Cloning of a gap junctional protein from vascular smooth muscle and expression in two-cell mouse embryos.

Gap junctional proteins (connexins) form aqueous channels that enable direct cell-cell transfer of ions and small molecules. The distribution and conductance of gap junction channels in cardiac muscle determine the pattern and synchrony of cellular activation. However, the capacity for smooth muscle to restrict contractile events temporally and spatially suggests that cell-cell coupling or its regulation may be decidedly different in this tissue. We isolated a cDNA from vascular smooth muscle which encodes a connexin (Mr 43,187) structurally homologous to cardiac connexin43. Vascular smooth muscle connexin43 mRNA was expressed prominently in smooth muscle tissues, cultured vascular myocytes, and arterial endothelial cells. A model for functional expression of connexins was developed in two-cell B6D2 mouse embryos. Microinjection of in vitro transcribed vascular smooth muscle connexin43 mRNA was shown to be sufficient to induce intercellular coupling in previously uncoupled blastomeres. Through the construction of two deletion mutants of connexin43, we also show that the formation of cell-to-cell connections does not depend upon a predicted cytoplasmic region within 98 residues of the carboxyl terminus. Finally, the identification of connexin43 in smooth muscle and endothelial cells provides supporting evidence for the existence of heterocellular coupling between cells of the vascular intima.

Identification of major autolytic cleavage sites in the regulatory subunit of vascular calpain II. A comparison of partial amino-terminal sequences to deduced sequence from complementary DNA.

Purified calpain II from vascular smooth muscle is a heterodimer consisting of catalytic (Mr = 76,000) and regulatory (Mr = 30,000) subunits. In the presence of Ca2+, the regulatory subunit undergoes stepwise autolysis resulting in enzyme activation. By slowing autoproteolysis, we identified major autolytic intermediates of the regulatory subunit. Gas-phase sequencing of the regulatory subunit and its autolytic fragments revealed that the NH2-terminus of the Mr = 30,000 form was blocked, whereas each fragment yielded a unique amino acid sequence, suggesting that autolysis proceeds in an NH2- to COOH-terminal direction. By comparison of actual amino acid sequences of autolytic cleavage intermediates to the full sequence deduced from cDNA, we have identified the major autolytic cleavage sites. Three different peptide bonds were cleaved, with neutral amino acids predominating on both sides of the peptide bond hydrolyzed. Importantly, leucine or isoleucine was identified in the second position upstream from the cleavage site in all three autolytic sequences. The presence of an upstream leucine residue in the autolytic cleavage sequence is reminiscent of the structure of potent microbial and synthetic peptide inhibitors of calpain.

Distribution of isoelectric variants of the 17,000-dalton myosin light chain in mammalian smooth muscle.

Isoelectric focusing of purified vascular smooth muscle myosin revealed two variants of the 17,000-dalton light chain subunits. The isoelectric points of the light chain variants were determined to be 4.13 (LC17a) and 4.19 (LC17b). Tryptic peptide maps of the two species of light chain generated by reverse-phase high performance liquid chromatography disclosed small but obvious differences in peptide composition while amino acid analyses of the variants were quite similar. Two-dimensional electrophoresis of extracts from various mammalian smooth muscles revealed tissue-specific differences in the relative content of LC17a and LC17b. Vascular (aorta, carotid, and pulmonary artery) muscles and tracheal smooth muscle contained both light chain variants while smooth muscle of the gastrointestinal tract (stomach and jejunum) contained LC17a only. The actin-activated Mg2+-ATPase activities of both phosphorylated and nonphosphorylated stomach (LC17b = 0) and aortic (LC17b = 40%) myosins were compared. In the presence of saturating tropomyosin, a 2-fold difference in Vmax was measured: phosphorylated, aortic, 0.119 +/- 0.009 versus stomach, 0.239 +/- 0.012 mumol of PO4 liberated/min/mg of myosin; nonphosphorylated, aortic, 0.065 +/- 0.004 versus stomach, 0.123 +/- 0.004 mumol of PO4 liberated/min/mg of myosin. In addition, the Vmax of myosin subfragment-1 ATPase from bovine aortic, pulmonary artery, and stomach myosins (LC17b contents, 40, 20, and 0%, respectively) was found to decrease in direct proportion to the LC17b content. Our results suggest that isoforms of the 17,000-dalton light chain subunits of mammalian smooth muscle myosin could play an important role in modulating actomyosin ATPase activity.

The effects of caldesmon on smooth muscle heavy actomeromyosin ATPase activity and binding of heavy meromyosin to actin.

Caldesmon was purified to homogeneity from both chicken gizzard and bovine aortic smooth muscles. Caldesmon purified from bovine aorta was slightly larger than caldesmon purified from chicken gizzards (Mr = 140,000) when the two were compared electrophoretically. Caldesmon bound tightly to actin saturating at a molar ratio of 1 caldesmon monomer per 6.6 actin monomers. Ca2+-calmodulin appeared to reduce the affinity of caldesmon for actin. Caldesmon was also a potent inhibitor of heavy actomeromyosin ATPase activity producing a maximal effect at a ratio of 1 caldesmon monomer per 7-10 actin monomers. This effect was also antagonized by Ca2+-calmodulin. While caldesmon inhibited heavy actomeromyosin ATPase activity, it greatly enhanced binding of both unphosphorylated and phosphorylated heavy meromyosin to actin in the presence of MgATP, reducing the Kd for binding by a factor of 40 for each form of heavy meromyosin. Although we did identify a Ca2+-calmodulin-stimulated "caldesmon kinase" activity in caldesmon preparations purified under nondenaturing conditions, we observed no effect of phosphorylation (2 mol of PO4/mol of caldesmon) on the capacity to inhibit heavy actomeromyosin ATPase activity. Our results suggest that caldesmon could serve some role in smooth muscle function by enhancing cross-bridge affinity while inhibiting actomyosin ATPase activity.

Plasma lactoferrin reflects granulocyte activation in vivo.

N-formyl-met-leu-phe (FMLP) causes polymorphonuclear leukocytes (PMN) to secrete and become "sticky" in vitro. We related these events to in vivo FMLP-induced neutropenia. FMLP was intravenously administered to anesthetized rabbits in doses ranging from 0.01 microgram to 1.0 microgram. Controls received phosphate-buffered saline (PBS), the diluent for FMLP. Blood pressure, respiratory rate, and arterial Po2 were monitored. High and intermediate doses of FMLP caused a dramatic but transient decrease in blood pressure and an increase in respiratory rate. Prior to FMLP infusion, plasma lactoferrin level was 6.4 +/- 4.1 micrograms/ml, and the absolute granulocyte account (AGC) was 2008 +/- 1229 (mean +/- SD). There was a positive linear correlation between AGC and plasma lactoferrin level prior to injection of FMLP (R2 = 0.74, p less than 0.01). At 1 min after FMLP injection, the percent change in AGC decreased as an exponential function of dose to as low as 10% of baseline (R2 = 0.86, p = 0.002) and plasma concentration of lactoferrin increased as an exponential function of dose to as high as 30 micrograms/ml (R2 = 0.84, p = 0.006). Thus, FMLP-induced neutropenia is associated with increased levels of plasma lactoferrin, suggesting that PMN are induced to degranulate in vivo.