Immunolocalization of sarcolemmal dihydropyridine receptor and sarcoplasmic reticular triadin and ryanodine receptor in rabbit ventricle and atrium.

The subcellular distribution of sarcolemmal dihydropyridine receptor (DHPR) and sarcoplasmic reticular triadin and Ca2+ release channel/ryanodine receptor (RyR) was determined in adult rabbit ventricle and atrium by double labeling immunofluorescence and laser scanning confocal microscopy. In ventricular muscle cells the immunostaining was observed primarily as transversely oriented punctate bands spaced at approximately 2-micron intervals along the whole length of the muscle fibers. Image analysis demonstrated a virtually complete overlap of the staining patterns of the three proteins, suggesting their close association at or near dyadic couplings that are formed where the sarcoplasmic reticulum (SR) is apposed to the surface membrane or its infoldings, the transverse (T-) tubules. In rabbit atrial cells, which lack an extensive T-tubular system, DHPR-specific staining was observed to form discrete spots along the sarcolemma but was absent from the interior of the fibers. In atrium, punctate triadin- and RyR-specific staining was also observed as spots at the cell periphery and image analysis indicated that the three proteins were co-localized at, or just below, the sarcolemma. In addition, in the atrial cells triadin- and RyR-specific staining was observed to form transverse bands in the interior cytoplasm at regularly spaced intervals of approximately 2 micron. Electron microscopy suggested that this cytoplasmic staining was occurring in regions where substantial amounts of extended junctional SR were present. These data indicate that the DHPR codistributes with triadin and the RyR in rabbit ventricle and atrium, and furthermore suggest that some of the SR Ca2+ release channels in atrium may be activated in the absence of a close association with the DHPR.

Increased caveolin-3 levels in mdx mouse muscles.

The density of skeletal muscle caveolae is increased in Duchenne muscular dystrophy and its genetic homologue, the mdx mouse. This structural change is significant as it may indicate muscle regeneration. We identified in mdx mouse tibialis anterior muscles significantly increased levels of caveolin-3, the chief protein in muscle caveolae, and reduced levels of neuronal nitric oxide synthase, an enzyme regulated by caveolin-3. Similar changes occurred in the corresponding mRNA levels. These data suggest that induction of caveolin-3 occurs and this may at least partly be responsible for increased number of caveolae, altered nNOS-caveolin cycle, and regeneration of dystrophic muscles.

Azidobutyryl clentiazem, a new photoactivatable diltiazem analog, labels benzothiazepine binding sites in the alpha 1 subunit of the skeletal muscle calcium channel.

[3H]Azidobutyryl clentiazem, a new photoactivable diltiazem derivative, has a higher binding affinity than azidobutyryl diltiazem. It can be covalently incorporated into the alpha 1 subunit of the skeletal muscle calcium channel by UV irradiation, which allows the benzothiazepine binding site to be determined. The photolabeled alpha 1 subunit and its proteolytic fragments were analyzed with a panel of sequence-directed antibodies. The results suggest that the linker region between segment S5 and S6 of domain IV is involved in benzothiazepine binding. This site is different from the dihydropyridine and verapamil binding sites.

Identification of 1,4-dihydropyridine binding domains within the primary structure of the alpha 1 subunit of the skeletal muscle L-type calcium channel.

Calcium channel blockers are drugs that bind to the alpha 1 subunit of L-type calcium channels and selectively inhibit ion movements through these channels. Determination of the mechanism of channel blockade requires localization of drug-binding sites within the primary structure of the receptor. In this study the 1,4-dihydropyridine-binding site of the membrane bound receptor has been identified. The covalently labeled receptor was purified and digested with trypsin. The labeled peptide fragments were immunoprecipitated with sequence-directed antibodies. The data indicate the existence of at least three distinct dihydropyridine-binding domains within the primary structure of the alpha 1 subunit.

Receptors for calcium antagonists.

Calcium antagonists have been divided into 3 different subclasses represented by nifedipine, verapamil and diltiazem. These drugs have different pharmacologic effects and are not interchangeable. Previous studies suggested that all calcium antagonists bind to a 170 kd polypeptide (now called the alpha 2 subunit of the voltage-dependent calcium channel). The apparent molecular weight of this polypeptide characteristically decreased from 170 to 140 kd upon disulfide reduction as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recent studies demonstrated that calcium antagonists bind to a previously unrecognized 165 kd polypeptide (alpha 1 subunit) that does not change its electrophoretic mobility on disulfide reduction. Because of their similar molecular weights, the 2 polypeptides may overlap each other on polyacrylamide gels. The primary structure of both polypeptides clearly shows, however, that they are different from each other and only the alpha 1 subunit has the features expected of an ion channel.

Receptor pharmacology of calcium entry blocking agents.

The mechanism of action of calcium channel modulators, a class of drugs that includes 3 chemical groups--1,4-dihydropyridines, phenylalkylamines and benzothiazepines--has been extensively reviewed. The best known representatives of these 3 groups are nifedipine, verapamil and diltiazem, respectively. These drugs bind reversibly, stereospecifically and with high affinity to both the membrane-bound and the purified receptor complex. Non-dihydropyridines allosterically regulate dihydropyridine binding. This has been shown by using (-) [3H]202-791 and (+) [3H]PN200-110 as labeled ligands. The purified receptor complex that possesses binding sites for all 3 chemical groups is likely to be related to the voltage-dependent calcium channel. As the result of a drug-receptor interaction, voltage-dependent calcium channels are either activated or inactivated. The drugs that activate channels act by promoting long-lasting channel openings. The drugs that inhibit calcium channels, the calcium entry-blocking agents, act by preventing channel openings upon membrane depolarization. A complex pharmacologic, electrophysiologic, biochemical, immunologic and molecular genetic approach is required to determine the molecular mechanism of action of calcium channel modulators. Clinically, calcium entry-blocking agents are recommended for the treatment of angina pectoris, hypertension, posthemorrhagic cerebral vasospasm, supraventricular tachycardia, migraine and asthma and the protection of the ischemic myocardium.

Induction of neuronal type nitric oxide synthase in skeletal muscle by chronic electrical stimulation in vivo.

Fast-twitch skeletal muscles contain more neuronal-type nitric oxide synthase (nNOS) than slow-twitch muscles because nNOS is present only in fast (type II) muscle fibers. Chronic in vivo electrical stimulation of tibialis anterior and extensor digitorum longus muscles of rabbits was used as a method of inducing fast-to-slow fiber type transformation. We have studied whether an increase in muscle contractile activity induced by electrical stimulation alters nNOS expression, and if so, whether the nNOS expression decreases to the levels present in slow muscles. Changes in the expression of myosin heavy chain isoforms and maximum velocity of shortening of skinned fibers indicated characteristic fast-to-slow fiber type transformation after 3 wk of stimulation. At the same time, activity of NOS doubled in the stimulated muscles, and this correlated with an increase in the expression of nNOS shown by immunoblot analysis. These data suggest that nNOS expression in skeletal muscle is regulated by muscle activity and that this regulation does not necessarily follow the fast-twitch and slow-twitch pattern during the dynamic phase of phenotype transformation.

Angiotensin and bradykinin metabolism by peptidases identified in skeletal muscle.

We have identified angiotensin-converting enzyme, neutral endopeptidase-24.11, and aminopeptidase M in a purified glycoprotein fraction of rabbit skeletal muscle membranes. The identification was based on substrate specificity and sensitivity to selective inhibitors. Angiotensin I metabolism was due to angiotensin-converting enzyme-mediated conversion to angiotensin II and neutral endopeptidase-24.11-mediated conversion to angiotensin(1-7). Bradykinin was degraded by angiotensin-converting enzyme and neutral endopeptidase-24.11; angiotensin II by neutral endopeptidase-24.11; and angiotensin III by neutral endopeptidase-24.11 and aminopeptidase M. Thus, the effects of angiotensins and kinins on skeletal muscle blood flow and metabolism may be regulated by local angiotensin-converting enzyme, neutral endopeptidase-24.11, and aminopeptidase M.

Angiotensin and bradykinin metabolism by peptidases identified in cultured human skeletal muscle myocytes and fibroblasts.

Angiotensin (ANG) and kinin metabolizing enzymes, angiotensin-converting enzyme (ACE; EC 3.4.15.1), neutral endopeptidase-24.11 (NEP-24.11; EC 3.4.24.11), and aminopeptidase M (AmM; EC 3.4.11.2), have recently been identified in a purified skeletal muscle glycoprotein fraction. We have analyzed the cellular localization of these enzymes. In cultured human skeletal muscle adult myoblasts, myotubes, and fibroblasts, kinins and angiotensins were metabolized by NEP-24.11 and AmM but not by ACE. NEP-24.11 degraded ANG II, ANG III. and bradykinin (BK) and converted ANG I to the active metabolite ANG(1-7). ANG III was converted to the novel ANG IV metabolite [des-Arg1]ANG III by AmM. These data suggest that, due to their abundance in the body, skeletal muscle myocytes and fibroblasts may play a major role in modulation of the systemic and local effects of angiotensins and kinins. This role could be particularly important in individuals receiving treatment with ACE inhibitors.

Identification of the increased expression of monocyte chemoattractant protein-1, cathepsin S, UPIX-1, and other genes in dystrophin-deficient mouse muscles by suppression subtractive hybridization.

The lack of dystrophin results in muscular dystrophy characterized by degeneration, inflammation, and partial regeneration of skeletal muscles. The fate of these muscles may be determined by the extent of adaptation to the defect and the efficiency of regeneration that is affected by inflammatory cells. We have used suppression subtractive hybridization and quantitative Northern blot analysis to identify differentially expressed genes. Increased expression of murine monocyte chemoattractant protein-1 (JE/MCP-1), cathepsin S, UPIX-1, nmb, cathepsin B, and lysozyme M mRNAs were identified in 2-month-old mdx mouse leg muscles. UPIX-1 is a novel gene. Although it was not expressed in control muscles, it was expressed in control brain, heart, and spleen. JE/MCP-1 and cathepsin S proteins in mdx muscles, as well as JE/MCP-1 protein in the serum of mdx mice were also detected. JE/MCP-1 may be responsible for attraction of inflammatory cells, and cathepsin S, a potent elastolytic protease, may contribute to the remodeling of the extracellular matrix that is required for the migration of these cells to the injured muscles.

Mg(2+)-ATPase from rabbit skeletal muscle transverse tubules is 67-kilodalton glycoprotein.

There exists a Mg(2+)-ATPase in transverse tubules which has properties that are very different from other ATPases located in skeletal muscle cells. Several groups have suggested that a 100-kDa glycoprotein is the molecular entity responsible for this Mg(2+)-ATPase activity. In this study we have extended the methods utilized in the purification of this integral membrane glycoprotein. Although the apparent pI (isoelectric point) of this protein is pH 5, most of the net charge is due to the presence of sialic acid on the associated glycan chains. After these residues are removed, the behavior of this protein on an anion exchange column changes dramatically, allowing it to be further purified. Using a combination of the earlier procedures (Kirley, T.L. (1988) J. Biol. Chem. 263, 12682-12689 and Kirley, T. L. (1991) Biochem. J. 278, 375-400.) and the one reported here, purification to specific activities of approximately 400,000 mumol of ATP hydrolyzed/mg of protein/h were obtained and all traces of the 100-kDa protein were removed. The digitonin-solubilized Mg(2+)-ATPase appears to be a dimer of two identical 67-kDa subunits as assessed by high performance size exclusion chromatography. A single diffuse protein band is observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis at approximately 67 kDa, which reduced to a single tight protein band at 52 kDa after deglycosylation with the following unique N-terminal amino acid sequence: Ala-Lys-Lys-Val-Leu-Pro-Leu-Leu-Leu-Pro- Pro-Leu-Val-X-Ala-Ala-Leu-Gly-Leu-Ala-X-Phe. Therefore, the Mg(2+)-ATPase appears to be an enzyme of very high specific activity, present in small quantities in transverse tubule membranes and unrelated to the known classes of ATPases present in skeletal muscle. The data reported here on the orientation of the transverse-tubule membrane preparations are consistent with the very recent report (Saborido, A., Moro, G., and Megias A. (1991) J. Biol. Chem. 266, 23490-23498) that the Mg(2+)-ATPase is an ecto-enzyme.

Photoaffinity labeling of the purified skeletal muscle calcium antagonist receptor by a novel benzothiazepine, [3H]azidobutyryl diltiazem.

Previous photoaffinity-labeling studies with [3H]azidopine, (+) [3H]PN200-110, and [3H]LU 49888 have demonstrated that 1,4-dihydropyridines (nifedipine-like drugs) and phenylalkylamines (verapamil-like drugs) bind exclusively to the 165-kDa alpha 1 subunit of skeletal muscle calcium channels. However, it has not been conclusively determined whether benzothiazepines (diltiazem-like drugs), which represent the third group of calcium antagonists, also bind to the alpha 1 subunit. Here we report data obtained with a newly developed benzothiazepine photoaffinity probe, [3H]azidobutyryl diltiazem. This drug competes with diltiazem for the benzothiazepine-binding site and, in purified calcium channel preparations, specifically labels the 165-kDa polypeptide which does not change its electrophoretic mobility upon disulfide reduction. These data show that benzothiazepines, just like 1,4-dihydropyridines and phenylalkylamines, bind to the alpha 1 subunit of the skeletal muscle calcium channels.

Characteristics of the phenylalkylamine binding site in canine cardiac sarcolemmal membranes.

We have investigated the phenylalkylamine binding site in canine cardiac sarcolemmal preparations using (-)-[3H]-desmethoxyverapamil as the labeled ligand. Radioligand binding experiments were carried out in 10 mM Hepes (Na+) buffer and 1 mM EGTA, at pH 7.4 and 20 degrees C. A single high affinity binding site for (-)-[3H]-desmethoxyverapamil was identified both by saturation and competition binding experiments. Several phenylalkylamine derivatives such as (-)-D600, (+)-D600, verapamil and (+)-desmethoxyverapamil completely inhibited (-)-[3H]-desmethoxyverapamil binding with the following order of potency: (-)-desmethoxyverapamil greater than (-)-D600 greater than verapamil greater than (+)-desmethoxyverapamil = (+)-D600. In contrast to this, ronipamil, a new long acting phenylalkylamine derivative, produced only a 70% inhibition. Diltiazem also completely inhibited (-)-[3H]-desmethoxyverapamil binding to canine cardiac sarcolemma while nifedipine displaced only 70% of binding. (-)-[3H]-desmethoxyverapamil binding was also inhibited by Ca++ and Mg++. These data suggest the presence of a saturable, reversible and stereoselective phenylalkylamine binding site in canine cardiac sarcolemmal preparations which may be a receptor for the phenylalkylamine Ca++ channel inhibitors.

Purification of putative Ca2+ channel protein from rabbit skeletal muscle. Determination of the amino-terminal sequence.

A putative Ca2+ channel protein was purified from rabbit skeletal muscle transverse tubules with the combined use of lectin affinity chromatography and ion-exchange chromatography, followed by sucrose density gradient centrifugation. The major component of the purified preparation detected by sodium dodecyl sulfate-gel electrophoresis was a protein of 150 kDa when reduced with 20 mM dithiothreitol and a 191-kDa protein when treated with 20 mM N-ethylmaleimide. Therefore, this protein appears to be identical with the alpha subunit previously described (Curtis, B. M., and Catterall, W. A. (1984) Biochemistry 23, 2113-2118). This protein was purified by preparative sodium dodecyl sulfate-gel electrophoresis, followed by electroelution and/or electroblotting, and its amino acid composition and NH2-terminal sequence were determined. The NH2-terminal sequence is: NH2-Glu-Pro-Phe-Pro-Ser-Ala-Val-X-Ile-Lys-Ser-X-Val-X-Lys-Met-Gln-.