Cardiac substances that influence blood-pressure. I. Identification of the agent that lowers blood-pressure in anaesthetized rats

Extracts of the atrium of the mammalian heart contain a natriuretic factor which may be associated with the atrium-specific granules. It has often been observed that the intravenous injection of a crude atrial extract into anaesthetized rats, causes a transient decrease in blood-pressure. In rabbits, this activity is present in stored aqueous extracts prepared from both atrial and ventricular tissue. The hypotensive activity, which can be readily separated from the natriuretic factor, is mainly due to the presence of adenosine and its derivatives, of which 5'-adenosine monophosphate is the major contributor. However, an extract from rabbit atrial muscle, carefully prepared under stringent conditions, caused a rapid and striking increase in blood-pressure, an activity that could not be detected in ventricular tissue.     

Lysophospholipase activity in rabbit skeletal muscle

Skeletal muscle contains appreciable lysophospholipase activity which is differentiated by muscle type with red muscle subcellular fractions having greater activity than the corresponding white ones.     

Effect of stimulation frequency on intracellular Na+ activity in rat atrial muscle

Mean intracellular Na+ activity (aNai) was measured in rat left atrial muscle stimulated at increasing frequencies between 0 and 12 Hz. Low-pass filtered signals from conventional and ion-selective microelectrodes were used to determine aNai. Preparations were bathed in a low Ca2+ (0.1 mM) Krebs-Henseleit buffer containing 1.0 mM Mn2+ to abolish contractile motion and permit stable impalements. Under these conditions, aNai increased progressively with frequency from 5.8 +/- 1.5 mM at 0 Hz to a maximum of 12.7 +/- 2.1 mM, which was observed at 10, 11, or 12 Hz. Further increases in frequency exceeded the effective refractory period, and aNai tended to decrease. These data suggest that aNai can be approximately doubled in rat atrial muscle by increasing the depolarization rate from 0 to 10-12 Hz, a range that has been shown to elicit a two- to three-fold elevation in Na+-pump activity in similar preparations.     

Phosphoenolpyruvate hydrolase activity of rabbit muscle pyruvate kinase.

Rabbit muscle pyruvate kinase catalyzes the hydrolysis of P-enolpyruvate at the same active site which catalyzes the physiologically important kinase reaction. The hydrolase activity is lower than the kinase activity by a factor of at least 10(3). There are specific monovalent cation and divalent cation requirements. No other cofactors are required. The relative activation of the pyruvate kinase for the hydrolase reaction is: Ni(II) greater than Co(II) greater than Mg(II) greater than Mn(II). This parallels the rates of nonenzymatic hydrolysis of P-enolpyruvate (Benkovic, S.J., and Schray, K.J. (1968) Biochemistry 7, 4097-4102). The pH rate profiles of the hydrolase and kinase reactions activated by Ni(II) and Co(II) are similar, suggesting common features in their mechanisms. In contrast to the kinase reaction, the reaction velocity of the hydrolase increases at high Co(II) concentrations indicating a second mode for hydrolysis.     

Regulation of atrial natriuretic factor receptors by angiotensin II in rat vascular smooth muscle cells

Atrial natriuretic factor (ANF) is actively involved in the control of blood pressure and fluid homeostasis as a physiological antagonist of the renin-angiotensin system. To evaluate a possible interaction between ANF and angiotensin II (Ang-II) receptors, we investigated the effect of long term pretreatment (18 h) of rat cultured vascular smooth muscle cells with Ang-II. Binding of 125I-labeled ANF and cyclic GMP production induced by ANF were measured. After preincubation of the cells with Ang-II (1, 10, and 100 nM), the number of ANF binding sites (Bmax) was decreased by 30, 59, and 71%, respectively, with a slight decrease of the Kd values. Sar1-Ile8-Ang-II (100 nM), a specific Ang-II receptor antagonist, totally inhibited the down-regulation induced by Ang-II (10 nM). Moreover, the regulatory effect of Ang-II on ANF receptors appeared more slowly as compared to ANF homologous receptor regulation. Ang-II pretreatment did not desensitize but increased cyclic GMP production elicited by ANF, implying that only the number of non-guanylate cyclase-coupled receptors was affected. These findings, which were not observed with 100 nM of epinephrine, norepinephrine, histamine, serotonin, and Arg-vasopressin, demonstrate a specific and functional link between ANF and Ang-II receptors. This study also shows that the regulation of ANF receptors is heterogeneous, providing new evidence of multiple classes of ANF receptors.     

Contractile activity enhances the synthesis of hexokinase II in rat skeletal muscle

An 11-fold increase in hexokinase activity and the hexokinase II isoform was found in rat tibialis anterior muscle after 7 days of chronic, low-frequency stimulation. In vivo labeling studies showed that this increase in enzyme protein content was related to an approx. 30-fold increase in [35S] methionine incorporation.     

The influence of testosterone on the alkaline proteolytic activity in rat skeletal muscle

The effects of testectomy and subsequent administration of testosterone propionate on the activity of the alkaline proteinases in rat skeletal muscle were investigated. Castration of the mature rat was followed by a short-term delay in protein accretion in skeletal muscle tissue as measured by the protein/DNA ratio and was paralleled by a 2–3 fold increase in specific activity of the alkaline proteinase(s). This increase of proteolytic activity was equally significant when expressed relative to μg DNA. Although the gain in body weight was significantly lower in the castrated rats, nevertheless the protein/DNA ratio in muscle after 6 weeks approximated the values of sham-operated control rats without normalization of the proteolytic activity.Treatment of the castrated rats with testosterone propoinate resulted in restoring normal levels of previously elevated levels of alkaline proteolytic activity in muscle tissue. The normalization of enzyme activity as well as protein accretion in muscle was dose-dependent. Treatment of the rats with a low dose (0.1 mg/day) of testosterone propionate failed to restore the proteolytic activity, but led to a small increase of the protein/DNA ratio as well as to a progressive increase in body weight. These data indicate a regulatory role of testosterone in the adaptive behaviour of the alkaline proteolytic system in rat skeletal muscle.     

Capsaicin-sensitive nerves influence the release of atrial natriuretic factor by atrial stretch in the rat.

Although many factors may modulate the release of atrial natriuretic factor (ANF), the primary mechanism has been demonstrated to be atrial stretch. Recent studies have led to the suggestion that the peptidergic innervation of the heart, through the release of peptides, may be involved in the control of ANF secretion. We have examined the influence of chronic capsaicin treatment on three models of atrial stretch that release ANF. This treatment inhibited ANF released through in vivo blood volume expansion and through balloon inflation in the right atrium of in vitro isolated perfused hearts. Immunohistochemical and electron microscopical analysis confirmed the absence of innervation of the heart by calcitonin gene related peptide and substance P immunoreactive nerve fibres and apparent lack of effect on atrial granules in capsaicin treated rats. We conclude that capsaicin-sensitive cardiac innervation is a component modulating the release of ANF, stimulated by atrial stretch in the rat.     

A comparison of the influence of potassium and ammonium ions on the phosphofructokinases from rabbit muscle and rat erythrocytes.

Phosphofructokinases from rat erythrocytes and rabbit muscle have been compared in their kinetic behavior with respect to monovalent cation activation and ATP inhibition. Both ammonium and potassium ions affect the muscle enzyme in a two-fold manner: they act both as activators and effectors. On the other hand only ammonium exerts the two-fold effects on the erythrocyte enzyme, while the potassium ions activate without affecting cooperativity. The lower ATP inhibition of muscle phosphofructokinase may be partially explained by the action of potassium ions on the cooperative behavior of the enzyme. The differences between the phosphofructokinases from erythrocytes and muscle in the potassium type-II activation and ATP inhibition represent an organ specifity. Furthermore, the inhibition constants for 2, 3-bisphosphoglycerate differ by 10-fold between the two enzymes.     

A primer independent activity of rabbit muscle phosphorylase b.

Rabbit muscle phosphorylase b was found to be capable of forming protein bound alpha-1,4 glucosyl chains upon incubation of the enzyme with appropriate concentrations of glucose-1-phosphate with no primer addition (unprimed synthesis). This activity would only be present in a small fraction of the total muscle phosphorylase b activity, as judged from the high concentrations of enzyme which are required to demonstrate the occurrence of unprimed synthesis. Polyacrylamide gel electrophoresis shows the presence of a phosphorylase isoenzyme capable of accepting glucosyl moieties, giving rise to a glucosylated protein enzymatically active in the chain lengthening of its own glucan.     

Classes of thiols that influence the activity of the skeletal muscle calcium release channel

The skeletal muscle Ca(2+) release channel/ryanodine receptor (RyR1) is a prototypic redox-responsive ion channel. Nearly half of the 101 cysteines per RyR1 subunit are kept in a reduced (free thiol) state under conditions comparable with resting muscle. Here we assessed the effects of physiological determinants of cellular redox state (oxygen tension, reduced (GSH) or oxidized (GSSG) glutathione, and NO/O(2) (released by 3-morpholinosydnonimine)) on RyR1 redox state and activity. Oxidation of approximately 10 RyR1 thiols (from approximately 48 to approximately 38 thiols/RyR1 subunit) had little effect on channel activity. Channel activity increased reversibly as the number of thiols was further reduced to approximately 23/subunit, whereas more extensive oxidation (to approximately 13 thiols/subunit) inactivated the channel irreversibly. Neither S-nitrosylation nor tyrosine nitration contributed to these effects. The results identify at least three functional classes of RyR1 thiols and suggest that 1) the channel may be protected from oxidation by a large reservoir of functionally inert thiols, 2) the channel may be designed to respond to moderate oxidative stress by a change in activation setpoint, and 3) the channel is susceptible to oxidative injury under more extensive conditions.     

Baroreflex modulation of muscle sympathetic nerve activity during cold pressor test in humans

The purpose of this project was to test the hypothesis that baroreceptor modulation of muscle sympathetic nerve activity (MSNA) and heart rate is altered during the cold pressor test. Ten subjects were exposed to a cold pressor test by immersing a hand in ice water for 3 min while arterial blood pressure, heart rate, and MSNA were recorded. During the second and third minute of the cold pressor test, blood pressure was lowered and then raised by intravenous bolus infusions of sodium nitroprusside and phenylephrine HCl, respectively. The slope of the relationship between MSNA and diastolic blood pressure was more negative (P < 0.005) during the cold pressor test (-244.9 +/- 26.3 units x beat(-1) x mmHg(-1)) when compared with control conditions (-138.8 +/- 18.6 units x beat(-1) x mmHg(-1)), whereas no significant change in the slope of the relationship between heart rate and systolic blood pressure was observed. These data suggest that baroreceptors remain capable of modulating MSNA and heart rate during a cold pressor test; however, the sensitivity of baroreflex modulation of MSNA is elevated without altering the sensitivity of baroreflex control of heart rate.     

Absence of diamine oxidase activity from rabbit and rat lungs.

To study the presence of diamine oxidase (DAO) activity in any tissue with putrescine as the substrate, it is necessary to use inhibitors to block all pathways that could further metabolize gamma-aminobutyraldehyde, which is the product of enzyme reaction. It is also necessary to inhibit any enzyme that may convert putrescine into higher polyamines. By this approach it was observed that lung tissue of both rat and rabbit exhibited no DAO activity. DAO activity was observed in the rat and rabbit intestine, the former showing 3 times as much activity as the latter. The other potential pathways of putrescine metabolism are of no consequence in the rat and rabbit intestine and lungs.     

The thermogenic activity of rat brown adipose tissue and rabbit white muscle Ca2+-ATPase

The Ca2+-ATPase (SERCA) found in vesicles derived from the sarco/endoplasmic reticulum vesicles of rats brown adipose tissue and rabbit white muscle were identified by gel electrophoresis, Western blot, electron microscopy and immunolabeling with gold particles. In both tissues, the isoform found was SERCA 1. The Ca2+ affinity of the fat SERCA 1 was different from the muscle isoform. The degree of uncoupling is estimated measuring the ratio between Ca2+ transport and ATP cleaved. In brown fat vesicles the degree of uncoupling varied depending on the Ca2+ concentration of the medium. This was not observed in vesicles derived from muscle. At all Ca2+ concentrations tested, the uncoupling was not related to Ca2+ leakage from the membrane and was far more pronounced in fat than in muscle vesicle. When a Ca2+ gradient was formed across the vesicles membrane the heat released during ATP hydrolysis varied between 22 and 26 Kcal/mol in both fat and muscle vesicles but in the absence of a gradient the heat released was 17 Kcal/mol in fat and 12 Kcal/mol in muscle. The data reported indicate that the SERCA 1 of brown adipocytes is far more thermogenic than the white muscle SERCA 1, and suggest that, in addition to storing Ca2+ inside the endoplasmic reticulum, the SERCA 1 may represent a source of heat production contributing to the thermogenic function of brown adipose tissue.     

Signal-transduction pathways that regulate smooth muscle function. II. Receptor-ion channel coupling mechanisms in gastrointestinal smooth muscle

Regulation of membrane ion channels by second messengers is an important mechanism by which gastrointestinal smooth muscle excitability is controlled. Receptor-mediated phosphorylation of Ca(2+) channels has been known for some time; however, recent findings indicate that these channels may also modulate intracellular signaling. The plasmalemma ion channels may also function as a point of convergence between different receptor types. In this review, the molecular mechanisms that link channel function and signal transduction are discussed. Emerging evidence also indicates altered second-messenger modulation of the Ca(2+) channel in the pathophysiology of smooth muscle dysmotility.     

Domain interaction in rabbit muscle pyruvate kinase. II. Small angle neutron scattering and computer simulation

The effects of ligands on the structure of rabbit muscle pyruvate kinase were studied by small angle neutron scattering. The radius of gyration, RG, decreases by about 1 A in the presence of the substrate phosphoenolpyruvate, but increases by about the same magnitude in the presence of the allosteric inhibitor phenylalanine. With increasing pH or in the absence of Mg2+ and K+, the RG of pyruvate kinase increases. Hence, there is a 2-A difference in RG between two alternative conformations. Length distribution analysis indicates that, under all experimental conditions which increase the radius of gyration, there is a pronounced increase observed in the probability for interatomic distance between 80 and 110 A. These small angle neutron scattering results indicate a "contraction" and "expansion" of the enzyme when it transforms between its active and inactive forms. Using the alpha-carbon coordinates of crystalline cat muscle pyruvate kinase, a length distribution profile was calculated, and it matches the scattering profile of the inactive form. These observations are expected since the crystals were grown in the absence of divalent cations (Stuart, D. I., Levine, M., Muirhead, H., and Stammers, D. K. (1979) J. Mol. Biol. 134, 109-142). Hence, results from neutron scattering, x-ray crystallographic, and sedimentation studies (Oberfelder, R. W., Lee, L. L.-Y., and Lee, J.C. (1984) Biochemistry 23, 3813-3821) are totally consistent with each other. With the aid of computer modeling, the crystal structure has been manipulated in order to effect changes that are consistent with the conformational change described by the solution scattering data. The structural manipulation involves the rotation of the B domain relative to the A domain, leading to the closure of the cleft between these domains. These manipulations resulted in the generation of new sets of atomic (C-alpha) coordinates, which were utilized in calculations, the result of which compared favorably with the solution data.