Swine Carotid Artery Research Articles

Dependence of ATP consumption on cross-bridge phosphorylation in swine carotid smooth muscle.

1. Ca(2+)-dependent phosphorylation of the myosin regulatory light chain (MRLC) initiates cross-bridge cycling and contraction in smooth muscle. A four-state cross-bridge model, in which Ca(2+)-dependent phosphorylation is the only proposed regulatory mechanism, can predict the mechanical output of the swine carotid media. Our aims were to determine whether ATP consumption rates and the economy of force maintenance are regulated functions of MRLC phosphorylation as predicted by the model. 2. Steady-state force and oxygen consumption were measured in medial rings of swine carotid arteries activated with depolarizing solutions and agents capable of maintaining a wide range of steady-state myoplasmic Ca2+ and MRLC phosphorylation levels. 3. Suprabasal ATP consumption increased almost linearly with MRLC phosphorylation and exhibited a hyperbolic increase with active stress, as predicted. 4. The economy of stress maintenance fell with increases in suprabasal phosphorylation. 5. In absolute terms the energetic cost of covalent regulation by cross-bridge phosphorylation was small, although it may be a significant fraction of the ATP consumption associated with contraction.

Evaluation of annexin V as a platelet-directed thrombus targeting agent

Annexin V is a human phospholipid binding protein (M r 36,000) that binds with high affinity to activated platelets in vitro. We studied the biodistribution and thrombus binding of annexin V in rabbit and swine models of fully occlusive arterial thrombi formed 1–2 h prior to injection of annexin V. Iodinated annexin V was cleared from blood in a rapid early phase ( t 1 2 = 6.4 min, 76% of radioactivity) and a slower late phase ( t 1 2 = 71 min, 24% of radioactivity). Organ uptake was highest in the kidney and spleen and lowest in heart and skeletal muscle. Thrombus/blood uptake ratios were (mean ± SEM): 6.39 ± 1.80 for rabbit iliac artery, 6.97 ± 1.45 for swine carotid artery, and 7.68 ± 1.70 for swine femoral artery (all p values < 0.01 versus control artery); a control protein, ovalbumin, showed an uptake ratio of 0.59 ± 0.08 in swine femoral artery thrombi. These results indicate that annexin V is useful as an agent for selective targeting of platelet-containing thrombi.

Regulation of Ca(2+)-dependent ATPase activity in detergent-skinned vascular smooth muscle

We measured force, actin-activated myosin adenosinetriphosphatase (ATPase) activity, and myosin light-chain (MLC) phosphorylation levels in Triton X-100 detergent-skinned media of swine carotid arteries. Pseudo-ATPase activity composed of MLC kinase and phosphatase activities contributed maximally 12% to steady-state tissue ATPase activity. An increase in the Ca2+ concentration ([Ca2+]) induced an increase in force, MLC phosphorylation, and actin-activated myosin ATPase activity; this protocol was defined as the force development phase of contraction. Force maintenance was defined as the state induced by decreasing the [Ca2+] after a maximal contraction. Lowering the [Ca2+] decreased MLC phosphorylation to levels similar to those measured during force development at each [Ca2+]. In contrast, force remained at elevated levels while actin-activated myosin ATPase activity fell to significantly lower levels than those measured during the development phase for each [Ca2+]. We suggest that the significantly lower actin-activated myosin ATPase activity observed during a state of elevated force, compared with the development phase of a contraction, is evidence of slowly cycling latch bridges.

Phorbol ester-induced contractions of swine carotid artery are supported by slowly cycling crossbridges which are not dependent on calcium or myosin light chain phosphorylation.

This study determined if phorbol ester-induced contraction of vascular smooth muscle requires calcium-dependent myosin light chain (MLC) phosphorylation and, if not, whether the mechanical characteristics of the contraction in terms of stiffness and crossbridge cycling are similar to those during a calcium- and MLC phosphorylation-dependent contraction. Carotid arterial strips were exposed to 1.0 microM phorbol 12,13-dibutyrate (PDBu) in the presence of normal physiological salt solution (PSS) or after calcium depletion in calcium-free PSS and compared with contraction elicited by calcium-containing 110 mM KCl-PSS. PDBu induced maximal stress in both the presence and absence of calcium. While there was a temporal correlation between MLC phosphorylation and shortening velocity during KCl depolarization, shortening velocity was dissociated from MLC phosphorylation during PDBu stimulation. The stress-stiffness relationship was not different during KCl and PDBu stimulation, suggesting similar crossbridge interactions even though MLC phosphorylation levels were significantly different. These results demonstrate that PDBu-induced contraction of the swine carotid artery is not dependent on calcium or MLC phosphorylation. We suggest the possibility that activation of a calcium-independent PKC isoform may result in the expression of an inherent level of actin-activated myosin ATPase activity resulting in the slow development of stress.

Cyclic nucleotide-dependent regulation of Mn2+ influx, [Ca2+]i, and arterial smooth muscle relaxation.

Elevations in cyclic nucleotide levels can decrease myoplasmic [Ca2+] ([Ca2+]i) and thereby induce arterial smooth muscle relaxation. We evaluated whether cyclic nucleotide-induced reductions in [Ca2+]i are caused by 1) decreased Ca2+ influx or 2) increased Ca2+ sequestration or efflux. Swine carotid medial tissues were loaded with fura-2, and Ca2+ influx was estimated from the quenching rate of 360-nm fluorescence after addition of extracellular Mn2+. Histamine stimulation or high KCl depolarization increased Mn2+ influx, [Ca2+]i, and contractile force. The Ca2+ channel blocker diltiazem attenuated histamine- or KCl-induced increases in Mn2+ influx, [Ca2+]i, and force. Addition of forskolin (which increases cAMP) or nitroglycerin (which increases cGMP) attenuated histamine-induced increases in Mn2+ influx, [Ca2+]i, and force. Addition of forskolin or nitroglycerin also relaxed KCl depolarized tissues; however, Mn2+ influx and [Ca2+]i remained high. These results suggest that Mn(2+)-induced quenching of 360-nm fluorescence is an estimate of Ca2+ influx in the intact swine carotid artery. These results also suggest that cyclic nucleotides can relax swine arterial smooth muscle by at least two mechanisms: 1) reduction of [Ca2+]i primarily induced by decreases in Ca2+ influx and 2) uncoupling force from [Ca2+]i without changing Ca2+ influx or [Ca2+]i.

Nitrovasodilators relax arterial smooth muscle by decreasing [Ca2+]i and uncoupling stress from myosin phosphorylation.

Elevations in guanosine 3',5'-cyclic monophosphate concentration ([cGMP]) are proposed to induce arterial smooth muscle relaxation by either 1) decreasing myoplasmic [Ca2+] ([Ca2+]i), 2) decreasing the [Ca2+]i sensitivity of phosphorylation, or 3) uncoupling force from myosin phosphorylation. We evaluated the importance of each of these mechanisms by measuring changes in [cGMP], aequorin- and fura-2-estimated [Ca2+]i, myosin light chain phosphorylation, and stress in histamine-stimulated swine carotid arteries. In tissues submaximally stimulated with 3 microM histamine, nitroprusside (NP) induced a proportional decrease in myoplasmic [Ca2+] and myosin phosphorylation, suggesting that the relaxation was at least partially induced by decreases in [Ca2+]i without a change in the [Ca2+]i sensitivity of phosphorylation. In tissues maximally stimulated with 10 microM histamine, NP and nitroglycerin produced significant relaxations that were not associated with significant sustained reductions in [Ca2+]i or myosin phosphorylation. With both submaximal and maximal histamine stimulation, nitrovasodilators produced more substantial relaxation than that expected from the nitrovasodilator-induced reduction in myosin phosphorylation. These results suggest that nitrovasodilators relax histamine-stimulated swine arterial smooth muscle by at least two mechanisms: 1) reducing [Ca2+]i, an effect observed in submaximally stimulated tissues, and 2) uncoupling of stress from myosin phosphorylation.

Histamine activates whole cell K + currents in swine carotid arterial smooth muscle cell

1. The effects of histamine on whole cell K + currents were evaluated in cultured smooth muscle cells that were derived from explants of swine carotid artery. These cells exhibited cytoplasmic protein and agonist-induced calcium responses typical of noncultured smooth muscle cells. 2. Upon obtaining whole cell configuration, depolarization from a holding potential of −80 mV elicited a slowly inactivating voltage-dependent outward current that was sensitive to tetraethylammonium and 4-aminopyridine. 3. These K + currents were at least partially Ca 2+ dependent since they were inhibited by extracellular bath application of verapamil or by substitution of external Ca 2+with Co 2+. 4. The addition of extracellular histamine increased the outward current both in the presence and absence of extracellular Ca 2+ and this response was blocked by (±)chlorpheniramine. 5. We conclude that swine carotid arterial smooth muscle cells exhibit voltage and calcium dependent potassium current and that these currents are modified by histamine stimulation.

Na(+)-Ca2+ exchange, myoplasmic Ca2+ concentration, and contraction of arterial smooth muscle.

Na(+)-Ca2+ exchange is proposed to be an important regulator of myoplasmic intracellular Ca2+ concentration ([Ca2+]i) and contraction in vascular smooth muscle. We investigated the role of Na(+)-Ca2+ exchange in regulating [Ca2+]i in swine carotid arterial tissues that were loaded with aequorin to allow simultaneous measurement of [Ca2+]i and force. Reversal of Na(+)-Ca2+ exchange, by reduction of extracellular Na+ concentration ([Na+]o) to 1.2 mM, induced a large increase in aequorin-estimated [Ca2+]i and a low [Ca2+]i sensitivity. The contraction induced by 1.2 mM [Na+]o was partially caused by depolarization and opening of L-type Ca2+ channels because 10 microM diltiazem partially attenuated the 1.2 mM [Na+]o-induced increases in [Ca2+]i. High dose ouabain (10 microM), a putative endogenous Na+,K(+)-ATPase inhibitor, increased both [Ca2+]i and force. However, the increases in [Ca2+]i and force were mostly blocked by 10 microM phentolamine, suggesting the predominant effect of ouabain was to increase norepinephrine release from nerve terminals. In the presence of 10 microM phentolamine, 10 microM ouabain slightly accentuated 1 microM histamine-induced increases in [Ca2+]i and force. The ouabain dose necessary to induce contraction in the absence of phentolamine was significantly less than the ouabain dose necessary to accentuate histamine-induced contractions in the presence of phentolamine. These results suggest that Na(+)-Ca2+ exchange exists in swine arterial smooth muscle. These data also suggest that ouabain (which should increase [Na+]i and inhibit Na(+)-Ca2+ exchange) primarily enhances contractile function in the swine carotid artery by releasing catecholamines from nerve terminals; direct action of Na+,K(+)-ATPase inhibitors on smooth muscle appears to occur only with very high doses.

Relaxation, [Ca2+]i, and the latch-bridge hypothesis in swine arterial smooth muscle

During vascular smooth muscle relaxation, myosin light-chain phosphorylation values decrease to resting values more rapidly than do stress values. Because phosphorylation is proportionally low, the latch-bridge hypothesis predicts that stress during relaxation should be predominantly carried by latch bridges. I evaluated the mechanical properties of latch bridges by changing tissue length and measuring myoplasmic Ca2+ concentration ([Ca2+]) with aequorin during relaxation of swine carotid medial tissues. Stress production was predicted with the latch-bridge model of Hai and Murphy, in which the measured aequorin [Ca2+] signal is the only determinant of stress. The aequorin-based latch-bridge model predicted relaxation induced by removal of the histamine stimulation. However, when tissues were relaxed by removal of extracellular Ca2+ or Ca(2+)-channel blockers in the continued presence of histamine, the aequorin-based model modestly underestimated the resulting relaxation. This underestimation was most likely caused by a small increase in the [Ca2+] sensitivity of phosphorylation since a model with an altered [Ca2+] sensitivity of phosphorylation more accurately predicted the resulting relaxation. The time course of relaxation in swine carotid artery was not substantially altered when the tissue was either briefly stretched or shortened and then returned to the original length. Because stretch should detach cross bridges, I modified the aequorin-based latch-bridge model to account for stretch-induced cross-bridge detachment. Because [Ca2+] values were slightly above resting values both before and after the stretch, the model predicted that phosphorylated cross bridges could reattach, be dephosphorylated, and form new latch bridges. The model predicted relaxation except during the first few seconds after stretch. These results suggest that latch-bridge reattachment is not necessary to explain the majority of the response to stretch during relaxation. The rate-limiting step for relaxation appears to be removal of [Ca2+] and not latch-bridge detachment.

Protein kinase C activation and myosin light chain phosphorylation in 32P-labeled arterial smooth muscle

Experiments using 32P-labeled strips of swine carotid artery medial smooth muscle were performed to define the relative contribution of myosin light chain (MLC) phosphorylation as an activation mechanism mediating contractile responses stimulated by phorbol dibutyrate (PDB). Tryptic phosphopeptide mapping of phosphorylated MLC indicated that near-maximal force responses were associated with increases in functional MLC phosphorylation of less than 10% of the total MLC content following tonic (45 min) stimulation by PDB. Significant phosphorylation of MLC residues, consistent with the specificity of protein kinase C, occurred in response to high concentrations of PDB (greater than 0.1 microM). Histamine (10 microM)-induced MLC phosphorylation after 2 min (72.5% of total MLC) or 45 min (61.7%) was restricted to serine residues on peptides thought to contain serine19. Although agonist (histamine)-induced responses were eliminated under conditions of Ca2+ depletion, near-maximal force in response to 10 microM PDB (89.4% of a standard KCl response) was associated with monophosphorylation of less than 9% of the total MLC on peptides interpreted as containing serine19. A substantial fraction of this was localized to threonine residues. The quantitative analysis of the relation between PDB-stimulated force and the residues in MLC phosphorylated supports the concept that PDB stimulation results in activation of arterial smooth muscle cross bridges by MLC-phosphorylation-independent mechanisms.

Force-length dependence of arterial lamellar, smooth muscle, and myofilament orientations.

Force generation by contractile elements of arterial tissue can be affected by alterations in their alignment with shortening. The orientation and morphometry of smooth muscle (SM) myofilaments, medial lamellae, and elastic laminae were examined as a function of length in intact swine carotid arteries or strips. Intimal-medial tissue strips were fixed during isometric contractions at lengths (L) defined with respect to the optimal length for force generation (Lo). The average orientation of SM cells in two perpendicular planes remained parallel to the long axis of the tissue at all lengths, but the absolute value of angular deviations increased with shortening. Tissue lengthening was associated with decreased folding of the elastic laminae. This decrease in waviness was quantified by a stretch index (SI). Ultrastructural observations indicated that the myofilament absolute angular deviation was greater than that for the cellular alignment. For arteries fixed in situ while constricted, SI was least in the periintimal laminae and increased in the peripheral laminae. The average decrease in force-generating capacity on shortening from Lo to 0.6 Lo attributed to increasing SM and myofilament angular deviations was calculated to be 7%.

Ba2+ induces contraction in swine carotid artery by mobilizing intracellular Ca2+.

Recent data suggest that Ba2+ activates the smooth muscle contractile apparatus. However, in vitro studies indicate that Ba2+ activates Ca2+ calmodulin-dependent enzymes only at very high concentrations. These observations might suggest that Ba2+ activates the contractile apparatus by a mechanism independent of myosin phosphorylation. We tested this hypothesis using intact and Triton X-100 skinned swine carotid medial strips. In intact tissues, Ba2+ stimulated dose-dependent contractions in the absence of extracellular Ca2+. However, in skinned tissues, Ba2+ induced much lower stress development at concentrations more than three orders of magnitude greater than needed for Ca2+-induced contraction. Ba2+ also failed to maintain stress in skinned tissues previously contracted with 4 microM Ca2+. The Ba2+-induced contraction in intact strips was associated with gradual increases in myosin phosphorylation from a basal level of approximately 5% to a sustained level of 30.0 +/- 0.8%. Partial intracellular calcium depletion with 100 microM histamine, 25 mM caffeine, and 5 mM ethyleneglycol-bis(beta-aminoethyl-ether)-N,N'-tetraacetic acid (EGTA) failed to abolish Ba2+-induced contractions. However, repeated contractions with Ba2+ in the absence of extracellular Ca2+ led to decreases in stress development. These observations suggest that Ba2+ induces stress development by mobilizing intracellular Ca2+ that was not released by histamine and caffeine.

Apparent dissociation between myosin light chain phosphorylation and maximal velocity of shortening in KCl depolarized swine carotid artery: effect of temperature and KCl concentration.

Stimulation of swine carotid artery medial fibers with 110 mM KCl at 37 degrees C results in increases in myosin light chain (MLC) phosphorylation levels and maximal shortening velocity (Vo) during the period of stress development. During the period of stress maintenance, MLC phosphorylation levels and Vo are not maintained, but fall to suprabasal levels, resulting in a correlation between MLC phosphorylation and Vo and suggesting that MLC phosphorylation regulates Vo. This study identifies other conditions of KCl depolarization of swine carotid medial fibers in which this relationship between MLC phosphorylation and Vo is altered. A decrease in temperature from 37 degrees to 23 degrees C results in a similar magnitude of stress maintenance in response to 110 mM KCl and similar levels of MLC phosphorylation, but a reduction in Vo by approximately 50%. This differential effect of temperature on Vo and MLC phosphorylation results in a downward shift in the slope of the regression line describing the relationship between these two parameters. Decreasing [KCl] to 40 mM in the stimulating solution results again in similar magnitudes of maintained stress. MLC phosphorylation levels are not transient, but maintained at a constant value and Vo is transient at levels approximately 50% of those at 110 mM KCl at 37 degrees C. This results in a complete lack of correlation between MLC phosphorylation and Vo. Thus, stress can be developed to equal magnitudes under differing activation conditions with dissimilar patterns of MLC phosphorylation and Vo. Therefore, there is not a strict relationship between MLC phosphorylation and Vo in all cases.

High force development and crossbridge attachment in smooth muscle from swine carotid arteries.

In experiments designed to achieve maximal activation, the active force/cell cross-sectional area in tissues prepared from the swine carotid media was 6.7 +/- 0.3 (sd) X 10(5) N/m5. This value exceeds that reported for other vertebrate muscle cells and is striking because of the low smooth muscle myosin content. The hypothesis that high force generation may, in part, reflect an increase in the crossbridge duty cycle, i.e., the fraction of the cycle during which force is generated, was tested by determining the rate of force redevelopment after a step shortening and the ration of the load-bearing capacity of the contractile system to the developed stress during the course of isometric contractions. Maximal crossbridge cycling rates estimated by the rate of force redevelopment occurred 30 seconds after the onset of a high K+-induced contraction, and decreased thereafter, although the load-bearing capacity or maximum active stress was maintained. These results from isometric experiments support the hypothesis and provide further evidence that attached, non-cycling crossbridges contribute to force maintenance in tonically contracting arterial smooth muscle.

Role of Ca2+ and myosin light chain phosphorylation in regulation of smooth muscle

The time course of phosphorylation of the 20,000-dalton myosin light chain (LC 20) was determined during contraction and relaxation in K+- and histamine-stimulated medial strips of swine carotid arteries. Resting LC 20 phosphorylation levels of 0.15 mol P/mol LC 20 rapidly increased to peak values of 0.6-0.7 mol P/mol LC 20 after stimulation and then declined significantly, although stress continued to rise to a stable steady-state maximum. LC 20 dephosphorylation after agonist washout preceded the decline in isometric stress. Over the entire contraction-relaxation cycle, phosphorylation was correlated with shortening velocity and not with developed stress. The maximum shortening velocity with no external load (Vo) was directly proportional to LC 20 phosphorylation (r = 0.986). The data indicate that LC 20 phosphorylation is necessary for cross-bridge cycling leading to shortening or stress development but that stress can be maintained by additional mechanisms. We suggest that dephosphorylation of an attached cross bridge in the presence of Ca2+ arrests the cycle, forming an attached, noncycling cross bridge.

Myosin light chain phosphorylation associated with contraction in arterial smooth muscle

The hypothesis that Ca2+ initiates contraction in smooth muscle by activating an endogenous myosin light chain kinase (MLCK) that phosphorylates the 20,000 dalton light chain (LC 20) of myosin was tested in tissues prepared from the media of swine carotid arteries. Unstimulated tissues with low levels of tone exhibited low levels of phosphorylated LC 20. On stimulation with a high-K+ physiological salt solution containing 1.6 mM CaCl2, LC 20 phosphorylation increased to 0.6 mol P/mol LC 20 within 30 s. This increase preceded force development, which required 2-4 min to attain a maximum steady-state value of 3.34 +/- 0.15 (SE) X 10(5) N/m2. These results support the hypothesis, as the stimulus was submaximal for the preparation. However, LC 20 phosphorylation declined significantly from its peak value before steady-state force was attained, reaching near control levels after 10 min of stimulation. The results suggest that Ca2+-stimulated LC 20 phosphorylation is an important physiological control mechanism but that additional factors are involved in the maintenance of tonic isometric force.

Tight binding of arterial myosin to skeletal F-actin.

A study of the K+-activated myosin ATPase activity, which was measured at high ionic strength in the absence of divalent cations, permitted estimates of the actin-myosin interaction under conditions where (i) myosin-myosin interactions were prevented, (ii) the actin-myosin interaction could be studied in the presence of ATP, and (iii) variation in myosin light chain phosphorylation did not alter smooth muscle myosin ATPase activity. A comparison of myosins isolated from swine carotid arteries and mixed (leg and back) rabbit skeletal muscle was conducted in the presence and absence of rabbit skeletal actin. It was found that (i) arterial myosin, like skeletal myosin, exhibited hyperbolic kinetics for ATP hydrolysis, (ii) specific ATPase activities were significantly higher for skeletal myosin, (iii) saturating concentrations of actin appear to totally inhibit the arterial myosin ATPase activity, but only partially inhibit skeletal myosin activity, (iv) the free actin concentration required for half-maximal inhibition was significantly lower for the arterial myosin ATPase activity than for the skeletal myosin activity; (v) unlike skeletal actomyosin, arterial actomyosin exhibits tight binding characteristics in the presence of ATP, (vi) the binding stoichiometry for arterial myosin to skeletal F-actin was 2 mol of actin monomer/mol of myosin. These observations reveal differences in the interaction of arterial and skeletal myosin with actin, and may in part, explain the high force-generating characteristics of arterial smooth muscle.

家畜頸動脈の抗原性除去に関する免疫薬理学的研究

To obtain better arterio grafts, we prepared three samples from adventitia of swine carotid arteries, and their antigenicity was studied with immunological procedures. Sample I was prepared from swine carotid arteries, by eliminating the extraneous fat and connective tissues. Sample II was prepared from Sample I by treatment with chymotrypsin, and Sample III was prepared from Sample II by treatment with 3% glutaraldehyde for 14 days. From the three samples, the water soluble protein fractions were extracted, and abbreviated as E-I, E-II and E-III, respectively. Immunopharmacological tests in guinea pigs and rabbits including active and passive anaphylaxis, passive cutaneous anaphylaxis, Schultz-Dale test and Arthus's phenomenon among these extracts revealed cross reactions between E-I and E-II. Also, precipitin test and Ouchterlony's method, precipitation was observed between E-I and E-II, and it was suggested that there might be at least a common antigen between E-I and E-II. The rabbits which had been implanted subcutaneously at the back with the minced Sample I evoked severe reactions and died after intravenous injection of E-I at the 3rd or 5th month after implantation. No reaction was observed between Sample III and E-III in the same experiment. From the above results, it was suggested that the antigenicity of swine carotid arteries could be eliminated by the treatment with chymotrypsin and glutaraldehyde.