Cornelis van Breemen

Excitation-Contraction Coupling in Rabbit Aorta Studied by the Lanthanum Method for Measuring Cellular Calcium Influx

Lanthanum inhibits 45Ca efflux from internally injected squid axons. Evidence for La3+ blockade of Ca2+ fluxes across the cell membrane was also obtained for aortic smooth muscle: 1mM La3+ completely inhibited contractions which otherwise resulted from adding Ca2+ to calcium-free depolarizing solutions, and 2 mM La3+ caused a 50% inhibition of the late 45Ca efflux. Ca2+ bound extracellularly could be displaced by La3+, and the binding sites preferred La3+ over Ca2+. We could thus use La3+ to eliminate extracellular bound Ca2+ from our calcium-influx measurements as follows: after exposure of the aortic strips to experimental solutions labeled with 45Ca, the extracellular Ca2+ label was displaced by putting the strips in a calcium-free solution containing 2 mM La3+ for 60 minutes. The loss of intracellular Ca2+ was minimized during this hour by the La3+ blockade of Ca2+ membrane fluxes. Tissue weight, 45Ca, and total Ca2+ were then measured using standard techniques. Studies employing this new method showed that depolarization by high K+, Na+ replacement, and high pH activate smooth muscle contraction by stimulating Ca2+ influx. Low pH and La3+ block Ca2+ influx. Norepinephrine initiates aortic contractions by release of intracellularly sequestered Ca2+. Angiotensin and histamine appear to release Ca2+ from this same fraction.

Norepinephrine and GTP-γ-S increase myofilament Ca2+ sensitivity in α-toxin permeabilized arterial smooth muscle

Summary A new method for preparing permeabilized smooth muscle fibers from rabbit mesenteric artery has been developed using α-toxin, a transmembrane pore-making exo-protein produced by Staphylococcus aureus. After α-toxin treatment the fibers developed tension as a function of Ca 2+ concentration (EC 50 = 890 nM). But they could not contract without added ATP, indicating ATP is permeable. When the sarcoplasmic reticulum was loaded with 5×10 −7 M Ca 2+ solution, NE induced a transient contraction in 2 mM EGTA 0 M Ca 2+ solution and a transient and maintained contraction in 5×10 −7 M Ca 2+ solution. GTP-γ-S, a non-hydrolyzable analogue of GTP, substituted for NE in producing these contractile effects. The analysis of the relationship between Ca 2+ and maintained tension revealed that NE and GTP-γ-S cause increases in Ca 2+ sensitivity of myofilament shifting the EC 50 to 280 nM and 160 nM, respectively. We conclude that NE or GTP-γ-S causes an increase in myofilament Ca 2+ sensitivity and that G protein may be involved in receptor signal transduction system. α-Toxin is a useful tool to permeabilize the smooth muscle tissue to ions and small molecules without any damage of receptor and signal transduction system.

Evidence for two separate Ca2+ pathways in smooth muscle plasmalemma

SummaryThe activation of rabbit aortic smooth muscle was studied by two most widely used vascular smooth muscle stimulants: α-adrenoceptor activation by norepinephrine (NE) and high-K+ depolarization. This was studied by measurements of isometric contractions and net as well as unidirectional Ca2+ fluxes. These parameters showed markedly differential sensitivities towards two smooth muscle inhibitors used in this study: D 600 and amrinone. By choosing an appropriate concentration of D 600 or amrinone, Ca2+ uptake or Ca2+ influx induced by high K+ or NE could be selectively inhibited. Furthermore, by using unidirectional flux measurements it was demonstrated that Ca2+ influx stimulated by NE and high K+ were additive in nature. The data from the addivity experiment exclude the interpretation of a common Ca2+ pathway with two separate mechanisms for opening it. The data on three criteria employed in this study provide evidence for the existence of two independent Ca2+ pathways, one for each mode of activation, for Ca2+ influx known to be associated with these contractions.

Superficial buffer barrier function of smooth muscle sarcoplasmic reticulum

In smooth muscle the superficial sarcoplasmic reticulum accumulates a portion of the Ca2+ that enters cells through the plasmalemma and thus functions as a buffer barrier to Ca2+ entry into the myoplasm (superficial buffer barrier or SBB). In this review Cornelis van Breemen, Qian Chen and Ismail Laher summarize experimental support for the SBB, and discuss data indicating that: (1) contraction is related more to the rate than extent of Ca2+ entry; (2) refilling of sarcoplasmic reticulum from the extracellular space is mediated by Ca2+ influx and Ca2+ pumping by the sarcoplasmic reticulum Ca2+ pump; (3) the superficial sarcoplasmic reticulum unloads Ca2+ to the extracellular space by a multi step process that involves sequentially the opening of Ca2+ and inositol 1,4,5-trisphosphate [Ins(1,4,5,)P3] sensitive channels and Ca2+ extrusion by Na(+)-Ca2+ exchange; (4) the SBB generates a peripheral Ca2+ gradient; (5) Ca(2+)-mobilizing receptor agonists generate Ins(1,4,5)P3 which short circuits the SBB to increase the effectiveness of Ca2+ influx in raising [Ca2+]i and consequently increase smooth muscle contraction. A physiologically regulated SBB is thought to enhance the informational content of Ca2+ signalling and support variable reduction of smooth muscle tone. Pharmacological modulation of Ca2+ transport in the superficial sarcoplasmic reticulum therefore presents an alternative means of controlling smooth muscle tone dependent on Ca2+ entry.

Relative contributions of Ca2+ influx and cellular Ca2+ release during drug induced activation of the rabbit aorta

SummaryUsing the “La-method” for measuring45Ca entry into smooth muscle cells we were able to show that the slow phase of a norepinephrine induced contraction is dependent on Ca2+ influx. Similarly Ca2+ influx was stimulated during the slow phase of angiotensin II and histamine induced contractions. The large rapid initial phases of these three types of drug induced aortic contractions were dependent on a common intracellular Ca2+ store which was rapidly exhausted in the presence of extracellular La3+.Relaxation of the aortic strips was not dependent on loss of Ca2+ from the cells indicating the presence of intracellular Ca2+ binding sites capable of lowering the cytoplasmic (Ca2+) to below 10−7 M. These latter sites are distinct from the intracellular Ca store released by norepinephrine, angiotensin and histamine.

Agonist induced release of intracellular Ca2+ in the rabbit aorta.

SummaryThe effects of hormonal agonists (norepinephrine, angiotensin, and histamine) on45Ca efflux from the rabbit aorta were studied using a Ca-EGTA buffered efflux medium. Each caused a transient stimulation of efflux rate which probably reflected the release of an intracellular45Ca store. The size of the stimulation of efflux correlated with the size of the initial rapid phase of contraction. The norepinephrine-sensitive intracellular Ca fraction was estimated to be greater than 21 μmoles/Kg wet tissue weight. This fraction is separate from intracellular Ca which is accumulated during relaxation. Evidence is presented for the lack of cyclic nucleotide involvement in the release of Ca2+, and possible alternative modes of coupling are discussed.

Long-Term Doxycycline Is More Effective Than Atenolol to Prevent Thoracic Aortic Aneurysm in Marfan Syndrome Through the Inhibition of Matrix Metalloproteinase-2 and -9

β-Blockers, eg, atenolol, are the cornerstone therapy for thoracic aortic aneurysm (TAA) in patients with Marfan syndrome; however, continued aortic dilatation has been reported. We have demonstrated that matrix metalloproteinase (MMP)-2 and -9 were upregulated during progression of TAA in Marfan syndrome, accompanied with degenerated elastic fibers and vasomotor dysfunction. We hypothesized that doxycycline, a nonspecific inhibitor of MMPs, would ameliorate TAA by attenuating elastic fiber degeneration and improving vasomotor function. A well-characterized mouse model of Marfan syndrome (Fbn1C1039G/+) was used. Mice were untreated (n=40), given doxycycline (0.24g/L, n=30), or given atenolol (0.5g/L, n=30) in drinking water at 6 weeks of age. The Fbn1+/+ mice served as control (n=40). At 3, 6, and 9 months, aortic segments from the ascending, arch, and descending portions were used to obtain the “average” value of the whole thoracic aorta. TAA was prevented in the doxycycline group, whereas mild aneurysm was evident in the atenolol group. Doxycycline improved elastic fiber integrity, normalized aortic stiffness, and prevented vessel weakening. The impairment of vasocontraction and endothelium-dependent relaxation in the untreated and atenolol groups were improved by doxycycline. The upregulation of transforming growth factor-β in the Marfan aorta was suppressed by doxycycline. Doxycycline augmented expression ratios of tissue inhibitors of MMP to MMPs. Intraperitoneally injected neutralizing antibodies against MMP-2 and -9 yielded similar effects to doxycycline. We concluded that long-term treatment with doxycycline, through the inhibition of MMP-2 and -9, is more effective than atenolol in preventing TAA in Marfan syndrome by preserving elastic fiber integrity, normalizing vasomotor function, and reducing transforming growth factor-β activation.

The mechanism of phenylephrine-mediated [Ca2+]i oscillations underlying tonic contraction in the rabbit inferior vena cava

1 We characterized the mechanisms in vascular smooth muscle cells (VSMCs) that produce asynchronous, wave‐like Ca2+ oscillations in response to phenylephrine (PE). Confocal imaging was used to observe [Ca2+]i in individual VSMCs of intact inferior vena cava (IVC) from rabbits. 2 It was found that the Ca2+ waves were initiated by Ca2+ release from the sarcoplasmic reticulum (SR) via inositol 1,4,5‐trisphosphate‐sensitive SR Ca2+ release channels (IP3R channels) and that refilling of the SR Ca2+ store through the sarcoplasmic‐endoplasmic reticulum Ca2+‐ATPase (SERCA) was required for maintained generation of the repetitive Ca2+ waves. 3 Blockade of L‐type voltage‐gated Ca2+ channels (L‐type VGCCs) with nifedipine reduced the frequency of PE‐stimulated [Ca2+]i oscillations, while additional blockade of receptor‐operated channels/store‐operated channels (ROCs/SOCs) with SKF96365 abolished the remaining oscillations. Parallel force measurements showed that nifedipine inhibited PE‐induced tonic contraction by 27% while SKF96365 abolished it. This indicates that stimulated Ca2+ entry refills the SR to support the recurrent waves of SR Ca2+ release and that both L‐type VGCCs and ROCs/SOCs contribute to this process. 4 Application of the Na+‐Ca2+ exchanger (NCX) inhibitors 2′,4′‐dichlorobenzamil (forward‐ and reverse‐mode inhibitor) and KB‐R7943 (reverse‐mode inhibitor) completely abolished the nifedipine‐resistant component of [Ca2+]i oscillations and markedly reduced PE‐induced tone. 5 Thus, we conclude that each Ca2+ wave depends on initial SR Ca2+ release via IP3R channels followed by SR Ca2+ refilling through SERCA. Na+ entry through ROCs/SOCs facilitates Ca2+ entry through the NCX operating in the reverse mode, which refills the SR and maintains PE‐induced [Ca2+]i oscillations. In addition some Ca2+ entry through L‐type VGCCs and ROCs/SOCs serves to modulate the frequency of the oscillations and the magnitude of force development.

Loss of Elastic Fiber Integrity and Reduction of Vascular Smooth Muscle Contraction Resulting From the Upregulated Activities of Matrix Metalloproteinase-2 and -9 in the Thoracic Aortic Aneurysm in Marfan Syndrome

Thoracic aortic aneurysm (TAA) is the life-threatening complication of Marfan syndrome (MFS), a connective tissue disorder caused by mutations in the fibrillin-1 gene. TAA is characterized by degradation of elastic fiber, suggesting the involvement of matrix metalloproteinase (MMP)-2 and -9, the activation of which is regulated by TIMP (tissue inhibitor of MMP) types 1 and 2. We hypothesized that MMP-2 and -9 were upregulated during TAA formation in Marfan syndrome, causing loss of elastic fibers and structural integrity. We studied mice, from 3 to 12 months, heterozygous for a mutant Fbn1 allele encoding a cysteine substitution in fibrillin-1 (Fbn1C1039G/+, designated as “Marfan” mice) (n=120), the most common class of mutation in Marfan syndrome. The littermates, Fbn1+/+ served as controls (n=120). In Marfan aneurysmal thoracic aorta, mRNA and protein expression of MMP-2 and -9 were detected at 3 months and peaked at 6 months of age, accompanied by severe elastic fiber fragmentation and degradation. From 3 to 9 months, the MMP-2/TIMP-2 ratio increased by 43% to 63% compared with the controls. Dilated thoracic aorta demonstrated increased elasticity but distention caused a pronounced loss of contraction, suggesting weakening of the aortic wall. Breaking stress of the aneurysmal aorta was 70% of the controls. Contraction in response to depolarization and receptor stimulation decreased in the aneurysmal thoracic aorta by 50% to 80%, but the expression of α-smooth muscle actin between the 2 strains was not significantly different. This report demonstrates the upregulation of MMP-2 and -9 during TAA formation in Marfan syndrome. The resulting elastic fiber degeneration with deterioration of the aortic contraction and mechanical properties may explain the pathogenesis of TAA.

Transient Receptor Potential Channel 6–Mediated, Localized Cytosolic [Na+] Transients Drive Na+/Ca2+ Exchanger–Mediated Ca2+ Entry in Purinergically Stimulated Aorta Smooth Muscle Cells

The Na+/Ca2+ exchanger (NCX) is increasingly recognized as a physiological mediator of Ca2+ influx and significantly contributes to salt-sensitive hypertension. We recently reported that Ca2+ influx by the NCX (1) is the primary mechanism of Ca2+ entry in purinergically stimulated rat aorta smooth muscle cells and (2) requires functional coupling with transient receptor potential channel 6 nonselective cation channels. Using the Na+ indicator CoroNa Green, we now directly observed and characterized the localized cytosolic [Na+] ([Na+]i) elevations that have long been hypothesized to underlie physiological NCX reversal but that have never been directly shown. Stimulation of rat aorta smooth muscle cells caused both global and monotonic [Na+]i elevations and localized [Na+]i transients (LNats) at the cell periphery. Inhibition of nonselective cation channels with SKF-96365 (50 &mgr;mol/L) and 2-amino-4-phosphonobutyrate (75 &mgr;mol/L) reduced both global and localized [Na+]i elevations in response to ATP (1 mmol/L). This effect was mimicked by expression of a dominant negative construct of transient receptor potential channel 6. Selective inhibition of NCX-mediated Ca2+ entry with KB-R7943 (10 &mgr;mol/L) enhanced the LNats, whereas the global cytosolic [Na+] signal was unaffected. Inhibition of mitochondrial Na+ uptake with CGP-37157 (10 &mgr;mol/L) increased both LNats and global cytosolic [Na+] elevations. These findings directly demonstrate NCX regulation by LNats, which are restricted to subsarcolemmal, cytoplasmic microdomains. Analysis of the LNats, which facilitate Ca2+ entry via NCX, suggests that mitochondria limit the cytosolic diffusion of LNats generated by agonist-mediated activation of transient receptor potential channel 6–containing channels.