior descending coronary artery flow by 49 to 68%, MpH by 0.58 to 0.76 and myocardial contractile force by 26 to 43%, and increased ST segment (surface electrocardiogram) by 3.2 to 11.7 mV. Lidocaine (injected i.v. 30 min after partial occlusion) decreased heart rate, blood pressure and myocardial contractile force, and attenuated the decrease in MpH during ischemia. Lidocaine in doses of 2, 5 and 10 mg/kg restored the myocardial [H+], that had been increased by partial occlusion, by 23, 38 and 50%, respectively. Even in the paced heart, lidocaine (10 mg/kg) attenuated the myocardial acidosis, although the degree of attenuation was smaller (36%). Partial occlusion elevated the ST segment even in the presence of 5 or 10 mg/kg of lidocaine. In the nonischemic heart, however, lidocaine (2, 5 or 10 mg/kg) did not change in MpH. It is concluded that lidocaine attenuates the myocardial acidosis during ischemia, and the primarily important mechanism of pH attenuation is not a decrease in heart rate.

Effect of diltiazem on subcellular distribution of lysosomal enzymes, high-energy phosphate metabolism and mechanical function in the ischemic heart was studied. Ischemia was induced by lowering the afterload pressure of the perfused working rat heart. The activities of cathepsin D, beta,N-acetylglucosaminidase and acid phosphatase were determined in the nonsedimentable and sedimentable fractions after centrifugation of the tissue extract to assess the subcellular distribution of lysosomal enzymes. After ischemia, decreases in the mechanical function and the tissue level of high-energy phosphates were observed. In addition, ischemia caused subcellular redistribution of lysosomal enzymes from the lysosomes to the cytoplasm. Reperfusion of the ischemic heart did not restore the mechanical function and the level of high-energy phosphates completely. Diltiazem (2.21 X 10(-6), 1.11 X 10(-5) and 2.21 X 10(-5) M) was provided for the heart 5 min before the onset of ischemia. Diltiazem preserved high-energy phosphates in the ischemic heart, and inhibited the subcellular redistribution of lysosomal enzymes being caused by ischemia, depending on its concentration. Reperfusion after ischemia with diltiazem recovered the mechanical function that had been decreased by ischemia. These results may indicate that diltiazem can protect the myocardium against ischemic damage.

The effect of diltiazem on ischemic myocardial acidosis was studied in the canine heart whose left anterior descending coronary artery was partially occluded to reduce coronary flow to about one-third (partial occlusion). Myocardial pH was measured by use of a micro glass pH electrode. The pH decreased from 7.53 to 6.93 by partial occlusion and appeared to reach a steady state within 30 min. Saline or drugs were injected i.v. 30 min after partial occlusion. The decreased pH increased spontaneously by 34% of the total reduction of pH in the next 60 min after saline injection. Diltiazem (100 micrograms/kg) potentiated the increase in pH; the pH increased by 76% 60 min after the injection. Propranolol (1 mg/kg) also potentiated the increase in pH that had been decreased by partial occlusion. The relation between pH decrease and the tissue levels of metabolites was also studied. The reduction of myocardial pH from 7.5 to 6.8 was accompanied by a 2-fold increase in the tissue lactate content and by decreases in the ATP and creatine phosphate contents. There was a significant correlation between the hydrogen ion concentration calculated from the pH and each of the lactate, ATP and creatine phosphate contents. The present study indicates that diltiazem attenuates ischemic myocardial acidosis.

The effect of diltiazem (d-cis-diltiazem) on the ischemic myocardium was compared with that of l-cis-diltiazem, an optical isomer having less potent calcium channel-blocking action, in the isolated, perfused working rat heart. Ischemia decreased mechanical function and tissue levels of ATP and creatine phosphate, and increased tissue levels of nonesterified fatty acids (NEFA), AMP and lactate. Reperfusion did not restore mechanical function, but restored incompletely the levels of metabolites (except NEFA) that had been altered by ischemia. The ischemia-induced changes in NEFA were prevented by d-cis-diltiazem completely and by l-cis-diltiazem incompletely. Other metabolic changes induced by ischemia were attenuated by d-cis-diltiazem but not by l-cis-diltiazem. In heart pretreated with d-cis- or l-cis-diltiazem, both the mechanical function and the levels of metabolites recovered during reperfusion, the degree of recovery with both drugs being similar. These results indicate that not only d-cis-diltiazem but also l-cis-diltiazem has an anti-ischemic action probably due to inhibition of the tissue NEFA accumulation. These results also suggest that the mechanism of the protective effect of d-cis-diltiazem on the ischemic myocardium is not entirely due to the calcium channel-blocking action. Treatment with low Ca2+ (1.0 mM CaCl2) also attenuated the ischemia-induced changes. The interval between reoxygenation and start of function in the reperfused heart that had been treated with low Ca2+ was significantly longer than that with d-cis- or l-cis-diltiazem. The effect of these isomers to shorten this interval may contribute to their common anti-ischemic action.

The effects of dl-propranolol, D-(-)-(4-nitrophenyl)-2-isopropylaminoethanol (INPEA) and L-(+)-INPEA on the hemodynamic and metabolic responses to isoproterenol were investigated in dogs anesthetized with pentobarbital. Isoproterenol (200 micrograms/kg i.p.) was injected 15 min after an i.v. injection of propranolol or INPEA, or their dextro-isomers. Isoproterenol increased heart rate, myocardial contractile force (determined by a strain gauge arch), blood glucose, blood lactate and plasma free fatty acids, and decreased diastolic blood pressure. dl-Propranolol (1 or 3 mg/kg) or D-(-)-INPEA (5 or 15 mg/kg) inhibited all the responses to isoproterenol. d-Propranolol (1 or 3 mg/kg) did not inhibit the isoproterenol-induced responses of heart rate, myocardial contractile force and plasma free fatty acids, but it inhibited the isoproterenol-induced responses of diastolic blood pressure, blood glucose and blood lactate, although the inhibition was small. Similar results were obtained by L-(+)-INPEA; L-(+)-INPEA (5 or 15 mg/kg) did not inhibit the isoproterenol-induced responses of heart rate, myocardial contractile force and plasma free fatty acids, but it inhibited the isoproterenol-induced responses of diastolic blood pressure, blood glucose and blood lactate, although the degree of inhibition was small. These results indicate that dextro-isomers of propranolol and INPEA may have a weak beta-2 adrenoceptor antagonistic action.