Urodilatin (CDD/ANP-95-126) is not biologically inactivated by a peptidase from dog kidney cortex membranes in contrast to atrial natriuretic peptide/cardiodilatin (alpha-hANP/CDD-99-126).

Atrial natriuretic peptide (CDD/ANP-99-126) is rapidly inactivated by a membrane preparations from dog kidney cortex. Inactivation occurs by cleavage of the ring structure in the position between Cys-105 and Phe-106. A unique proteolytic product separated by HPLC on reverse-phase column appears as a single peak which elutes prior the intact peptide. In contrast, CDD/ANP-95-126 (urodilatin) which is released from the kidney is not destroyed by proteolysis using an identical membrane preparation.

Relaxation of smooth muscle by cardiodilatin/atrial natriuretic peptide is inhibited by cAMP-dependent phosphorylation.

Cardiodilatins/atrial natriuretic peptides (CDD/ANP) exhibit a common amino acid sequence: Arg101-Arg102-Ser103-Ser104. Cyclic AMP-dependent phosphorylation of Ser104 of atrial peptides with [gamma-32P]ATP enables rapid identification of cardiac hormones. The biological activity of in vitro phosphorylated cardiodilatin (CDD-28/alpha-hANP) is dramatically altered compared to the unphosphorylated peptide: the vaso-relaxant effect of cardiodilatin 28 is inhibited upon phosphorylation.

Calcium-requirement for activation of skinned vascular smooth muscle from spontaneously hypertensive (SHRSP) and normotensive control rats.

The aim of this study was to clarify whether the increased vascular tone in spontaneous hypertension of rats is due to a change of the calcium-sensitivity of the contractile proteins themselves. In skinned rat tail artery rings from SHRSP and WKY rats the calcium-requirement for half maximal activation (3.6 X 10(-6)M Ca++ for both rat strains) as well as relaxation half times (1.45 +/- 0.43 min, SHRSP and 1.63 +/- 0.48 min, WKY) were found to be identical. The extent of myosin phosphorylation in the contracted and in the relaxed state did not differ between SHRSP and WKY. It is concluded that changes at the level of the contractile proteins are not involved in the increased vascular tone of SHRSP essential hypertension.

Atrial natriuretic peptide-like immunoreactive cells in the guinea pig inner ear.

Using specific antibodies against cardiodilatin/atrial natriuretic peptide (CDD/ANP) in a conventional immuno-histochemical method (PAP) we located ANP/CDD-like immuno-reactive cells related to the secretory area, to the sensory and to the neuronal area in the compartments of the inner ear (cochlea, utricle/ampulla, and endolymphatic sac). Immunoreactive cells were unevenly distributed in the different compartments as well as within the cochlear space. Our findings suggest that ANP/CDD may play a role in the local control of fluid and electrolyte homeostasis of the inner ear. ANP/CDD-binding sites and ANP/CDD-like immunoreactivity in the inner ear may also indicate that the peptide has an additional paracrine and/or autocrine function in the organ.

Force generated by non-cycling crossbridges at low ionic strength in skinned smooth muscle from Taenia coli.

In skinned smooth muscle preparations of Taenia coli from guinea pig the cyclic actin-myosin-interaction is inhibited to resting levels whereas the force remains high when in the contracted muscle the ionic strength of the incubation solutions is lowered from 65 mM to 18 mM. In accordance with a strongly depressed ATPase activity under these conditions the isotonic shortening velocity is drastically decreased suggesting that crossbridges are not cycling (latch-state). Since the Ca2+ concentration is kept constant (4 X 10(-5) M) and the phosphorylation of the myosin light chain (LC) is found to be unchanged it must be concluded that the formation of latch-bridges is not necessarily dependent on a change in the Ca2+ concentration or the state of phosphorylation.

Effect of inorganic phosphate on the Ca2+ sensitivity in skinned Taenia coli smooth muscle fibers. Comparison of tension, ATPase activity, and phosphorylation of the regulatory myosin light chains.

Inorganic phosphate (Pi) decreases maximal tension in contracted skeletal and heart muscle fibers. We investigated the effects of 10 mM Pi on the force-calcium relationship in Triton X-100-skinned Taenia coli smooth muscle fibers. Isometric force measurements show that the calcium sensitivity of the force depends on the phosphate concentration. Furthermore 10 mM Pi relaxes the fibers more at intermediate than at high calcium ion concentrations: At pCa 4.5 tension decreases in the presence of 10 mM Pi by approximately 12% but it decreases 70% at pCa 6.17. Removal of phosphate partially reverses the relaxation. Simultaneous determination of actomyosin ATPase activity and force (Güth, K., and J. Junge, 1982, Nature (Lond.), 300:775-776) shows that the ATPase activity does not correlate with the changes in force. In the presence of Pi, tension decreases more than the ATPase activity. The level of phosphorylation of the 20,000-D regulatory myosin light chain is not changed in the presence or absence of 10 mM Pi. The results are discussed in terms of slowly or noncycling myosin crossbridges formed at lower calcium concentrations, which contribute to the force development but not to the ATPase activity. These crossbridges are considered to be dissociated in the presence of phosphate.

Isolation and elucidation of some functional properties of the "mute" catalytic subunit of cAMP-dependent protein kinase.

A mute isoenzyme of type II cAMP-dependent protein kinase from rat muscle has been reported that is released from the regulatory subunit by cAMP but remains inactive until combination with heat- and acid-stable modulator has occurred. This enzyme has now been obtained in isolation free of the normal catalytic subunit using affinity chromatography with both an ATP analog (Blue Dextran/Sepharose) and a protein substrate analog (Kemptide/CH-Sepharose). Separation can be effected in both cases before activation of the mute enzyme. Affinity of the mute enzyme for Blue Dextran--a ligand specific for the dinucleotide fold in this kinase--is somewhat higher than that of the normal enzyme. Conversely, before reaction with the modulatory protein the mute enzyme will not bind at all to Kemptide/CH-Sepharose, where the normal enzyme elutes at 50 mM KCl. When pretreated with the modulatory protein and so activated, mute enzyme binds to Kemptide with a very high affinity and can only be eluted using a natural substrate (phosphorylase kinase), up to 500 mM salt being ineffective. The modulator thus appears to act through alteration of the protein substrate binding site on the enzyme.

Phosphorylation of the myosin light chains and satellite proteins in detergent-skinned arterial smooth muscle.

Isoelectric focusing of extracts prepared from detergent-skinned porcine carotid artery showed that contraction was associated with phosphorylation of the regulatory myosin light chains and two additional proteins of the same apparent molecular weight (20,000). These two proteins, previously described as satellites, did not appear to be artifactually derived from the phosphorylated light chains during electrophoresis. That is, each of the phosphorylated proteins migrated as separate and distinct proteins when subjected to a second cycle of isoelectric focusing. Moreover, relaxation of skinned fibers was associated with dephosphorylation of the light chains and both satellites. These findings suggest that the satellites may represent varients of the light chains per se, or another regulatory protein which is reversibly phosphorylated and dephosphorylated during contraction and relaxation of vascular smooth muscle.

Isolation of the catalytic subunits of cyclic AMP-dependent protein kinases from different mammalian tissues on the basis of charge differences of their subunits.

Both, the experimental data and a literature survey presented reveal common charge differences in the subunit composition of cAMP-dependent protein kinases from a variety of mammalian tissues. In general, the holoenzymes (type I and II) focus at pH 4.5-5.5, the cAMP-binding regulatory subunit below pH 4, whereas catalytic subunits are found at pH 6.7-9.1. For the purification of the catalytic subunits, the anionic holoenzymes of a variety of rat and rabbit organs as well as mouse and human-derived tissue culture cells were adsorbed to DEAE-cellulose. The catalytic subunit from the particular isoenzyme were selectively eluted at an appropriate ionic strength depending on the isoenzyme elution pattern by small amounts of cAMP. Extraction of tissues with Triton X-100 increased enzyme yield. Improved elution of the enzyme was accomplished by 8-(4-aminobutyl)amino-cAMP instead of cyclic AMP. Carboxymethyl-cellulose chromatography leads to both, concentration and, if necessary, to further purification of the enzyme.

Skinned smooth muscle: time course of force and ATPase activity during contraction cycle.

The time course of ATPase activity and force has been determined during contraction and relaxation in skinned (hyperpermeable) anterior byssus retractor muscle, ABRM, of Mytilus edulis and compared with corresponding measurements on skinned taenia coli of guinea-pigs. Following a calcium-induced contraction, lowering the [Ca++] to 10(-8) M rapidly reduces ATPase activity within 2 min to resting levels while force declines only to about 30-50% of maximal tension within the same time. Thus slow relaxation is due to a 'catch-like-state' which is common to different kinds of smooth muscles and can be reduced with cAMP in ABRM and by Pi in taenia coli.

Phosphorylation and dephosphorylation of the natriuretic peptide urodilatin (CDD-/ANP-95-126) and the effect on biological activity.

Urodilatin (CDD-/ANP-95-126), a new peptide hormone from human urine, is comprised of the same amino acid sequence as cardiodilatin (CDD-99-126/alpha-hANP) except for N-terminal extention by four amino acid residues. The presence of the recognition sequence Arg101-Arg-Ser-Ser104 for the cyclic AMP-dependent protein kinase enables rapid phosphorylation in the Ser104-position. Phosphorylation of urodilatin is associated with decreased vasorelaxant potency, while dephosphorylation of "phospho-urodilatin" by acidic phosphatase completely restores bioactivity.

Effect of Ca2+-independent myosin light chain kinase on different skinned smooth muscle fibers.

In various skinned smooth muscle fiber preparations, (porcine carotid artery, rat tail artery, chicken gizzard and Taenia coli from guinea pig) a Ca2+-independent myosin light chain kinase (MLCK) initiated a contraction in absence of Ca2+. While the Ca2+ insensitive MLCK was effective on the vertebrate smooth muscles it did not act on the invertebrate skinned skeletal muscle preparation from Limulus and anterior byssus retractor muscle from Mytilus edulis. The results indicate that in vertebrate smooth muscles phosphorylation is sufficient for activation and that there is no obligatory role for an additional mechanism in initiation of contraction.

Effect of calmodulin, Ca2+, and cAMP protein kinase on skinned tracheal smooth muscle.

The characteristics of contraction and relaxation of membrane skinned smooth muscle from guinea pig trachealis muscle are described. Micromolar Ca2+ elicited reproducible contractions in Mg-ATP salt solution at 20 degrees C. The speed of contraction was much faster at 30 and 37 degrees C, enabling cumulative concentration-response curves to be obtained. At these temperatures, a progressive increase in basal tension occurred in the absence of Ca2+. This tension was active and developed more rapidly at pH 6.7 than at pH 7.0. Calmodulin (0.1-10 microM) greatly increased the speed of contraction and lowered the threshold Ca2+ concentration ([Ca2+]) required to initiate contraction from 0.13 to 0.02 microM Ca2+. Trifluoperazine antagonized responses to Ca2+. Thiophosphorylation with adenosine 5'-O-(3-thiotriphosphate) produced maximum tension development, which was Ca2+-independent. This effect was reversible. The results are compatible with myosin-linked regulation of contraction in which a Ca2+ X calmodulin complex activates myosin light chain kinase to phosphorylate myosin. The catalytic subunit of cAMP-dependent protein kinase strongly inhibited tension development and slowly relaxed fibers contracted with threshold [Ca2+] consistent with an action via phosphorylation of myosin light chain kinase. This effect was extremely slow compared with the rate of relaxation by Ca2+ withdrawal or with relaxation of intact smooth muscle by beta-adrenergic agonists.

Evidence for a "mute" catalytic subunit of cyclic AMP-dependent protein kinase from rat muscle and its mode of activation.

An isoenzyme of the catalytic subunit of type II cyclic AMP-dependent protein kinase from rat muscle is reported which coelutes with the classical catalytic subunit but differs from it in isoelectric point (pI 8.7 vs pI 9.1) and is enzymmatically inactive. After reaction with a heat- and acid-stable component of the protein kinase modulator fraction from the same tissue, the "mute" isoenzyme displays a high activity when assayed on isoelectric focusing gels. This activation process does not occur through proteolytic degradation and is not characteristic of a turnover-type reaction. The data imply direct interaction between the isoenzyme and a modulating protein which may subsequently be separated from the enzyme without reversal of the activation. The modulator protein thus appears to act as a template, inducing a conformational change. The implications of such a mute isoenzyme and its control through small modulator proteins are discussed.

Temporal relationship between force, ATPase activity, and myosin phosphorylation during a contraction/relaxation cycle in a skinned smooth muscle.

The temporal relationship between myosin phosphorylation, contractile force and ATPase activity was studied in skinned preparations from the guinea-pig Taenia coli. When free Calcium concentration [( Ca2+]) was increased from pCa (-log[Ca2+]) 9 to pCa 4.5 at low calmodulin concentration (0.05 microM), ATPase activity and myosin light-chain phosphorylation rose quickly, while the increase in force and stiffness was delayed. The time-course of tension increase was faster at higher calmodulin concentrations (5 microM), although the maximal level of phosphorylation was unchanged. Lowering the calcium concentration from pCa 4.5 to pCa 9 at the plateau of contraction caused a rapid decrease in ATPase activity and in myosin phosphorylation, while force and stiffness decayed more slowly. The force decay could be accelerated by inorganic phosphate. These results suggest that, during contraction, force may be produced actively by phosphorylated and ATP-splitting crossbridges, but may be maintained by dephosphorylated crossbridges which cycle slowly. However, force could also be modulated by calmodulin and inorganic phosphate in a manner not involving an alteration in the extent of myosin phosphorylation.

Myosin isoenzymes of vascular smooth and cardiac muscle in the spontaneously hypertensive and normotensive male and female rat: a comparative study.

Cardiac hypertrophy in hypertensive subjects, its biochemical markers, and functional consequences are of great clinical importance but still unclear. We observed a shift of the ventricular isomyosin of adult spontaneously hypertensive (H) rats of both sexes to the V3 form and a decreased myofibrillar ATPase activity in the H animals when compared to normotensive (N) controls. Compared to the male H rats, age-matched female H animals revealed a lower blood pressure, the same or even an elevated magnitude of cardiac hypertrophy, a different ventricular isomyosin pattern, and a higher myofibrillar ATPase activity. In female H rats the V1 and V3 isomyosins were equally distributed (35% V1 and 35% V3), but in male H animals the V3 was predominant (24% V1 and 45% V3). The Ca2+-regulated Mg2+-dependent myofibrillar ATPase of the rat ventricle correlated positively with the amount of V1 when measured at pCa 5 (maximum activation). At submaximum Ca2+-concentrations (pCa 6.9-5.9) the myofibrillar ATPase activities were not changed with the proportion of V1. The cooperativity of the Ca2+-activation of the myofibrillar ATPase increased with increasing amount of V1 (Hill-coefficient 3.7 with 100% V1) and decreased with increased proportion of V3 (Hill-coefficient 1.3 at 45% V3). Two myosin isoenzymes were detected in the aorta of rats, a slow migrating (S2) and a fast migrating (S1) form having both a higher mobility than the ventricular isomyosins. Only one band was observed in the portal vein, which revealed the same mobility as S2. Hypertension did not change the appearance of these vascular smooth muscle isomyosins neither in male nor in female animals.

Degradation of porcine brain natriuretic peptide (pBNP-26) by endoprotease-24.11 from kidney cortical membranes.

Porcine brain natriuretic peptide of 26 amino acid residues (pBNP-26) is inactivated by endoprotease-24.11 (EC 3.4.24.11) of kidney cortical membranes. In contrast to human alpha atrial natriuretic peptide/cardiodilatin (ANP/CDD) showing a single major cleavage within the disulfide-linked loop between Cys and Phe in position 7 and 8, pBNP-26 is cleaved at several sites. Although both pBNP-26 and ANP/CDD exhibit Cys-Phe peptide bonds at the corresponding positions this bond is not cleaved in BNP-26.

The heart is the center of a new endocrine, paracrine, and neuroendocrine system.

This review indicates that the heart is a polypeptide-producing organ which should be classified among the traditional endocrine tissues. Cardiac hormones have only been known for a few years, the discovery of their endocrine functions, however, occurred in the 1950ies when Gauer, Henry and Kisch observed specific physiological and morphological features of the heart atria indicative of an endocrine activity. Because of their basic effects many target organs involved in the regulation of body fluid pressure and composition are related to this endocrine organ located in the atrial appendages of the heart. The compact endocrine portion of the heart is built up by myoendocrine cells which form the functional endocrine units and produce a variety of polypeptide hormones called cardiodilatin (CDD) or atrial natriuretic polypeptide (ANP), which belong to one family. Also, co-storage of a partially homologous regulatory polypeptide called brain natriuretic polypeptide (BNP) occurs, as has been determined by immunohistochemistry and radioimmunoassay. CDD and/or BNP are found in numerous organs where they exert paracrine and neurocrine functions, e.g., in the brain, peripheral nervous system, kidney, and adrenal medulla. In these organs, a differential post-translational processing of cardiac polypeptides is observed, resulting in different functional activities according to discriminating receptor interactions and degrading metabolism. Some of the extra-auricular sites of synthesis and storage of CDD-like peptides are briefly mentioned. In summary the heart constitutes the center of a multilocal and multifunctional system of specific cardiac polypeptides of endocrine, paraneuronal, and neuronal character.

Chromatographic separation of two heterogeneous forms of the catalytic subunit of cyclic AMP-dependent protein kinase holoenzyme type I and type II from striated muscle of different mammalian species.

Electrophoretically homogeneous preparations of catalytic subunit (C) of cAMP-dependent protein kinase isolated according to two different procedures from holoenzyme type I and type II from rabbit and from holoenzyme type II from rat skeletal muscle and from bovine cardiac muscle can be separated on carboxymethyl cellulose or on a Mono S column (Pharmacia) by salt gradient elution into two enzymatically active peaks called A and B, which do not interconvert on rechromatography. Cochromatography of peak A fractions or of peak B fractions derived from both holoenzymes respectively yields single enzyme peaks in each case, thus indicating that both represent different entities, which were named CA and CB. The separate character of both enzyme forms is supported by the fact that CB under all conditions is degraded faster by the C-specific protease (E. Alhanaty et al. (1981) Proc. Natl. Acad. Sci. USA 78, 3492-3495) than CA, a phenomenon which is enhanced in both enzyme forms by substrate (Kemptide). The separation of both subtypes from each other is probably based on differences in isoelectric values (delta pH less than or equal to 0.5 units). The reason for the charge difference is not presently known. CA and CB do not differ significantly in their phosphate content. No differences between CA and CB have been detectable so far with respect to their migration in SDS gels, kinetic behavior regarding both substrates and cosubstrate, pH dependence, inhibition by regulatory subunits of holoenzyme type I (rabbit skeletal muscle) and of type II (bovine cardiac muscle), and inhibition by specific-heat and acid-stable inhibitor-modulator. The peptide pattern of both forms after limited proteolysis exhibits small differences.