Ca2+-antagonists inhibit the N-methyltransferase-dependent synthesis of phosphatidylcholine in the heart.

Evidence indicates that, in addition to the L-type Ca2+ channel blockade, Ca2+-antagonists target other functions including the Ca2+-pumps. This study was conducted to test the possibility that the reported inhibition of heart sarcolemmal (SL) and sarcoplasmic reticular (SR) Ca2+-pumps by verapamil and diltiazem could be due to drug-induced depression of phosphatidylethanolamine (PE) N-methylation which modulates these Ca2+-transport systems. Three catalytic sites individually responsible for the synthesis of PE monomethyl (site I), dimethyl (site II) and trimethyl (phosphatidylcholine (PC), site III) derivates were examined in SL and SR membranes by employing different concentrations of S-adenosyl-L-methionine (AdoMet). Total methyl group incorporation into SL PE, in vitro, was significantly depressed by 10(-6)-10(-3) M verapamil or diltiazem at site III. The catalytic activity of site I was inhibited by 10(-3) M verapamil only, whereas the site II activity was not affected by these drugs. The inhibition induced by verapamil or diltiazem (10(-5) M) was associated with a depression of the Vmax value without any change in the apparent affinity for AdoMet. Both drugs decreased the SR as well as mitochondrial PE N-methylation at site III. A selective depression of site III activity was also observed in SL isolated from hearts of rats treated with verapamil in vivo. Furthermore, administration of [3H-methyl]-methionine following the treatment of animals with verapamil, reduced the synthesis of PC by N-methyltransferase. Verapamil also depressed the N-methylation-dependent positive inotropic effect induced by methionine in the isolated Langendorff heart. Both agents depressed the SL Ca2+-pump and although diltiazem also inhibited the SR Ca2+-pump, verapamil exerted a stimulatory effect. In addition, verapamil decreased SR Ca2+-release. These results suggest that verapamil and diltiazem alter the cardiac PE N-methyltransferase system. This action is apparently additional to the drugs' effect on L-type Ca2+ channels and may serve as a biochemical mechanism for the drugs' inhibition of the cardiac Ca2+-pumps and altered cardiac function.

Sequence of alterations in subcellular organelles during the development of heart dysfunction in diabetes.

Although changes in different subcellular organelles such as myofibrils, sarcoplasmic reticulum (SR), mitochondria and sarcolemma (SL), as well as in heart function have been reported to occur in chronic diabetes, their inter-relationships and functional significance are poorly understood. In order to gain information on this aspect, diabetes in rats was induced by an intravenous injection of streptozotocin and animals were assessed hemodynamically at 15-27 days. Ventricular tissue from several diabetic animals was pooled, subcellular organelles were isolated and their biochemical activities determined. Significant depressions in cardiac contractile and relaxation were observed to be associated with decreases in myofibrillar Ca(2+)-stimulated ATPase and SR Ca(2+)-pump activities at 21 days from the induction of diabetes. Likewise, the SL Na+-Ca2+ exchange and Ca(2+)-channel density were decreased at 21 days but the affinity of SL Ca(2+)-channels was increased in the diabetic heart. The SL Ca(2+)-pump and Na+-K+ ATPase activities were depressed at 18 and 24 days, respectively. Both alpha- and beta- adrenoceptor densities in SL were decreased at 27 days whereas no changes in mitochondrial function were observed at these early stages of diabetes. The SL low affinity Ca(2+)-binding was decreased while the low affinity Ca(2+)-ATPase activity was increased at 18 days following the induction of diabetes. These results indicate that SL defects precede those in SR, myofibrils or mitochondria and suggest that abnormalities in Ca(2+)-handling as well as interaction of Ca2+ with myofilaments in cardiomyocytes may lead to the development of heart dysfunction in chronic diabetes.

Mechanisms of inotropic responses of the isolated rat hearts to vanadate.

In view of the invariable development of insulin resistance in different types of cardiovascular diseases, considerable attention has been focused on vanadate because of its ability to exert insulin-like effects in the body. Since vanadate, like insulin, has been shown to exert a beneficial effect in diabetic cardiomyopathy, this study was undertaken to examine the mechanisms of its action on the heart. Vanadate, at 5-10 microM concentrations, produced a positive inotropic effect in the isolated perfused rat heart, whereas at higher concentrations (20 microM), it decreased the contractile force development. The positive inotropic effect of 10 microM vanadate was not affected by the pretreatment of animals with reserpine as well as the presence of propranolol or phenoxybenzamine in the perfusion medium. The increase in contractile force development due to vanadate at low (0.3-0.6 mM) concentrations of Ca2+ was markedly augmented, but this agent produced a negative inotropic action at high concentrations of Ca2+ (2.0-3.0 mM). Preperfusion of hearts with verapamil enhanced the positive inotropic effect of vanadate whereas hearts preperfused with ouabain, low sodium or amiloride showed negative inotropic effects of vanadate. Vanadate was found to inhibit sarcoplasmic reticular Ca(2+)-pump and sarcolemmal Ca(2+)-pump as well as Na(+)-K(+)-ATPase activities but the sarcolemmal effects were evident at lower concentrations in comparison to that on the sarcoplasmic reticulum. The actions of vanadate on membrane Ca2+ transport and ATPase systems were specific since this agent exerted no effect on sarcolemmal Na(+)-Ca2+ exchange or myofibrillar ATPase activities. In isolated cardiomyocytes suspended in buffer containing 0.5 or 1.0 mM Ca2+, vanadate increased the intracellular concentration of Ca2+; this increase in intracellular Ca2+ was more pronounced at 0.5 mM Ca2+. These results indicate that increased intracellular concentration of Ca2+ due to inhibition of sarcolemmal Na(+)-K(+)-ATPase and sarcolemmal Ca(2+)-pump may be the primary mechanism of the positive inotropic action of vanadate in the heart. It is suggested that vanadate may serve as an inotropic agent and that this mechanism may contribute towards its beneficial effects on cardiac dysfunction in different cardiovascular diseases.

Characterization of adenylyl cyclase in heart sarcolemma in the absence or presence of alamethicin.

Alamethicin is commonly used as an agent for unmasking the latent enzyme activities in vesicular membrane preparations; however, relatively little is known about the effect of this agent on the characteristics of adenylyl cyclase in heart sarcolemma. By employing rat heart sarcolemmal preparation, we observed 5 to 6 fold increase in adenylyl cyclase activity upon treatment with alamethicin. Kinetic experiments using various concentrations of MgATP revealed that the increase in adenylyl cyclase activity in alamethicin treated membranes was associated with an increase in Vmax as well as affinity of the substrate for the enzyme. Dose-responses of the control and alamethicin-treated preparations to various activators of adenylyl cyclase revealed that the sensitivity of the enzyme to forskolin, NaF and GppNHp, was markedly increased upon treating sarcolemma with alamethicin. The activation of adenylyl cyclase by forskolin was also enhanced by increasing the concentration of alamethicin in the incubation medium. Furthermore, there was a greater increase in adenylyl cyclase activity with different concentrations of Mn2+ in the presence of alamethicin. These results suggest that alamethicin treatment alters the characteristics of adenylyl cyclase in addition to unmasking the enzyme activity in the purified sarcolemmal vesicular preparation.

Effects of some L-carnitine derivatives on heart membrane ATPases.

In order to understand the role of carnitine metabolites in the genesis of cellular dysfunction and damage due to myocardial ischemia, the effects of 1-100 microM L-carnitine, acetylcarnitine, propionylcarnitine, and palmitoylcarnitine were investigated on rat heart sarcolemmal, sarcoplasmic reticular, and mitochondrial ATPase activities. Palmitoylcarnitine, unlike acetylcarnitine, propionylcarnitine and carnitine, produced marked inhibitory actions on sarcolemmal Na,K-ATPase and Ca2(+)-stimulated ATPase, as well as sarcoplasmic reticular Ca2(+)-stimulated ATPase activities; Na,K-ATPase was most sensitive. Although palmitoylcarnitine, unlike carnitine or its short-chain fatty-acid derivatives, also depressed sarcolemmal Ca2+ ATPase or Mg2+ ATPase, sarcoplasmic reticular Mg2+ ATPase, and mitochondrial Mg2+ ATPase, mitochondria were less sensitive in comparison to other organelles. Myofibrillar Ca2(+)-stimulated ATPase was slightly inhibited by very high concentrations of palmitoylcarnitine only. It is suggested that the observed depression of the sarcolemmal Na(+)-pump system by low concentrations of long-chain acyl derivatives of carnitine may contribute towards the pathogenesis of arrhythmias due to myocardial ischemia. Furthermore, the inhibition of Ca2(+)-pump mechanisms in the sarcolemmal and sarcoplasmic reticular membranes by relatively high concentrations of palmitoylcarnitine may result in the occurrence of intracellular Ca2+ overload and subsequent cell damage, as well as cardiac dysfunction due to myocardial ischemia.

Depression in cardiac contractile force induced by cholesterol.

Cholesterol, combined with albumin, induced rapid functional changes in the isolated perfused rat heart. Under constant perfusion pressure, the decline in contractile force and the rate of development of force due to cholesterol was accompanied by an increase in resting tension and a decrease in coronary flow. Such effects were not produced by albumin alone. Depression in contractile force and increase in resting tension were also evident when coronary flow was kept constant; these effect of cholesterol were dependent on time and dose. The findings support the view that cholesterol may affect cardiac function independently of the atherosclerotic lesion in the hypercholesterolemic state.

Stimulation of phospholipid N-methylation by isoproterenol in rat hearts.

Phosphatidylethanolamine (PtdEtn) N-methyltransferase activities were studied in rat heart sarcolemmal and sarcoplasmic reticular fractions after a single intraperitoneal injection of isoproterenol (0.5-5.0 mg/kg). Three active sites (I, II, and III) for PtdEtn N-methylation were assayed by measurement of [3H]methyl group incorporation from 0.055, 10, and 150 microM S-adenosyl-L-[methyl-3H]methionine into membrane PtdEtn molecules. Total methylation activity for catalytic site I of both sarcolemma and sarcoplasmic reticulum was stimulated within 2 minutes by isoproterenol in a dose-dependent manner. Although the increased methyltransferase activity in sarcoplasmic reticulum was normalized at 10 minutes, the enzyme activity in sarcolemma was normalized at 5 minutes but was again increased at 10-30 minutes after isoproterenol injection. No changes in response to isoproterenol were seen for site II and III N-methylation activities in either membrane. Individual N-methylated phospholipids (phosphatidyl-N-monomethylethanolamine, phosphatidyl-N,N-dimethylethanolamine, and phosphatidylcholine), which specifically formed at each site, showed similar behavior. Pretreatment of the animals with a beta-blocking drug, atenolol, for 2 days prevented the isoproterenol-induced changes in hemodynamic parameters and sarcolemmal methylation without affecting the enhanced methylation activities in sarcoplasmic reticulum. In vitro addition of cyclic AMP-dependent protein kinase (catalytic subunit) plus Mg-ATP enhanced methyltransferase activities in sarcolemma and sarcoplasmic reticulum from control hearts by 2.7- and 2.3-fold, respectively; however, under the same in vitro conditions, only about 20% activation was seen in both subcellular membranes isolated from the heart of isoproterenol-injected animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Modification of sarcolemmal phosphatidylethanolamine N-methylation during heart hypertrophy.

To evaluate changes in heart sarcolemmal phosphatidylethanolamine (PE) N-methylation, left ventricular hypertrophy was induced in rabbits by banding the abdominal aorta for 4, 8, 14, and 22 wk. The degree of cardiac hypertrophy did not change over the period of time studied. Three catalytic sites involved in the sequential methyl transfer reactions were examined by assaying the incorporation of radiolabeled methyl groups from S-adenosyl-L-methionine (0.055, 10, and 150 microM) into sarcolemmal PE molecules under optimal conditions. Total N-methylation activity at all three sites was significantly increased at 4 wk, unaltered at 8 and 14 wk, and depressed at 22 wk after banding the aorta. Similar biphasic changes were seen for the individual methylated lipid products (monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, and phosphatidylcholine) specifically formed at each catalytic site. At all three sites, alterations in PE N-methylation at 22 wk were associated with changes in Vmax values without any change in the apparent affinity for S-adenosyl-L-methionine. In contrast to sarcolemma, a significant increase of the PE N-methylation activity at sites I and III was observed in the sarcoplasmic reticular (microsomal) fraction from 22-wk hypertrophied hearts; the increase in site II was not significant. On the other hand, no changes in the N-methylation activity of the mitochondrial fraction were seen at 22 wk after banding. These findings indicate the occurrence of biphasic alterations in the sarcolemmal PE N-methylation activity during the presence of a stable degree of hypertrophy.

Hercon technology for transdermal delivery of drugs.

The Hercon controlled drug delivery technology is based on a multi-layered laminated polymeric structure, in which a layer of vinyl chloride copolymer or terpolymer containing the drug is sandwiched between two or more layers of polymeric films. The drug is released from the device at a controlled rate by a process of diffusion through the reservoir and one of the outer layers, which can function as a rate controlling membrane. This basic technology has been successfully utilized for the development and commercialization of Nitroglycerin Transdermal System (NTS, Bolar Pharmaceutical Co., Inc). In vitro and in vivo investigations of transdermal delivery of different other drugs from the Hercon polymeric devices have indicated the feasibility of using this system to meet a variety of therapeutic needs.

Changes in immunoreactive somatostatin in brain following lidocaine-induced kindling in rat.

The kindling phenomenon has become a useful model for studying epileptogenesis. The present authors have previously reported increased levels of immunoreactive somatostatin (IR-SRIF) in various regions of the brain of electrically-amygdaloid kindled (EAK) rats. In this study, an examination was made of immunoreactive somatostatin in pharmacologically-kindled (PK) rats. Sixteen male Sprague-Dawley rats were injected intraperitoneally (i.p.) with a subthreshold dose of lidocaine (60 mg/kg), once daily. Once the kindling phenomenon was established, kindled rats (7), non-kindled rats (9) and controls (6) were sacrificed by microwave irradiation. Another group of 5 rats was injected with a single suprathreshold dose of lidocaine (110 mg/kg) and killed 10 min after the resultant seizure. Various brain areas were removed and assayed for immunoreactive somatostatin in kindled rats. Immunoreactive somatostatin was significantly greater than in controls in the amygdala (56%; P less than 0.02), entorhinal + piriform cortex (50%; P less than 0.05) and hypothalamus (29%; P less than 0.02). In non-kindled rats, immunoreactive somatostatin increased only in the amygdala (58%; P less than 0.02). No difference was found in the immunoreactive somatostatin content of rats injected with an suprathreshold dose of lidocaine compared to controls. The alteration of immunoreactive somatostatin, in both lidocaine-kindled and electrically-amygdaloid kindled rats suggests a possible role of this neuropeptide in kindling.

Behavioral changes in rat following perinatal exposure to ethanol.

Behavioral changes following pre- and postnatal ethanol administration were examined in rat pups of various ages. A significant increase in locomotor activity occurred at all ages (16, 22 and 30 days) examined. Nose poking activity in 22-day-old pups and standing activity in 30-day-old pups were also significantly higher following perinatal ethanol exposure. No significant change in grooming behavior was observed. The overactivity in rat pups following perinatal ethanol exposure is consistent with the frequently observed hyperactivity in fetal alcohol syndrome children.

The effect of concussion on cerebral uptake of 2-deoxy-D-glucose in rat.

The effect of concussion on 2-deoxy-D-glucose uptake by various regions of rat brain was investigated. The relative uptake of 2-deoxy-D-glucose was measured at 10 and 20 min post-concussion. The uptake did not change significantly from control rats at 20 min post-concussion. This may be reflective of transient nature of concussion. The uptake was higher in cortex and lower in subcortical structures at 10 min post-concussion. These changes are similar to those reported in spreading cortical depression. Spreading cortical depression may thus be a component of concussion mechanism.

Prenatal exposure to ethanol alters [3h]naloxone binding in rat striatum.

Pregnant rats were administered ethanol from day 8 of pregnancy until the day of delivery. Control rats were given isocaloric replacement of sucrose. [3H]Naloxone binding was determined in the striatum of pups of various ages in both groups. The binding was significantly lower in the pups of ethanol treated mothers than in pups of control mothers at all ages examined. The findings suggests an interference in the normal development of striatal receptors in rats prenatally exposed to ethanol.

Concussion in rats causes an immediate change in occupancy but not affinity of hypothalamic cholinergic receptors.

Experimental concussion in rats is associated with subsequent enhanced binding in vitro of certain ligands to hypothalamic tissue but not to other brain regions. Scatchard analysis shows that the number of specific binding sites for [3H]quinuclidinylbenzilate increases within 3 s after concussion with no significant change in binding affinity. The apparent increase in receptors is likely the result of a deficit in binding of endogenous ligand. We propose that concussion produces a pressure wave that deforms cholinergic and possibly other receptors selectively in the hypothalamus, resulting in a transient deficit in transmitter binding and interruption of neuronal circuits concerned with the state of consciousness.

The uptake and distribution of 14-C-mescaline in different organs of developing rat.

Rats of 1,4,8,12,20, and 60 days postnatal age were injected ip with 14-C-mescaline (50 nCi/g). The levels of mescaline and its deaminated metabolite, 3,4,5-trimethoxyphenylacetic acid, were examined in the brain, liver, heart, spleen, lung, and kidney at 30, 60, 90, and 120 min. Mescaline was rapidly taken up by all the organs examined. In general, the organs of younger rats accumulated much larger amounts than those of adult animals. Brain concentrated the lowest amounts in comparison with other tissues. In the brain, the uptake was the highest in 1-day-old rats and decreased with age. The disappearance of mescaline in various organs was comparatively slower in younger animals than in 20-day or older rats. Rats immediately after birth and uptake was the highest in 1-day-old rats and decreased with age. The disappearance of mescaline in various organs was comparatively slower in younger animals than in 20-day or older rats. Rats immediately after birth and up to 20 days of age metabolized mescaline less efficiently than adults. From the data, it appears that the blood-brain barrier for mescaline develops gradually with age but is not completely impermeable in adults.