Summary The actions of cyclic AMP are subject to several levels of post-receptor modulation in cardiac tissue. Isoproterenol and prostaglandin E1 both stimulate cAMP accumulation, but only isoproterenol causes activation of particulate cAMP-dependent protein kinase, leading to activation of phosphorylase kinase and glycogen phosphorylase, and inhibition of glycogen synthase. Through the use of isolated, adult ventricular myocytes, we have determined that the hormone-specific activation of glycogen phosphorylase is due to subcellular compartmentation of cAMP. There is some evidence that cyclic nucleotide phosphodiesterases, whose activity is stimulated by alpha1-adrenergic agonists in isolated myocytes, may have a role in compartmentation. Phosphoinositide hydrolysis is stimulated by alpha, and muscarinic agonists, presumably leading to activation of protein kinase C, which in turn has multiple effects on hormone-sensitive adenylate cyclase.Abbreviations cAMP
Adenosine-3,5-Cyclic Monophosphate
- cGMP
Guanosine-3,5-Cyclic Monophosphate
- Gi, GS
Guanine nucleotide-binding proteins linked to inhibition and stimulation, respectively, of adenylate cyclase
- GTP
Guanosine-5-triphosphate
- PDE
Cyclic Nucleotide Phosphodiesterase
- PGE1
Prostaglandin E1相似文献
Transgenic animals provide a model system to elucidate the role of specific proteins in development. This model is now being used increasingly in the cardiovascular system to study cardiac growth and differentiation. During cardiac myocyte development a transition occurs from hyperplastic to hypertrophic growth. In the heart the switch from myocyte proliferation to terminal differentiation is synchronous with a decrease in c-myc mRNA abundance. To determine whether c-myc functions to regulate myocyte proliferation and/or differentiation, we examined the in vivo effect of increasing c-myc expression during fetal development and of preventing the decrease in c-myc mRNA expression that normally occurs during myocyte development. The model system used was a strain of transgenic mice exhibiting constitutive expression of c-myc mRNA in cardiac myocytes throughout development. Increased c-myc mRNA expression is associated with both atrial and ventricular enlargement in the transgenic mice. This increase in cardiac mass is secondary to myocyte hyperplasia, with the transgenic hearts containing greater than twice as many myocytes as nontransgenic hearts. The results of this study indicate that constitutive expression of c-myc mRNA in the heart during development results in enhanced hyperplastic growth, and suggest a regulatory role for the c-myc protooncogene in cardiac myogenesis. 相似文献
Summary The following studies were undertaken to develop a cultured uterine myocyte model which would allow further clarification
of the adrenergic signal transduction mechanisms utilized by these myocytes. After mechanical removal of the endometrium,
rabbit uterine myoctes were isolated by an overnight enzymatic disaggregation using collagenase and DNase I. The isolated
myocytes were maintained in culture in 75-cm2 flasks containing Waymouth's MB 751/1 medium-10% fetal bovine serum along with 10−8M estradiol, penicillin, streptomycin, and Fungizone. The phase contrast and electron micrographic appearance of these cells
was consistent with that previously reported for smooth muscle myocytes in culture. Immunocytochemical studies utilizing monoclonal
anti-alpha-smooth muscle actin antibodies confirmed the presence of smooth muscle actin in these cultured myocytes. Western
blot studies similarly confirmed the presence of alpha-smooth muscle actin in rabbit myometrial tissue and the cultured myocytes,
both the primary and F1 generation. After prelabeling the myocytes with [3H]inositol, adrenergic stimulation experiments demonstrated alpha-1 receptor mediated stimulation of inositol phosphates.
Beta receptor stimulation experiments confirmed cAMP production in these cultured myocytes, and the ability of clonidine,
an alpha-2 agonist, to inhibit forskolin stimulated cAMP production confirmed the presence of functional alpha-2 adrenergic
receptors in these myocytes. In conclusion, these cultured rabbit uterine myocytes have provided an in vitro model which can
be utilized to further clarify the adrenergic receptor signal transduction mechanisms in genital tract smooth muscle.
This research was supported by grant HD-22063 from the National Institutes of Health, Bethesda, MD. 相似文献
17β-estradiol propionate was found to reduce the gap junctional communication in a concentration range similar to that of
testosterone propionate, in primary cultures of rat Sertoli cells and cardiac myocytes. Uncoupling was reversible on washing
out and occurred without concomitant rise in the intracellular calcium concentration.
Esterification was a prerequisite for the activity of extracellularly applied steroid compounds (for example, testosterone
was ineffective even at external concentrations up to 100 μm, whereas its intracellular application at 1 μm totally interrupted intercellular communication), but their uncoupling efficiency did not depend on the nature of the ester
chain nor on its position on the steroid nucleus. The derivatives of two other androgen hormones (derivatives of the androstane
nucleus) were also efficient as junctional uncouplers. Among five steroid molecules belonging to the pregnane family, only
one (pregnanediol diacetate) interrupted the junctional communication. Neither cholic acid nor cholesteryl acetate or ouabain
showed this effect. Altogether, no correlation with the presence or position of double bonds nor with the trans- or cis-fusion of the A and B rings could be recognized.
These results suggest that this reversible, nondeleterious uncoupling effect of steroids is independent of the shape of the
molecules and is more probably related to their size and liposolubility, that condition their insertion into the lipid bilayer.
Their incorporation into the membrane could disturb the activity of the membrane proteins by a physical mechanism.
Received: 10 April 1995/Revised: 27 October 1995 相似文献
We investigated the effect of 10–8 M noradrenaline (NA) on [Ca2+], and electrical activity of single myocytes of guinea-pig ventricular myocardium loaded with Indo 1-AM. Membrane potential was recorded by means of the patch electrode and patch amplifier set to the current clamp mode. Cells were stimulated at a rate of 30/min by 3 ms pulses of the current injected through the recording electrode. Superfusion of NA resulted in slight shortening of action potentials (APs), increase in rate of rise and amplitude of the respective Ca2+ transients, and appearance of secondary Ca2+ transients of two kinds: 1. appearing before repolarisation of AP and decay of the preceding Ca2+ transient were completed and 2. appearing between the APs. We named them early after-transients (EAT) and delayed after-transients (DAT), respectively. Without any additional intervention EATS caused some prolongation of APs duration and DATs resulted in subthreshold delayed after-depolarisations (DADS). When sarcolemmal K+ conductance was decreased by tetraethylammonium (TEA) in the patch electrode or 20 M BaCl2 in the Tyrode solution, EATs initiated early after depolarizations (EADs) and DATs initiated suprathreshold DADs triggering full-sized APs. Superfusion of 30.0 mM Na+ (replaced with LiCl) resulted in reduction of AP duration by -70% and appearance of DATs. Also, the frequent multiple oscillations of Ca 2+ concentration were often observed. Neither DATs nor the oscillations had any affect on electrical activity of the cells. Their electrogenicity could not be increased by TEA or 20.0 M Ba2+. EATs and DATs and their respective EADs and DADs could not be initiated by NA or low Na+ superfusion in the cells pretreated with 2 × 10–7 M thapsigargin, a selective blocker of Ca2+-ATPase of sarcoplasmic reticulum (SR). We conclude that in contrast to the current hypothesis, EADs can be initiated by Ca2+ released early in the cardiac cycle from the overloaded SR, and that electrogenicity of both types of Ca2+ oscillations critically depends on the sarcolemmal K+ conductance. 相似文献
The published experimental data and existing concepts of cellular regulation of respiration are analyzed. Conventional, simplified considerations of regulatory mechanism by cytoplasmic ADP according to Michaelis-Menten kinetics or by derived parameters such as phosphate potential etc. do not explain relationships between oxygen consumption, workload and metabolic state of the cell. On the other hand, there are abundant data in literature showing microheterogeneity of cytoplasmic space in muscle cells, in particular with respect to ATP (and ADP) due to the structural organization of cell interior, existence of multienzyme complexes and structured water phase. Also very recent experimental data show that the intracellular diffusion of ADP is retarded in cardiomyocytes because of very low permeability of the mitochondrial outer membrane for adenine nucleotidesin vivo. Most probably, permeability of the outer mitochondrial membrane porin channels is controlled in the cellsin vivo by some intracellular factors which may be connected to cytoskeleton and lost during mitochondrial isolation. All these numerous data show convincingly that cellular metabolism cannot be understood if cell interior is considered as homogenous solution, and it is necessary to use the theories of organized metabolic systems and substrate-product channelling in multienzyme systems to understand metabolic regulation of respiration. One of these systems is the creatine kinase system, which channels high energy phosphates from mitochondria to sites of energy utilization. It is proposed that in muscle cells feed-back signal between contraction and mitochondrial respiration may be conducted by metabolic wave (propagation of oscillations of local concentration of ADP and creatine) through cytoplasmic equilibrium creatine and adenylate kinases and is amplified by coupled creatine kinase reaction in mitochondria. Mitochondrial creatine kinase has experimentally been shown to be a powerful amplifier of regulatory action of weak ADP fluxes due to its coupling to adenine nucleotide translocase. This phenomenon is also carefully analyzed.It is easier to explain biochemistry in terms of transport than it is to explain transport in terms of biochemistry. P. Mitchell The Ninth Sir Hans Krebs Lecture, Dresden, July 2, 1978. 相似文献
1. 1. Isolated cardiac myocytes of perch, Perca fluviatilis, were kept in culture conditions for 1–2 months at 12 or 22°C. In the culture most myocytes flattened, lost their spindle-shaped morphology, protruded pseudopod-like branches and many of them started visible contractions in 1–2 weeks and continued beating for several months. Myocytes did not divide in the sparse cell population used. Typical intracellular structures could be seen in electron micrographs still after 1–2 months, but the sarcoplasmic organization became gradually more irregular in the culture.
2. 2. Beat rates showed linear temperature relationship on the Arrhenius plot. Myocytes cultivated at 22°C showed higher frequencies and slightly less dependence on temperature than myocytes cultivated at 12°C (apparent activation energies (Ea) 86 and 107 kJ/mol, respectively).
3. 3. Temperature dependence of frequencies was related to the presence of added serum or adrenergic agonists: β-adrenergic agonists increased the frequencies and rendered the cells less dependent on temperature; apparent activation energy was 43 kJ/mol for isoprenaline or adrenaline and 108 kJ/mol for noradrenaline and control group.
4. 4. Heat tolerance was greater in myocytes cultivated at 22°C than in myocytes cultivated at 12°C, and the change in tolerance appeared in 12 h after the alteration of culture temperature and the increased tolerance was persistent after that.
5. 5. It is suggested, that the processes of quick heat-hardening and of slower but persistent heat resistance acclimation developed in these cells in culture conditions but not the capacity acclimation, which seems to be dependent on adrenergic regulation of beat rate.
A close spatial relationship between specific granules containing atrial natriuretic factor (ANF) and microtubules was demonstrated in primary cultures of neonatal rat cardiac myocytes. For the detection of specific granules and microtubules, the myocytes were double immunolabelled with antibodies against -ANF and -tubulin and examined by conventional fluorescence or laser scanning confocal microscopy. In addition, the ultrastructural distribution of specific granules was demonstrated by electron microscopy. In the atrial myocytes, ANF was stored in numerous specific granules that were mainly localized in the perinuclear sarcoplasm. In the ventricular myocytes, however, a minority of the cells (10%) exhibited limited ANF immunoreactivity after 4 days in culture. Microtubules were present throughout the sarcoplasm of the myocytes. They were most densely packed in the perinuclear regions. Depolymerization of the microtubules with nocodazole was followed by dispersal of ANF immunostaining both in the atrial myocytes and in the ventricular myocytes exhibiting ANF immunoreactivity. When the microtubules were allowed to recover, the perinuclear distribution of specific granules, as seen in non-treated myocytes, reappeared. Measurements of secreted immunoreactive ANF by radioimmunoassay revealed that the secretion of ANF from atrial myocytes into the medium was significantly reduced following nocodazole treatment, whereas a similar decrease in secretion from ventricular myocytes was not observed. These findings indicate that ANF-containing specific granules are closely associated with microtubules within the myocytes. It is suggested that secretion of ANF from the atrial myocytes, in contrast to the ventricular myocytes, is microtubule-dependent. 相似文献
To examine whether basic fibroblast growth factor (bFGF) administered to the heart by perfusion can improve cardiac resistance to injury we employed an isolated rat heart model of ischemia-reperfusion injury and determined the extent of functional recovery in bFGF-treated and control hearts. Global ischemia was simulated by interruption of flow for 60 min. Recovery of developed force of contraction (DF), recorded after reestablishment of flow for 30 min, reached 63.8±1.5% and 96.5±3.5% of preischemic levels in control and bFGF-treated hearts (10 g/heart), respectively, indicating that bFGF induced significantly improved recovery of mechanical function. Recoveries of the rates of contraction or relaxation were also significantly improved in bFGF-treated hearts. Extent of myocardial injury, assessed by determination of phosphocreatine kinase in the effluent, was reduced as a result of bFGF treatment. As a first step towards understanding the mechanism and direct cellular target(s) of bFGF-induced cardioprotection, we investigated its fate after perfusion. Perfusion of 10 g bFGF/heart resulted in a 4-fold increase in bFGF associated with the heart compared to control levels, as estimated by biochemical fractionation and immunoblotting. Immunofluorescent staining of the bFGF-perfused hearts revealed intense anti-bFGF staining in association with blood vessels as well as the periphery of cardiomyocytes, suggesting that the latter may be a target for direct bFGF action. In conclusion, our findings of bFGF-induced increases in cardiac resistance to, and improved functional recovery from, ischemia-reperfusion injury indicate that bFGF may have clinical applications in the treatment of ischemic heart disease. 相似文献
Summary Postnatal rat heart cells in culture enriched with respect to muscle cells were obtained by either high density seeding or
by the replating technique. [3H]Thymidine incorporation to DNA and the enzymatic pattern of cytoplasmic and lysosomal enzymes have been studied as a function
of the culture’s age, of seeding density, and replating. It was shown that (a) replating maintains predominance of myocyte
population for at least 2 wk in culture; (b) heavy seeding density allows homogeneous myocyte population for the 1st wk in
culture; and (c) the enzyme profile of the culture may serve as an indicator for the type of cell population in culture and
its state of differentiation.
This study was done as partial fulfilment of the M.Sc. thesis in Biochemistry (SY). Supported by grants from The Chief Scientist,
Ministry of Health, State of Israel; The Ministry of Education and Sciences, State of Niedersachssen (FRG); and The Foundation
for Heart Research from Mr. and Mrs. D. Vidal-Madjar, Paris, France. 相似文献