Intact Muscle Preparations Research Articles

Effects of pH and temperature on myocardial calcium-activation in Pterygoplichthys (catfish).

Fish are chronically exposed to a wide range of temperatures and acidic environments. Fish hearts have to therefore adapt to these changes in order to maintain contractility. Myofibrillar responsiveness to Ca2+ is exquisitely sensitive to both temperature and pH in mammalian myocardium. To evaluate myofilament calcium-activation, we chemically skinned ventricular myocardium from catfish (Pterygoplichthys). A decrease in pH from 7.5 to 6.8, irrespective of temperature change, shifted the calcium-force curve towards higher calcium concentrations without affecting maximal Ca(2+)-activated force. The contractile elements are therefore sensitive to changes in pH. In intact muscle preparations the active twitch force was decreased with increasing temperature (10-22 degrees C). However, the sensitivity of the myofilaments to Ca2+ was independent of temperature. These data suggest a possible role of the sarcoplasmic reticulum (SR) in mediating the effects of temperature. The response of intact muscle preparations to changes in temperature is therefore not likely due to temperature-dependent changes in myofilament calcium responsiveness.

Voltage dependence of Ca2+ sparks in intact cerebral arteries.

Ca2+ sparks have been previously described in isolated smooth muscle cells. Here we present the first measurements of local Ca2+ transients ("Ca2+ sparks") in an intact smooth muscle preparation. Ca2+ sparks appear to result from the opening of ryanodine-sensitive Ca2+ release (RyR) channels in the sarcoplasmic reticulum (SR). Intracellular Ca2+ concentration ([Ca2+]i) was measured in intact cerebral arteries (40-150 micron in diameter) from rats, using the fluorescent Ca2+ indicator fluo 3 and a laser scanning confocal microscope. Membrane potential depolarization by elevation of external K+ from 6 to 30 mM increased Ca2+ spark frequency (4. 3-fold) and amplitude (approximately 2-fold) as well as global arterial wall [Ca2+]i (approximately 1.7-fold). The half time of decay ( approximately 50 ms) was not affected by membrane potential depolarization. Ryanodine (10 microM), which inhibits RyR channels and Ca2+ sparks in isolated cells, and thapsigargin (100 nM), which indirectly inhibits RyR channels by blocking the SR Ca2+-ATPase, completely inhibited Ca2+ sparks in intact cerebral arteries. Diltiazem, an inhibitor of voltage-dependent Ca2+ channels, lowered global [Ca2+]i and Ca2+ spark frequency and amplitude in intact cerebral arteries in a concentration-dependent manner. The frequency of Ca2+ sparks (<1 s-1 . cell-1), even under conditions of steady depolarization, was too low to contribute significant amounts of Ca2+ to global Ca2+ in intact arteries. These results provide direct evidence that Ca2+ sparks exist in quiescent smooth muscle cells in intact arteries and that changes of membrane potential that would simulate physiological changes modulate both Ca2+ spark frequency and amplitude in arterial smooth muscle.

Dissimilar Action of Two Cyclic Adenosine‐Monophosphate Analogues on the Sodium Current in Intact Rat Papillary Muscle

In intact papillary muscles from rat we have found with the loose-patch-clamp technique an increase of the fast cardiac sodium current (INa+) by isoproterenol (ISO). In this study we have tested two membrane permeable analogues of the intracellular second messenger cyclic adenosine-monophosphate (cAMP) to investigate the intracellular pathway: 8-Br-cAMP (50 microM) and the newer developed Sp-5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole- 3',5'-cyclic-monophosphorothioate (5,6-DCl-cBiMPS, 20 microM). The availability of INa+ was determined with test pulses to +/- 0 mV every 3.5 seconds after 2.5-second conditioning between -130 mV and -50 mV and a holding potential at the resting potential of the cell under examination, and after wash-in of either compound. The peak currents were fit to a Boltzmann equation, and expressed by the maximal attainable current INa+,max, the mid-point potential V1/2, and a steepness parameter alpha. Values are given by mean +/- SEM. 8-Br-cAMP showed a significant shift of the availability curve in the hyperpolarized direction (V1/2 = -82 +/- 2 mV vs -86 +/- 2 mV, n = 5, P < 0.05) with only minor changes of INa+,max and alpha. In contrast, 5,6-DCl-cBiMPS had no significant effect on V1/2 but increased INa+,max by 8% +/- 2% versus control (n = 5, P < 0.05). In an intact muscle preparation we have found that 5,6-DCl-cBiMPS has a similar effect as that observed with the beta-adrenergic agonist ISO (100 nM), whereas 8-Br-cAMP exhibited a dissimilar action.(ABSTRACT TRUNCATED AT 250 WORDS)

Tyrosine kinase pathways and the regulation of smooth muscle contractility

Epidermal growth factor (EGF) mediates three tyrosine kinase-dependent smooth muscle response paradigms, two of which comprise a rapid increase in muscle tension and one of which is characterized by an agonist-mediated reduction in sensitivity to other agents. The three types of response are mediated via distinct signal transduction pathways, and marked tissue and species variation have been observed, even for a single growth factor agonist. Vasoactive agents, such as angiotensin II and vasopressin, that act via G protein-coupled receptors can also work via tyrosine kinase pathways to cause contraction in some of the same intact smooth muscle preparations that contract in response to EGF. In this review, Morley Hollenberg discusses the tyrosine kinase-modulated signal transduction pathways for EGF and also agonists that act via G protein-coupled receptors, and hypothesizes that there may be an intermediary non-receptor tyrosine kinase that may serve as a point of convergence for the contractile actions of these agents in smooth muscle.

The acute actions of growth factors in smooth muscle systems

Work over the past six to eight years has established that mitogenic polypeptides such as epidermal growth factor-urogastrone (EGF-URO) or platelet-derived growth factor (PDGF), commonly referred to as ‘growth factors’, can have rapid (seconds to minutes) regulatory actions in a variety of smooth muscle systems. With EGF-URO as a prototype example, this article describes three distinct smooth muscle response paradigms, two of which (type A and type B) comprise a rapid (seconds to minutes) increase in muscle tension) and one of which (type C) is characterized by an EGF-URO-mediated reduction in sensitivity to other agonists. The quite distinct signal transduction pathways for the three types of response paradigms are outlined, and the marked tissue and species variation in the types of smooth muscle responses that can be observed, even for a single growth factor agonist, are summarized. The article also illustrates that G-protein-coupled vasoactive agents such as angiotensin-II and vasopressin, which can act as ‘growth factors’ in cultured cell systems, can also work via tyrosine kinase pathways to cause contraction in some of the same intact smooth muscle preparations that contract in response to growth factors such as EGF-URO. Attention is drawn to the fact that many so-called ‘growth factors’, quite apart from stimulating cell division and differentiation, may in many instances act as rapid localized paracrine/autocrine regulators of tissues such as smooth muscle. It is also pointed out that some of the tyrosine kinase-modulated signal pathways usually associated with the mitogenic action of ‘growth factors’ may be involved not only in the rapid effects of agents such as EGF-URO in smooth muscle but also in the contractile actions of G-protein-linked agonists.

Amino acid loss during volume regulatory decrease in cultured chick heart cells

Mechanisms of volume regulation in hyposomotically treated cultured chick heart cell preparations were studied using optical, biochemical, and nuclear magnetic resonance methods. This approach afforded the resolution of time-dependent responses that might ordinarily be obscured by the complex morphology of intact cardiac muscle preparations. In hyposmotic solutions, cells swelled to a peak volume within 3 min and slowly regulated toward original volume (regulatory volume decrease, RVD). Upon return of the cells to isosmotic solution following hyposmotic treatment, the cells shrank to a steady-state volume that was substantially less than the initial volume in control solution. A vigorous RVD could also be elicited by hyposmotic swelling under Cl(-)-free conditions. Measurement of both inorganic cation loss via atomic absorption spectroscopy and organic solute loss via 1H-nuclear magnetic resonance and high-pressure liquid chromatographic techniques revealed that the RVD observed following exposure to hyposomotic solutions was mediated in part by a substantial loss of taurine, glutamate, aspartate, and glycine as well as loss of inorganic ions (Na+,K+). The hyposmotically activated transport of amino acids was also associated with the production of glutamate and aspartate. The volume regulatory release and production of amino acids have significant implications for the metabolic and functional integrity of cardiac cells.

Ca(2+)-dependent heat production under basal and near-basal conditions in the mouse soleus muscle.

1. The rate of energy expended for the clearance of sarcoplasmic Ca2+ by sarcoreticular Ca2+ uptake process(es), plus the concomitant metabolic reactions, was evaluated from measurements of resting heat production by mouse soleus muscle before and after indirect inhibition of Ca2+ uptake by sarcoplasmic reticulum (SR). 2. Direct inhibition of the Ca2+, Mg(2+)-ATPase of SR membrane in intact muscle preparations exposed to the specific inhibitor 2,5-di(tert-butyl-1,4-benzohydroquinone (tBuBHQ) slowly increased the rate of heat production (E). Indirect inhibition of SR Ca2+ uptake was obtained by reducing sarcoplasmic Ca2+ concentration (Ca2+i) as a consequence of reducing Ca2+ release from the SR using dantrolene sodium. This promptly decreased E by 12%. Exposure of the preparations to an Mg(2+)-enriched environment (high Mg2+) or to the chemical phosphatase 2,3-butanedione monoxime (BDM), two other procedures aimed at decreasing SR Ca2+ release, also acutely decreased E, by 20 and 24%, respectively. 3. Subthreshold-for-contracture depolarization of the sarcolemma achieved by increasing extracellular K+ concentration to 11.8 mM induced a biphasic increase of E: an initial peak to 290% of basal E, followed by a plateau phase at 140% of basal E during which resting muscle tension was increased by less than 3%. Most, if not all, of the plateau-phase metabolic response was quickly suppressed by dantrolene or high Mg2+ or BDM. Another means of increasing SR Ca2+ cycling was to partially remove the calmodulin-dependent control of SR Ca2+ release using the calmodulin inhibitor W-7. The progressive increase in E with 30 microM-W-7 was largely reduced by dantrolene or high Mg2+ or BDM. 4. In the presence of either dantrolene or BDM to prevent the effect of W-7 on SR Ca2+ release, exposure of the muscle to W-7 acutely suppressed about 3% of E. This and the above results confirm that the plasmalemmal, calmodulin-dependent Ca(2+)-ATPase, although a qualitatively essential part of the Ca2+i homeostatic system of the cell, can only be responsible for a very minor part of the energy expenditure devoted to the homeostasis of Ca2+i. Active Ca2+ uptake by SR which, at least in the submicromolar range of Ca2+i, is expected to be responsible for most of this Ca(2+)-dependent energy expenditure, might dissipate up to 25-40% of total metabolic energy in the intact mouse soleus under basal and near-basal conditions.

The isoproterenol-induced chloride current and cardiac resting potential

Isoproterenol can induce potentially arrhymthogenic depolarizations of the resting membrane of single guinea-pig ventricular myocytes [ 1]. This effect on resting potential of single guinea-pig ventricular myocytes differs qualitatively from results obtained by others from intact cardiac muscle preparations [ 2–5] and also differs from our experience with single dog ventricular myocytes. We performed experiments on dog and guinea-pig ventricular myocytes in an attempt to clarify the effects of isoproterenol on the resting potential of mammalian ventricular myocytes. Voltage recordings with 40–60 mΩ 3 m potassium chloride filled microelectrodes revealed an isoproterenol-induced depolarization of 4.3 ± 1.0 mV in guinea-pig but no depolarization in dog myocytes. Activation of an outwardly rectifying chloride current is responsible for the isoproterenol-induced depolarization of guinea-pig ventricular myocytes [ 6–9]. Our whole cell patch clamp recordings consistently revealed such a current in guinea-pig cells but always failed to demonstrate an insoproterenol-induced chloride current in dog myocytes under identical conditions. In contrast to single cells, isoproterenol did not depolarize intact guinea-pig papillary muscle when potential was recorded with 40–60 mΩ 3 m KCl filled electrodes. Furthermore, we saw no depolarization in single guinea-pig myocytes when recording electrodes did not contain chloride. We conclude that: (1) despite activation of a chloride current, isoproterenol does not significantly depolarize guinea-pig ventricular muscle unless the driving force for the current at the resting potential is increased by elevating [Cl] i above physiological levels, and (2) an isoproterenol-induced chloride current, although demonstrable in guinea-pig ventricular cells, is not present in healthy dog ventricular cells.

Physiological and pathophysiological modulation of calcium signaling in myocardial cells.

The relationship between changes in intracellular Ca2+ transients and isometric contractions has been assessed in intact cardiac muscle preparations, superficial cells of which have been microinjected with the Ca(2+)-sensitive bioluminescent protein aequorin. Regulation of myocardial contractility by physiological and pathophysiological intervention is achieved by either (1) modulation of intracellular Ca2+ mobilization, or (2) modulation of Ca2+ sensitivity of myofibrils, or both. Regulation of contractility by changes in heart rate a well established frequency-force relationship that plays an important role in the cardiac pumping function in situ is mainly achieved by mechanism (1), other mechanisms becoming involved depending on the range of frequency of stimulation. The length-dependent regulation of contractility (length-tension relationship in vitro or Frank-Starling's law, or ventricular function curve in situ) is achieved essentially by mechanism (2). Catecholamines promote mechanism (1) through activation of beta- and/or alpha-adrenoceptors, alpha-adrenoceptor stimulation being much less effective than beta-stimulation in this respect. beta-Adrenoceptor stimulation decreases, while alpha-stimulation may increase the Ca(2+)-sensitivity of contractile proteins. Subsequent to exposure of muscle preparations to Ca2+ free solution, a prominent and reversible dissociation of force of contraction from Ca2+ transients was produced when the [Ca2+]0 was gradually returned to the level of the normal Krebs-Henseleit solution [( Ca2+]0 = 2.5 mM). The aequorin-injected multicellular intact myocardial cell preparation provides an excellent experimental paradigm through which to address the physiological, pharmacological and pathophysiological modulation of E-C coupling in mammalian cardiac muscle. The subcellular mechanism involved, especially in the pathophysiological modulation of Ca2+ signaling process in myocardial cells, awaits further study.

An intact cremaster muscle preparation for studying the microcirculation by in vivo microscopy

We describe here in detail a new preparation which has the advantage of limiting muscle dissection drastically and of preserving all the vascular interconnections intact

Ethanol inhibits electrically-induced calcium transients in isolated rat cardiac myocytes

The fluorescent Ca 2+ indicator fura-2 was used to follow cytosolic Ca 2+ transients during excitation-contraction coupling in suspensions of isolated rat heart cells induced to beat synchronously by electrical field stimulation. The Ca 2+ transient reached a maximum at about 30 ms after application of the electrical stimulus and then relaxed to the basal level over the following 200 ms. Treatment of the myocytes with 0.25 to 2.0% ethanol (40 to 340 m m) caused a decrease in the peak of the Ca 2+ transient, with no apparent change in the time to peak. This effect of ethanol occurred progressively over a period of about 1 min before a new stable state was achieved. At 1% ethanol the peak Ca 2+ level was reduced by 50%. Ethanol reversed the stimulatory effect of isoproterenol on peak Ca 2+ and at high levels of ethanol the β-adrenergic agonist no longer caused any enhancement of the Ca 2+ transient. Ethanol did not cause any marked change in the basal Ca 2+ level between beats. The effects of ethanol were readily reversible. These results suggest that the negative inotropic effect of ethanol observed in intact cardiac muscle preparations may result in part from interference with the Ca 2+ fluxes responsible for excitation-contraction coupling in ventricular myocytes.

Effects of 2,3-butanedione monoxime on the contractile activation properties of fast- and slow-twitch rat muscle fibres.

1. The effects of 2,3-butanedione monoxime (BDM, 0.2-10 mmol/l) have been examined at different temperatures on calcium transients (measured with aequorin) and isometric force in intact bundles of fibres from soleus (slow-twitch) and extensor digitorum longus (EDL; fast-twitch) muscles of the rat and on Ca2+-activated isometric force of mechanically skinned soleus and EDL fibres. Ca2+ release and uptake by the sarcoplasmic reticulum has also been investigated directly on skinned EDL fibres at 1 mmol/l BDM at 23 degrees C. 2. BDM bound calcium with low affinity (KCaBDMapp = 55.9 +/- 4.3 M-1 at 22 degrees C, pH 7.10, ionic strength approximately 155 mmol/l) and did not affect the kinetics and quantum yield of the Ca2+-induced aequorin luminescence. 3. BDM reversibly reduced both force- and Ca2+-dependent aequorin light during the twitch and tetanus of all intact-muscle preparations in a dose-related manner. Twitch responses of soleus fibres were more sensitive to BDM than were EDL fibres. Higher concentrations of BDM were needed to abolish the tetanic force response than the twitch response in both muscle types. 4. The initial rate of rise and the decay rate constants of twitches in both soleus and EDL muscles were increased in the presence of BDM. Accordingly, a higher frequency of stimulation was required to produce fusion of the tetanus. This phenomenon could be explained by the reduced magnitude of the Ca2+ transient. 5. BDM reduced maximal Ca2+-activated force development in mechanically skinned soleus and EDL muscle fibres in a dose-dependent fashion. This depression of force was less sensitive to BDM than were the light transients in intact fibres. Furthermore, BDM affected EDL skinned fibres to a greater extent than soleus skinned fibres, in contrast with its effects on intact fibres. 6. At concentrations of BDM greater than 2.5 mmol/l, BDM significantly decreased the sensitivity of the contractile apparatus to calcium. The relative force-pCa (= -log [Ca2+]) curves for both soleus and EDL skinned fibres were shifted to the right (i.e. to higher calcium concentrations) in a dose-related manner. 7. BDM (10 mmol/l) slowed maximal Ca2+-activated force development of skinned preparations of both fibre types at all temperatures investigated (4-24 degrees C). 8. BDM reduced stiffness in skinned preparations maximally activated by calcium in proportion to the reduction in the isometric force response. BDM also inhibited rigor force of all fibres but only if it was present before the onset of rigor.(ABSTRACT TRUNCATED AT 400 WORDS)

Changes in membrane potential and phosphoinositides during α1-adrenoceptor stimulation in smooth muscle cells of guinea-pig taenia caeci

The effect of a submaximal concentration of adrenaline (3–5 μM) was studied in taenia caeci smooth muscle cells. Membrane potential hyperpolarization was observed in intact muscle preparations and this response could be separated into two phases, depending on the state of a membrane-bound calcium compartment. The effect of α 1-adrenergic stimulation was also measured by [ 3H]inositol incorporation into phospholipid and inositol phosphate fractions of taenia cell suspensions both in the absence and presence of 2.5 mM extracellular calcium. In the absence of extracellular calcium the inositol phospholipids increased within 15 s after stimulation, followed by enhanced inositol phosphates. With calcium present there was a biphasic increase in the phosphatidylinositol 4,5-bisphosphate (PIP 2) fraction with a simultaneous release of inositol phosphates. Lithium ions affected the incorporation of label into the lipids but not into the inositol phosphate fractions. These findings suggest that, in taenia caeci cells, α 1-adrenergic-induced membrane hyperpolarization resulting in muscle relaxation is associated with changes in the PIP 2 content.

Temperature-dependent calcium sensitivity changes in skinned muscle fibres of rat and toad.

Single mechanically skinned muscle fibres of different types (fast- and slow-twitch mammalian; slow and twitch amphibian) were successively activated in solutions of various Ca2+ concentrations at different temperatures. An increase in temperature from 5 to 22 degrees C reversibly shifted the isometric steady-state force-pCa curves towards higher Ca2+ concentration for individual fibres of each of the muscle types. A further increase in temperature to 35 degrees C in mammalian fibres resulted in an additional decrease in Ca2+ sensitivity. The temperature dependence of Ca2+ sensitivity was greater in the 'faster' fibre types: fast-twitch greater than slow-twitch; twitch greater than slow. The maximum isometric force response, P0, of both rat and toad skinned fibres was found to be strongly dependent on temperature below 22 degrees C. No detectable force could be induced by Ca2+ in mammalian muscle fibres at 0-1 degree C while in toad fibres P0 decreased by about 90% when temperature dropped from 20 to 0 degree C. Since in mechanically skinned fibres of other amphibians (Bufo bufo, Rana species) P0 is only marginally affected it is likely that the P0-temperature relations are indicative of the range of temperature over which the muscles are normally functional. The P0-temperature relations of skinned muscle fibres closely resembled the P0-temperature relations of tetanically stimulated intact muscle preparations from the same species of animals suggesting that the contractile apparatus is mainly responsible for the variation of force response with temperature in intact muscle.

Effects of isoprenaline on the contraction-relaxation cycle in the cat trachea.

Effects of isoprenaline (Isop) on the contractile properties of the smooth muscle cells of cat trachea were investigated using intact and chemically skinned muscle preparations and an isometric tension recording method. In the intact muscle preparations, Isop 3 X 10(-10) or 3 X 10(-9) M significantly suppressed the amplitude of tonic contractions evoked by acetylcholine (ACh) 10(-7) M or 10(-5) M, respectively. Following treatment of the tissue with Ca2+-free 2 mM EGTA-containing solution after depletion of stored Ca2+ with caffeine, 2.5 mM Ca2+ was applied for 5 min (procedure 1), and subsequently 10 mM caffeine was applied in Ca2+-free 2 mM EGTA containing solution. The object was to estimate the amount of stored Ca2+ during procedure 1 from the amplitude of the caffeine (10 mM)-induced contraction (procedure 2). Isop, applied during procedure 1, did not affect the amplitude of the caffeine-induced contraction; however, when applied during procedure 2, this agent (10(-8)M) significantly suppressed the amplitude of the caffeine-induced contraction to about 90% of the control value. ACh (10(-5)M), applied during procedure 1, evoked phasic and tonic contractions. Isop (10(-8)M), applied simultaneously with ACh (10(-5)M), suppressed the amplitude of the ACh-induced contraction yet increased the amplitude of contraction evoked by the subsequent application of caffeine 10 mM (procedure 2). Effects of conditioning application of ACh (10(-7) or 10(-5)M) on the caffeine-induced contraction were observed in the presence or absence of Isop during procedure 2. When ACh 10(-5)M was used, subsequent application of caffeine 10 mM evoked no mechanical response, in control conditions. However, after the pretreatment of the tissue with Isop during procedure 2, the amplitude of the ACh (10(-5)M)-induced contraction was not affected, yet the subsequent application of caffeine (10 mM) evoked minute but discrete contractions, indicating that Isop did enhance the sequestration of free Ca2+ into the storage sites. In the saponin-treated skinned muscles, the minimum concentration of Ca2+ required to produce contraction was 1 X 10(-7)M, and the maximum contraction was obtained with 1 X 10(-5)M Ca2+. Isop (10(-6)M) had no effect on the relationship between free-Ca2+ and the amplitude of the contraction. However, simultaneous application of high concentrations of cyclic AMP (10(-4)M) and cyclic AMP-dependent protein kinase (50 micrograms ml-1) significantly suppressed contractions evoked by 3 X 10(-7) or 10(-5)M Ca2+. 8 These results indicate that Isop suppresses the contraction evoked by various agonists in the cat trachea, mainly through sequestration of Ca2+ into the intracellular storage sites, rather than by direct or indirect (through cyclic AMP) actions on the contractile proteins.

Adrenergic and serotonergic regulation of skeletal muscle metabolism in rat. I. The effects of adrenergic and serotonergic antagonists on the regulation of muscle amino acid release, glycogenolysis, and cyclic nucleotide levels.

The biochemical mechanisms of serotonergic and adrenergic action on skeletal muscle cyclic nucleotide, glycogen, and amino acid metabolism have been investigated in intact rat epitrochlaris skeletal muscle preparations. Endogenous catecholamine levels in these preparations were 28.6 +/- 2.1 pg/mg of muscle. Release of these catecholamines by tyramine produced a 25% inhibition of alanine and glutamine release. Pretreatment of animals in vivo with 6-hydroxydopamine depleted catecholamine content by 85%. On incubation, preparations from these pretreated animals showed no effect of tyramine on amino acid metabolism. Serotonin (10(-5) M) and epinephrine (10(-5) M) inhibited alanine and glutamine release equally in preparations from 6-hydroxydopamine-pretreated as compared to control rats. Adrenergic antagonists such as dl-propranolol (10(-8)-10(-6) M), oxprenolol (10(-8)-10(-6) M), and practolol (10(-6)-10(-4) M) blocked equally the inhibition of alanine and glutamine release, prevented the stimulations of muscle cAMP levels, phosphosphorylase a formation, and the depletion of muscle glycogen produced by either epinephrine or serotonin. In contrast, serotonergic antagonists such as methysergide (10(-8)-10(-6) M) and cyproheptadine (10(-8)-10(-6) M) blocked the inhibition of alanine and glutamine release, the stimulations of muscle cAMP levels and phosphorylase a formation, and the decreased muscle glycogen content effected by serotonin but not by epinephrine. Incubation of muscles with both epinephrine and serotonin together produced additive stimulation of muscle cAMP levels, but not of the inhibition of alanine and glutamine release. These data indicate that the action of these agonists on skeletal muscle protein and amino acid, glycogen, and cyclic nucleotide metabolism proceeds directly via separate and discrete serotonergic and adrenergic receptor-adenylyl cyclase mechanisms in skeletal muscle.

Effects of parathyroid hormone on skeletal muscle protein and amino acid metabolism in the rat.

Because prominent skeletal muscle dysfunction and muscle wasting are seen in both chronic uremia and in primary hyperparathyroidism, and because markedly elevated parathyroid hormone levels occur in both disorders, potential effects of parathyroid hormone on skeletal muscle protein, amino acid, and cyclic nucleotide metabolism were studied in vitro using isolated intact rat epitrochlearis skeletal muscle preparations. Intact bovine parathyroid hormone and the synthetic 1-34 fragment of this hormone stimulated the release of alanine and glutamine from muscle of control but not from chronically uremic animals. This stimulation was dependent upon the concentration of parathyroid hormone added: At 10(5) ng/ml parathyroid hormone increased alanine release 84% and glutamine release 75%. Intracellular levels of alanine and glutamine were not altered by parathyroid hormone. Increasing concentrations of the 1-34 polypeptide decreased [(3)H]leucine incorporation into protein of muscles from both control and uremic animals. Using muscles from animals given a pulse-chase label of [guanido-(14)C]arginine in vivo, parathyroid hormone increased the rate of loss of (14)C label from acid-precipitable protein during incubation and correspondingly increased the rate of appearance of this label in the incubation media. Parathyroid hormone increased muscle cAMP levels by 140% and cGMP levels by 185%, but had no effect on skeletal muscle cyclic nucleotide phosphodiesterase activities as assayed in vitro. Adenylyl cyclase activity in membrane preparations from control but not uremic rats was stimulated by parathyroid hormone in a concentration-dependent fashion. However, no stimulation of guanylyl cyclase activity was noted by parathyroid hormone, although stimulation by sodium azide was present. Incubation of muscles with added parathyroid hormone produced a diminished responsiveness towards epinephrine or serotonin regulation of amino acid release and cAMP formation in the presence compared to the absence of parathyroid hormone. In the absence of parathyroid hormone, detectable inhibition of alanine and glutamine release was produced by 10(-9) M epinephrine, whereas in the presence of parathyroid hormone (1,000 ng/ml) inhibition of alanine and glutamine release required 10(-6) M or greater epinephrine. Resistance to cyclic AMP action as well as inhibition of cyclic AMP formation by parathyroid hormone was found. Preincubation of rat sarcolemma with 1-34 parathyroid hormone produced a decreased activity of the isoproterenol-stimulable adenylyl cyclase activity but there was no apparent change in the concentration of isoproterenol required for one-half maximal and maximal stimulation of the enzyme. These findings suggest that high levels of parathyroid hormone have direct effects on skeletal muscle protein, amino acid, and cyclic nucleotide metabolism in muscle of normal but not uremic animals. Treatment with these high levels of parathyroid hormone in vitro appears to reproduce in normal muscle, the metabolic deficits and loss of hormone responsiveness observed in muscle of chronically uremic animals. It is therefore possible that direct effects of parathyroid hormone on skeletal muscle may account in part for the muscle dysfunction and wasting of primary hyperparathyroidism and chronic uremia.

Uniform sarcomere shortening behavior in isolated cardiac muscle cells.

We have observed the dynamics of sarcomere shortening and the diffracting action of single, functionally intact, unattached cardiac muscle cells enzymatically isolated from the ventricular tissue of adult rats. Sarcomere length was measured either (a) continuously by a light diffraction method or (b) by direct inspection of the cell's striated image as recorded on videotape or by cinemicroscopy (120--400 frames/s). At physiological levels of added CaCl2 (0.5--2.0 mM), many cells were quiescent (i.e., they did not beat spontaneously) and contracted in response to electrical stimulation (less than or equal to 1.0-ms pulse width). Sarcomere length in the quiescent, unstimulated cells (1.93 +/- 0.10 [SD] micrometers), at peak shortening (1.57 +/- 0.13 micrometers, n = 49), and the maximum velocity of sarcomere shortening and relengthening were comparable to previous observations in intact heart muscle preparations. The dispersion of light diffracted by the cell remained narrow, and individual striations remained distinct and laterally well registered throughout the shortening-relengthening cycle. In contrast, appreciable nonuniformity and internal buckling were seen at sarcomere lengths < 1.8 micrometers when the resting cell, embedded in gelatin, was longitudinally compressed These results indicate (a) that shortening and relengthening is characterized by uniform activation between myofibrils within the cardiac cell and (b) that physiologically significant relengthening forces in living heart muscle originate at the level of the cell rather than in extracellular connections. First-order diffracted light intensity, extremely variable during sarcomere shortening, was always greatest during midrelaxation preceding the onset of a very slow and uniform phase of sarcomere relengthening.

Relaxation of mammalian single cardiac cells after pretreatment with the detergent Brij‐58.

1. The influence of load and activation on relaxation of heart muscle has been studied. 2. Cardiac cells devoid of functioning sarcolemma were isolated from rat and cat ventricular myocardium. Pretreatment with the detergent Brij-58 destroyed residual sarcoplasmic reticulum function. In order to analyse the mechanical properties of relaxation in these cells, a new miniature transducer was designed which could measure force by feedback sensing (resolution of 1 microgram). Contraction was induced by ionophoretically released calcium ions. Activation, sequestration of calcium and loading conditions could be controlled independently. 3. The time course of relaxation was shown to be governed by the amount of calcium released, and unlike intact preparations from rat or cat heart (but like those from frog), to be independent of load and of alterations in load. 4. We conclude that relaxation of the cardiac contractile system is determined basically by an activation-dependent mechanism, which is masked by load dependence in intact muscle preparations with a well developed calcium sequestering membraneous system.

Alanine and glutamine synthesis and release from skeletal muscle. II. The precursor role of amino acids in alanine and glutamine synthesis.

The synthesis and release of alanine and glutamine have been studied in the intact rat epitrochlaris skeletal muscle preparation. Aspartate, cysteine, leucine, valine, methionine, isoleucine, serine, theronine, and glycine increased significantly the formation and release of alanine from muscle. Cysteine, leucine, valine, methionine, isoleucine, tyrosine, lysine, and phenylalanine increased the rate of glutamine synthesis. Only ornithine, arginine, and tryptophan were without effect on the synthesis of either alanine or glutamine. Half-maximal stimulation of alanine and glutamine formation by added amino acids was observed with concentrations ranging between 0.5 and 1.0 mM. Increases in alanine and glutamine formation were not accompanied by changes in pyruvate production or glucose uptake. The progressive decline in alanine and glutamine synthesis noted on prolonged incubation was prevented by the addition of amino acids to the incubation medium. Stimulation of alanine synthesis by added amino acids was unaffected by inhibition of glycolysis with iodoacetate. Inhibition of alanine aminotransferase with aminooxyacetate significantly decreased alanine formation. Pyruvate and ammonium chloride did not increase further the rate of either alanine or glutamine formation above that produced by added amino acids. These data indicate that most amino acids are precursors for alanine and glutamine synthesis in skeletal muscle. A general mechanism is presented for the de novo formation of alanine from amino acids in skeletal muscle, and the importance of proteolysis for the supply of amino acid precursors for alanine and glutamine synthesis is discussed.