Effect of a myosin regulatory light chain mutation K104E on actin-myosin interactions.

Familial hypertrophic cardiomyopathy (FHC) is the most common cause of sudden cardiac death in young individuals. Molecular mechanisms underlying this disorder are largely unknown; this study aims at revealing how disruptions in actin-myosin interactions can play a role in this disorder. Cross-bridge (XB) kinetics and the degree of order were examined in contracting myofibrils from the ex vivo left ventricles of transgenic (Tg) mice expressing FHC regulatory light chain (RLC) mutation K104E. Because the degree of order and the kinetics are best studied when an individual XB makes a significant contribution to the overall signal, the number of observed XBs in an ex vivo ventricle was minimized to ∼20. Autofluorescence and photobleaching were minimized by labeling the myosin lever arm with a relatively long-lived red-emitting dye containing a chromophore system encapsulated in a cyclic macromolecule. Mutated XBs were significantly better ordered during steady-state contraction and during rigor, but the mutation had no effect on the degree of order in relaxed myofibrils. The K104E mutation increased the rate of XB binding to thin filaments and the rate of execution of the power stroke. The stopped-flow experiments revealed a significantly faster observed dissociation rate in Tg-K104E vs. Tg-wild-type (WT) myosin and a smaller second-order ATP-binding rate for the K104E compared with WT myosin. Collectively, our data indicate that the mutation-induced changes in the interaction of myosin with actin during the contraction-relaxation cycle may contribute to altered contractility and the development of FHC.

The spatial distribution of actin and mechanical cycle of myosin are different in right and left ventricles of healthy mouse hearts.

The contraction of the right ventricle (RV) expels blood into the pulmonary circulation, and the contraction of the left ventricle (LV) pumps blood into the systemic circulation through the aorta. The respective afterloads imposed on the LV and RV by aortic and pulmonary artery pressures create very different mechanical requirements for the two ventricles. Indeed, differences have been observed in the contractile performance between left and right ventricular myocytes in dilated cardiomyopathy, in congestive heart failure, and in energy usage and speed of contraction at light loads in healthy hearts. In spite of these functional differences, it is commonly believed that the right and left ventricular muscles are identical because there were no differences in stress development, twitch duration, work performance, or power among the RV and LV in dogs. This report shows that on a mesoscopic scale [when only a few molecules are studied (here three to six molecules of actin) in ex vivo ventricular myofibrils], the two ventricles in rigor differ in the degree of orientational disorder of actin within in filaments and during contraction in the kinetics of the cross-bridge cycle.

Possible changes in luminal surface charge densities of small intestine membrane in the 4-7.4 pH range exhibit varied influence on the absorption rate constants of the ionized and un-ionized species of sulfadiazine in rats.

The overall apparent first-order rate constants (Kab) of small intestinal absorption of sulfadiazine were determined in rats in situ at various pHs (4.01-7.42) of the recirculation fluids at 32 degrees. The purpose of the study was to verify our hypothesis that the rate-determining step for the absorption of sulfonamides involves the formation of an "activated complex" consisting of a transient association of the sulfonamide molecules with the surface protein of the microvillus membrane. The Kab versus Fu (where Fu is the fraction of un-ionized sulfadiazine at a given pH) profile prepared according to the equation Kab = Ki+Fu(Ku-Ki), where Ki and Ku are the first-order rate constants of absorption of the ionized and un-ionized species, respectively, was clearly resolvable into two linear segments: one based on the data in the 4.01-6.43 pH range and the other based on the data in the 6.43-7.42 pH range. In view of the fact that the combined molar concentration of aspartic acid and glutamic acid in the microvillus membrane protein is about twice that of arginine and lysine, the resolution of the data into two linear segments suggested that the negative charge density of the microvillus surface membrane protein in the 6.43-7.42 pH range is greater than that in the 4.01-6.43 pH range. The Ku and Ki values of sulfadiazine were determined in each pH range. It was noted that Ku was greater than Ki in each pH range, and that Ki in the 4.01-6.43 pH range was greater than that in the 6.43-7.42 pH range.(ABSTRACT TRUNCATED AT 250 WORDS)

Elucidation of the role of hydrophobic bonding in influencing intestinal absorption of model sulfonamides and revealing possible mechanism of drug absorption in rat model.

A recirculation technique was used to study the first-order kinetics of intestinal absorption of un-ionized sulfadiazine, sulfamerazine, and sulfamethazine in rats in situ at 32, 35, and 38 degrees C. The absorption rate constant (Kab) of each sulfonamide increased with increase in temperature and, at each temperature, Kab was the highest for sulfamethazine and the lowest for sulfadiazine. Applying the activated complex formation theory, the energy of activation (Ea), free energy of activation (delta F*), enthalpy of activation (delta H*), and entropy of activation (delta S*) of absorption were determined for the sulfonamides to gain some insight into the mechanism of their intestinal absorption. The high values of delta F* indicated that the barrier for sulfonamide absorption was great. For each drug, the value of delta H* was positive and that the delta S* negative. However, delta H* and delta S* were the highest for sulfamethazine and the lowest for sulfadiazine, thus revealing the influence of hydrophobic bonding in increasing Kab of the sulfonamides with the increase in methyl group content of their molecules. By considering the facts that (1) the microvillus membrane of the intestinal absorptive cells regulates the rate of passive absorption of drugs, (2) the microvillus membrane is rich in proteins, which are located external to the membrane and exposed to the intestinal fluid, and (3) hydrophobic bonding contributes to the activation parameters of absorption, it was postulated that the activated complex formed in the absorption process consisted of a transient association of the sulfonamide molecules with some protein component of the microvillus membrane.

Effects of chronic ethanol ingestion on tissue elimination-phase kinetics of procainamide in rats.

The effects of chronic ethanol ingestion on the pharmacokinetics of procainamide in various tissues were studied. Ethanol-treated rats received ethanol at 4 g/kg/day for an initial 7 days and then at 8 g/kg/day for the subsequent 21 days; control rats received isocaloric sucrose. After a single intravenous dose, the semilogarithmic procainamide concentration-time profiles observed in hearts and kidneys of both groups of rats were similar to the previously reported biexponential profiles of procainamide concentration in blood. This finding indicates a rapid distribution equilibrium of drug in both blood and these highly perfused tissues. The profiles of drug concentration in thigh muscle and fat of both groups of rats exhibited a drug-uptake phase during the initial 25-min period followed by a monoexponential decline in drug concentration. For all tissues, the slopes (beta values) of the curves of the drug concentration versus time were calculated on the basis of elimination-phase data, except for fat of control rats where the predominant elimination phase was not discernible. The beta values in hearts and thigh muscles of ethanol-treated rats were significantly higher than those in the corresponding tissues of control rats. These results are evaluated in light of previously reported effects of the same ethanol treatment on the distribution pharmacokinetics and the steady-state partition coefficients of the drug in these tissues. Possible mechanisms are proposed to account for these effects on the basis of the known diverse effects of chronic ethanol ingestion on the cellular compositions of individual organs and tissues.

Effects of chronic ethanol ingestion on pharmacokinetics of procainamide in rats.

Blood level studies were carried out in rats to determine the effects of chronic ethanol ingestion on the distribution pharmacokinetic parameters and tissue steady-state partition coefficients of procainamide. The ethanol-treated rats received 4g/kg of ethanol daily for 28 days in Treatment A and 4 g/kg of ethanol for an initial 7 days, followed by 8 g/kg of ethanol for the subsequent 21 days in Treatment B; the control rats received isocaloric sucrose in the respective groups. As determined from two-compartment analysis of the blood level data, both ethanol treatments significantly decreased the distribution clearance (CLd; k12Vdc) and the apparent first-order rate constant for drug transfer from the central compartment to the tissue compartment (k12) of procainamide without affecting the total body clearance of drug (CL) or the apparent volumes of distribution of drug in the body at steady state (Vdss) and at pseudo-equilibrium (Vd beta). Additionally, the apparent volume of distribution of the drug in the central compartment (Vdc) was 57-62% greater due to both ethanol treatments. Furthermore, the steady-state partition coefficients of the drug were found to be significantly lower in heart and kidneys and greater in fat of the ethanol-treated rats (Treatment B) as compared with those in the control rats. Possible mechanisms are proposed to account for these various effects in light of the known effects of chronic ethanol ingestion on the chemical composition of cell membranes of tissues and organs.

Influence of temperature and hydrophobic group-associated icebergs on the activation energy of drug decomposition and its implication in drug shelf-life prediction.

The Ea values of aspirin hydrolysis, as a result of hydronium-ion catalysis, intramolecular-nucleophilic catalysis, and hydroxyl-ion catalysis, were significantly different from each other when determined in the 30-40, 45-55, and 60-70 degrees C ranges. The different Ea values were attributed to differences in both delta H* and delta S*, which could be accounted for by the various activated complexes formed in the hydrolysis of aspirin for each mechanism and the disruptive effect of temperature on the iceberg structures of water present around the phenyl group and the methyl group of aspirin at 42 and 58 degrees C, respectively. A linear relationship observed between the calculated "differential" enthalpy and entropy values, with a slope (compensation temperature) value of about 307 degrees K, supported a role for icebergs associated with hydrophobic groups in the formation of the activated complexes. This study illustrates that the predicted shelf life of a drug at room temperature could be erroneous if estimated from a single Ea value which is calculated from the decomposition rate constants determined at widely spaced temperatures in the range of 10-70 degrees C, using the Arrhenius relationship.

Effects of phenobarbital on the distribution pharmacokinetics and biological half-lives of model nonmicrosomal enzyme metabolizable sulfonamides in rats.

The pharmacokinetics of sulfisoxazole and sulfanilamide were studied in control rats and in rats treated for 5 days with a daily 100 mg/kg ip dose of phenobarbital. These drugs represent the organic anionic and nonionized drugs, respectively, whose nonmicrosomal enzymatic metabolisms were unstimulated by phenobarbital. Sulfisoxazole showed the characteristics of a two-compartment open model. However, its biological half-life and the apparent distribution volume of the central compartment were significantly lower and the intercompartmental transport rate constants and the urinary excretion rate constant were significantly greater, in phenobarbital treated rats than in control rats. The apparent steady-state distribution volume of sulfisoxazole was smaller in the phenobarbital treated rats at the 90% confidence level. Sulfanilamide showed characteristics of a one-compartment model in both the control and phenobarbital treated rats, but none of the pharmacokinetic parameters of the compound in the phenobarbital treated rats were significantly different from those in the control rats.

Pharmacokinetic evidence for possible renal accumulation of model organic anions in rats.

The urinary excretion and blood level kinetics of p-methylbenzoylformic acid (I) after intravenous infusion for 1 hr were studied in rats. The determined first-order half-lives were compared with those determined in studies in which a single intravenous dose of I was administered rapidly to rts that previously were infused with normal saline for 1 hr. While the blood t 1/2 or body clearance of I determined in the 1-hr infusion studies was similar to that determined in the single intravenous dose studies, the urinary of t 1/2 of I determined in the 1-hr infusion studies was significantly greater than that determined in the single intravenous dose studies. In infusion studies where the half-lives of I were determined in the presence of renal tubular secretion inhibitor, DL-tropic acid (II), the ratio of (urinary t 1/2)II present/(urinary t 1/2)II absent was almost twice the ratio of (blood t 1/2)II present/(blood t 1/2)II absent. The urinary t 1/2 value of I determined after infusion for only 10 min was intermediate between values obtained in the single intravenous dose studies and the 1-hr infusion studies. These data provide pharmacokinetic evidence to support the hypothesis that I and other organic anions temporarily accumulate in the surface-lying renal tubular cells after a single intravenous dose, but they tend to penetrate into the deeper renal tubular cells upon intravenous infusion, with depth of penetration increasing with increasing infusion time.

Rationale for apparent differences in pharmacokinetic aspects of model compounds determined from blood level data and urinary excretion data in rats.

Results of studies carried out in rats for model compounds, D-(-) mandelic acid, benzoylformic acid, and some of their para-alkylated homologs, showed that their biological half-lives determined from the elimination phase of urinary excretion data were longer than those determined from the elimination phase of blood level data. With compounds that followed multicompartment open models, the initial distributive phase (alpha-phase) noted from the blood level data was not detected from the urinary excretion data. Based on the analysis of half-life data obtained in the absence and presence of DL-tropic acid (a competitive renal tubular secretion inhibitor of these compounds), it is proposed that, besides the shortness of the alpha-phase period, the factor accounting for these compounds is their retention and/or detention in the renal tubular membranes during their tubular secretion. Furthermore, it is proposed that the renal tubular membranes do not constitute a part of the central or peripheral compartment. Examples are cited to show that, where studies are reported for the same drugs in the same human subjects in the same laboratory, drugs usually exhibit longer biological half-lives when urinary excretion data rather than blood level data are used.

Utilization of model compounds to evaluate effects of slight chemical modifications on their distribution. Pharmacokinetic parameters in rats and mechanisms inferred for their transmembrane transport.

The pharmacokinetics of each of the model compounds benzoylformic acid (I), p-methylbenzoylformic acid (II), p-ethylbenzoylformic acid (III), D-(-)-mandelic acid (IV), D-(-)-p-methylmandelic acid (V), D-(-)-p-ethylmandelic acid (VI), and D-(-)-p-isopropylmandelic acid (VII) were studied in rats to determine the influence of slight chemical modifications of the compounds on their distribution pharmacokinetic parameters in rats. The effects of the specific chemical modifications considered were those of the less than CHOH group of IV against the less than C=0 group of I, the para-alkylation of I and IV, and the branched alkyl group (isopropyl) against the straight chain alkyl groups of the homologs of IV. While the disappearance of I from the blood was describable by the three-compartment open model, that of IV was describable by the two-compartment open model. The apparent volume of distribution of the central compartment (V1) for IV was smaller than that for I, but the volume of the peripheral compartment (V2) for IV was greater than that (V2 + V3) for I. The disappearance of V, VI, and VII from the blood was also describable by the two-compartment open model, but the apparent V1 and V2 for these compounds were lower than those for the parent compound, IV. However, the disappearance of II and II from the blood was describable by a one-compartment open model. Evaluation of the appropriate distribution pharmacokinetic parameters suggested that the peripheral compartment for the anions of these compounds consisted of moderately perfused tissues and that the transmembrane transport of these organic anions between the central and peripheral compartments occurs by diffusion mainly through the aqueous membrane pores, which are lined with polar portions of membrane proteins and/or phospholipids. The possible increased hydrophobic bonding between the alkyl groups of these compounds and the hydrophobic groups of the proteins and/or phospholipids of the membrane pores is implicated to decrease the distribution of the para-alkylated homologs into the peripheral compartments and, consequently, diminish the volumes of their peripheral compartments. The heteroporosity of the membranes of the tissues of the central compartment is proposed as the reason for the diminished volume of the central compartment for V and VI as compared to that of IV or VII.