Acute intravenous exposure to silver nanoparticles during pregnancy induces particle size and vehicle dependent changes in vascular tissue contractility in Sprague Dawley rats.

The use of silver nanoparticles (AgNP) raises safety concerns during susceptible life stages such as pregnancy. We hypothesized that acute intravenous exposure to AgNP during late stages of pregnancy will increase vascular tissue contractility, potentially contributing to alterations in fetal growth. Sprague Dawley rats were exposed to a single dose of PVP or Citrate stabilized 20 or 110nm AgNP (700µg/kg). Differential vascular responses and EC50 values were observed in myographic studies in uterine, mesenteric arteries and thoracic aortic segments, 24h post-exposure. Reciprocal responses were observed in aortic and uterine vessels following PVP stabilized AgNP with an increased force of contraction in uterine artery and increased relaxation responses in aorta. Citrate stabilized AgNP exposure increased contractile force in both uterine and aortic vessels. Intravenous AgNP exposure during pregnancy displayed particle size and vehicle dependent moderate changes in vascular tissue contractility, potentially influencing fetal blood supply.

Cardiac Ischemia Reperfusion Injury Following Instillation of 20 nm Citrate-capped Nanosilver.

BACKGROUND: Silver nanoparticles (AgNP) have garnered much interest due to their antimicrobial properties, becoming one of the most utilized nano-scale materials. However, any potential evocable cardiovascular injury associated with exposure has not been reported to date. We have previously demonstrated expansion of myocardial infarction after intratracheal (IT) instillation of carbon-based nanomaterials. We hypothesized pulmonary exposure to Ag core AgNP induces a measureable increase in circulating cytokines, expansion of cardiac ischemia-reperfusion (I/R) injury and is associated with depressed coronary constrictor and relaxation responses. Secondarily, we addressed the potential contribution of silver ion release on AgNP toxicity. METHODS: Male Sprague-Dawley rats were exposed to 200 µl of 1 mg/ml of 20 nm citrate-capped Ag core AgNP, 0.01, 0.1, 1 mg/ml Silver Acetate (AgAc), or a citrate vehicle by intratracheal (IT) instillation. One and 7 days following IT instillation the lungs were evaluated for inflammation and the presence of silver; serum was analyzed for concentrations of selected cytokines; cardiac I/R injury and coronary artery reactivity were assessed. RESULTS: AgNP instillation resulted in modest pulmonary inflammation with detection of silver in lung tissue and alveolar macrophages, elevation of serum cytokines: G-CSF, MIP-1α, IL-1ß, IL-2, IL-6, IL-13, IL-10, IL-18, IL-17α, TNFα, and RANTES, expansion of I/R injury and depression of the coronary vessel reactivity at 1 day post IT compared to vehicle treated rats. Silver within lung tissue was persistent at 7 days post IT instillation and was associated with an elevation in cytokines: IL-2, IL-13, and TNFα and expansion of I/R injury. AgAc resulted in a concentration dependent infarct expansion and depressed vascular reactivity without marked pulmonary inflammation or serum cytokine response. CONCLUSIONS: Based on these data, IT instillation of AgNP increases circulating levels of several key cytokines, which may contribute to persistent expansion of I/R injury possibly through an impaired vascular responsiveness.

Vascular Tissue Contractility Changes Following Late Gestational Exposure to Multi-Walled Carbon Nanotubes or their Dispersing Vehicle in Sprague Dawley Rats.

Multi-walled carbon nanotubes (MWCNTs) are increasingly used in industry and in nanomedicine raising safety concerns, especially during unique life-stages such as pregnancy. We hypothesized that MWCNT exposure during pregnancy will increase vascular tissue contractile responses by increasing Rho kinase signaling. Pregnant (17-19 gestational days) and non-pregnant Sprague Dawley rats were exposed to 100 µg/kg of MWCNTs by intratracheal instillation or intravenous administration. Vasoactive responses of uterine, mesenteric, aortic and umbilical vessels were studied 24 hours post-exposure by wire myography. The contractile responses of the vessel segments were different between the pregnant and non-pregnant rats, following MWCNT exposure. Maximum stress generation in the uterine artery segments from the pregnant rats following pulmonary MWCNT exposure was increased in response to angiotensin II by 4.9 mN/mm2 (+118%), as compared to the naïve response and by 2.6 mN/mm2 (+40.7%) as compared to the vehicle exposed group. Following MWCNT exposure, serotonin induced approximately 4 mN/mm2 increase in stress generation of the mesenteric artery from both pregnant and non-pregnant rats as compared to the vehicle response. A significant contribution of the dispersion medium was identified as inducing changes in the contractile properties following both pulmonary and intravenous exposure to MWCNTs. Wire myographic studies in the presence of a Rho kinase inhibitor and RhoA and Rho kinase mRNA/protein expression of rat aortic endothelial cells were unaltered following exposure to MWCNTs, suggesting absent/minimal contribution of Rho kinase to the enhanced contractile responses following MWCNT exposure. The reactivity of the umbilical vein was not changed; however, mean fetal weight gain was reduced with dispersion media and MWCNT exposure by both routes. These results suggest a susceptibility of the vasculature during gestation to MWCNT and their dispersion media-induced vasoconstriction, predisposing reduced fetal growth during pregnancy.

Role of PKCalpha and PKCiota in phenylephrine-induced contraction of rat corpora cavernosa.

Constriction of the penile vasculature prevents erection and is largely mediated by physiological agonists. We hypothesized that protein kinase C (PKC) may act as a regulator of penile vascular tone. Studies were designed to identify PKC isoforms present and to investigate their roles in phenylephrine-induced muscle contraction in the isolated rat corpora cavernosa. We demonstrated the presence of PKCalpha, beta, gamma, epsilon, delta, eta, and iota in rat corpora cavernosa and a subcellular distribution, which favored a membrane association for PKCalpha, beta, delta, and iota. Phenylephrine (3 microM) generated an active stress of 9.6 +/- 1.5 mN/mm2 and was associated with a significant increase of PKCalpha and PKCiota immunoreactivity in the particulate fraction. The amount of PKCalpha and PKCiota in the particulate fraction rose by 36 +/- 4.4 and 51 +/- 2.2% with phenylephrine stimulation. Furthermore, the phenylephrine concentration-response curve was potentiated in the presence of phorbol 12-myristate13-acetate (PMA) (0.1 microM), a PKC activator (EC50: phenylephrine 1.0 +/- 0.8 microM vs phenylephrine + PMA 0.3 +/- 0.1 microM) and inhibited in the presence of chelerythrine chloride (30 microM), a PKC inhibitor (EC50: phenylephrine 1.0 +/- 0.8 microM vs phenylephrine + chelerythrine chloride 5.7 +/- 2.4 microM). Based on these results, we suggest a potential role for PKCalpha and PKCiota in phenylephrine-induced smooth muscle tone of the rat cavernosum.

Vasoconstriction, RhoA/Rho-kinase and the erectile response.

Recent studies have suggested that contraction of the smooth muscle in the cavernosal arterioles and in the walls of the cavernosal sinuses is maintained by the RhoA/Rho-kinase signaling pathway. However, this contraction activity must be overcome to permit the vasorelaxation essential for erection. We postulate that nitric oxide (NO) causes erection primarily by inhibiting the RhoA/Rho-kinase pathway. The following will discuss evidence in support of the important role of Rho-kinase-mediated vasoconstriction in the nonerect penis and how NO overrides this Rho-kinase-mediated vasoconstriction to permit vasodilation and erection.

Cross-bridge regulation by Ca(2+)-dependent phosphorylation in amphibian smooth muscle.

A covalent regulatory mechanism involving Ca(2+)-dependent cross-bridge phosphorylation determines both the number of cycling cross bridges and cycling kinetics in mammalian smooth muscle. Our objective was to determine whether a similar regulatory mechanism governed smooth muscle contraction from a poikilothermic amphibian in a test of the hypothesis that myosin regulatory light chain (MRLC) phosphorylation could modulate shortening velocity. We measured MRLC phosphorylation of Rana catesbiana urinary bladder strips at 25 degrees C in tonic contractions in response to K+ depolarization, field stimulation, or carbachol stimulation. The force-length relationship was characterized by a steep ascending limb and a shallow descending limb. There was a rapid rise in unloaded shortening velocity early in a contraction, which then fell and was maintained at low rates while high force was maintained. In support of the hypothesis, we found a positive correlation of the level of myosin phosphorylation and an estimate of tissue shortening velocity. These results suggest that MRLC phosphorylation in amphibian smooth muscle modulates both the number of attached cross bridges (force) and the cross-bridge cycling kinetics (shortening velocity) as in mammalian smooth muscle.

PI3-kinase/Akt modulates vascular smooth muscle tone via cAMP signaling pathways.

Phosphatidylinositol 3-kinase (PI3-kinase) activates protein kinase B (also known as Akt), which phosphorylates and activates a cyclic nucleotide phosphodiesterase 3B. Increases in cyclic nucleotide concentrations inhibit agonist-induced contraction of vascular smooth muscle. Thus we hypothesized that the PI3-kinase/Akt pathway may regulate vascular smooth muscle tone. In unstimulated, intact bovine carotid artery smooth muscle, the basal phosphorylation of Akt was higher than that in cultured smooth muscle cells. The phosphorylation of Akt decreases in a time-dependent manner when incubated with the PI3-kinase inhibitor, LY-294002. Agonist (serotonin)-, phorbol ester (phorbol 12,13-dibutyrate; PDBu)-, and depolarization (KCl)-induced contractions of vascular smooth muscles were all inhibited in a dose-dependent fashion by LY-294002. However, LY-294002 did not inhibit serotonin- or PDBu-induced increases in myosin light chain phosphorylation or total O(2) consumption, suggesting that inhibition of contraction was not mediated by reversal or inhibition of the pathways that lead to smooth muscle activation and contraction. Treatment of vascular smooth muscle with LY-294002 increased the activity of cAMP-dependent protein kinase and increased the phosphorylation of the cAMP-dependent protein kinase substrate heat shock protein 20 (HSP20). These data suggest that activation of the PI3-kinase/Akt pathway in unstimulated smooth muscle may modulate vascular smooth muscle tone (allow agonist-induced contraction) through inhibition of the cyclic nucleotide/HSP20 pathway and suggest that cyclic nucleotide-dependent inhibition of contraction is dissociated from the myosin light chain contractile regulatory pathways.

Effect of Rho-kinase inhibition on vasoconstriction in the penile circulation.

A recent report from this laboratory (Chitaley K, Wingard C, Webb R, Branam H, Stopper V, Lewis R, and Mills T. Nature Medicine 7: 119-122, 2001) showed that inhibition of Rho-kinase increased the erectile response (intracavernosal pressure and mean arterial pressure) by a process that does not require nitric oxide or cGMP. The present study investigated whether vasoconstrictor agents, which are active in the penis, act via the Rho-kinase pathway. Western analysis revealed RhoA and Rho-kinase protein in the penis. Treatment with the selective Rho-kinase inhibitor Y-27632 significantly increased the magnitude of the erectile response. Intracavernous administration of endothelin-1 (ET-1; 50 pmol) or methoxamine (10 microg/kg) reduced the erectile response to autonomic stimulation. If Y-27632 was given before ET-1 or methoxamine, the vasoconstrictor effect was reduced, and intracavernosal pressure and mean arterial pressure remained elevated. However, when given after methoxamine, Y-27632 had a reduced vasodilatory effect, and Y-27632 had no vasodilatory effect when given after ET-1. These findings suggest that ET-1 and methoxamine increase Rho-kinase activity in the cavernous circulation and support the hypothesis that the vasoconstriction that maintains the penis in the nonerect state is mediated, in part, by the Rho-kinase pathway.

Erection and NO override the vasoconstrictive effect of alpha-adrenergic stimulation in the rat penile vasculature.

Studies in this laboratory are designed to determine the effects of vasoconstrictor agents on the erectile response in rats. We have previously demonstrated that the vasoconstrictor effect of endothelin-1 (ET-1) is sharply reduced by erection and by nitric oxide (NO) administration. The present study was performed to determine if vasoconstriction, resulting from alpha-adrenergic stimulation, is altered by erection and NO. During continuous monitoring of corpus cavernosum pressure (CCP) and mean arterial pressure (MAP), erection was induced by electrical stimulation of the autonomic ganglion for the innervation of the penis. When the alpha-adrenergic agonist methoxamine (METH, 10 microg/kg) was injected before erection (ie, into the non-erect penis), the subsequent erectile response (CCP/MAP) was significantly reduced from 0.68+/-0.03 before METH to 0.34+/-0.08 after METH. Injection of METH into the erect penis (ie, during erection) reduced the vasoconstrictor action of METH; CCP/MAP was 0.74+/-0.02 before METH and 0.55+/-0.05 after METH (P<0.05). The vasoconstrictor action of METH was slightly reduced when given in conjunction with NOR-1, a NO donor drug; CCP/MAP was 0.70+/-0.05 before METH, 0.55+/-0.09 after METH but this change was not significant. These results demonstrate that the response to alpha-adrenergic stimulation is attenuated during erection in response to ganglionic stimulation. Furthermore, it appears that NO, produced during erection, may serve to override agonist-induced vasoconstriction. These results support our hypothesis that NO acts to directly stimulate relaxation of cavernous smooth muscle and to inhibit the vasoconstrictor actions of agents like ET-1 and alpha-adrenergic agonists including norepinephrine.

Endothelin-1-induced vasoconstriction is inhibited during erection in rats.

Recent evidence indicates that endothelin-1 (ET-1) might be a principal vasoconstrictor in the penis. We report that ET-1 injection into the cavernous sinuses before erection sharply reduced the magnitude of subsequent erections. Corpus cavernosum pressure-to-mean arterial pressure ratios (CCP/MAP), with maximal ganglionic stimulation, were 0.62 +/- 0.05 before ET-1 injection and 0.31 +/- 0.05 after, indicating that ET-1 acted as a vasoconstrictor. When ET-1 was injected during a maximal neurally induced erection, the ability of ET-1 to attenuate subsequent erections was diminished (CCP/MAP 0.75 +/- 0.02 before ET-1, 0.61 +/- 0.03 after). At submaximal stimulation voltages, injection of ET-1 during erection also attenuated its vasoconstrictive effect. Similarly, when ET-1 was injected during erection induced by intracavernosal injection of the nitric oxide (NO) donor NOR-1, subsequent erections were not significantly suppressed (CCP/MAP 0.53 +/- 0.04 before ET-1, 0.45 +/- 0.04 after). These findings that ET-1-induced vasoconstriction is attenuated during erection are consistent with the hypothesis that NO mediates erection both by initiating pathways that cause smooth muscle relaxation and by inhibiting the vasoconstrictive actions of ET-1.

Antagonism of Rho-kinase stimulates rat penile erection via a nitric oxide-independent pathway.

Relaxation of the smooth muscle cells in the cavernosal arterioles and sinuses results in increased blood flow into the penis, raising corpus cavernosum pressure to culminate in penile erection. Nitric oxide, released from non-adrenergic/non-cholinergic nerves, is considered the principle stimulator of cavernosal smooth muscle relaxation, however, the inhibition of vasoconstrictors (that is, norepinephrine and endothelin-1, refs. 5-9) cannot be ignored as a potential regulator of penile erection. The calcium-sensitizing rho-A/Rho-kinase pathway may play a synergistic role in cavernosal vasoconstriction to maintain penile flaccidity. Rho-kinase is known to inhibit myosin light chain phosphatase, and to directly phosphorylate myosin light-chain (in solution), altogether resulting in a net increase in activated myosin and the promotion of cellular contraction. Although Rho-kinase protein and mRNA have been detected in cavernosal tissue, the role of Rho-kinase in the regulation of cavernosal tone is unknown. Using pharmacologic antagonism (Y-27632, ref. 13, 18), we examined the role of Rho-kinase in cavernosal tone, based on the hypothesis that antagonism of Rho-kinase results in increased corpus cavernosum pressure, initiating the erectile response independently of nitric oxide. Our finding, that Rho-kinase antagonism stimulates rat penile erection independently of nitric oxide, introduces a potential alternate avenue for the treatment of erectile dysfunction.

Receptor-specific influence of endothelin-1 in the erectile response of the rat.

Specific receptor antagonists were used to examine the role of endothelin-1 (ET-1) in the erectile response of the rat. In these studies, intact rats were cannulated to permit the continuous recording of mean arterial pressure (MAP) and intracavernosal pressure (CCP). Erection was induced by electrical stimulation of the autonomic ganglion, which regulates blood flow to the penis. The animals were subjected to intracavernosal injection with vehicle only (Cont) or with an antagonist to the endothelin-A receptor (ET(A)) or to the endothelin-B receptor (ET(B)). Blockade of the ET(A) or the ET(B) had no effect on the erectile response (CCP/MAP) during maximal ganglionic stimulation. When ET-1 was injected into Cont rats, there was a marked vasoconstriction with a sharp rise in MAP and a decline in CCP as the cavernosal arterioles constricted and limited inflow. The injection of the ET(A) antagonist prevented the vasoconstriction after ET-1 injection into Cont rats, whereas blockade of the ET(B) had no effect on the vasoconstrictive effect to ET-1. Similar results were obtained during submaximal ganglionic stimulation. With minimal levels of ganglionic stimulation, ET-1 injection led to a moderated degree of vasodilation in the presence of the ET(A) antagonist. The ET(B) antagonist failed to alter the CCP response during minimal stimulation, but it did have a marked effect on the MAP response to ET-1 injection. The results of these studies confirm that cavernosal tissue of the rat penis is highly responsive to ET-1. However, the failure of the ET-1 antagonists to affect penile erection in response to ganglionic stimulation reflects a minimal role of ET-1 in the erectile response in the rat.

cGMP-mediated phosphorylation of heat shock protein 20 may cause smooth muscle relaxation without myosin light chain dephosphorylation in swine carotid artery.

Nitrovasodilators such as nitroglycerine, via production of nitric oxide and an increase in [cGMP], can induce arterial smooth muscle relaxation without proportional reduction in myosin light chain (MLC) phosphorylation or myoplasmic [Ca2+]. These findings suggest that regulatory systems, other than MLC phosphorylation and Ca2+, partially mediate nitroglycerine-induced relaxation. In swine carotid artery, we found that a membrane-permeant cGMP analogue induced relaxation without MLC dephosphorylation, suggesting that cGMP mediated the relaxation. Nitroglycerine-induced relaxation was associated with a reduction in O2 consumption, suggesting that the interaction between phosphorylated myosin and the thin filament was inhibited. Nitroglycerine-induced relaxation was associated with a 10-fold increase in the phosphorylation of a protein on Ser16. We identified this protein as heat shock protein 20 (HSP20), a member of a family of proteins known to bind to thin filaments. When homogenates of nitroglycerine-relaxed tissues were centrifuged at 6000 g, phosphorylated HSP20 preferentially sedimented in the pellet, suggesting that phosphorylation of HSP20 may increase its affinity for the thin filament. We noted that a domain of HSP20 is partially homologous to the 'minimum inhibitory sequence' of skeletal troponin I. The peptide HSP20110-121, which contains this domain, bound to actin-containing filaments only in the presence of tropomyosin, a characteristic of troponin I. High concentrations of HSP20110-121 abolished Ca2+-activated force in skinned swine carotid artery. HSP20110-121 also partially decreased actin-activated myosin S1 ATPase activity. These data suggest that cGMP-mediated phosphorylation of HSP20 on Ser16 may have a role in smooth muscle relaxation without MLC dephosphorylation. HSP20 contains an actin-binding sequence at amino acid residues 110-121 that inhibited force production in skinned carotid artery. We hypothesize that phosphorylation of HSP20 regulates force independent of MLC phosphorylation via binding of HSP20 to thin filaments and inhibition of cross-bridge cycling.

Phosphorylation events associated with cyclic nucleotide-dependent inhibition of smooth muscle contraction.

Activation of cyclic nucleotide-dependent signaling pathways leads to relaxation of bovine carotid artery smooth muscle contractions and is associated with increased phosphorylation of the small heat shock-related protein (HSP20). Previous reports have shown that human umbilical artery smooth muscle is uniquely resistant to cyclic nucleotide-dependent relaxation, and HSP20 is not phosphorylated. In this investigation, we determined the phosphorylation events associated with cyclic nucleotide-dependent inhibition of smooth muscle contraction. In carotid artery, activation of cyclic nucleotide-dependent signaling pathways inhibited contractile responses to serotonin but did not inhibit myosin light chain phosphorylation or oxygen consumption. The inhibition of contraction was associated with increases in HSP20 phosphorylation. In umbilical artery, activation of cyclic nucleotide-dependent signaling pathways did not inhibit serotonin-induced contraction or myosin light chain phosphorylation. The lack of contractile inhibition in umbilical artery was not associated with significant increases in HSP20 phosphorylation. In conclusion, cyclic nucleotide-dependent contractile inhibition is independent of the inhibition of myosin light chain phosphorylation or oxygen consumption but does correlate with increased HSP20 phosphorylation.

Inhibition of Ca2+-dependent contraction in swine carotid artery by myosin kinase inhibitors.

Experiments were designed to examine the efficacy of the MLCK inhibitors wortmannin and ML-9 in intact smooth muscle to determine whether contractile agonists can induce a Ca(2+) and myosin light chain phosphorylation-independent contraction. Both wortmannin and ML-9 reduced active stress in a dose-dependent manner. Both inhibitors interfered with Ca2+ mobilization in either the K(+)-depolarized or agonist activated swine carotid media at concentrations greater than 10 microM. Wortmannin reduced MRLC phosphorylation and stress to resting levels in stimulated tissues while Ca2+ remained above resting levels. There was no evidence for Ca2+ and MRLC phosphorylation-independent stress generation in swine arterial smooth muscle.

Energetic cost of activation processes during contraction of swine arterial smooth muscle.

1. The objective of this study was to partition the increase in ATP consumption during contraction of swine carotid arterial smooth muscle estimated from suprabasal oxygen consumption (suprabasal JO2) and lactate release (Jlactate) into a component associated with cross-bridge cycling (JX) and one reflecting activation (JA). 2. Two experimental approaches-varying length under constant activation, and varying activation at a long length (1.8 times the optimal length for force development (Lo)) where force generation is minimal-revealed a linear dependence of JO2 and activation energy (JA) on cross-bridge phosphorylation. Protocols inducing a large increase in myosin regulatory light chain (MRLC) phosphorylation at 1.8 Lo resulted in significant elevations of JO2 and marked reductions in the economy of force maintenance. Our evidence suggests that this is primarily due to the increased cost of cross-bridge phosphorylation. 3. The extrapolated estimate of JA during maximal K(+)-induced depolarization made by varying length was 16%, while at 1.8 Lo it was 33% of the suprabasal JO2 at Lo. Calculated activation energies ranged from 17 to 45% of the suprabasal JO2 at Lo and from 72 to 87% of the suprabasal JO2 at 1.8 Lo under stimulation conditions that varied steady-state MRLC phosphorylation from 15 to 50%. 4. The results suggest that the kinetics of cross-bridge phosphorylation-dephosphorylation can rival those of cross-bridge cycling during isometric contractions in swine arterial smooth muscle.

Dependence of force on length at constant cross-bridge phosphorylation in the swine carotid media.

1. The dependence of force (F) on length (L) in smooth muscle remains uncertain since (i) it is influenced by changes in activation (myosin light chain phosphorylation), (ii) no anatomical reference length for the contractile unit is available, (iii) the length at which optimum force is generated (L(o)) exhibits a broad, flat optimum, and (iv) the presence of an extensive connective tissue network makes it difficult to stretch tissues without damage. 2. A swine carotid medial ring preparation prepared by removal of the adventitia and endothelium could be stretched to 1.8 L(o) without decreasing active force generation on return to shorter lengths. 3. A highly reproducible mechanically defined reference length, L(o), was obtained by fitting force-length data between 0.3 and 1.6 L(o) with a third-order polynomial where L = L(o) when dF/dL = 0. 4. Activation as assessed by myosin regulatory light chain (MRLC) phosphorylation increased with length in 100 microM histamine-stimulated tissues from 0.6 to 1.8 L(o). 5. Activation was constant in K(+)-depolarized and field-stimulated tissues from 1.0 to 1.8 L(o) allowing determination of the descending limb of the force-length relation to be assessed independently of activation. 6. The slope of the descending limb of the force-length relation was linear except at very long lengths, which often produced tissue damage. The slope was not statistically different from that estimated for sarcomeres in vertebrate skeletal muscle. 7. The medial ring preparation and the procedures used to define the reference length provide advantages for the measurement of length-dependent variables.

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.

Energetics of crossbridge phosphorylation and contraction in vascular smooth muscle.

Ca(2+)-dependent crossbridge phosphorylation is the primary mechanism governing crossbridge cycling in smooth muscle. A four-state crossbridge model in which phosphorylation is the only proposed regulatory mechanism was successful in predicting the mechanical properties of the swine carotid media including latch (sustained force with reduced crossbridge cycling). This model also predicts that the ATP consumption of crossbridge phosphorylation is approximately equal to that of crossbridge cycling and that ATP consumption will rise hyperbolically with increases in steady-state force. This review shows these predictions to be consistent with the available energetics data for the carotid media. The absolute energetic cost of covalent regulation is modest and less than the energy savings associated with latch. However, covalent regulation should reduce the total mechanical efficiency of smooth muscle relative to striated muscle.